René Pires

The Role of Particular Strains of Neisseria meningitidis in Meningococcal Arthritis, Pericarditis, and Pneumonia

The clinical presentations of meningococcal diseases other than meningitis or meningococcemia may lead to erroneous diagnosis. Although several reports have described unusual meningococcal diseases, the Neisseria meningitidis strains involved in these forms have been poorly characterized. In this study, meningococcal arthritis and pericarditis were confirmed by isolation of N. meningitidis and/or detection of meningococcal DNA in synovial or pericardial fluid, respectively, and meningococcal pneumonia was detected by isolation of N. meningitidis from blood. From 1999 through 2002, meningococcal disease was bacteriologically confirmed in 26 cases of arthritis, 6 cases of pericarditis, and 33 cases of pneumonia by the National Reference Center for the Meningococci in Paris. We found a statistically significant association between strains of serogroup W135, mostly of the clonal complex ET-37, and arthritis. Pneumonia was most frequently diagnosed in patients aged >70 years, and 54.5% of the strains belonged to serogroup W135, although these strains had heterogeneous phenotypes. Bacteremia is a key step in the pathophysiology of meningococcal disease and precedes any form of invasive infection.

Neisseria meningitidis Strains Isolated from Invasive Infections in France (1999–2002): Phenotypes and Antibiotic Susceptibility Patterns

Infections due to Neisseria meningitidis are a major public health concern. In France, during 1999-2002, a total of 2167 clinical isolates of N. meningitidis from invasive infections were studied at the National Reference Center for Meningococci (Paris). Serogroup B strains were the most common (58%), followed by serogroup C strains (29%) and serogroup W135 strains (8%). Various phenotypes were observed, reflecting heterogeneity in the meningococcal population. Strains were susceptible to antibiotics currently used for treatment and chemoprophylaxis of meningococcal infections. However, the prevalence of meningococci with reduced susceptibility to penicillin is increasing. Such strains were heterogeneous and accounted for approximately 30% of isolates during this period, warranting continued surveillance of this phenomenon.

A model of meningococcal bacteremia after respiratory superinfection in influenza A virus-infected mice

We developed a model of sequential influenza A virus (IAV)-Neisseria meningitidis serogroup C (Nm) infection in BALB/c mice. Mice infected intranasally with a sublethal IAV dose (260 pfu) were superinfected intranasally with Nm. Fatal meningococcal pneumonia and bacteremia were observed in IAV-infected mice superinfected with Nm on day 7, but not in those superinfected on day 10. The susceptibility of mice to Nm superinfection was correlated with the peak interferon-gamma production in the lungs and decrease in IAV load. After Nm challenge, both IAV-infected and uninfected control mice produced the inflammatory cytokines interleukin (IL)-1 and IL-6. However, IL-10 was detected in susceptible mice superinfected on day 7 after IAV infection, but not in resistant mice. This model of dual IAV-Nm infection was also used to evaluate the role of bacterial virulence factors in the synthesis of the capsule. A capsule-defective mutant was cleared from the lungs, whereas a mutant inactivated for the crgA gene, negatively regulating expression of the pili and capsule, upon contact with host cells, retained invasiveness. Therefore, this model of meningococcal disease in adult mice reproduces the pathogenesis of human meningococcemia with fatal sepsis, and is useful for analyzing known or new genes identified in genomic studies.

A modified gel filtration technique producing an unusual exclusion volume of IgM: a simple way of preparing monoclonal IgM

The vast majority of monoclonal IgM proteins is eluted just before the total volume of the column when filtered through G200 Sephadex or S200 Sephacryl gels equilibrated in a 0.005 M phosphate buffer but eluted with 0.05 M phosphate buffer containing 1.7 M NaCl. This unusual behaviour in low-ionic buffer is probably due to the poor solubility of IgM in diluted buffers. It allows a 1-step purification procedure under mild conditions and is suitable for both large and small scale preparations.

NON-IMMUNE VH-BINDING SPECIFICITY OF HUMAN PROTEIN FV

The specificity of human F(ab)‐binding Protein Fv (previously called Protein F), a sialoprotein released into the digestive tract mainly during hepatitis, was investigated with fragments or chains of monoclonal immunoglobulins. Protein Fv bound an unreduced H‐chain dimer of a monoclonal human IgA2m(I) molecule but neither the corresponding L‐chain dimer, nor several Bence‐Jones molecules. Using enzymatic subfragments of F(ab)μ(, or F(ab′)2γ, a significant binding was observed with Fv fragments (VH + VL), while Fb fragments (CHI+CL) were inactive. Taken altogether, these results prove that the structure recognized by Protein Fv is located in the VH domain. This structure probably involves discontinuous epitopes linked by a disulphide bond, which are destroyed by combined reduction and dissociation. Protein Fv does not interfere with the antigen‐binding site, since there was no reciprocal inhibition with the antigen‐antibody reaction.

IgM reassociation in the absence of J-chain

Human monoclonal IgM pentamers with different biophysical properties (euglobulin, pseudoglobulin or cryoglobulin) were reduced and reassociated in the absence of J-chain. Reassembly occurred for 50-82% of the monomers. The reassociated molecules consisted of covalent oligomers and pentamers. The deficiency of J-chain (estimated to be less than 0.17% of normal) was shown by alkaline-urea overloaded gel electrophoresis followed by silver staining. The addition of exogenous J-chain, from polymeric IgA or IgM, did not significantly modify the reassembly ratio. Thus J-chain does not seem to be an absolute requirement for IgM polymerization.

HIGH AFFINITY SERUM-DERIVED FAB FRAGMENTS AS ANOTHER SOURCE OF ANTIBODIES IN THE GUT LUMEN OF BOTH NEONATES AND ADULTS

The authors have investigated the presence of serum‐derived immunoglobulin G (IgG) fragments in the human intestine at various ages, these fragments possibly representing another source of antibodies in addition to secretory IgA (SIgA). Fab fragments of the γ isotype were found to be the major molecular form of immunoglobulins in the meconium (median value: 3.7 mg/g of stools), as compared with Fabα (75 μg/g) and IgM (2.6 μg/g). These fragments provided by molecules of the maternal serum displayed a strong antibody activity to the tetanus toxoid and were also found in the stools of 1‐week‐old babies fed with formula milk. The release of serum antibodies into the digestive lumen occurs largely via hepatobiliary secretions, as suggested by the presence of IgG antitoxins in the bile of children operated on extrahepatic biliary atresia. In adults, the Fab antitoxins were also detected in most stool extracts. Affinity of these molecules was found to be similar to that of their serum counterpart with a Ka of 2.1×1010 M1 (median value). These mucosal antibody fragments, associated with the normal pathway of serum IgG catabolism, could provide additional immune defences against pathogens, and be of importance to supplement an immature or deficient secretory immune system.

Non-Immune Binding of Human Protein Fv to Immunoglobulins from Various Mammalian and Non-Mammalian Species

Reactivity of the secretory protein Fv with immunoglobulins (Ig) from various species of vertebrates was investigated. Binding was observed throughout all taxonomic classes: mammalian, avian, reptilian, amphibian and fish. Contrasting with tins wide spectrum, no significant binding was found with most mammalian ungulates, such as horse (Perissodactl), cow, sheep and goat (Artiodactyls), Nevertheless disruption of’the hydrogen bonds of Ig allowed these non‐reactive molecules lo bind. Such a conserved reactivity dining evolution, and our previous data on the effect of the cleavage of the intra‐chain disulphide bonds, suggest that protein Fv recognizes a discontinuous framework structure involving both the FRI and FR3 regions in the variable domain of the heavy chain of Ig.

An Endogenous Superantigen in the Rat Gut Lumen as a Model to Study the Role of Human Protein-Fv

Protein‐Fv (pFv) is a human B superantigen which can bind the variable domain (VH) of the heavy chain of immunoglobulins (Igs) and enhance the effector functions of secretory antibodies in the gut lumen. This study describes a rat molecule related to pFv by a similar specificity to the human VH3 domain. Investigation of the content of different gut segments shows that the rat pFv is usually hidden by its binding to local Igs to form macromolecular complexes similar to the immune fortresses described in normal humans. Furthermore, the pFv level generally increases from the jejunum to the colon in parallel with the decreasing water dilution, and finally a discontinuous presence can occur along the digestive tract. Detection of this molecule in the fetus proves its non‐microbial origin. Variations of the release of pFv, according to the breeding and to littermating, suggest the influence of external factors. This animal model allows the development of a study of the functions of pFv in vivo using a current laboratory species and has already provided evidence that the synthesis of this important molecule of the secretory immune system is regulated by environmental factors.

ACTIVATION OF THE CLASSICAL PATHWAY OF COMPLEMENT BY NON-IMMUNE COMPLEXES OF IMMUNOGLOBULINS WITH HUMAN PROTEIN FV (FV FRAGMENT-BINDING PROTEIN)

Protein Ev, a human sialoprotein recently described in the stools of patients suffering from liver diseases, binds the variable domain of the heavy chains of immunoglobulins. We show here that preincubation of this protein with monoclonal human IgG1 and IgM activates the complement cascade by forming nonimmune complexes, as evidenced by haemolysis inhibition of antibody‐coated sheep erythrocytes. As negative controls, no inhibition was observed after incubation either with immunoglobulins or with protein Fv alone, and with the protein Ev‐depleted medium. Activation was due to the binding of immunoglobulins with protein Fv, as shown by inhibition of protein Fv‐induced agglutination of the sensitized erythrocytes in the absence of complement. Activation of the classical pathway was demonstrated both by using a buman IgG4 or F(ab)2 fragments unable to activate C1q, and by Western blot analysis of the cleavage of C4 in human serum. These results confirm that protein Fv‐binding mimicks antigen‐antibody reactions, and suggest its involvement in hepatitis‐associated vasculitis and in local lesions of some infiammatory gut diseases.