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Inducible gene regulation methods are indispensable in diverse biological applications, yet many of them have severe limitations in their applicability. These include inducer toxicity, a limited variety of organisms the given system can be used in, and side effects of the induction method. In this study, a novel inducible system, the RuX system , was created using a mutant ligand-binding domain of the glucocorticoid receptor (CS1/CD), used together with various genetic elements such as the Gal4 DNA-binding domain or Cre recombinase. The RuX system is shown to be capable of over 1000-fold inducibility, has flexible applications, and is offered for use in cell cultures., Competing Interests: The authors declare no conflict of interest., (Copyright © 2023 Anne Meinzinger et al.)

Ca 2+ is a second messenger that regulates a variety of cellular responses in bone, including osteoblast differentiation. Mutations in trimeric intracellular cation channel B (TRIC-B), an endoplasmic reticulum channel specific for K + , a counter ion for Ca 2+ flux, affect bone and cause a recessive form of osteogenesis imperfecta (OI) with a still puzzling mechanism. Using a conditional Tmem38b knock out mouse, we demonstrated that lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. At the cellular level, delayed osteoblast differentiation and decreased collagen synthesis were found consequent to the Ca 2+ imbalance and associated with reduced collagen incorporation in the extracellular matrix and poor mineralization. The impaired SMAD signaling detected in mutant mice, and validated in OI patient osteoblasts, explained the osteoblast malfunction. The reduced SMAD phosphorylation and nuclear translocation were mainly caused by alteration in Ca 2+ calmodulin kinase II (CaMKII)-mediated signaling and to a less extend by a lower TGF-β reservoir. SMAD signaling, osteoblast differentiation and matrix mineralization were only partially rescued by TGF-β treatment, strengthening the impact of CaMKII-SMAD axes on osteoblast function. Our data established the TRIC-B role in osteoblasts and deepened the contribution of the CaMKII-SMAD signaling in bone., Competing Interests: Declaration of Competing Interest The authors have declared that no conflict of interest exists., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The recent identification of homozygous WNT1 mutations in individuals with osteogenesis imperfecta type XV (OI-XV) has suggested that WNT1 is a key ligand promoting the differentiation and function of bone-forming osteoblasts. Although such an influence was supported by subsequent studies, a mouse model of OI-XV remained to be established. Therefore, we introduced a previously identified disease-causing mutation (G177C) into the murine Wnt1 gene. Homozygous Wnt1 G177C/G177C mice were viable and did not display defects in brain development, but the majority of 24-week-old Wnt1 G177C/G177C mice had skeletal fractures. This increased bone fragility was not fully explained by reduced bone mass but also by impaired bone matrix quality. Importantly, the homozygous presence of the G177C mutation did not interfere with the osteoanabolic influence of either parathyroid hormone injection or activating mutation of LRP5, the latter mimicking the effect of sclerostin neutralization. Finally, transcriptomic analyses revealed that short-term administration of WNT1 to osteogenic cells induced not only the expression of canonical WNT signaling targets but also the expression of genes encoding extracellular matrix modifiers. Taken together, our data demonstrate that regulating bone matrix quality is a primary function of WNT1. They further suggest that individuals with WNT1 mutations should profit from existing osteoanabolic therapies., (© 2021. The Author(s).)

Objective: The human matrilin-3 T303M (in mouse T298M) mutation has been proposed to predispose for osteoarthritis, but due to the lack of an appropriate animal model this hypothesis could not be tested. This study was carried out to identify pathogenic mechanisms in a transgenic mouse line by which the mutation might contribute to disease development., Methods: A mouse line carrying the T298M point mutation in the Matn3 locus was generated and features of skeletal development in ageing animals were characterized by immunohistology, micro computed tomography, transmission electron microscopy and atomic force microscopy. The effect of transgenic matrilin-3 was also studied after surgically induced osteoarthritis., Results: The matrilin-3 T298M mutation influences endochondral ossification and leads to larger cartilage collagen fibril diameters. This in turn leads to an increased compressive stiffness of the articular cartilage, which, upon challenge, aggravates osteoarthritis development., Conclusions: The mouse matrilin-3 T298M mutation causes a predisposition for post-traumatic osteoarthritis and the corresponding knock-in mouse line therefore represents a valid model for investigating the pathogenic mechanisms involved in osteoarthritis development., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Mutations in the gene ANO5 , encoding for the transmembrane protein Anoctamin 5 (Ano5), have been identified to cause gnathodiaphyseal dysplasia (GDD) in humans, a skeletal disorder characterized by sclerosis of tubular bones, increased fracture risk and fibro-osseous lesions of the jawbones. To better understand the pathomechanism of GDD we have generated via Crispr/CAS9 gene editing a mouse model harboring the murine equivalent (Ano5 p.T491F) of a GDD-causing ANO5 mutation identified in a previously reported patient. Skeletal phenotyping by contact radiography, μCT and undecalcified histomorphometry was performed in male mice, heterozygous and homozygous for the mutation, at the ages of 12 and 24 weeks. These mice did not display alterations of skeletal microarchitecture or mandible morphology. The results were confirmed in female mice and animals derived from a second, independent clone. Finally, no skeletal phenotype was observed in mice lacking ~40% of their Ano5 gene due to a frameshift mutation. Therefore, our results indicate that Ano5 is dispensable for bone homeostasis in mice, at least under unchallenged conditions, and that these animals may not present the most adequate model to study the physiological role of Anoctamin 5., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors.)

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Na v 1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Na v 1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Na v 1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Na v 1.5 in which the SIV motif is deleted (ΔSIV), sodium current (I Na ) recordings from the lateral membrane revealed a SIV-motif-independent I Na . Since immunostaining has suggested that Na v 1.5 is expressed in transverse (T-) tubules, this remaining I Na might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Na v 1.5 N-terminus, which may explain the SIV-motif independent I Na at the lateral membrane of cardiomyocytes. Aim: To address the role of α1-syntrophin in regulating the I Na at the lateral membrane of cardiac cells. Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral I Na was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining I Na was still recorded. In addition, cell-attached I Na recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels. Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Na v 1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Na v 1.5 channels, however, remains open.

The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. In this work, we have tested this strategy in vivo with the aim to generate two conditional knock-in mice for missense mutations in the Impad1 and Clcn7 genes causing two different skeletal dysplasias. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* and the Clcn7 exon 7 and an inverted exon 7* containing the point mutations were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon. The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation. Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Similarly, the phenotype of homozygous Clcn7 floxed mice overlaps with Clcn7 knock-out mice. Expression studies by qPCR and RT-PCR demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. Thus, the skeletal phenotypes in both murine strains were not caused by the missense mutations, but by aberrant splicing. Our data demonstrate that the Cre mediated genetic switch strategy should be considered cautiously for the generation of conditional knock-in mice., Competing Interests: M. Capulli, R. Costantini, A. Maurizi, C. Paganini, L. Monti, A. Forlino, A. Teti, A. Rossi do not report disclosures. S. Sonntag and D. Shmerling are affiliated to PolyGene AG. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials.

Aim: Deletion of QKP1507-1509 amino-acids in SCN5A gene product, the voltage-gated Na + channel Nav1.5, has been associated with a large phenotypic spectrum of type 3 long QT syndrome, conduction disorder, dilated cardiomyopathy and high incidence of sudden death. The aim of this study was to develop and characterize a novel model of type 3 long QT syndrome to study the consequences of the QKP1507-1509 deletion., Methods and Results: We generated a knock-in mouse presenting the delQKP1510-1512 mutation (Scn5a +/ΔQKP ) equivalent to human deletion. Scn5a +/ΔQKP mice showed prolonged QT interval, conduction defects and ventricular arrhythmias at the age of 2 weeks, and, subsequently, structural defects and premature mortality. The mutation increased Na + window current and generated a late Na + current. Ventricular action potentials from Scn5a +/ΔQKP mice were prolonged. At the age of 4 weeks, Scn5a +/ΔQKP mice exhibited a remodeling leading to [Ca 2+ ] i transients with higher amplitude and slower kinetics, combined with enhanced SR Ca 2+ load. SERCA2 expression was not altered. However, total phospholamban expression was higher whereas the amount of Ca 2+ -calmodulin-dependent kinase II (CaMKII)-dependent T17-phosphorylated form was lower, in hearts from 4-week-old mice only. This was associated with a lower activity of CaMKII and lower calmodulin expression. In addition, Scn5a +/ΔQKP cardiomyocytes showed larger Ca 2+ waves, correlated with the presence of afterdepolarizations during action potential recording. Ranolazine partially prevented action potential and QT interval prolongation in 4-week-old Scn5a +/ΔQKP mice and suppressed arrhythmias., Conclusion: The Scn5a +/ΔQKP mouse model recapitulates the clinical phenotype of mutation carriers and provides new and unexpected insights into the pathological development of the disease in patients carrying the QKP1507-1509 deletion., (Copyright © 2018. Published by Elsevier Ltd.)

Aims: The histidine-rich calcium-binding protein (HRC) Ser96Ala variant has previously been identified as a potential biomarker for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. Herein, the role of this variant in cardiac pathophysiology is delineated through a novel mouse model, carrying the human mutation in the homologous mouse position., Methods and Results: The mouse HRC serine 81, homologous to human HRC serine 96, was mutated to alanine, using knock-in gene targeting. The HRC-Ser81Ala mice presented increased mortality in the absence of structural or histological abnormalities, indicating that early death may be arrhythmia-related. Indeed, under stress-but not baseline-conditions, the HRC-Ser81Ala mice developed ventricular arrhythmias, whilst at the cardiomyocyte level they exhibited increased occurrence of triggered activity. Cardiac contraction was decreased in vivo, ex vivo, and in vitro. Additionally, Ca2+ transients and SR Ca2+ load were both reduced suggesting that cytosolic Ca2+ overload is not the underlying proarrhythmic mechanism. Interestingly, total SR Ca2+ leak was increased in HRC-Ser81Ala cardiomyocytes, without an increase in Ca2+ spark and wave frequency. However, Ca2+ wave propagation was significantly slower and the duration of the associated Na/Ca exchange current was increased. Moreover, action potential duration was also increased. Notably, Ca2+/Calmodulin kinase II (CaMKII) phosphorylation of the ryanodine receptor was increased, whilst KN-93, an inhibitor of CaMKII, reduced the occurrence of arrhythmias., Conclusions: The homologous mutation Ser81Ala in HRC in mice, corresponding to Ser96Ala in humans, is associated with sudden death and depressed cardiac function. Ventricular arrhythmias are related to abnormal Ca2+ cycling across the SR. The data further support a role for CaMKII with the perspective to treat arrhythmias through CaMKII inhibition., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2017. For permissions, please email: journals.permissions@oup.com.)

Background: Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization., Objective: The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes., Methods: SAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice., Results: Action potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice., Conclusion: These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome., (Copyright © 2015 Heart Rhythm Society. Published by Elsevier Inc. All rights reserved.)

Expression of the transmembrane glycoprotein CD98 (encoded by SLC3A2) is increased in intestinal inflammatory conditions, such as inflammatory bowel disease (IBD), and in various carcinomas, yet its pathogenetic role remains unknown. By generating gain- and loss-of-function mouse models with genetically manipulated CD98 expression specifically in intestinal epithelial cells (IECs), we explored the role of CD98 in intestinal homeostasis, inflammation, and colitis-associated tumorigenesis. IEC-specific CD98 overexpression induced gut homeostatic defects and increased inflammatory responses to DSS-induced colitis, promoting colitis-associated tumorigenesis in mice. Further analysis indicated that the ability of IEC-specific CD98 overexpression to induce tumorigenesis was linked to its capacity to induce barrier dysfunction and to stimulate cell proliferation and production of proinflammatory mediators. To validate these results, we constructed mice carrying conditional floxed Slc3a2 alleles and crossed them with Villin-Cre mice such that CD98 was downregulated only in IECs. These mice exhibited attenuated inflammatory responses and resistance to both DSS-induced colitis and colitis-associated tumorigenesis. Together, our data show that intestinal CD98 expression has a crucial role in controlling homeostatic and innate immune responses in the gut. Modulation of CD98 expression in IECs therefore represents a promising therapeutic strategy for the treatment and prevention of inflammatory intestinal diseases, such as IBD and colitis-associated cancer.

Conventional approaches to produce transgenic mice recurrently yield unpredictable patterns and levels of transgene expression, a situation calling for the development of new techniques to overcome these drawbacks in the context of overexpression studies. Here we present an efficient method for rapid and reproducible transgenesis using the recombinase mediated cassette exchange (RMCE) (Bouhassira et al.: Blood 90:3332-3344, 1997) procedure. A lox511-EGFP-TK/neo-loxP cassette was placed under the control of the endogenous mouse beta-actin promoter. Heterozygous mice revealed strong and ubiquitous EGFP expression throughout embryogenesis and adulthood. Reproducibly, the same expression pattern was obtained with RMCE when it was used to replace the EGFP-harboring cassette by ECFP or placental alkaline phosphatase (PLAP) reporter genes (DePrimo et al.: Transgenic Res 5:459-466, 1996). Furthermore, the RMCE procedure proved efficient as well in embryonic stem (ES) cells as directly in zygotes. Our results demonstrate ubiquitous expression of floxed transgenes in the endogenous beta-actin locus and they support the general use of the beta-actin locus for targeted transgenesis., (Copyright 2005 Wiley-Liss, Inc)

Mice devoid of PrP are resistant to scrapie and fail to replicate the agent. Introduction of transgenes expressing PrP into such mice restores susceptibility to scrapie. We find that truncated PrP devoid of the five copper binding octarepeats still sustains scrapie infection; however, incubation times are longer and prion titers and protease-resistant PrP are about 30-fold lower than in wild-type mice. Surprisingly, brains of terminally ill animals show no histopathology typical for scrapie. However, in the spinal cord, infectivity, gliosis, and motor neuron loss are as in scrapie-infected wild-type controls. Thus, while the region comprising the octarepeats is not essential for mediating pathogenesis and prion replication, it modulates the extent of these events and of disease presentation.

Aim of the Study: An allergen-reduced dietary intervention programme with strict dietary requirements was implemented over the first four months of life in an unselected population-based infant cohort and compared to a non-intervention cohort (the ZUFF study). Recommendations for the dietary programme in the intervention cohort were extended, but not strictly implemented, until the end of month six. The intervention was based on breastfeeding, a moderate whey hydrolysate formula (pHF), and delayed introduction of weaning foods with a high allergenicity. This study was a prospective, controlled, and unblinded study, the first to assess the effects of an allergen-reduced, pHF-based early nutritional programme in a broad unselected infant population. Because overall healthy development of the infant is a major objective of any nutritional programme, the study evaluated the effects of the dietary intervention on infant growth and general health status rather than specific allergic manifestations. Part I of this paper gave results for nutritional behaviour only, and Part II gives results for growth and general health status during the intervention period through the sixth month of life., Methods: Assignment of study infants was to demographically comparable intervention (Z) or control (FF) cohorts according to place of birth. In the intervention cohort (Z=564), the recommended dietary regimen was breastfeeding and--if exclusive breastfeeding was not possible--supplementation with a moderately hydrolysed, allergen-reduced infant formula (pHF). Weaning foods were delayed until four months of age or later in case of weaning foods with high allergenicity. In the control cohort (FF=566), there was no specific intervention. Imbalances between cohorts in confounding (adjuvant) factors that could influence health-related outcomes were integrated as covariates into the logistic regression of the main analyses. Growth parameters included weight, length, head circumference, BMI, and Z scores (SDS). General health status was assessed by clinically significant findings in gastrointestinal, respiratory, or skin symptoms., Results: Growth at 6 weeks and at 3 and 6 months was similar for Z and FF. Significantly fewer Z than FF infants had clinically noteworthy health findings at 3 months (Z=27% versus FF=37%, odds ratio=0.63, CI=0.48-0.82) and 6 months (Z=33% versus FF=49%, odds ratio=0.51, CI= 0.40-0.66). This corresponds to a 30 % reduction in overall health concerns at 6 months for the intervention cohort. At 3 and 6 months, differences between cohorts in most measures of general health status were strongly influenced by a lower incidence of skin symptoms in the Z cohort. Within FF, there were fewer exclusively breastfed (eBF) infants with health problems at 3 months compared with those who were partially (pBF) or non-breastfed (nBF) (eBF=31%, pBF=40%, nBF=39%, p< 0.05). In contrast, in the Z intervention cohort, the number of infants with health concerns was similar for exclusively breastfed infants and for those in whom mother's milk was supplemented or replaced by pHF (eBF=29%, pBF=25%, nBF=26%, ns). In a subanalysis of overall health findings in infants without a family risk of allergies, there were again significantly fewer Z than FF infants with any health or any skin problem., Conclusion: An allergen-reduced dietary recommendation that includes a moderate whey hydrolysate infant formula (pHF) has no negative effects on growth parameters up to 6 months of life in an infant population unselected for atopic risk. The dietary intervention produced improvements in general health status when compared with a control cohort that received infant formula with unhydrolysed proteins (IF), and high allergenic weaning foods at an earlier age. The difference between cohorts was principally due to fewer adverse skin findings. (ABSTRACT TRUNCATED)

Background: The best nutritional option for newborn infants is mother's milk. However, some newborn babies may not be exclusively breastfed during the first months of life, potentially leading to reduced overall health status and the early onset of allergic diseases in some infants. Considerable research has been devoted to the development and assessment of infant nutrition programmes, particularly to the prevention of allergies in high-risk infants. However, equal numbers of infants with and without an elevated familial risk of allergies will eventually develop allergic diseases. Therefore, optimizing nutritional programmes for the early infant population as a whole is an important--but as yet insufficiently studied--area of investigation. Moreover, although safe and effective nutrition must primarily support healthy development of the infant, few studies have evaluated the overall health benefits of nutritional interventions, but have focussed on specific allergic manifestations. In animal models, an allergen-reduced moderate whey hydrolysate formula (pHF, Nestlé Beba HA) induces the development of oral tolerance towards cow's milk proteins, without inducing sensitization. In infants with a high risk for allergies, pHF formulae reduce the early onset of allergic disease during the first 5 years of life by approximately 50% compared with a dietary regimen of unaltered proteins. At present, very little is known about the overall health benefits of such a dietary intervention on the unselected infant population as a whole., Aim of the Study: The aim of our prospective, controlled study was to investigate the overall health benefits of an allergen-reduced nutritional programme in a newborn infant population unselected for atopic risk factors. The population in our study was as comparable as possible to the general population of healthy newborn infants. Our study included exclusive breastfeeding, use of a moderate whey hydrolysate formula (pHF, Nestlé Beba HA) if infant formula was needed, and delayed introduction of low-allergenic weaning foods. The study included assessments of compliance with the dietary programme, and evaluated nutritional habits, growth, and overall health status for 24 months. The health evaluation included allergic manifestations but did--by porpose--not define or evaluate them specifically. Part I of this paper gives results for nutritional habits during the first 6 months of life, Part II gives results for growth and general health status for the same time period, Part III will present feeding habits during the second half of the first year of life, and Part IV will present results to 24 months of age. The complete study report is published as a supplement to this journal., Methods: Nutritional assignment was to demographically comparable intervention (Z) or control (FF) cohorts according to the infant's place of birth. In the intervention cohort (Z, n = 564), the recommended dietary regimen was breastfeeding and/or the pHF formula, with no weaning food before 4 months of age. In the control cohort (FF, n = 566), there was no intervention. Longitudinal diet groups, defined for 4 months, excluding dropouts and noncompliants, were exclusive breastfeeding (eBF, Z, n = 201, FF, n = 162), partial breastfeeding (pBF, Z, n = 222, FF, n = 311), or non-breastfeeding (nBF, Z, n = 43, FF, n = 62). Imbalances between groups and cohorts in confounding factors that could influence health-related symptoms were integrated as covariates into the main analyses using logistic regression. Nutritional surveillance was carried out using continuous prospective monitoring., Results: The overall rate of breastfeeding, irrespective of partial or exclusive breastfeeding or the additional use of weaning foods, was similar in both cohorts at 4 and 6 months. However, from ages 3 to 6 months, significantly more Z than FF infants were exclusively breastfed (p < 0.05), and weaning foods were introduced at a significantly later age in Z t

To substitute for exocrine pancreatic insufficiency, patients with cystic fibrosis (CF) take pancreatic enzymes (PE) originating from porcine pancreas. Five different pancreatic enzyme preparations used by our patients contained 0.5-1.4 microg selenium per g tablet. In patients taking PE in doses that were gradually increased to improve fat absorption during a 48-month period, the effects of PE dose on erythrocyte selenium-dependent glutathione peroxidase (SeGSH-Px) activities and plasma selenium concentrations were studied. At baseline, erythrocyte SeGSH-Px activities were significantly lower in patients (p=.01), while plasma selenium concentrations did not differ between patients and healthy subjects. When PE dose and, consequently, selenium intake from PE was increased, erythrocyte SeGSH-Px activities (p < .001) and plasma selenium concentrations (p=.02) increased. Changes in SeGSH-Px activities during the initial 8 months correlated with those in selenium intake from PE (r=0.67, p < .001). Plasma selenium concentrations plateaued at 12 months and erythrocyte SeGSH-Px activities did so at 36 months, when patients had reached SeGSH-Px activities similar to those of healthy subjects. At 48 months, patients took an average lipase dose of 17400 U x kg(-1) x d(-1) and selenium dose from PE of 0.53 microg x kg(-1) x d(-1). We conclude that selenium content of PE preparations has a significant effect on SeGSH-Px activity in patients with CF. This form of selenium supply needs to be taken into account when selenium supplements are given to patients with CF.

The physiological role of prion protein (PrP) remains unknown. Mice devoid of PrP develop normally but are resistant to scrapie; introduction of a PrP transgene restores susceptibility to the disease. To identify the regions of PrP necessary for this activity, we prepared PrP knockout mice expressing PrPs with amino-proximal deletions. Surprisingly, PrP lacking residues 32-121 or 32-134, but not with shorter deletions, caused severe ataxia and neuronal death limited to the granular layer of the cerebellum as early as 1-3 months after birth. The defect was completely abolished by introducing one copy of a wild-type PrP gene. We speculate that these truncated PrPs may be nonfunctional and compete with some other molecule with a PrP-like function for a common ligand.

Unlabelled: The study evaluates faecal immunoreactive lipase (IRL) measurement in spot stool samples as an index of exocrine pancreatic function in patients with cystic fibrosis (CF). Stool samples (211) from 183 healthy volunteers (age range: 2 days-14.2 years) showed a normal log distribution of IRL values with a median concentration of 71.4 micrograms/g (range: 0.53-4160 micrograms/g). In 156 stool samples from 58 patients with proven CF, the median IRL concentration of 0.4 microgram/g (range: 0.003-107 micrograms/g) was significantly lower (P < 0.001) than that of normal controls. In healthy controls, IRL levels were age related with significantly higher levels (P < 0.001) shortly after birth compared to older children. Stimulation of the exocrine pancreas by oral milk feeding resulted in a significant (P < 0.001) increase in a faecal IRL concentration. Faecal IRL concentrations in meconium were very low and of the same magnitude as in patients with CF., Conclusion: Faecal IRL determination had a high diagnostic sensitivity (87%) and excellent diagnostic specificity (97%) in patients with CF. A negative test result (PVneg. 99%) virtually excluded CF under screening conditions.

The prion, the transmissible agent that causes spongiform encephalopathies such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease, is believed to be devoid of nucleic acid and to be identical to PrPSc (prion protein: scrapie form), a modified form of the normal host protein PrPC (prion protein: cellular form) which is encoded by the single copy gene Prnp. The 'protein only' hypothesis proposes that PrPSc, when introduced into a normal host, causes the conversion of PrPC into PrPSc; it therefore predicts that an animal devoid of PrPC should be resistant to prion diseases. The authors generated homozygous Prnp(o/o) ('PrP knockout') mice and showed that, after inoculation with prions, these mice remained free from scrapie for at least two years while wild-type controls all died within six months. There was no propagation of prions in the Prnp(o/o) animals. Surprisingly, heterozygous Prnp(o/+) mice, which express PrPC at about half the normal level, also showed enhanced resistance to scrapie despite high levels of infectious agent and PrPSc in the brain at an early stage. After introduction of murine PrP transgenes, Prnp(o/o) mice became highly susceptible to mouse--but not to hamster--prions, while the insertion of Syrian hamster PrP transgenes rendered the mice susceptible to hamster prions but much less susceptible to mouse prions. These complementation experiments enabled the application of reverse genetics. The authors prepared animals transgenic for genes encoding PrP with amino terminal deletions of various lengths and found that PrP that lacks 48 amino proximal amino acids (which comprise four of the five octa repeats of PrP) is still biologically active.

Vitamin C status and possible associations with the disease process in cystic fibrosis (CF) patients were investigated. Plasma vitamin C concentrations in patients from two different mid-European populations (Swiss, n = 62; Austrian, n = 60) taking no or low-dose vitamin C from multivitamin supplements did not differ from each other or from control subjects (n = 34). Vitamin C concentrations decreased with age (5.05 mumol.L-1, y-1). When followed up for 12 mo, patients had the highest plasma vitamin C concentrations in February and the lowest in May and August (P < 0.01); the decrease in vitamin C was accompanied by increases in plasma malondialdehyde (P < 0.001) and tumor necrosis factor alpha concentrations (P < 0.01). During supplementation with vitamin E for 2 mo or beta-carotene for 12 mo vitamin C concentrations did not change. They correlated inversely with white blood cell count (r = -0.36, P = 0.008), bands (r = -0.36, P = 0.02), alpha 1-acid glycoprotein (r = -0.45, P = 0.002), interleukin 6 (r = -0.46, P = 0.0006), and neutrophil elastase/alpha 1-proteinase inhibitor complexes (r = -0.34, P = 0.02). In patients with vitamin C concentrations < 40 mumol/L, all indexes of inflammation were relatively high, whereas those with concentrations > 80 mumol/L (upper quartile of control subjects) showed clearly lower values. These results are consistent with the hypothesis that by scavenging oxygen free radicals vitamin C interacts with an inflammation-amplifying cycle of activation of alveolar macrophages and neutrophils, release of proinflammatory cytokines and oxygen free radicals, and inactivation of antiproteases.

In a cross-sectional survey, plasma concentrations of alpha- and gamma-tocopherol, alpha- and beta-carotene (cis and trans isomers), lycopene, and retinol were determined by reversed-phase HPLC, and ratios of plasma alpha-tocopherol to cholesterol were calculated in 208 Swiss individuals ages 0.4-38.7 years. The influence of age, sex, and season of sampling was studied. Age was a significant predictor of all plasma concentrations except alpha-carotene. No sex-related differences were observed. Season of sampling affected alpha-tocopherol and retinol (higher in winter) and gamma-tocopherol and cholesterol concentrations (higher in winter and spring than in the other seasons). After correction for seasonal influences, age differences were 0.24 micromol/L per year for alpha-tocopherol, 0.04 micromol/L per year for retinol, and 0.04 micromol/L per year for cholesterol concentrations; ratios of plasma alpha-tocopherol to cholesterol were not affected by age. We constructed age-specific reference intervals from the regression line and a multiple of the standard deviation. Separate regression equations are presented for seasons with low and high values.

Lung inflammation in cystic fibrosis (CF) is associated with an increased release from activated neutrophils of oxidants and proteinases. Free radical generation is not efficiently neutralized, and the major anti-proteinase, alpha 1-proteinase inhibitor (alpha 1-PI) is thought to be oxidatively inactivated. We hypothesized that enhanced antioxidant protection could represent an additional long-term strategy to attentuate the host inflammatory response. The effect on plasma neutrophil elastase/alpha 1-PI (NE/alpha 1-PI) complex levels (as a marker of lung inflammation) and plasma malondialdehyde concentrations (as a marker of lipid peroxidation) of additional oral beta-carotene supplementation was studied in 33 CF patients who had already received long-term vitamin E supplementation. In the presence of a more than 10-fold increase in plasma beta-carotene concentrations (mean +/- SEM) (0.09 +/- 0.01 to 1.07 +/- 0.19 mumol/L; p < 0.0001), a small increase in plasma alpha-tocopherol concentrations (23.8 +/- 1.31 to 28.4 +/- 1.81 mumol/L; p = 0.02), and a more than 50% decrease in plasma malondialdehyde concentrations (1.00 +/- 0.07 to 0.46 +/- 0.03 mumol/L; p < 0.0001), plasma NE/alpha 1-PI complex levels decreased from 102.2 +/- 16.0 to 83.0 +/- 10.4 micrograms/L; (p = 0.02). Plasma retinol concentrations increased (1.05 +/- 0.06 to 1.23 +/- 0.07 mumol/L; p = 0.0001) due to conversion of beta-carotene to retinol, which could have contributed to the decrease in NE/alpha 1-PI complex levels. Based on these results, we speculate that efficient antioxidant supplementation could attenuate lung inflammation in CF.

Biochemical vitamin E deficiency and low plasma lipids are frequent findings in patients with cystic fibrosis (CF). The response to a single oral dose of all-rac-alpha-tocopheryl acetate [100 IU (100 mg)/kg body wt] was studied over 24 h in 25 CF patients with exocrine pancreatic insufficiency and in 23 healthy individuals. Patients received pancreatic enzymes together with the vitamin E test dose. At baseline, plasma alpha-tocopherol concentrations correlated with cholesterol concentrations; both were lower in patients than in control subjects, as were erythrocyte alpha-tocopherol concentrations (all P < 0.0001). Plasma and erythrocyte alpha-tocopherol concentrations were significantly higher than baseline concentrations from 3 and 6 h onward, respectively, and peaked most frequently at 6 and 12 h, respectively, in both patients and control subjects. Maximum increases and areas under the concentration time curves for plasma alpha-tocopherol concentrations were smaller in patients than in control subjects (P < 0.0001). When ratios of plasma alpha-tocopherol to cholesterol (to correct for differences in cholesterol concentrations) or erythrocyte alpha-tocopherol concentrations were applied, patients were shown to respond as efficiently as control subjects. On the basis of these results, we recommend vitamin E supplements in doses high enough to achieve vitamin E status in CF patients well within the range of healthy individuals; these supplements should be given with appropriate amounts of pancreatic enzymes. However, for long-term supplementation much lower doses than those used in this test situation may be sufficient.

To investigate the efficacy of three different vitamin E preparations for optimizing vitamin E status in cystic fibrosis (CF patients long-term, 29 patients (aged 0.7-29.8 y) were randomly assigned to receive 400 IU of either RRR-alpha-tocopherol (A: 268 mg, n = 10) or all rac-alpha-tocopheryl acetate as a fat-soluble (B: 400 mg, n = 10) or water-miscible preparation (C: 400 mg, n = 9) and were followed for 6 wk. In the whole study group, plasma alpha-tocopherol concentrations increased from baseline (10.5 +/- 4.6 micromol/L) to 3 wk (25.7 +/- 6.5 micromol/L; P < 0.001), but not further between 3 and 6 wk; concentrations at 3 and 6 wk did not differ from those of age-matched control subjects (23.6 +/- 3.9 micromol/L). There was no significant difference in the increase from baseline to 6 wk among preparations A (17.75 +/- 8.43 micromol/L), B (14.0 +/- 9.4 micromol/L), and C (15.5 +/- 7.1 micromol/L). Because of differences in body weight, the dose administered ranged from 5.5 to 47.4 IU x kg-1 x d-1; it correlated positively with the increase in plasma alpha-tocopherol concentrations (P < 0.001). There was no significant difference in the increase in plasma alpha-tocopherol concentrations between patients with CF-associated liver disease (n = 8) who received 10.2 +/- 3.8 IU x kg-1 x d-1 and those without liver disease taking comparable doses. We conclude that CF patients can be efficiently supplemented with 400 IU/d of any one of the three vitamin E preparations and plasma values of healthy control subjects can be achieved.

Antioxidants such as vitamin E protect unsaturated fatty acids of LDL against oxidation. In the ex vivo model used, LDL was exposed to Cu2+ ions, a potent prooxidant capable of initiating the oxidation of LDL. The lag time, indicating the delay of conjugated diene formation in LDL due to antioxidant protection, was measured in 54 cystic fibrosis (CF) patients with plasma alpha-tocopherol levels below (Group A, n = 30) or above (Group B, n = 24) 15.9 mumol/L (mean - 2 SD of Swiss population). Patients were reevaluated after 2 months on 400 IU/d of oral RRR-alpha-tocopherol. In group A, alpha-tocopherol concentrations in LDL increased significantly from 3.2 +/- 1.6 mol/mol LDL to 8.2 +/- 2.8 mol/mol (P < 0.001) and lag times increased from 79 +/- 33 min to 126 +/- 48 min (P < 0.001), whereas in the vitamin E sufficient group B no further increase neither in LDL alpha-tocopherol concentrations or in lag times was observed. LDL oleic acid concentrations were higher, and linoleic acid concentrations were lower in patients than in controls. After efficient vitamin E supplementation, lag times were positively related to LDL alpha-tocopherol (P < 0.01) and negatively to LDL linoleic and arachidonic acid content (P < 0.001). The maximum rate of oxidation correlated positively with linoleic and arachidonic acid concentrations, as did the maximum conjugated diene absorbance. These results indicate that LDL resistance to oxidation is impaired in vitamin E deficient CF patients but can be normalized within 2 months when alpha-tocopherol is given in sufficient amounts. Linoleic and arachidonic acid content exhibit a major influence on the LDL resistance to oxidation.

The aim of this study was to determine the efficacy of long-term oral beta-carotene supplementation for correcting impaired beta-carotene status in cystic fibrosis patients. Thirty-five patients (2.3-30.5 years of age) with coefficients of fat absorption of 46-96% (median 88%) received beta-carotene 0.5 mg/kg daily and were followed over a 16-month treatment period. Baseline plasma beta-carotene concentrations in patients (mean +/- SD, 0.09 +/- 0.06 mumol/l) were significantly lower than those of age-matched controls (0.86 +/- 0.56 mumol/l) (p < 0.0001). Concentrations increased rapidly and reached a plateau at or before 3 weeks that was maintained throughout the study period. Values obtained at 3 weeks (0.89 +/- 0.64 mumol/l) were significantly higher (p < 0.0001) than those at baseline and did not differ from controls. Plasma retinol and alpha-tocopherol concentrations increased during the observation period, but remained within normal ranges. Plasma retinyl palmitate, which was below the detection limit in all but one patient at baseline, did not increase. Thus oral beta-carotene supplementation is effective and normalizes beta-carotene status of cystic fibrosis patients without evidence of significant side effects.

We investigated the effect of correcting beta-carotene deficiency in cystic fibrosis (CF) patients on two parameters of lipid peroxidation. The resistance to oxidation of low density lipoprotein (LDL) was measured by the lag time preceding the onset of conjugated diene formation during exposure to copper(II) ions, and lipid peroxide formation was quantitated by malondialdehyde concentrations in plasma (TBA/HPLC method). Simultaneously, alpha-tocopherol and beta-carotene concentrations were determined in LDL and in plasma. Thirty-four CF patients were investigated before and after 3 months of oral beta-carotene supplementation. Beta-carotene concentrations increased (p < 0.0001) in plasma (mean +/- SD) (0.09 +/- 0.06 vs. 1.07 +/- 0.86 mumol/l) and in LDL (0.02 +/- 0.02 vs. 0.31 +/- 0.28 mol/mol), without significant changes in alpha-tocopherol, either in plasma (24.7 +/- 5.9 vs. 25.4 +/- 7.6) or in LDL (8.47 +/- 2.95 vs. 9.05 +/- 4.13). Lag times, being shorter (p < 0.05) in patients than in controls, increased from 48.5 +/- 21.3 to 69.1 +/- 27.9 min (p < 0.001) and plasma MDA concentrations, being greater (p < 0.0001) in patients than in controls, decreased from 0.95 +/- 0.32 to 0.61 +/- 0.15 mumol/l (p < 0.0001). At 3 months, lag times and MDA concentrations did not any longer differ between patients and controls. These data suggest that excess lipid peroxidation occurring in beta-carotene deficiency can be limited and normalized during efficient beta-carotene supplementation in CF patients.

Since 1976, various activity indices for Crohn's disease have been developed but none has been suitable for use in the paediatric age group. Therefore, the German-Swiss Study Group on Crohn's Disease in Children and Adolescents decided to develop their own paediatric Crohn's disease activity index (PCDAI) by multiple regression analysis of prospectively collected data. The result was a simple index consisting of two clinical (appetite, number of stools/week) and four laboratory variables (erythrocyte sedimentation rate, serum iron and alpha 2-globulin concentrations and bands as percentage of white blood cells). Applying the index to patients who were followed-up, it could be demonstrated that the changes in PCDAI inversely reflected the changes in weight and that the surgical removal of the inflamed parts of the gut reduced the disease activity index to levels comparable to those obtained in patients after successful, exclusively conservative, treatment. Low disease activity was maintained for at least three years.

The second child of healthy unrelated parents presented with chronic diarrhoea since the age of two months, initially associated with non-characteristic liver involvement. Recurrent infections, severe failure to thrive and various metabolic deficiencies complicated the further course, as well as profuse watery diarrhoea with elevated regulatory gut peptides, responding only to somatostatin analog treatment. At 22 months of age, intermittent cholestasis with permanently normal serum gamma-glutamyltransferase was evident. The child died of fulminant purulent meningitis at the age of three years six months. Liver histology showed intrahepatic cholestasis, bile duct paucity with focal proliferation as well as slight portal and intralobular fibrosis. The clinical, biochemical and histopathological findings were indicative of Byler's disease.

Polyunsaturated fatty acids of biomembranes are a major target of lipid peroxidation. In vitamin E deficiency an efficient delivery of a high oral loading dose of all-rac-alpha-tocopheryl acetate to erythrocyte membranes could provide an early onset antioxidative effect. We investigated short-term changes in erythrocyte alpha-tocopherol after a single oral dose of 100 mg all-rac-alpha-tocopheryl acetate/kg in 10 vitamin E-deficient cystic fibrosis (CF) patients. Over 24 h, erythrocyte alpha-tocopherol increased 68% to 420% of preloading concentrations. With two exceptions, peak values were achieved 12 or 24 h after administration, which was 3-18 h later than peak plasma concentrations. Separate median-based curve estimates for the changes in erythrocyte alpha-tocopherol for five patients with and five without associated cholestatic liver disease were obtained. Cross-sectional test results revealed significantly lower erythrocyte alpha-tocopherol for the 9- and 24-h observations for patients with cholestatic liver disease compared with those without. Oral all-rac-alpha-tocopheryl acetate can be rapidly incorporated into erythrocyte membranes in vitamin E-deficient CF patients.

Summarizing the development of experiences and discussions of the last 20 years, a working group of the ESPGAN has recently updated the criteria of diagnosis of coeliac disease. The reliable finding of the initial typical mucosal lesion in the untreated patient is a hallmark of the disease and, if followed by an unequivocal clinical response to a gluten free diet, can be considered as final evidence for the diagnosis. Gluten challenges should be limited to cases without this evidence. High levels of antigliadin- and antireticulin- or or antiendomysium antibodies are supportive for the diagnosis but cannot substitute biopsy proof of the disease. The importance of a very long term strict gluten free diet in coeliac disease is established by recent evidence showing that in such cases there is no increased risk of malignancy.

The paper presents a discussion of the definition, the indications and some of the difficulties and complications of total long-term parenteral alimentation in infants and children. The problems of protein quality, the inadequacy of the E/T ratio, the quantities and quality of carbohydrates and the metabolic complications due to inappropriate electrolyte and mineral salts composition are reviewed. It is pointed out that the optimal amounts of some of the components used are still under investigation, that there seems to be no imperative reason not to use glucose as the sole carbohydrate in this age group and that most of the possible long-term sequelae and complications of total long-term parenteral alimentation will have to be looked for by prospective studies of the children treated.

In the mature human newborn infant, only pancreatic alpha-amylase shows a very low but highly inducible activity. Other digestive enzymes, although not yet at adult levels, increase rapidly with age. Intestinal absorptive functions develop already during early and mid-pregnancy and are mostly at adult levels in the newborn infant. Fat absorption is usually low in the premature infant and increases rapidly during the first month.

Protein electrophoresis in a 9-yr-old Libyan boy suffering from intestinal alpha chain disease showed a conspicuous peak in the gamma region. Examination in immunoelectrophoresis with monospecific antisera against IgA revealed a quantitatively important monoclonal gradient. The heavy chain protein in serum, urine and duodenal juice was further analysed immunologically in double diffusion, antigen-antibody crossed electrophoresis and biochemically in gel filtration experiments and SDS PAGE. By these techniques alpha 1 chain aggregates of different molecular weight could be found. Immunofluorescence examination of small intestine biopsies showed abundant plasma cells containing alpha heavy chains but no light chains.

In the pediatric hospitals and clinics of Zurich, St. Gallen, Winterthur, Aarau and Lucerne, 488 cases of coeliac disease have been found in children born between 1960 and 1978. The diagnosis was established clinically and confirmed by intestinal biopsy in each case. The data of coeliac patients diagnosed before 1966 and after 1975 has proven to be unreliable for statistical evaluation and were omitted from our calculations. During the years 1966-1975, 354 patients with confirmed coeliac disease and 412733 live births were registered. This corresponds, for the symptomatic coeliac disease, to an incidence of 1:1165 in the Northeastern part of Switzerland. A statistical analysis of the data confirmed its validity and the lack of trends during the period of study.

The criteria for the diagnosis of coeliac disease are the evidence of a well characterized and typical, albeit not pathognomonic lesion of the proximal small bowel mucosa in untreated patients as well as the unequivocal rapid response to a gluten free diet (GFD). The instruction and the motivation of the patients in GFD aims at an early and positive establishment of dietary habits, based on an adequate choice of gluten free foods. As coeliac disease is genetically determined, GFD should be maintained life long. The results of long term evaluations of relapses in coeliac patients confirm this postulate by demonstrating that only 6.5% of the patients show no mucosal alteration upon gluten ingestion, even after years on GFD, and that another group (12-18%) will deteriorate very slowly over years. Routine gluten challenges in patients, whose initial diagnosis was established according to the discussed criteria, seem therefore unjustified, as they represent a serious challenge of established dietary and life habits for the vast majority of patients without any positive issue for them.

P-Amino-benzoic acid (PABA) is split specifically by pancreatic chymotrypsin from the synthetic tripeptide N-benzoyl-L-tyrosyl-PABA. The urinary excretion of absorbed PABA serves as an index for exocrine pancreatic function. The peptide (0.015 g/kg) was administered orally to 20 controls (aged between 5 months and 16 years), 6 patients with exocrine pancreatic insufficiency caused by cystic fibrosis (CF), and 9 newborn infants. In the controls the mean 6-hour PABA recovery was 58.5% (+/- 11.2 SD). Recovery in patients with CF was lower (P less than 0.001) with no overlap. In newborn infants the mean 6-hour PABA recovery was 23.4 (+/- 17.7 SD); overlapping in 3 instances with the results in CF patients. This simple, noninvasive test thus appears promising and merits further investigation in younger infants, especially newborns.

In a clinical survey, the symptoms, associated diseases, and local and metabolic complications of Crohn's disease are discussed. Medical treatment (with SASP, corticosteroids, and immunosuppressives), as well as treatment for malnutrition, is of special importance.