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.

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.

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.

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.

[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.

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.

[Further study on heterogeneic basis of complement C8 beta deficiency].

OBJECTIVE: In Caucasian population, the most common molecular basis for C8 beta deficiency s a single C to T transition in exon 9 of C8 beta gene resulting in a stop codon. In previous family studies, two individuals were identified with C8 beta complete deficiency and were found to be only heterozygous for this mutation. This study was conducted by the present authors in search of other possible causes for these two C8 beta deficient individuals. METHODS: Using direct DNA sequence analysis of all exon-specific PCR products of the C8 beta gene from these two C8 beta deficient patients and their descendants. RESULTS: Two other C to T transitions at base 298 and 388 in exon 3 were detected, which could also create a termination codon. The descendants from one of the deficient patients were also analysed for the mutations, and it could be demonstrated that the two C to T mutations in exons 9 and 3 are segregating independently. CONCLUSION: These two mutations, which create a termination codon, are sufficient to explain the complete C8 beta deficiency in both patients.

The role of Fcgamma receptor polymorphisms and C3 in the immune defence against Neisseria meningitidis in complement-deficient individuals.

Individuals with either a late (C5-9) complement component deficiency (LCCD) or properdin deficiency are at increased risk to develop meningococcal disease, often due to serogroups W135 and Y. Anti-meningococcal defence in both LCCD persons and properdin-deficient individuals without bactericidal antibodies depends mainly on phagocytosis. Three types of opsonin receptors are involved in phagocytosis by polymorphonuclear cells (PMN). These represent the polymorphic FcgammaRIIa (CD32) and FcgammaRIIIb (CD16b) receptors, and the C3 receptor CR3 (CD11b/CD18). When the distribution of FcgammaRIIa and FcgammaRIIIb allotypes was assessed in 15 LCCD and in 15 properdin-deficient patients with/without previous meningococcal disease, we found the combination of FcgammaRIIa-R/R131 with FcgammaRIIIb-NA2/NA2 allotypes to be associated with previous meningococcal disease (odds ratio 13.9, Fisher's test P = 0.036). No such relation was observed in the properdin-deficient patients. The importance of FcgammaRIIa allotypes was also demonstrated using in vitro phagocytosis assays. PMN from FcgammaRIIa-R/R131 homozygous donors internalized IgG2 opsonized meningococci W135 significantly (P < 0.05) less than PMN from FcgammaRIIa-H/H131 donors. When properdin-deficient serum was tested, it was observed that reconstitution with properdin resulted in enhanced PMN phagocytosis of the W135 meningococci (P = 0.001). This enhanced phagocytosis was parallelled by an increase in C3 deposition onto the opsonized meningococci W135 (r = 0.6568, P = 0. 01). We conclude that the occurrence of meningococcal disease in LCCD patients is associated with certain FcgammaR allotypes. Properdin-deficient individuals are susceptible to meningococcal disease because of an insufficient C3 deposition on the surface of meningococci, resulting in insufficient phagocytosis.

Genetic basis of human complement C8 alpha-gamma deficiency.

Deficiency of the alpha-gamma subunit of the eighth component of complement (C8alpha-gammaD) is frequently associated with recurrent neisserial infections, especially meningitis caused by Neisseria meningitidis. We here report the molecular basis of C8alpha-gammaD in two unrelated Japanese subjects. Screening all 11 exons of the C8alpha gene and all 7 exons of the C8gamma gene and their boundaries by exon-specific PCR/single-strand conformation polymorphism demonstrated aberrant single-stranded DNA fragments in exon 2 of C8alpha gene in case 1 and in exons 2 and 9 of C8alpha gene in case 2. Nucleotide sequencing of the amplified DNA fragments in case 1 revealed a homozygous single-point mutation at the second exon-intron boundary, inactivating the universally conserved 5' splice site consensus sequence of the second intron (IVS2+1G-->T). Case 2 was a compound heterozygote for the splice junction mutation, IVS2+1G-->T, and a nonsense mutation at Arg394 (R394X). R394X was caused by a C to T transition at nucleotide 1407, the first nucleotide of the codon CGA for Arg394, leading to a stop codon TGA. No mutations were detected in the C8gamma gene by our method. Our results indicate that the pathogenesis of C8alpha-gammaD might be caused by heterogeneous molecular defects in the C8alpha gene.

Characterization of soluble terminal complement complex assembled in C8beta-deficient plasma and serum.

Sera genetically deficient in either the alpha-gamma or the beta-subunit of complement component C8 virtually lack haemolytic activity. We have studied the formation and the structural organization of the soluble terminal complement complex (TCC) assembled in these sera following activation with cobra venom factor (CVF). The TCC concentration in the activated C8alpha-gamma and C8beta-deficient samples was 0.2% and 4%, respectively, when compared with zymosan-activated normal serum. TCC was purified from the activated C8beta-deficient samples by affinity chromatography and analysed by immunoblotting and enzyme immunoassay. No C8beta was detected in one TCC preparation, while 7% of the normal level was present in the other. The level of the other terminal components, including that of C8alpha-gamma, was normal. The ability of C8alpha-gamma to promote the assembly of TCC in the presence of a limited amount of C8beta or in the apparent absence of this subunit was confirmed using purified components, by mixing C5b6 and either of the purified C8 subunits together with C7 and C9. These data show that soluble TCC can be formed in C8beta-deficient sera that contain little or no C8beta.

Terminal complement component deficiencies in Japan.

From the serological screening for complement component deficiencies, we found 2 subjects with inherited C5 deficiency (C5D), 4 with C6D, 6 with C7D, 4 with C81 (alpha-gamma subunit) D and 138 with C9D among 145,640 healthy Japanese blood donors. Recently, the genetic bases of some of the C6D, C7D, C81D and C9D Japanese individuals were elucidated using an exon-specific PCR-SSCP method followed by direct sequencing of the target exons.

C5b-7 and C5b-8 precursors of the membrane attack complex (C5b-9) are effective killers of E. coli J5 during serum incubation.

The finding that C9-deficient sera (C9D) can kill serum sensitive strains of Gram-negative bacteria by us and other investigators, questions the role of C9 in the membrane attack complex as necessary for cell death. In these studies we have demonstrated that C5b-8 complexes generated on E. coli J5 during incubation in C9-depleted and C9-neutralized sera are effective in killing Gram-negative bacteria. In the same study, we extended our investigations to show that the deposition of C5b-7 complexes (from C8-deficient [C8D], C8 depleted and C8-neutralized sera) is also effective in killing Gram-negative bacteria. In all cases, these studies demonstrated that when E. coli J5 was incubated with C8D, C9D and pooled normal human serum [PNHS], deposited C5b-9 complexes from PNHS produced more killing than C5b-7 or C5b-8 complexes alone. These experiments clearly demonstrated that C5b-7 and C5b-8 complexes are bactericidal and that multimeric C9 within C5b-9 is not an absolute requirement for inner membrane damage and cell death of Gram-negative bacteria.

Heterozygous C8beta complement deficiency does not predispose to meningococcal disease.

We have identified 42 Russian patients with homozygous C8beta complement component deficiency, all of whom had experienced at least one episode of systemic meningococcal disease. About 90% of these individuals have a C --> T exchange in exon 9, leading to a premature stop codon. If, like the homozygous-deficient state, heterozygous C8beta deficiency constitutes a risk factor for meningococcal disease, it would be expected to be detected with increased frequency among individuals suffering from this disease. Using allele-specific polymerase chain reaction (PCR), we studied 153 consecutive patients with meningococcal disease admitted to the Moscow Hospital for Infectious Diseases to determine the frequency of C8 null allele. No individuals with heterozygous C --> T exchange were identified among these 153 patients, despite the fact that seven persons were detected who had homozygous C8beta deficiency, caused by the same C --> T exchange in exon 9, and one patient who had C7 component deficiency. Thus, heterozygous deficiency, although more frequent than homozygous deficiency in the general population, does not result in a substantial increase in susceptibility to meningococcal disease.

Molecular, genetic, and functional analysis of homozygous C8 beta-chain deficiency in two siblings.

UNLABELLED: C8 deficiency is associated with an increased susceptibility to neisserial infections. We present a case of an 11 year old boy who suffered from infection with Neisseria meningitidis. Medical history of the patient and his family (n = 5) did not indicate any previous immunodeficiency symptoms. Results from the analysis of phagocyte and lymphocyte functions were within the normal range. No hemolytic activities of the classical (CH50) and the alternative (APH50) pathways of complement were measurable, and SC5b-9 protein complexes could not be detected in the patient's plasma. Further analysis by highly sensitive ELISA and functional assays revealed a complete deficiency of C8. Upon the reconstitution with purified C8 total hemolytic activity could be restored. SDS-PAGE and Western blot analysis established a deficiency of the C8 beta chain. Genetic analysis at the genomic DNA level demonstrated the common C-T mutation in exon 9 of the C8B gene. Family analysis presented the older sister with non-detectable function of C8 in serum, both parents with about half-normal C8 titres, and the younger sister with normal C8 function. The parents and both sisters were asymptomatic, although the older of the sisters presented with the same complete C8 beta-chain deficiency as the patient described. IN CONCLUSION: the common C-T mutation in the C8B genes is the genetic basis of C8 beta-chain deficiency in two members of this Bosnian family.