Mycoplasma pneumoniae Compared to Streptococcus pneumoniae Avoids Induction of Proinflammatory Epithelial Cell Responses despite Robustly Inducing TLR2 Signaling.

Mycoplasma pneumoniae and Streptococcus pneumoniae are the most common bacterial causes of pneumonia in children. The clinical characteristics of pneumonia differ significantly between the two bacteria. We aimed to elucidate the differences in pathogenesis between M. pneumoniae and S. pneumoniae by characterizing the respiratory epithelial cell immune response to both pathogens. Using primary human bronchial epithelial cells in air-liquid interface cultures, we observed lower production of the proinflammatory cytokines interleukin-6 (IL-6) and IL-8 in response to M. pneumoniae than to S. pneumoniae. In contrast to the differences in proinflammatory cytokine production, Toll-like receptor 2 (TLR2)-mediated signaling in response to M. pneumoniae was stronger than to S. pneumoniae. This difference largely depended on TLR1 and not TLR6. We found that M. pneumoniae, but not S. pneumoniae, also induced signaling of TLR10, a coreceptor of TLR2 that has inhibitory properties. M. pneumoniae-induced TLR10 signaling on airway epithelial cells was partially responsible for low IL-8 production, as blocking TLR10 by specific antibodies increased cytokine production. M. pneumoniae maintained Th2-associated cytokine production by epithelial cells, which concurs with the known association of M. pneumoniae infection with asthma. M. pneumoniae left IL-33 levels unchanged, whereas S. pneumoniae downregulated IL-33 production both under homeostatic and Th2-promoting conditions. By directly comparing M. pneumoniae and S. pneumoniae, we demonstrate that M. pneumoniae avoids induction of proinflammatory cytokine response despite its ability to induce robust TLR2 signaling. Our new findings suggest that this apparent paradox may be partially explained by M. pneumoniae-induced signaling of TLR2/TLR10.

Brain-derived Neurotrophic Factor (BDNF) and gray matter volume in bipolar disorder.

INTRODUCTION: Bipolar Disorder (BD) is a severe psychiatric condition characterized by grey matter (GM) volumes reduction. Neurotrophic factors have been suggested to play a role in the neuroprogressive changes during the illness course. In particular peripheral brain-derived neurotrophic factor (BDNF) has been proposed as a potential biomarker related to disease activity and neuroprogression in BD. The aim of our study was to investigate if serum levels of BDNF are associated with GM volumes in BD patients and healthy controls (HC). METHODS: We studied 36 inpatients affected by a major depressive episode in course of BD type I and 17 HC. Analysis of variance was performed to investigate the effect of diagnosis on GM volumes in the whole brain. Threshold for significance was P<0.05, Family Wise Error (FWE) corrected for multiple comparisons. All the analyses were controlled for the effect of nuisance covariates known to influence GM volumes, such as age, gender and lithium treatment. RESULTS: BD patients showed significantly higher serum BDNF levels compared with HC. Reduced GM volumes in BD patients compared to HC were observed in several brain areas, encompassing the caudate head, superior temporal gyrus, insula, fusiform gyrus, parahippocampal gyrus, and anterior cingulate cortex. The interaction analysis between BDNF levels and diagnosis showed a significant effect in the middle frontal gyrus. HC reported higher BDNF levels associated with higher GM volumes, whereas no association between BDNF and GM volumes was observed in BD. DISCUSSION: Our study seems to suggest that although the production of BDNF is increased in BD possibly to prevent and repair neural damage, its effects could be hampered by underlying neuroinflammatory processes interfering with the neurodevelopmental role of BDNF.

Distinct SagA from Hospital-Associated Clade A1 Enterococcus faecium Strains Contributes to Biofilm Formation.

Enterococcus faecium is an important nosocomial pathogen causing biofilm-mediated infections. Elucidation of E. faecium biofilm pathogenesis is pivotal for the development of new strategies to treat these infections. In several bacteria, extracellular DNA (eDNA) and proteins act as matrix components contributing to biofilm development. In this study, we investigated biofilm formation capacity and the roles of eDNA and secreted proteins for 83 E. faecium strains with different phylogenetic origins that clustered in clade A1 and clade B. Although there was no significant difference in biofilm formation between E. faecium strains from these two clades, the addition of DNase I or proteinase K to biofilms demonstrated that eDNA is essential for biofilm formation in most E. faecium strains, whereas proteolysis impacted primarily biofilms of E. faecium clade A1 strains. Secreted antigen A (SagA) was the most abundant protein in biofilms from E. faecium clade A1 and B strains, although its localization differed between the two groups. sagA was present in all sequenced E. faecium strains, with a consistent difference in the repeat region between the clades, which correlated with the susceptibility of biofilms to proteinase K. This indicates an association between the SagA variable repeat profile and the localization and contribution of SagA in E. faecium biofilms.

PerR controls peroxide- and iron-responsive expression of oxidative stress defense genes in Helicobacter hepaticus.

Chronic intestinal and hepatic colonization with the microaerophilic murine pathogen Helicobacter hepaticus can lead to a range of inflammatory diseases of the lower digestive tract. Colonization is associated with an active cellular immune response and production of oxygen radicals. During colonization, H. hepaticus needs to cope with and respond to oxidative stress, and here we report on the role of the H. hepaticus PerR-regulator (HH0942) in the expression of the peroxidase-encoding katA (HH0043) and ahpC (HH1564) genes. Transcription of katA and ahpC was induced by hydrogen peroxide, and by iron restriction of growth media. This iron- and hydrogen peroxide-responsive regulation of katA and ahpC was mediated at the transcriptional level, from promoters directly upstream of the genes. Inactivation of the perR gene resulted in constitutive, iron-independent high-level expression of the katA and ahpC transcripts and corresponding proteins. Finally, inactivation of the katA gene resulted in increased sensitivity of H. hepaticus to hydrogen peroxide and reduced aerotolerance. In H. hepaticus, iron metabolism and oxidative stress defense are intimately connected via the PerR regulatory protein. This regulatory pattern resembles that observed in the enteric pathogen Campylobacter jejuni, but contrasts with the pattern observed in the closely related human gastric pathogen Helicobacter pylori.

CYP21 and CYP21P variability in steroid 21-hydroxylase deficiency patients and in the general population in the Netherlands.

Steroid 21-hydroxylase deficiency is caused by defectiveness of the CYP21 gene. Such defects have presumably originated from interactions with the nearby CYP21P pseudogene during evolution. We studied these mechanisms by comparing the genetic variability of CYP21, CYP21P, and CYP21P/CYP21 hybrids (resulting from large-scale rearrangements) at eight mutation sites in a group of Dutch steroid 21-hydroxylase deficiency patients, their family members, and controls. The most common CYP21 defect in patients with salt-losing steroid 21-hydroxylase deficiency was a splice junction mutation in intron 2. The most common defect in the simple virilising form of the disease was ile72 --> asn. CYP21P showed considerable sequence variation in its central and 3' sections; the 5' section was constant. A single nucleotide (T) insert in exon 7 was found in all CYP21P genes. During the course of evolution, this was probably the third defect introduced into CYP21P after the splice junction mutation in intron 2 and the 8 bp deletion in exon 3. Gene conversions introducing CYP21-like sequences contribute to CYP21P variability. Such an event has occurred de novo in one family. A comparison of CYP21 and CYP21P mutations on the same chromosome shows that at least some of the small-scale gene conversions that supposedly transfer defects to CYP21 involve interaction between homologous chromosomes. The majority of the putative CYP21P-CYP21 transitions in hybrid genes appears to occur in a distinct zone that lies 5' of nucleotide 2108, which is further downstream than previously hypothesised. The other transitions lie upstream of nucleotide 999. Apparent 'large-scale' CYP21-CYP21P gene conversions lead to hybrid genes that are very similar to those found in CYP21 deletions, so these haplotypes have probably resulted from a meiotic double unequal crossover.

Aldosterone production despite absence or defectiveness of the CYP21 genes in two patients with salt-losing congenital adrenal hyperplasia caused by steroid 21-hydroxylase deficiency.

Aldosterone and cortisol were found in plasma samples from two patients with salt-losing congenital adrenal hyperplasia caused by steroid 21-hydroxylase deficiency. One patient had a CYP21 gene deletion on one chromosome and a mutation causing erroneous mRNA splicing on the other. The other patient had a CYP21 gene deletion on one chromosome and a large scale conversion of CYP21 to CYP21P on the other. All CYP21P-like genes in these patients were defective, since they carried a deleterious 8 bp deletion in the third exon. After HPLC purification of the patients' plasma samples, cortisol was no longer detectable in the radioimmunoassay, but aldosterone levels were still within or slightly above the normal reference range. Aldosterone dropped to very low levels after steroid replacement therapy had taken effect. In at least one of these patients, the genetic defect rules out normal functioning of the adrenocortical steroid 21-hydroxylase, which implies involvement of an alternative enzyme system.

A novel pathogenic taxon of the Mycobacterium tuberculosis complex, Canetti: characterization of an exceptional isolate from Africa.

In an attempt to characterize an unusual mycobacterial strain isolated from a 2-year-old Somali patient with lymphadenitis, we applied various molecular methods not previously used for the taxonomic classification of mycobacteria. This isolate, designated So93, did not differ from Mycobacterium tuberculosis in the biochemical tests and in its 16S rRNA sequence, but produced smooth and glossy colonies, which is highly exceptional for this species. This smooth phenotype was unstable and switched nonreversibly to a rough colony morphology with a low frequency. The two colony types were equally virulent for the guinea pig, exhibiting characteristic tuberculous disease. Both morphotypes had shorter generation times than the M. tuberculosis reference laboratory strain H37Rv and clinical isolates of M. tuberculosis and Mycobacterium bovis. Furthermore, the So93 isolate differed from all M. tuberculosis complex strains described thus far by having only a single copy of insertion sequence IS1081, an unusual composition of the direct repeat cluster, and a characteristic phenolic glycolipid and lipooligosaccharide. This glycolipid had previously been observed only in a smooth isolate of M. tuberculosis obtained in 1969 by Canetti in France. Analysis of the Canetti strain showed that it shared virtually all genetic properties characteristic of So93, distinguishing these two strains from the known M. tuberculosis complex taxa, M. tuberculosis, Mycobacterium africanum, M. bovis, and Mycobacterium microti. The natural reservoir, host range, and mode of transmission of the group of bacteria described in this paper are presently unknown. This study, partly based on not previously used molecular criteria, supports the idea that the established members within the M. tuberculosis complex and the newly described Canetti grouping should be regarded as a single species, which likely will be designated "M. tuberculosis".

Molecular basis of androgen insensitivity.

Mutations in the androgen receptor gene in 46,XY individuals can be associated with the androgen insensitivity syndrome, of which the phenotype can vary from a female phenotype to an undervirilized or infertile male phenotype. We have studied the androgen receptor gene of androgen insensitivity patients to get information about amino acid residues or regions involved in DNA binding and transcription activation. Genomic DNA was analysed by PCR-SSCP under two different conditions. Three new mutations were found in exon 1 of three patients with a female phenotype. A cytosine insertion at codon 42 resulted in a frameshift and consequently in the introduction of a premature stop at codon 171. Deletion of an adenine at codon 263 gave rise to a premature stop at codon 292. In both these cases, receptor protein was not detectable and hormone binding was not measurable. In a third patient, a guanine-to-adenine transition at codon 493 converted a tryptophan codon into a stop codon. Genital skin fibroblasts from this patient were not available. In exon 2 of the androgen receptor gene of a patient with receptor-positive androgen insensitivity, a cytosine-to-adenine transition, converting alanine 564 into an aspartic acid residue, resulted in defective DNA binding and transactivation. In three other receptor-positive androgen insensitivity patients no mutations were found with PCR-SSCP.

Comparative study of five different DNA fingerprint techniques for molecular typing of Streptococcus pneumoniae strains.

The aim of this study was to identify the strengths and weaknesses of five DNA fingerprint methods for epidemiological typing of Streptococcus pneumoniae. We investigated the usefulness of (i) ribotyping, (ii) BOX fingerprinting with the BOX repetitive sequence of S. pneumoniae as a DNA probe, (iii) PCR fingerprinting with a primer homologous to the enterobacterial repetitive intergenic consensus sequence, (iv) pulsed-field gel electrophoresis of large DNA fragments, and (v) restriction fragment end labeling to detect restriction fragment length polymorphism of small DNA fragments. Twenty-eight S. pneumoniae strains isolated from the blood and/or cerebrospinal fluid of 21 patients were analyzed. Genetic clustering among the 28 strains was independent of the DNA fingerprint technique used. However, the discriminatory power and the similarity values differed significantly among the individual techniques. BOX fingerprinting, pulsed-field gel electrophoresis, and restriction fragment end labeling provided the highest degree of discriminatory power. Furthermore, the ease with which computerized fingerprint analysis could be conducted also varied significantly among the techniques. Ribotyping, BOX fingerprinting, and restriction fragment end labeling were very suitable techniques for accurate computerized data analysis. Because of their high discriminatory potential and ease of accurate analysis, we conclude that BOX fingerprinting and restriction fragment end labeling are the most suitable techniques to type pneumococcal strains.

A practical approach to the detection of androgen receptor gene mutations and pedigree analysis in families with x-linked androgen insensitivity.

Androgen insensitivity syndrome (AIS) is an X-linked disorder in which defects in the androgen receptor gene have prevented the normal development of both internal and external male structures in 46,XY individuals. This survey reports the analysis of 11 AIS subjects. The androgen receptor gene of these subjects was analyzed using polymerase chain reaction (PCR)-single-strand conformation polymorphism analysis and sequencing or sequencing of PCR-amplified androgen receptor gene fragments alone. In total, 10 single base changes and one partial gene deletion were detected. Seven single base changes resulted in an amino acid change, one resulted in the introduction of a premature stop codon, one event represented a single base insertion resulting in a frame-shift, and one single base change affected a donor splice site. The androgen receptor protein in genital skin fibroblasts from several patients was studied with respect to molecular mass after immunoprecipitation and SDS-PAGE. Two patients expressed a truncated receptor protein in agreement with the established genomic mutation. Pedigree analysis was performed to identify possible carriers for the syndrome in families of AIS patients using single-strand conformation polymorphism and restriction site analysis of PCR products. In one case, the polymorphic (CAG)n(CAA) repeat in exon 1 encoding a polyglutamine stretch was used to identify the mutant allele in a family with X-linked partial androgen insensitivity before the identification of the actual genomic mutation. PCR-single-strand conformation polymorphism analysis proved to be a fast and reliable technique to screen for androgen receptor gene mutations and to study the androgen receptor gene of family members of AIS-affected individuals.

Family studies of the steroid 21-hydroxylase and complement C4 genes define 11 haplotypes in classical congenital adrenal hyperplasia in The Netherlands.

Two steroid 21-hydroxylase genes are normally present within the human major histocompatibility complex near the genes encoding the fourth component of complement (C4A and C4B). Steroid 21-hydroxylase is encoded by the CYP21 gene, while the highly homologous CYP21P gene is a pseudogene. We studied steroid 21-hydroxylase and complement C4 haplotypes in 33 Dutch patients (29 families) suffering form classical congenital adrenal hyperplasia (CAH) and in their 80 family members, and also in 55 unrelated healthy controls, using 21-hydroxylase and complement C4 cDNA probes. Eleven different haplotypes, defined in terms of gene deletions, gene duplications, conversions of CYP21 to CYP21P, and "long" and "short" C4 genes, were found. In 23% of the patients' haplotypes, the CYP21 gene was deleted; in 12%, it was converted into a CYP21P pseudogene. In the remaining 65%, the defect was apparently caused by a mutation not detectable by this method. The most common haplotype (with one CYP21 and one CYP21P gene) was significantly more often observed in patients with simple virilizing CAH than in those with salt-losing CAH. Comparison of the 21-hydroxylase haplotypes found in CAH patients from several countries shows evidence for considerable genetic variation between the groups studied.

A rare TaqI polymorphism in a human complement C4 gene is caused by an additional restriction site in the first intron.

We studied the configuration of the complement C4/CYP21 (steroid 21-hydroxylase) region of the human major histocompatibility complex in patients suffering from congenital adrenal hyperplasia (CAH) and in the general population in The Netherlands, using C4 and CYP21 probes and the restriction enzymes TaqI and Bg/II. We found a rare TaqI 3.9-kb restriction fragment in the mother of a CAH patient, and present evidence that this polymorphism is caused by an additional restriction site in the first intron of a complement C4 gene.

[Adrenogenital syndrome. II. Molecular biology]. / Het adrenogenitaal syndroom. II. Moleculaire biologie.

The adrenogenital syndrome (AGS) is usually caused by steroid 21-hydroxylase deficiency. Two steroid 21-hydroxylase genes are present within the major histocompatibility complex (MHC) on chromosome 6: an active gene (CYP21) and a pseudogene (CYP21P). Several types of mutations have been described; these mutations can be categorized as gene deletions, gene duplications, gene conversions and smaller mutations inside the gene. Some of these cause a defect in the CYP21 gene, possibly resulting in 21-hydroxylase deficiency. Apart from the intrinsic scientific value of these results, the methods applied become increasingly important in diagnostics.

[Adrenogenital syndrome. I. Introduction, enzymology and heredity]. / Het adrenogenitaal syndroom. I. Inleiding, enzymologie en erfelijkheid.

The adrenogenital syndrome (AGS) is a relatively common inherited metabolic disease, generally caused by a deficiency of the adrenocortical enzyme steroid 21-hydroxylase. This results in an insufficient biosynthesis of several important steroid hormones such as cortisol and aldosterone, and, on the other hand, in a strongly increased production of androgens (testosterone). In girls, virilization of the external genitals is usually seen. In some patients, severe salt loss occurs shortly after birth, and a life-threatening crisis may develop. Mild variants of the disease have also been described. Steroid 21-hydroxylase is encoded by a gene within the HLA complex on the short arm of chromosome 6. HLA typing thus allows the study of the hereditary transmission of several forms of the AGS. In addition, molecular biology at present opens new perspectives to fundamental and clinical genetic research.

Vitamin D metabolism in breast-fed infants and their mothers.

The aim of this longitudinal study was to examine vitamin D metabolism in exclusively breast-fed infants. The four common vitamin D metabolites--25-hydroxyvitamin D (25OHD), 1,25-dihydroxyvitamin D [1,25(OH)2D], 24,25-dihydroxyvitamin D [24,25(OH)2D], and 25,26-dihydroxyvitamin D [25,26(OH)2D]--as well as vitamin D binding protein (DBP) were determined simultaneously in mothers and their children from delivery to several months of age. Maternal blood samples, drawn approximately 6 wk before the expected date of delivery, were also analyzed. At delivery, total vitamin D metabolites in maternal and fetal plasma were closely correlated, maternal levels being higher. Unbound (free) vitamin D metabolite concentrations were higher in fetal than in maternal plasma, with the exception of free 1,25(OH)2D levels, which were equal. This suggests a rapid placental transfer of 1,25(OH)2D. 24,25(OH)2D and 25,26(OH)2D levels both in mothers and children were closely correlated with the precursor sterol 25OHD. For 1,25(OH)2D, no correlation could be demonstrated with any of the other vitamin D metabolites. DBP concentrations in maternal plasma at the time of delivery were about twice the mean adult reference value. In cord blood, DBP levels were in the lower part of the adult reference range. Maternal total 1,25(OH)2D levels, which were twice the reference mean during pregnancy, fell sharply after delivery but free 1,25(OH)2D levels much less. Analogous to the biochemical changes in the mother, the infants' DBP levels fell after birth, as a result of the sudden disappearance of the estrogen stimulus. At the same time, the mineral supply via the placenta was cut off.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma 1.25-dihydroxyvitamin D concentrations in preterm infants.

1.25-Dihydroxyvitamin D concentrations were measured in 10 preterm infants (mean gestational age 29 weeks, range 26-32; mean birthweight 1226 g, range 980-1700). Total parenteral nutrition was begun after birth and partial enteral feeding was started at 1 week of age. Total enteral feeding was achieved at a mean age of 26 days (range 16-47). The daily vitamin D3 intake was about 400 I. U. No clinical, chemical or radiological signs of rickets were observed. The mean 1.25-dihydroxyvitamin D concentration +/- SEM was 103.2 +/- 24.0 pmol/l at 1 week (range 9.6-252.0), 141.6 +/- 26.4 at 3 weeks (range 31.2-324.0), 153.6 +/- 21.6 at 6 weeks (range 67.2- 256.8), 165.6 +/- 24.0 at 9 weeks (range 74.4-307.2) and 153.6 +/- 21.6 at 12 weeks (range 76.8-268.8) postnatal age. The mean values at 6, 9 and 12 weeks were significantly higher (p resp. less than 0.01, less than 0.002 and less than 0.005) than in adults (88.8 +/- 7.2; n = 27). 1.25-Dihydroxyvitamin D concentrations were highly variable and did not correlate with 25-hydroxyvitamin D concentrations, plasma calcium and phosphorus concentrations and plasma alkaline phosphatase levels, nor with illness nor postnatal age. The data demonstrate that preterm infants are capable of producing high plasma levels of 1.25-dihydroxyvitamin D.

Interlaboratory variation of vitamin D1) metabolite measurements.

Interlaboratory variation of the measurement of 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D by six laboratories in the Netherlands was studied. Three different serum samples and two different standard solutions of each metabolite were assayed. Substantial interlaboratory variation was found for the measurement of serum samples. The mean interlaboratory CV's for the 25-hydroxy vitamin D, 24,25-dihydroxy vitamin D and 1,25-dihydroxy vitamin D assays in the three sera were 48%, 38% and 20% respectively. The measurement of standard solutions of all metabolites showed relative little variation (mean CV 8%). The small number of samples allowed no evaluation of intralaboratory variation. The much higher CV's of the measurement of serum samples, when compared to standard solutions, may be attributed to differences in extraction and purification procedures which are probably responsible for the presence of varying amounts of interfering substances during the final quantification of metabolites.