French national diagnostic and care protocol for Kawasaki disease.

Kawasaki disease (KD) is an acute vasculitis with a particular tropism for the coronary arteries. KD mainly affects male children between 6 months and 5 years of age. The diagnosis is clinical, based on the international American Heart Association criteria. It should be systematically considered in children with a fever, either of 5 days or more, or of 3 days if all other criteria are present. It is important to note that most children present with marked irritability and may have digestive signs. Although the biological inflammatory response is not specific, it is of great value for the diagnosis. Because of the difficulty of recognising incomplete or atypical forms of KD, and the need for urgent treatment, the child should be referred to a paediatric hospital as soon as the diagnosis is suspected. In the event of signs of heart failure (pallor, tachycardia, polypnea, sweating, hepatomegaly, unstable blood pressure), medical transfer to an intensive care unit (ICU) is essential. The standard treatment is an infusion of IVIG combined with aspirin (before 10 days of fever, and for a minimum of 6 weeks), which reduces the risk of coronary aneurysms. In case of coronary involvement, antiplatelet therapy can be maintained for life. In case of a giant aneurysm, anticoagulant treatment is added to the antiplatelet agent. The prognosis of KD is generally good and most children recover without sequelae. The prognosis in children with initial coronary involvement depends on the progression of the cardiac anomalies, which are monitored during careful specialised cardiological follow-up.

Early-onset granulomatous arthritis, uveitis and skin rash: characterization of skin involvement in Blau syndrome.

BACKGROUND: Blau syndrome (BS) is a rare monogenic autoinflammatory disease caused by NOD2 mutations. BS classically presents in early childhood as a triad of granulomatous polyarthritis, uveitis and skin involvement. Joint and ocular involvement have been characterized by several cohort studies but only very little data are available on skin lesions. OBJECTIVES: We aimed to provide a detailed clinical and microscopic analysis of skin manifestations and to study whether they may contribute to an early diagnosis. METHODS: We conducted a retrospective multicentre study in a French cohort of 21 patients diagnosed with genetically confirmed BS. RESULTS: Skin involvement was the first clinical manifestation of BS in 15/16 patients with dermatological manifestations. The presence of skin lesions was associated with significant shorter age at diagnosis (P = 0.03) and diagnostic delay (P = 0.04). Dermatological assessment allowed an earlier diagnosis (P = 0.001) and reduces the diagnostic delay (P = 0.007). Early skin lesions had a homogeneous, stereotypical clinical presentation, namely non-confluent erythematous or pigmented millimetric papules in 13/14(93%) patients. In contrast, skin lesions occurring during later disease stages had a more heterogeneous clinical presentation, including ichthyosiform dermatosis, panniculitis, livedoid lesions and vasculitis. Whatever their time of occurrence and the clinical aspect, all biopsied showed histologically presence of granuloma. CONCLUSION: Skin involvement in BS is the earliest clinical manifestation of the BS in the large majority of patients. The recognition of dermatological manifestations as granulomatous skin lesions and early dermatological expertise are the key to an early diagnosis of BS. In view of our results, it seems reasonable to propose a simplified view of skin lesions of BS in which the granuloma is the key structure.

[Mevalonate kinase deficiency in 2016]. / Le déficit en mévalonate kinase en 2016.

Mevalonate kinase deficiency is a rare, autosomal recessive, auto-inflammatory disease. This results from mutations in the gene MVK coding for the enzyme mevalonate kinase. This enzyme is involved in cholesterol and isoprenoids synthesis. Depending partially of the residual activity of the mevalonate kinase, the clinical spectrum realizes a continuum which extends from the mild phenotype of the hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) to a lethal form of mevalonic aciduria. The HIDS is characterized by recurrent episodes of fever with an intense inflammatory syndrome, accompanied with lymphadenopathy, abdominal pain, diarrhea, arthralgia, hepatomegaly, splenomegaly and skin rash. The first attack more frequently takes place in the first year of life, even during the neonatal period, where it can be confused with a maternofetal infection. There is furthermore in mevalonate aciduria a psychomotor retardation, a failure to thrive, a cerebellar ataxia, a dysmorphic syndrome and a reduction of the visual acuity. The diagnosis is based on the mevalonic aciduria during febrile attack. Genetics confirm the diagnosis in more than 80 % of the cases. The dosage of IgD, low sensitive and specific, has no interest. There is no reference treatment. The less severe forms can be treated by non-steroidal anti-inflammatory drugs or steroids during febrile attacks. The most severe patients can be treated by biotherapy: antagonists of IL-1, TNF-α and IL-6.

[Value of whole-body MRI in vertebral fractures]. / Intérêt de l'IRM corps entier dans les fractures vertébrales.

Chronic recurrent multifocal osteomyelitis (CRMO) is a rare autoinflammatory disease in children. Pathological vertebral fracture may be the first symptom revealing this disease. We describe the case of a 14-year-old boy, with no significant past medical history, who had a sudden dorsal pain after carrying a friend on his back. Plain radiographs and MRI showed fractures of the superior endplate of T5 and T6 associated with a mild degree of kyphosis. MRI allowed ruling out discitis. The diagnostic hypotheses raised were cancer (lymphoma, leukemia), Langerhans cell histiocytosis, osteogenesis imperfecta, and CRMO. A whole-body MRI (wbMRI) was performed and disclosed several clinically silent signal abnormalities in key sites of CRMO (pelvic bone and tibial metaphyses). We point out that CRMO should be systematically added to the list of possible diseases in case of vertebral fracture. In this perspective, wbMRI is a major noninvasive tool to assess the diagnosis of CRMO, and allows avoiding a bone biopsy in most cases.

[Tocilizumab: experience in a French rheumatological pediatric center]. / Traitement par tocilizumab : expérience d'un centre de rhumatologie pédiatrique.

Tocilizumab (TCZ) is an anti-interleukin-6-receptor antibody. The blockade of IL-6 is used as a strategy for the treatment of systemic juvenile idiopathic arthritis (S-JIA) and multicentric Castleman disease (MCD). In this study, we describe the tolerability profile of tocilizumab in eight children followed in a pediatric rheumatology department. Six patients were treated for S-JIA and two for a MCD. They received doses of TCZ between 8 and 12mg/kg of body weight depending on their disease. Infusions were received every 2-4 weeks. The mean duration of treatment was 32.9 months (14 months to 4.5 years). Clinical adverse events were all mild or moderate. No cases of macrophage activation syndrome and no anaphylactic reactions were reported. TCZ was never stopped for a clinical adverse event. Neutropenia was the most common biological adverse event, sometimes requiring dose adjustments. Thrombopenia, lymphopenia, and increased liver enzymes were reported as well, but treatment was not modified. All these biological adverse events were not complicated by any clinical manifestation. In conclusion, TCZ had a good tolerability profile in these eight patients with partial or total efficacy. Despite this advantageous profile, TCZ should be closely monitored because of the potential severity of adverse events. Moreover, long-term safety has still not been assessed.

[Management of monoarthritis in children]. / Conduite à tenir devant une monoarthrite chez l'enfant.

Joint swelling is common in childhood. The pattern of presentation, the duration and the location may reveal sometimes monoarthritis. A detailed clinical history, thorough clinical examination, and sometimes complementary tests are needed to reach the correct diagnosis. The single most important investigation in a child with acute monoarthritis is joint aspiration to rule out septic arthritis that may destroy the joint within few hours. Serum inflammatory markers, antinuclear antibody, tuberculosis testing, and imaging (in specific cases) play an important role in making the diagnosis. This article presents the clinical approach to the diagnosis of monoarthritis as well as the different causes of monoarthritis in children.

[Intravenous immunoglobulins in autoimmune and inflammatory disorders: beyond a simple substitution]. / Immunoglobulines intraveineuses dans les maladies auto-immunes et inflammatoires: au-delà d'une simple substitution.

Despite their widespread use since many years in autoimmune and inflammatory disorders, the mechanisms of action of IVIg have not been completely understood. These mechanisms depend on Fc and/or F(ab')2. IVIg interacts with the different components of the immune system: Fc receptors, complement, cytokines, T and B lymphocytes, dendritic cells, granulocytes and NK cells. Here, we discuss the recent advances in the understanding of the mechanisms of action of IVIg, in particular the importance of the sialylated Fc fragment. These advances maybe help us conceive better therapeutic strategies against autoimmune and inflammatory disorders.

Identification of vaccine candidates against serogroup B meningococcus by whole-genome sequencing.

Neisseria meningitidis is a major cause of bacterial septicemia and meningitis. Sequence variation of surface-exposed proteins and cross-reactivity of the serogroup B capsular polysaccharide with human tissues have hampered efforts to develop a successful vaccine. To overcome these obstacles, the entire genome sequence of a virulent serogroup B strain (MC58) was used to identify vaccine candidates. A total of 350 candidate antigens were expressed in Escherichia coli, purified, and used to immunize mice. The sera allowed the identification of proteins that are surface exposed, that are conserved in sequence across a range of strains, and that induce a bactericidal antibody response, a property known to correlate with vaccine efficacy in humans.

Comparison of secretion of a hepatitis C virus glycoprotein in Saccharomyces cerevisiae and Kluyveromyces lactis.

A C-terminally truncated form of the hepatitis C virus (HCV) putative envelope glycoprotein E2 was expressed in two yeast species, Saccharomyces cerevisiae and Kluyveromyces lactis, using a yeast signal peptide sequence to direct the viral glycoprotein to the endoplasmic reticulum (ER) pathway of secretion. Characterization of secreted E2 showed that the protein is endoglycosidase-H-sensitive in both yeasts. Moreover, in vivo inhibition of glycosylation with tunicamycin prevented secretion of E2 and showed that, of its 11 putative N-linked glycosylation sites, at least eight were core-glycosylated. Analysis of the heterologous glycoprotein by SDS-PAGE under nonreducing conditions and by gel filtration demonstrated the formation of multiple disulphides, which resulted in secretion of heterogeneous aggregates with an average molecular mass of 770-1000 kDa in both yeasts. However, variations were observed in the binding of the glycoprotein secreted by the two yeasts to a mannose-specific lectin, and also in its reactivity with anti-E2-specific antibodies. This denotes differences between the two yeasts in folding and/or modification of the E2 glycoprotein.

Integration of minitransposons for expression of the Escherichia coli elt genes at a preferred site in Salmonella typhimurium identifies a novel putative fimbrial locus.

An asd-complementing mini-Tn5 transposon was constructed for random insertion of the Escherichia coli LT enterotoxin genes (elt) into the genome of Deltaasd attenuated strains of Salmonella typhimurium. Transfer of the minitransposon to different S. typhimurium strains resulted in random integration only in strain chi4072, while in strain chi3987, which harbours the virulence plasmid, over 20% of the insertions occurred at the same site. Expression of elt was found to be highest in Salmonella isolates carrying the mini-Tn5 integrated at the preferred site, which was mapped to an uncharacterised region of the virulence plasmid. Sequence analysis of the integration site showed that it lies within an open reading frame with sequence similarity to E. coli leuO and contiguous to a novel fimbrial locus.

Levels of expression and immunogenicity of attenuated Salmonella enterica serovar typhimurium strains expressing Escherichia coli mutant heat-labile enterotoxin.

The effects of heterologous gene dosage as well as Salmonella typhimurium strain variability on immune response toward both the heterologous antigen, the nontoxic mutant of the Escherichia coli heat-labile enterotoxin LTK63, and the carrier Salmonella strain have been analyzed. Effects of a single integration into the host DNA and different-copy-number episomal vectors were compared in S. typhimurium delta cya delta crp delta asd strains of two different serotypes, UK-1 and SR-11. Expression of the enterotoxin in the different Salmonella isolates in vitro was found to vary considerably and, for the episomal vectors, to correlate with the plasmid copy number. LTK63-specific serum immunoglobulin G (IgG) and mucosal immunoglobulin A (IgA) antibodies were highest in mice immunized with the high-level-expression strain. High anti-LTK63 IgG and IgA titers were found to correspond to higher anti-Salmonella immunity, suggesting that LTK63 exerts an adjuvant effect on response to the carrier. Statistically significant differences in anti-LTK63 immune response were observed between groups of mice immunized with the attenuated delta cya delta crp UK-1 and SR-11 derivatives producing the antigen at the same rate. These data indicate that the same attenuation in S. typhimurium strains of different genetic backgrounds can influence significantly the immune response toward the heterologous antigen. Moreover, delivery of the LTK63 enterotoxin to the immune system by attenuated S. typhimurium strains is effective only when synthesis of the antigen is very high during the initial phase of invasion, while persistence of the S. typhimurium strain in deep tissues has only marginal influence.

Synthetic alleles at position 121 define a functional domain of human interleukin-1 beta.

The non-conservative substitution of the tyrosine residue at position 121 of human interleukin-1 beta (IL-1 beta) generates protein mutants showing strong reduction of the capacity to induce (a) prostaglandin E2 (PGE2) release from fibroblasts and smooth muscle cells, (b) murine T-cells proliferation and (c) activation of interleukin-6 (IL-6) gene expression. It is generally accepted that these functions are mediated by the type-I interleukin-1 receptor (IL-1RI). However, the mutant proteins maintain the binding affinity to the types-I and II IL-1 receptors, which is the same as the control IL-1 beta, suggesting that this amino acid substitution does not alter the structure of the molecule, except locally. Thus we have identified a new functional site of IL-1 beta different from the known receptor binding region, responsible for fundamental IL-1 beta functions. Moreover, we show that the same mutants maintain at least two hypothalamic functions, that is, the in vitro short-term PGE2 release from rat hypothalamus and the induction of fever in rabbits. This result suggests that there is yet another site of the molecule responsible for the hypothalamic functions, implying that multiple active sites on the IL-1 beta molecule, possibly binding to more than one receptor chain, trigger different signals.

Gastric antisecretory and anti-ulcer actions of IL-1 in rat involve different IL-1 receptor types.

Limited knowledge exists concerning the interleukin-1 (IL-1) receptor type (IL-1RT) mediating the potent antisecretory and gastro-protective actions of IL-1. In the present study, the gastric actions of IL-1 beta and two related mutant proteins, yIL-1 beta delta 4, an analogue that preferentially binds to IL-1-RTII, and mutant yIL-1 beta N7/Q, an analogue that has equal affinity as IL-1 beta for IL-1RTI and IL-1RTII, have been compared. Modulation of IL-1 gastric actions were also investigated using monoclonal antibody (MAb) preparations raised against IL-1RTI or IL-1RTII. In the pylorus-ligated rat, yIL-1 beta delta 4, yIL-1 beta N7/Q, and IL-1 beta (all at 1 microgram/kg ip) reduced gastric acid secretion (50, 79, and 78%, respectively), indicating the importance of IL-1RTII binding for antisecretory activity. This was further substantiated in experiments using the MAb preparations, which showed that IL-1 beta (1 microgram/kg ip) antisecretory activity was reversed by MAb IL-1RTII (10-50 micrograms/kg sc) but not by MAb IL-1RTI (50 micrograms/kg sc). In contrast, at dosages 10-fold higher (10 micrograms/kg ip) than that used in the study to inhibit acid secretion, IL-1 beta and yIL-1N7/Q equally reduced (approximately 80%) indomethacin-induced gastric damage, but yIL-1 beta delta 4 was ineffective. The results using yIL-1 beta delta 4 indicated that impairment of IL-1RTI binding capacity appeared to be paralleled by a decreased gastroprotective effect.(ABSTRACT TRUNCATED AT 250 WORDS)

A kluyveromyces lactis gene homologue to AAC2 complements the Saccaromyces cerevisiae op1 mutation.

A mutation (op1) in the Saccharomyces cerevisiae AAC2 gene, which codes for the most abundant ADP/ATP carrier isoform, results in lack of mitochondrial-dependent growth and in an as yet unexplained petite-negative phenotype. A gene from the petite-negative yeast Kluyveromyces lactis has been isolated by complementing in multicopy the op1 mutation of S. cerevisiae. This gene, designated KIAAC, can complement the petite-negative phenotype of op1 as well as its inability to grow on nonfermentable carbon sources. KIAAC contains a 915-base pair open reading frame coding for a protein of 305 amino acids which shows a high degree of identity to AAC2. The K. lactis ADP/ATP carrier also shares identity with other known ADP/ATP carrier sequences. In particular, the degree of identity of KIAAC is higher with the Neurospora crassa carrier (80.1%) than with AAC1 (76.6%). The nucleotide sequence upstream of the KIAAC coding region was found to contain a long DNA segment with no coding potential, but presenting features of highly regulated promoter sequences.

Induction of cytochrome P-450 by 5-methoxypsoralen in the yeast Saccharomyces cerevisiae.

Yeast cells (D7 strain) incubated in the presence of 5-methoxypsoralen (5-MOP) increase the activity of the monooxygenase system cytochrome P-450 dependent (cytochrome P-450 level and 7-ethoxycoumarin-O-diethylase activity). Northern analysis of cytochrome P-450 specific RNA shows that 5-MOP treatments induce an increase in mRNA. The induction of cytochrome P-450 appears to occur at the transcriptional level. The capacity of 5-MOP to induce the cytochrome P-450 system in eukaryotic cells, in which it is known to be involved in the metabolism of the psoralen, may decrease the availability of the compound for photo-induced genotoxic reactions, which may explain the good tolerance in patients.

Loop substitution as a tool to identify active sites of interleukin-1 beta.

By computer analysis of the amino acid sequence of human interleukin-1 beta (IL-1 beta) and of the human type I IL-1 receptor (IL-1RI), we have identified two hydropathically complementary peptides (Fassina, G., Roller, P. P., Olson, A. D., Thorgeirsson, S. S., and Omichinski, J. G. (1989) J. Biol. Chem. 264, 11252-11257) capable of binding to each other. The sequence of the IL-1 beta peptide corresponds to that of residues 88-99 (loop 7 of the crystal structure of mature IL-1 beta) of mature IL-1 beta, one of the exposed and highly charged regions of the molecule. The substitution of this loop with an amino acid sequence of the same length but different hydropathic profile generates a mutant with drastically reduced binding activity to IL-1RI. In contrast, the binding affinity to the type II IL-1R (IL-1RII) is the same as that of wild type IL-1 beta. The results show that 1) loop 7 is part of the binding site of IL-1 beta to IL-1RI, but not to IL-1RII. 2) The structure of the mutant protein is not grossly altered except locally at the position of the substituted loop. 3) The substitution of amino acids by site-directed mutagenesis of the loop 7 region generates mutants with binding affinity constants slightly lower than that of wild type IL-1 beta and not comparable to that of the loop substitution analogue. 4. All mutants analyzed, including the loop substitutions, are biologically active, confirming the structural integrity of the proteins. We propose a binding site in which the cooperation of several low energy bonds extended over a wide area results in a high affinity complex between IL-1 and the type I receptor.

Inhibition of yeast cytochrome P-450 by ammonium metavanadate.

Incubation of diploid D7 strain cells of Saccharomyces cerevisiae (grown in 20% glucose) in the presence of ammonium metavanadate (AMV) led to a decrease in the cytochrome P-450-dependent monooxygenase system (cytochrome P-450 level and 7-ethoxycoumarin O-deethylase). The electrophoretic analysis of microsomal fractions of yeast cells treated with metavanadate revealed a decrease in the intensity of the bands corresponding to a M(r) in the range of 51,000-58,000 Da compared with those observed in controls, i.e., cells grown in 20% glucose. Analysis of the cytochrome P-450 transcript showed that AMV treatment reduced the mRNA level. Our results suggest that AMV inhibits the yeast cytochrome P-450 system by acting at both the pre- and post-transcriptional levels.

Conceivable difference in the anti-inflammatory mechanisms of lipocortins 1 and 5.

Human recombinant lipocortins (LCT) 1 and 5 have been expressed in a yeast secretion vector and purified by ion exchange chromatography. The action of the proteins has been investigated in two models of experimental acute inflammation in the rat: carrageenin induced paw oedema and zymosan induced pleurisy. The effects of the proteins on PGE(2) release in vitro by rat macrophages stimulated with zymosan and on rat neutrophil chemotaxis induced by FMLP have also been assessed. LCT-1 significantly inhibited both paw swelling in carrageenin oedema and leukocyte migration in zymosan pleurisy. Moreover it showed a dose dependent, inhibitory effect on PGE(2) release. Neutrophil chemotaxis was only weakly affected by LCT-1. Conversely LCT-5 did not reduce carrageenin oedema and slightly inhibited PGE(2) release, but showed profound, dose dependent inhibitory activity on leukocyte migration in zymosan pleurisy and on neutrophil chemotaxis. These data suggest that LCT-1 acts mainly by interfering with arachidonic acid metabolism via the inhibition of phospholipase A(2). The anti-inflammatory activity of LCT-5, at variance with LCT-1, may be due to a direct effect on cell motility in addition to the interference with arachidonic acid metabolism.

Expression of cytochrome P450 in yeast after different chemical treatments.

In Saccharomyces cerevisiae a number of chemical agents induce synthesis of cytochrome P450. A cytochrome P450 gene has been well characterized in this yeast: CYP51, which codes for a constitutive enzyme involved in the 14 alpha-demethylation of lanosterol, a key step in the biosynthesis of ergosterol. In this work, we have analysed the level of transcription of the CYP51 gene in correlation with cytochrome P450 enzymatic activity after treatment with several chemical agents known to interact with cytochrome P450. Using as a probe a DNA fragment whose identity to the CYP51 gene was established by sequence analysis and mapping on chromosome VIII, a unique RNA species was observed in all treatment samples. The increased level found for this transcript in cells treated with ethanol, 20% glucose, phenobarbital or 5-methoxypsoralen correlates with the levels of induction in cytochrome P450 enzymatic activity measured in cells grown under the same conditions, indicating that induction of cytochrome P450 by these treatments is regulated at the transcriptional level.

Kluyveromyces lactis rDNA as a target for multiple integration by homologous recombination.

Gene targeting to a single chromosomal locus has been extensively used in Saccharomyces cerevisiae. In this study, we have analyzed targeting of a repetitive sequence, the 25S rDNA gene, to the chromosomal rDNA cluster of Kluyveromyces lactis by the use of a replacement vector. We have obtained K. lactis transformants carrying multiple copies of the replacement cassette inserted into the rDNA chromosomal locus. Analysis of several transformants has shown that the number of integrated copies could range from 4 to 40. Moreover, the distribution of integration sites within the rDNA locus was found to differ in most transformants. Single-copy integration at multiple sites, rather than multicopy integration at a very limited number of sites, was found to be the most frequent event. Also, in most transformants, integration sites were distributed at random as well as in an orderly fashion, i.e., in contiguous or alternate rDNA repeats, suggesting that amplification of the integrated sequences, rather than multiple integration events, may account for the copy number of insertions.