Göte Forsberg

Utility of the housekeeping genes 18S rRNA, beta-actin and glyceraldehyde-3-phosphate-dehydrogenase for normalization in real-time quantitative reverse transcriptase-polymerase chain reaction analysis of gene expression in human T lymphocytes.

The accuracy of 18S rRNA, β‐actin mRNA and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) mRNA as indicators of cell number when used for normalization in gene expression analysis of T lymphocytes at different activation stages was investigated. Quantitative real‐time reverse transcriptase‐polymerase chain reaction was used to determine the expression level of 18S rRNA, β‐actin mRNA, GAPDH mRNA and mRNA for six cytokines in carefully counted samples of resting human peripheral blood mononuclear cells (PBMCs), intestinal lymphocytes and PBMCs subjected to polyclonal T‐cell activation. The 18S rRNA level in activated and resting PBMCs and intestinal lymphocytes was essentially the same, while the levels of β‐actin and GAPDH mRNAs fluctuated markedly upon activation. When isolated γδTCR+, CD4+ and CD8+ subpopulations were studied, 18S rRNA levels remained unchanged after 21 h of activation but increased slightly after 96 h. In contrast, there was a 30–70‐fold increase of GAPDH mRNA/cell in these cell populations upon activation. Cytokine analysis revealed that only normalization to 18S rRNA gave a result that satisfactorily reflected their mRNA expression levels per cell. In conclusion, 18S rRNA was the most stable housekeeping gene and hence superior for normalization in comparative analyses of mRNA expression levels in human T lymphocytes.

Presence of Bacteria and Innate Immunity of Intestinal Epithelium in Childhood Celiac Disease

OBJECTIVE:Exposure to gliadin and related prolamins and appropriate HLA-DQ haplotype are necessary but not sufficient for contracting celiac disease (CD). Aberrant innate immune reactions could be contributing risk factors. Therefore, jejunal biopsies were screened for bacteria and the innate immune status of the epithelium investigated.METHODS:Children with untreated, treated, challenged CD, and controls were analyzed. Bacteria were identified by scanning electron microscopy. Glycocalyx composition and mucin and antimicrobial peptide production were studied by quantitative RT-PCR, antibody and lectin immunohistochemistry.RESULTS:Rod-shaped bacteria were frequently associated with the mucosa of CD patients, with both active and inactive disease, but not with controls. The lectin Ulex europaeus agglutinin I (UEAI) stained goblet cells in the mucosa of all CD patients but not of controls. The lectin peanut agglutinin (PNA) stained glycocalyx of controls but not of CD patients. mRNA levels of mucin-2 (MUC2), α-defensins HD-5 and HD-6, and lysozyme were significantly increased in active CD and returned to normal in treated CD. Their expression levels correlated to the interferon-γ mRNA levels in intraepithelial lymphocytes. MUC2, HD-5, and lysozyme proteins were seen in absorptive epithelial cells. β-defensins hBD-1 and hBD-2, carcinoembryonic antigen (CEA), CEA cell adhesion molecule-1a (CEACAM1a), and MUC3 were not affected.CONCLUSIONS:Unique carbohydrate structures of the glycocalyx/mucous layer are likely discriminating features of CD patients. These glycosylation differences could facilitate bacterial adhesion. Ectopic production of MUC2, HD-5, and lysozyme in active CD is compatible with goblet and Paneth cell metaplasia induced by high interferon-γ production by intraepithelial lymphocytes.

Over‐expression of interleukin 10 in mucosal T cells of patients with active ulcerative colitis

Ulcerative colitis (UC), a chronic inflammatory bowel disease, exhibits pronounced increase of T lymphocytes in the inflamed mucosa. To understand the role of intestinal T lymphocytes in the pathogenesis of UC their cytokine production in the mucosa was analysed. Intestinal T lymphocytes of UC, Crohns disease and control patients were analysed for cytokine mRNA levels by real‐time quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) directly after isolation without in vitro stimulation. Frequencies of cytokine positive cells were determined in UC and control colon by immunomorphometry. T lymphocytes in normal colon expressed interleukin (IL)‐2, interferon (IFN)‐γ, tumour necrosis factor (TNF)‐α and transforming growth factor (TGF)‐β1, but not IL‐4, IL‐5 or IL‐10. In UC, a highly significant increase in IL‐10 mRNA levels in T lymphocytes and an increased frequency of IL‐10 positive cells was seen in colon. IL‐10 mRNA levels were also elevated in T lymphocytes of the non‐inflamed ileum and correlated with disease activity at both locations. CD4+ T lymphocytes were the major source of IL‐10 mRNA. IL‐2, IFN‐γ and TNF‐α mRNA levels were decreased in colonic T lymphocytes, and virtually no IL‐2, IFN‐γ, TNF‐α or TGF‐β positive cells were detected in basal lymphoid aggregates. However, scattered IL‐10 positive cells were found here. Lamina propria outside the aggregates contained IL‐10‐, IFN‐γ, TNF‐α and TGF‐β but not IL‐2 positive cells. T cells of UC patients did not express IL‐4 or IL‐5. Taken, together the data suggest a generalized activation of IL‐10 producing CD4+ T cells along the intestine of UC patients. The local environment seems to determine the biological consequences of elevated IL‐10.

Proximal small intestinal microbiota and identification of rod-shaped bacteria associated with childhood celiac disease

OBJECTIVES:Alterations in the composition of the microbiota in the intestine may promote development of celiac disease (CD). Using scanning electron microscopy (SEM) we previously demonstrated that rod-shaped bacteria were present on the epithelium of proximal small intestine in children with CD but not in controls. In this study we characterize the microbiota of proximal small intestine in children with CD and controls and identify CD-associated rod-shaped bacteria.METHODS:Proximal small intestine biopsies from 45 children with CD and 18 clinical controls were studied. Bacteria were identified by 16S rDNA sequencing in DNA extracted from biopsies washed with buffer containing dithiothreitol to enrich bacteria adhering to the epithelial lining, by culture-based methods and by SEM and transmission electron microscopy.RESULTS:The normal, mucosa-associated microbiota of proximal small intestine was limited. It was dominated by the genera Streptococcus and Neisseria, and also contained Veillonella, Gemella, Actinomyces, Rothia, and Haemophilus. The proximal small intestine microbiota in biopsies from CD patients collected during 2004–2007 differed only marginally from that of controls, and only one biopsy (4%) had rod-shaped bacteria by SEM (SEM+). In nine frozen SEM+ CD biopsies from the previous study, microbiotas were significantly enriched in Clostridium, Prevotella, and Actinomyces compared with SEM− biopsies. Bacteria of all three genera were isolated from children born during the Swedish CD epidemic. New Clostridium and Prevotella species and Actinomyces graevenitzii were tentatively identified.CONCLUSIONS:Rod-shaped bacteria, probably of the indicated species, constituted a significant fraction of the proximal small intestine microbiota in children born during the Swedish CD epidemic and may have been an important risk factor for CD contributing to the fourfold increase in disease incidence in children below 2 years of age during that time.Am J Gastroenterol advance online publication, 15 September 2009; doi:10.1038/ajg.2009.524

Comparison of the cost-effectiveness of budesonide and sodium cromoglycate in the management of childhood asthma in everyday clinical practice

BACKGROUND Budesonide and sodium cromoglycate are both recommended as maintenance therapy for childhood asthma. OBJECTIVE To compare the cost-effectiveness of these two treatment strategies in clinical practice, in an open-label, pharmacoeconomic clinical trial. METHODS Health economics were evaluated in 138 children, ages 5 to 11 years, with unstable asthma not previously treated with corticosteroids or cromones. The asthma was stabilized during 4 to 6 weeks with budesonide 200 to 400 microg twice daily. The children were then randomly allocated to one of the two treatment strategies aiming at maintaining asthma control for 12 months; budesonide 400 microg/day (N = 69) or sodium cromoglycate 60 mg/day (N = 69). If asthma control was judged unsatisfactory, the doses were increased or the children were switched to the alternate treatment. RESULTS In children continuing on the same treatment, the degree of asthma control was similar in the two groups at study end. To maintain asthma control, 42% of cromoglycate children switched to budesonide, and then experienced a 14% increase in symptom-free days. No budesonide patient had to switch therapy because of lack of asthma control. Although not statistically significant, total annual cost per patient was 24% (Swedish kronor 4195; US

Aberrant extrathymic T cell receptor gene rearrangement in the small intestinal mucosa: a risk factor for coeliac disease?

487; Euro 485) lower in the budesonide than the cromoglycate group, mainly due to a lower cost for asthma medication. CONCLUSIONS A budesonide strategy for continued maintenance treatment, after an initial period of stabilizing treatment with budesonide, resulted in lower costs and less drug switches than did a strategy with sodium cromoglycate.

Celiac Disease : Effect of weaning on disease risk

Background: Coeliac disease is a small intestine enteropathy caused by permanent intolerance to wheat gluten. Gluten intake by patients with coeliac disease provokes a strong reaction by intestinal intraepithelial lymphocytes (IELs), which normalises on a gluten-free diet. Aim: To investigate whether impaired extrathymic T cell maturation and/or secondary T cell receptor (TCR) gene recombination in IELs are features of coeliac disease which could contribute to the failure of establishing tolerance to gluten. Methods: Expression levels of the four splice-forms of recombination activating gene-1 (RAG1) mRNA and preT α-chain (preTα) mRNA were determined in IEL-subsets of children with coeliac disease and controls. Frequencies of RAG1 expressing IELs were determined by immunomorphometry. Results: In controls, the RAG1-1A/2 splice-form selectively expressed outside the thymus, was dominant and expressed in both mature (TCR+) and immature (CD2+CD7+TCR−) IELs (∼8 mRNA copies/18S rRNA U). PreTα was expressed almost exclusively in CD2+CD7+TCR− IELs (∼40 mRNA copies/18S rRNA U). By contrast, RAG1 and preTα mRNA levels were low in patients with coeliac disease compared to controls, both with active disease and with inactive, symptom-free disease on a gluten-free diet (p values <0.01 for mature and <0.05 for immature IELs). Similarly, the frequencies of RAG1+ IELs were significantly lower in patients with coeliac disease compared to controls (p<0.001). Conclusions: Patients with coeliac disease appear to have an impaired capacity for extrathymic TCR gene rearrangement. This is an inherent feature, which probably plays a pivotal role in the failure to efficiently downregulate the T cell response to gluten.

Paradoxical coexpression of proinflammatory and down-regulatory cytokines in intestinal T cells in childhood celiac disease

From the weaning period and onwards the intestinal mucosa is exposed to an increasing number of antigens, e.g. food components and microorganisms. Of all the antigens that reach the systemic circulation from the gut lumen, only a minority are potentially harmful to humans and need to be defended against. The majority of intestinal antigens do not require a protective immune response, but may even be beneficial for the individual. Thus, the mucosal immune system must have the capacity to discriminate between when an appropriate protective immune response to harmful foreign antigens is required and when a muted or non-response is preferable. One important component of the latter is oral tolerance, which may be defined as a systemic hypo-responsiveness or non-responsiveness of mature T and B lymphocytes to antigen challenge after prior oral exposure to the antigen [1, 2]. Celiac disease (CD), or permanent gluten-sensitive enteropathy, develops because tolerance to ingested wheat gluten (gliadins and glutenins), and related proteins from rye and barley never develops, or is broken after it has developed. The disease is characterized by inflammation of the small intestine resulting in crypt hyperplasia, villous atrophy and flattening of the mucosa. Other characteristics are an increased number of intraepithelial lymphocytes (IELs) and lamina propria lymphocytes, increased serum concentrations of IgA antibodies towards gliadin and the autoantigen tissue transglutaminase. When gluten is withdrawn from the diet the mucosal Hernell O, Schmitz J (eds): Feeding during Late Infancy and Early Childhood: Impact on Health. Nestlé Nutr Workshop Ser Pediatr Program, vol 56, pp 27–42, Nestec Ltd., Vevey/S. Karger AG, Basel,