Marie-Louise Hammarström

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.

Increased expression of antimicrobial peptides and lysozyme in colonic epithelial cells of patients with ulcerative colitis.

The impact of chronic inflammation on the expression of human α‐defensins 5 and 6 (HD‐5, HD‐6), β‐defensins 1 and 2 (hBD‐1, hBD‐2) and lysozyme in epithelial cells of small and large intestine was investigated. Intestinal specimens from 16 patients with ulcerative colitis (UC), 14 patients with Crohns disease (CD) and 40 controls with no history of inflammatory bowel disease were studied. mRNA expression levels of the five defence molecules were determined in freshly isolated epithelial cells by real‐time quantitative RT‐PCR. Specific copy standards were used allowing comparison between the expression levels of the different defensins. HD‐5 and lysozyme protein expression was also studied by immunohistochemistry. Colonic epithelial cells from patients with UC displayed a significant increase of hBD‐2, HD‐5, HD‐6 and lysozyme mRNA as compared to epithelial cells in controls. Lysozyme mRNA was expressed at very high average copy numbers followed by HD‐5, HD‐6, hBD‐1 and hBD‐2 mRNA. HD‐5 and lysozyme protein was demonstrated in metaplastic Paneth‐like cells in UC colon. There was no correlation between hBD‐2 mRNA levels and HD‐5 or HD‐6 mRNA levels in colon epithelial cells of UC patients. Colonic epithelial cells of Crohns colitis patients showed increased mRNA levels of HD‐5 and lysozyme mRNA whereas ileal epithelial cells of Crohns patients with ileo‐caecal inflammation did not. Chronic inflammation in colon results in induction of hBD‐2 and α‐defensins and increased lysozyme expression. hBD‐1 expression levels in colon remain unchanged in colitis. The high antimicrobial activity of epithelial cells in chronic colitis may be a consequence of changes in the epithelial lining, permitting adherence of both pathogenic bacteria and commensals directly to the epithelial cell surface.

Probiotics during weaning reduce the incidence of eczema

A reduced microbial load early in life has been suggested to be linked to the increasing prevalence of allergic diseases in the industrialized world. Some studies have indicated that probiotics may be effective in the prevention of eczema. In vitro studies indicate that probiotics have immunomodulatory effects. In the present study, we evaluated the effects of feeding Lactobacillus F19 during weaning on the incidence of eczema and Th1/Th2 balance. In a double‐blind, placebo‐controlled randomized intervention trial, infants were fed cereals with (n = 89) or without Lactobacillus F19 (n = 90) from 4 to 13 months of age. We assessed the cumulative incidence of eczema at 13 months of age. The ratio of interferon‐γ (IFN‐γ) to interleukin 4 (IL4) mRNA expression levels in polyclonally stimulated peripheral blood T cells was used as a proxy for immune balance. Total and specific IgE serum levels were also assessed. The cumulative incidence of eczema at 13 months was 11% (4–17%, 95% CI) and 22% (13–31%, 95% CI) in the probiotic and placebo groups, respectively (p < 0.05). The number needed to treat was 9 (6.5–11.5, 95% CI). At 13 months of age, the IFN‐γ/IL4 mRNA ratio was higher in the probiotic compared with the placebo group (p < 0.05). In contrast, there were no differences between groups in serum concentrations of IgE. In summary, feeding Lactobacillus F19 during weaning could be an effective tool in the prevention of early manifestation of allergy, e.g., eczema. The higher Th1/Th2 ratio in the probiotic compared with the placebo group suggests enhancing effects of Lactobacillus F19 on the T cell‐mediated immune response.

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.

β-Defensin-3 and -4 in intestinal epithelial cells display increased mRNA expression in ulcerative colitis

mRNA expression of two recently described human β‐defensins (hBD‐3 and hBD‐4) in epithelial cells of normal small and large intestine and the impact of chronic intestinal inflammation on their expression levels was investigated. Intestinal specimens from patients with ulcerative colitis (UC), Crohns disease (CD) and controls with no history of inflammatory bowel disease were studied. hBD‐3 and hBD‐4 mRNAs were determined in freshly isolated epithelial cells by real‐time quantitative reverse transcription–polymerase chain reaction (QRT‐PCR) and by in situ hybridization. The effect of proinflammatory cytokines on hBD‐3 and hBD‐4 mRNA expression in colon carcinoma cells was also investigated. Purified epithelial cells of normal small and large intestine expressed both hBD‐3 and hBD‐4 mRNA, with higher expression levels of hBD‐3 mRNA. In situ hybridization revealed higher levels of mRNA expression in the crypt‐ compared to the villus/luminal‐compartment. Interferon (IFN)‐γ, but not tumour necrosis factor (TNF)‐α or IL‐1β, augmented hBD‐3 mRNA expression. None of these agents stimulated hBD‐4 expression. Colonic epithelial cells from patients with UC displayed a significant increase in hBD‐3 and hBD‐4 mRNA compared to epithelial cells of controls. In contrast, small intestinal epithelial cells from CD patients did not show increased expression levels compared to the corresponding control cells. Moreover, Crohns colitis did not show increased expression of hBD‐4 mRNA, while the data are inconclusive for hBD‐3 mRNA. We conclude that the chronic inflammatory reaction induced in the colon of UC patients enhances hBD‐3 and hBD‐4 mRNA expression in the epithelium, whereas in CD this is less evident.

Characterisation of mucosal lymphoid aggregates in ulcerative colitis: immune cell phenotype and TcR-γδ expression

BACKGROUND AND AIMS A histopathological feature considered indicative of ulcerative colitis (UC) is the so-called basal lymphoid aggregates. Their relevance in the pathogenesis of UC is, however, unknown. We have performed a comprehensive analysis of the immune cells in these aggregates most likely corresponding to the lymphoid follicular hyperplasia also described in other colitides. METHODS Resection specimens of UC and normal colon were analysed by immunomorphometry, immunoflow cytometry, and immunoelectron microscopy, using a large panel of monoclonal antibodies. RESULTS (1) In all cases of UC, colonic lamina propria contained numerous basal aggregates composed of lymphocytes, follicular dendritic cells, and CD80/B7.1 positive dendritic cells. (2) CD4+CD28−αβ T cells and B cells were the dominant cell types in the aggregates. (3) The aggregates contained a large fraction of cells that are normally associated with the epithelium: that is, γδ T cells (11 (7)%) and αEβ7 +cells (26 (13)%). The γδ T cells used Vδ1 and were CD4−CD8−. Immunoelectron microscopy analysis demonstrated TcR-γδ internalisation and surface downregulation, indicating that the γδ T cells were activated and engaged in the disease process. (4) One third of cells in the aggregates expressed the antiapoptotic protein bcl-2. CONCLUSIONS Basal lymphoid aggregates in UC colon are a consequence of anomalous lymphoid follicular hyperplasia, characterised by abnormal follicular architecture and unusual cell immunophenotypes. The aggregates increase in size with severity of disease, and contain large numbers of apoptosis resistant cells and activated mucosal γδ T cells. The latter probably colonise the aggregates as an immunoregulatory response to stressed lymphocytes or as a substitute for defective T helper cells in B cell activation. γδ T cells in the aggregates may be characteristic of UC.

Immunomorphologic studies of human decidua-associated lymphoid cells in normal early pregnancy.

Human decidual lymphocytes from early, normal pregnancy were characterized in situ with respect to ultrastructure and distribution of subsets. The ultrastructure of isolated decidual gamma delta T cells was also studied. CD45+ cells comprised 11 +/- 2% of all decidual cells. The majority were localized in large lymphoid cell clusters (LCC), near endometrial glands, or as intraepithelial lymphocytes (IEL) in glandular epithelium. The major cell populations in LCC were CD56+TCR-gamma delta+ cells, CD56+ cells, TCR-alpha beta+CD4+ cells, and TCR-alpha beta+CD8+ cells. All expressed activation markers (CD45RO, Kp43, and/or HML-1) and MHC class II Ag (HLA-DR, HLA-DP, and/or HLA-DQ). No B cells were found. Almost all IEL were activated TCR-gamma delta+ cells (CD56+ and CD56-). The glandular epithelial cells expressed heat shock protein 60 at the basolateral side facing the TCR-gamma delta+ IEL. Decidual lymphocytes displayed cytoplasmic processes, microvilli, characteristic cytoplasmic granules, and had intimate contact with neighboring cells. Lymphocytes in the outer rim of LCC and the stroma showed signs of cellular movement. Two main morphotypes of gamma delta T cells could be distinguished. One had single microvilli, membrane-bound granules, and nuclear inclusions. The other had many microvilli, nonmembrane-bound granules and cytoplasmic multivesicular bodies. Our data suggest that LCC are centers of immune reactivity where T and NK cells become activated. The activated cells may guard against infections and undue trophoblast invasion and/or be involved in modulating the local maternal immune system toward unresponsiveness against the semiallogeneic fetus.

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

Human gamma delta T cells that inhibit the in vitro growth of the asexual blood stages of the Plasmodium falciparum parasite express cytolytic and proinflammatory molecules.

The functional properties, regarding parasite growth inhibition in vitro, the cytotoxic potential and cytokine profiles of human γδ+ and αβ+ T cells, T‐cell lines and clones stimulated with Plasmodium falciparum‐antigen‐or T‐cell mitogen in vitro were investigated. Using reverse transcriptase‐polymerase chain reaction (RT‐PCR) and specific primers, mRNA for the cytolytic molecules perforin, granzyme A and B, Fas and Fas ligand (FasL) were detected in both the γδ‐ and the αβT cells. Despite this fact, only γδT cells inhibited, both Vδ1+ and Vδ2+, the in vitro growth of the asexual blood stages in a dose dependent manner. The inhibition required cell‐to‐cell contact and was not observed until the second parasite replication implied that the likely γδT‐cell target was the extracellular merozoite or schizont. The failure of αβT cells to inhibit the growth of the parasite suggests requirement of additional cytolytic molecules/signals or different receptor specificities exhibited by the γδT cells. Both the γδ‐ and αβT cells expressed mRNA for a large number of cytokines. Interferon (IFN)‐γ, interleukin (IL) IL‐5, IL‐6, IL‐8, tumour necrosis factor alpha (TNFα), tumour necrosis factor beta (TNF‐β)/lymphotoxin (LT) and T‐cell growth factor beta‐1 (TGF‐β1) were observed in all activated clones tested. No IL‐3 was detected, while IL‐1β, IL‐2, IL‐4, IL‐10 and GM‐CSF were variably expressed. In conclusion, our data show that γδT cells in malaria nonimmune individuals inhibit the asexual blood stages of P. falciparum malaria, while similarly activated αβT cells do not. Thus, it is likely that the γδT cells could play a mandatory role in the elimination of parasites and/or the regulation of the early immune response to malaria infection.