Is Still's disease still one disease? A case of Adult-onset Still's disease showing accumulation in the carotids and the large vessels of the legs on positron emission tomography: CT images.

Adult-onset Still's disease (AOSD) is known as a systemic inflammatory disease of unknown etiology and pathogenesis, characterized by fever, skin eruptions, systemic organ involvement, and arthralgias. AOSD is difficult to diagnose because of its heterogeneous clinical manifestations and prevalence (although more prevalent in the young, onset of AOSD after the age of 60 has also been described), and absence of pathognomonic clinical features. The disease also lacks a specific diagnostic test. To date, association studies between AOSD and HLA loci have failed to indentify a genetic predisposition. The recent publication of entirely different PET-CT manifestations found in three patients who were supposed to have the same disease (AOSD), as well as the surprisingly different PET-CT images of our AOSD patient (accumulation in the carotids and large vessels of the legs), raises our suspicion that AOSD is actually not one entity but a constellation of disorders whose varying underlying pathologies are now being revealed by new imaging techniques.

Antipituitary antibodies in dutch patients with idiopathic hypopituitarism.

BACKGROUND: Despite extensive research, in the majority of patients with isolated growth hormone deficiency (IGHD) and multiple pituitary hormone deficiency (MPHD), the cause of their clinical picture remains unknown. Recent articles suggest that some cases of idiopathic growth hormone deficiency might be explained by a silent form of autoimmune hypophysitis based on the presence of antipituitary antibodies (APA) at high titers (>1:8). METHODS: We collected clinical data and serum from 71 patients participating in the Dutch HYPOPIT study. APA screening in 40 IGHD patients and 31 MPHD patients was performed by an indirect immunofluorescence method. APA, when present, were related to clinical and morphological pituitary findings. RESULTS: APA were present at high titers in 7 of 31 MPHD patients (23%) and 1 of 40 IGHD patients (2.5%). Among APA-positive MPHD patients, apart from growth hormone deficiency, all patients of pubertal age had gonadotroph defi- ciency, all had thyroid hormone deficiency and 50% had ACTH deficiency. CONCLUSION: The high frequency of APA in our idiopathic MPHD population indicates that, in 23% of the patients diagnosed with idiopathic MPHD, the hormone deficiencies might actually be caused by a silent form of autoimmune hypophysitis. Screening for APA should therefore be considered in all patients with 'idiopathic' MPHD.

Facial and pituitary morphology are related in Dutch patients with GH deficiency.

OBJECTIVE: Classical GH deficiency (GHD) is associated with typical phenotypic features. We have analysed standardized photographs of 137 Caucasian patients with GHD, in order to examine the relations between auxological, biochemical, pituitary and facial morphometric features. PATIENTS AND MEASUREMENTS: We analysed pictures of 137 patients: 73 (55 Males/18 Females) with Isolated GHD and 64 (48 M/16 F) with multiple pituitary hormone deficiency (MPHD). Of each patient, standardized frontal and lateral digital pictures were taken and analysed using Adobe Photoshop 5.0. RESULTS: Canthal index (CI), the relative distance between the eyes, was related to pituitary morphology. Patients with an ectopic posterior pituitary (EPP) had significantly higher CI values than patients without EPP. We found CI > 39 to be a good cut-off value to select children with highest probability of having EPP. The combination of CI > 39 with the presence of hormonal deficiencies additional to GHD strongly predicted EPP: 93% of the patients with a CI > 39 and additional hormonal deficiencies had EPP, in contrast to 77% of the patients with additional hormonal deficiencies but a CI < 39, and 29% of the patients with none of these criteria (P = 0.0001). CONCLUSION: CI, measured on digital pictures, is associated with ectopia of the posterior pituitary and this might be caused by an altered midline development, affecting both the pituitary and the facial structures of GHD patients.

GH receptor d3 polymorphism in Dutch patients with MPHD and IGHD born small or appropriate for gestational age.

OBJECTIVE: GH acts through the GH receptor (GHR). The GHR gene contains a genetic polymorphism caused by a deletion of exon 3 (d3), with high frequency in the normal population. There is a continuing controversy whether the presence or absence of the exon 3 deletion (d3+ vs. d3-) affects the effect of GH in human growth. DESIGN, PATIENTS AND MEASUREMENTS: For 144 patients with idiopathic isolated GH deficiency (IGHD, n = 72) or multiple pituitary hormone deficiency (MPHD, n = 72), amplification of the region around exon 3 of the GHR gene was performed. Clinical data and response to GH treatment were compared between GHR d3+and d3- IGHD and MPHD patients born either small for gestational age (SGA) or appropriate for gestational age (AGA). RESULTS: IGHD patients born SGA had a significantly higher d3+frequency (82%) than IGHD patients born AGA (35%, P = 0.006). Within the group of IGHD patients born SGA, d3- patients showed a slightly better spontaneous catch up growth before start of GH treatment than d3+ patients (1.1 +/- 1.1 SD vs. 0.6 +/- 1.1 SDS, P = 0.040) There was no difference in patients first year's response to GH treatment between GHR d3+ and d3- patients. CONCLUSIONS: In IGHD and MPHD patients, response to GH treatment was independent of GHR genotype. GHR-d3 was significantly more frequent among IGHD patients born SGA. As we are the third to report an association between birth size and GHR d3 status, it is conceivable that the GHR-d3 might affect prenatal growth in IGHD patients by a yet unknown mechanism.

Prevalence and clinical significance of organ-specific autoantibodies in type 1 diabetes mellitus.

As diabetes mellitus type 1 (DM1) is associated with other autoimmune diseases, clinical tools are needed to diagnose and predict the occurrence of other autoimmune diseases in DM1. We performed a systematic search of the literature on the prevalence, and the diagnostic and prognostic significance of organ-specific autoantibodies in DM1, focusing on the most prevalent autoimmune diseases in DM1: Hashimoto's disease, autoimmune gastric disease, Addison's disease and coeliac disease. We found 163 articles that fulfilled our selection criteria. We analysed and compared the prevalence of autoantibodies in DM1 and control populations, studied the relation between antibody prevalence and age, gender, race and DM1 duration and studied the relation between the presence of autoantibodies and organ dysfunction. Because of the large variation in population characteristics and study design, a uniform conclusion on the relation of these autoantibody prevalences with age, gender, race, DM1 duration and target organ failure cannot be drawn easily. In addition, most studies reviewed used a cross-sectional design. Therefore, few data on the predictive value of the organ-specific antibodies in DM1 populations are present in these studies. Obviously, prospective studies are needed to fill this gap in knowledge. Despite these restrictions, the general picture from the present review is that the prevalence of the organ-specific autoantibodies is significantly higher in DM1 than in control populations. Given the relevant risk for organ failure in DM1 patients with autoantibodies against thyroid, gastric, adrenal and intestinal antigens, we recommend checking these autoantibodies in these patients at least once, for instance at the diagnosis of DM1. For detailed advice on assessing the different organ autoantibodies and function we refer to the summaries in the results section.

Aspergillus niger membrane-associated proteome analysis for the identification of glucose transporters.

BACKGROUND: The development of biological processes that replace the existing petrochemical-based industry is one of the biggest challenges in biotechnology. Aspergillus niger is one of the main industrial producers of lignocellulolytic enzymes, which are used in the conversion of lignocellulosic feedstocks into fermentable sugars. Both the hydrolytic enzymes responsible for lignocellulose depolymerisation and the molecular mechanisms controlling their expression have been well described, but little is known about the transport systems for sugar uptake in A. niger. Understanding the transportome of A. niger is essential to achieve further improvements at strain and process design level. Therefore, this study aims to identify and classify A. niger sugar transporters, using newly developed tools for in silico and in vivo analysis of its membrane-associated proteome. RESULTS: In the present research work, a hidden Markov model (HMM), that shows a good performance in the identification and segmentation of functionally validated glucose transporters, was constructed. The model (HMMgluT) was used to analyse the A. niger membrane-associated proteome response to high and low glucose concentrations at a low pH. By combining the abundance patterns of the proteins found in the A. niger plasmalemma proteome with their HMMgluT scores, two new putative high-affinity glucose transporters, denoted MstG and MstH, were identified. MstG and MstH were functionally validated and biochemically characterised by heterologous expression in a S. cerevisiae glucose transport null mutant. They were shown to be a high-affinity glucose transporter (K m = 0.5 ± 0.04 mM) and a very high-affinity glucose transporter (K m = 0.06 ± 0.005 mM), respectively. CONCLUSIONS: This study, focusing for the first time on the membrane-associated proteome of the industrially relevant organism A. niger, shows the global response of the transportome to the availability of different glucose concentrations. Analysis of the A. niger transportome with the newly developed HMMgluT showed to be an efficient approach for the identification and classification of new glucose transporters.

Identification, isolation and sequence of the Aspergillus nidulans xlnC gene encoding the 34-kDa xylanase.

The xlnC gene encoding the 34-kDa xylanase (X34) of Aspergillus nidulans (An) has been cloned and sequenced, as has its corresponding cDNA. xlnC contains nine introns and shows considerable similarity to the xynA and xylP xylanase-encoding genes of A. kawachii (Ak) and Penicillium chrysogenum (Pc), respectively. Analysis of xylanase production in An multicopy transformants showed elevated levels of X34 and increased total xylanase activity, but no elevated production of other xylanases. Northern analysis demonstrated transcriptional induction by xylan and repression by glucose.

Characterization of the pyruvate kinase-encoding gene (pki1) of Trichoderma reesei.

The pyruvate kinase-encoding gene (pki1) from Trichoderma reesei was isolated by hybridization to the corresponding Aspergillus nidulans pkiA gene. The 1614-bp nucleotide (nt) sequence of the cloned gene codes for a 538-amino-acid protein. The coding sequence contains a single intron of 246 nt at a position identical to that of intron E in the A. nidulans gene. The PKI protein shows extensive homology to the PKIs of A. nidulans and A. niger (67%) and Saccharomyces cerevisiae (59%). The 5' non-coding sequence contains a number of motifs typical for yeast glycolytic genes, but so far only rarely found in filamentous fungi.

CreA modulates the XlnR-induced expression on xylose of Aspergillus niger genes involved in xylan degradation.

The expression of the feruloyl esterase gene faeA, the alpha-glucuronidase gene aguA, the endoxylanase gene xlnB, and the beta-xylosidase gene xlnD from Aspergillus niger on xylose was studied in a wild-type strain and in a CreA mutant. A decrease in expression of all four genes was observed with increasing xylose concentrations in the wild-type strain, whereas expression levels in the CreA mutant were not influenced. The results in the wild type indicated that xylose concentrations higher than 1 mM resulted in repression of the expression of the xylanolytic genes tested mediated by the carbon catabolite repressor protein CreA. On xylose, the expression levels of the xylanolytic genes were therefore not only determined by induction via XlnR, but also by repression via CreA. The genes tested were not influenced to the same extent by XlnR or CreA, resulting in specific expression levels and patterns for each individual gene.

Purification and characterization of an alpha-L-rhamnosidase from Aspergillus niger.

An enzyme with alpha-L-rhamnosidase activity was purified by anion exchange chromatography from an Aspergillus niger commercial preparation. The alpha-L-rhamnosidase was shown to be N-glycosylated, and had a molecular mass of 85 kD on sodium dodecylsulfate-polyacrylamide gel electrophoresis of which approximately 12% was contributed by carbohydrate. The enzyme was optimally active at pH 4.5 and 65 degrees C. When tested towards p-nitrophenyl-alpha-L-rhamnopyranoside it showed Km and Vmax values of 2.9 mM and 20.6 U mg-1, respectively whereas it was inhibited competitively by L-rhamnose (Ki 3.5 mM). Substrate specificity studies showed alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucose. Moreover, the enzyme was able to release L-rhamnose from geranyl-beta-D-rutinoside and 2-phenylethyl-beta-D-rutinoside.

Characterization of galactosidases from Aspergillus niger: purification of a novel alpha-galactosidase activity.

An enzyme with beta-galactosidase activity and three proteins exhibiting alpha-galactosidase activity were purified from a culture filtrate of Aspergillus niger grown on arabinoxylan. beta-galactosidase, optimally active at pH 4 and 60-65 degrees C, was active against p-nitrophenyl-beta-D-galactopyranoside, lactose, and pectic galactan. It was not able to release galactose from sugar beet pectin or lemon pectin. Its action on pectic galactan was increased by the presence of beta-galactanase. The three forms of alpha-galactosidase activity that showed different molecular masses and pIs were found to have the same mass after deglycosylation with N-glycanase F and to be the same protein based on their N-terminal amino acid sequence data. The purified alpha-galactosidase was shown to be different from alpha-galactosidase A from A. niger. This confirmed the existence of at least two different alpha-galactosidases in A. niger. alpha-Galactosidase, optimally active at pH 4.5 and 50-55 degrees C, was active toward p-nitrophenyl-alpha-D-galactopyranoside, melibiose, raffinose, stachyose, and locust bean gum, on which substrate it exhibited synergism with beta-mannanase.

A clinical approach to pharmacogenetics.

Taking into account the high frequency of adverse drug reactions (ADRs) in the clinic and taking into account the growing knowledge of the genetic mechanisms underlying some of these ADRs, we believe that every clinician should know at least the basic principles of pharmacogenetics. However, our experience is that many clinicians are unaware of the potential contribution of pharmacogenetic testing and have not implemented this new modality in their daily practice. We present a case of Stevens-Johnson syndrome in a patient treated with carbamazepine. Following the pathways of clinical reasoning, we describe the possibilities of pharmacogenetic testing in the clinic (HLA-B*1502 and HLA-A*3101 in our patient). We describe the pharmacological and pharmacogenetic aspects relevant for the clinician's daily practice (the existence of ADR subtypes, cytochrome P450, drug-drug interactions, genetic variations, CYP450 and HLA genotyping). Based on the Dutch top 100 of most prescribed drugs, we provide data on CYP450 and HLA genotypes relevant to those 100 most commonly used drugs. We discuss the availability and costs of pharmacogenetic testing, show a calculation of the 'number needed to genotype' and, based on these data, we propose a decision model for pharmacogenetic testing by clinicians.

Association analysis of ten candidate genes in a large multinational cohort of small for gestational age children and children with idiopathic short stature (NESTEGG study).

BACKGROUND: Fetal growth failure has been associated with an increased risk of hypertension, cardiovascular disease and diabetes in adulthood. Exploring the mechanisms underlying this association should improve our understanding of these common adult diseases. PATIENTS AND METHODS: We investigated 225 SNPs in 10 genes involved in growth and glucose metabolism (GH1, GHR, IGF1, IGF1R, STAT5A, STAT5B, MAPK1, MAPK3, PPARγ and INS) in 1,437 children from the multinational NESTEGG consortium: 345 patients born small for gestational age who remained short (SGA-S), 288 who showed catch-up growth (SGA-Cu), 410 idiopathic short stature (ISS) and 394 controls. We related genotype to pre- and/or postnatal growth parameters, response to growth hormone (if applicable) and blood pressure. RESULTS: We found several clinical associations for GH1, GHR, IGF1, IGF1R, PPARγ and MAPK1. One SNP remained significant after Bonferroni's correction: IGF1R SNP rs4966035's minor allele A was significantly more prevalent among SGA and associated with smaller birth length (p = 0.000378) and birth weight (weaker association), independent of gestational age. CONCLUSION: IGF1R SNP rs4966035 is significantly associated with birth length, independent of gestational age. This and other associations suggest that polymorphisms in these genes might partly explain the phenotype of short children born SGA and children with ISS.

Personalized approach to growth hormone treatment: clinical use of growth prediction models.

The goal of growth hormone (GH) treatment in a short child is to attain a fast catch-up growth toward the target height (TH) standard deviation score (SDS), followed by a maintenance phase, a proper pubertal height gain, and an adult height close to TH. The short-term response variable of GH treatment, first-year height velocity (HV) (cm/year or change in height SDS), can either be compared with GH response charts for diagnosis, age and gender, or with predicted HV based on prediction models. Three types of prediction models have been described: the Kabi International Growth Hormone Study models, the Gothenburg models and the Cologne model. With these models, 50-80% of the variance could be explained. When used prospectively, individualized dosing reduces the variation in growth response in comparison with a fixed dose per body weight. Insulin-like growth factor-I-based dose titration also led to a decrease in the variation. It is uncertain whether adding biochemical, genetic or proteomic markers may improve the accuracy of the prediction. Prediction models may lead to a more evidence-based approach to determine the GH dose regimen and may reduce the drug costs for GH treatment. There is a need for user-friendly software programs to make prediction models easily available in the clinic.

Long-term follow-up of organ-specific antibodies and related organ dysfunction in type 1 diabetes mellitus.

OBJECTIVE: Diabetes mellitus type 1 (DM1) is associated with other autoimmune disorders. To our knowledge, there are no longitudinal data considering the long-term clinical relevance of organ-specific antibodies (OS-Ab) in DM1 patients. We performed a long-term retrospective longitudinal study in order to investigate the presence and diagnostic accuracy (positive predictive value: PPV and negative predictive value: NPV) of OS-Ab in DM1 patients. RESEARCH DESIGN AND METHODS: In a retrospective longitudinal study, the presence of OS-Ab and related organ function were analysed in 396 DM1 patients (184 F/212 M, age 44 ± 13 years, age at onset of DM1 21 ± 13 years), with a median follow-up time of 23 ± 10 years. RESULTS: OS-Ab frequencies at baseline were: antibodies against thyroglobulin (Tg-Ab) 4.3%, antibodies against thyroid peroxidase (TPO-Ab) 8.1%, Tg- and/or TPO-Ab 10.4%, antibodies against parietal cells (PCA) 5.8% and antibodies against adrenal cortex (ACA) 0.5%. The occurrence of (sub)clinical hypothyroidism was higher in patients with Tg-Ab (47%) or TPO-Ab (42%) than in those without these antibodies (6.2 and 5.1%, respectively, p<0.001). PPV and NPV for Tg-Ab were 0.60 and 0.88, respectively, for TPO -Ab 0.54 and 0.91. Also in patients with PCA, organ dysfunction occurred more often (61%) than in patients without PCA (9.7%, p<0.001). PPV for PCA was 0.61 and NPV 0.90. NPV and PPV for ACA could not be calculated because of the low prevalence. CONCLUSION: Long-term follow-up of 396 DM1 patients shows that the presence of thyroid antibodies and/ or parietal cell antibodies is clearly associated with dysfunction of the corresponding organ.

Structure of the Aspergillus nidulans pyruvate kinase gene.

The complete nucleotide sequence of the Aspergillus nidulans pyruvate kinase gene, including its flanking sequences, is presented. The gene has a 1,578 bp coding sequence that encodes a protein of 526 amino acids; the latter is strongly homologous to the pyruvate kinases found in Saccharomyces cerevisiae (66%) and mammals (53%). The gene is interrupted by seven introns, three of which are in a conserved position compared to those present in the mammalian pyruvate kinase genes sequenced thus far. A fourth intron within the mononucleotide binding fold domain is in a conserved position with respect to the position of an intron within the NAD+ binding region of maize ADH I. The transcription start site has been determined; a major site of transcription was found 80 bp before the translation initiation codon. The promoter region of the A. nidulans pyruvate kinase gene contains no direct homologies with the TATA or CCAAT sequences in the expected region (30-70 bp) before the transcription initiation site. However, extended CT-enriched regions are found in the promotor region, similar to what has been observed in genes that are highly expressed in Saccharomyces cerevisiae and filamentous fungi.

Isolation and transformation of the pyruvate kinase gene of Aspergillus nidulans.

The Aspergillus nidulans pyruvate kinase gene was isolated by heterologous hybridization using the corresponding yeast gene as a probe. A 2.9 kb EcoRI/BamHI fragment, which exclusively hybridized to the yeast gene, was subcloned in pBR322. This clone was used to transform an A. nidulans pkiA deletion mutant to PKI+. The analysis of transformants with respect to the kind of integration revealed about 80% homologous integration--55% by a double cross-over event (type III integration), 25% by a single cross-over event (type I integration). Type II transformants (20%) that arise by non-homologous integration have not been further characterized with respect to the sites of integration. A direct correlation between the number of copies of the gene integrated into the genome and the measured pyruvate kinase activity was found after growth ona glycolytic carbon source. From this, it was concluded that the 2.9 kb EcoRI/BamHI fragment contains the complete pyruvate kinase structural gene, including the promoter region. However, after growth on a gluconeogenic carbon source, the regulation of gene expression was found to be disturbed. On acetate an increase in activity per gene copy (0.2 IU) was found in the transformants, as compared with wild-type levels. It is suggested that the pyruvate kinase gene is regulated by negative control, and that some sequences involved in this regulation are missing in the cloned fragment.

The Aspergillus niger transcriptional activator XlnR, which is involved in the degradation of the polysaccharides xylan and cellulose, also regulates D-xylose reductase gene expression.

Screening of an Aspergillus niger differential cDNA library, constructed by subtracting cDNA fragments of a xlnR loss-of-function mutant from wild-type cDNA fragments, resulted in the cloning of the gene encoding D-xylose reductase (xyrA). Northern blot analysis using an A. niger wild-type strain, a xlnR multiple-copy strain and a xlnR loss-of-function mutant confirmed that the xyrA gene is regulated by XlnR, the transcriptional activator of the xylanolytic enzyme system in A. niger. D-xylose reductase catalyses the NADPH-dependent reduction of D-xylose to xylitol, which is the first step in D-xylose catabolism in fungi. Until now, XlnR was shown to control the transcription of genes encoding extracellular hydrolytic enzymes involved in cellulose and xylan degradation. In the present study, we show that A. niger is able to harmonize its sugar metabolism and extracellular xylan degradation via XlnR by regulating the expression of XyrA.

Isolation and characterization of the Aspergillus niger pyruvate kinase gene.

The Aspergillus niger gene encoding pyruvate kinase was cloned by heterologous hybridization using a fragment from the corresponding yeast gene as a probe. The primary structure of the gene, including 5' and 3' flanking sequences, was determined. The structural part of the A. niger pkiA gene is 2054 bp long and is interrupted by seven putative introns. Splicing of the intron sequences results in an open reading frame of 1578 bp, encoding a protein of 526 amino-acid residues and a molecular weight of 58,130 Da. Extensive homology is found with pyruvate kinase from A. nidulans; only 33 amino acids are different between both proteins. Transformation experiments using the pyrA gene as a selection marker and the subcloned pkiA gene as a co-transforming marker led to increased levels of pyruvate kinase. Analysis of the transformants showed that in none of the transformants integration had occurred at the pkiA locus. Predominantly co-integration of the pyrA- and the pkiA-containing plasmids was found in the cases examined.

Arabinoxylan degradation by fungi: characterization of the arabinoxylan-arabinofuranohydrolase encoding genes from Aspergillus niger and Aspergillus tubingensis.

The genes encoding the enzyme arabinoxylan arabinofuranohydrolase, which releases L-arabinose from arabinoxylan, have been cloned from the closely related fungi Aspergillus niger and Aspergillus tubingensis and were shown to be functional in A. niger. Integration of multiple copies in the genome resulted in over-expression of the enzymes. The arabinofuranohydrolases encoded comprise 332 amino acids and have 94% amino acid identity. Their primary structure is not related to those of other alpha-L-arabinofuranosidases, except for a low similarity with XYLC, a bacterial alpha-L-arabinofuranosidase from Pseudomonas fluorescens which acts on oat spelt xylan. The axhA expression pattern in A. niger differed from that of abfB, since it was strongly induced by birchwood xylan and much less by L-arabitol or L-arabinose. Furthermore, Northern analysis revealed that axhA expression was de repressed in creAd mutants and carbon catabolite repressed by D-glucose.