Combining strains of lactic acid bacteria may reduce their toxin and heavy metal removal efficiency from aqueous solution.

AIMS: The primary objective of this study was to compare the removal of cadmium, lead, aflatoxin B1 and microcystin-LR from aqueous solution by Lactobacillus rhamnosus GG, L. rhamnosus LC705, Propionibacterium freudenreichii shermanii JS and Bifidobacterium breve Bbi99/E8, separately and in combination. METHODS AND RESULTS: The removal of toxins and heavy metals was assessed in batch experiments. The removal of all compounds was observed to be strain specific. The removal of lead by a combination of all the strains used was observed to be lower than could be predicted from the removal by single strains (P < 0.05). A similar trend was also observed with the other compounds studied. CONCLUSIONS: The results show that the toxin-removal capacity of a combination of strains of lactic acid bacteria is not the sum of their individual capacities. Therefore, pure single strains should be used when the goal is to remove single compounds. The use of combinations of strains may be beneficial when several compounds are removed together. This needs to be studied in future experiments. SIGNIFICANCE AND IMPACT OF THE STUDY: Lactic acid bacteria have been identified as potent tools for the decontamination of heavy metals, cyanotoxins and mycotoxins. The results of this study should be considered when selecting combinations of bacteria for the simultaneous removal of several toxic compounds.

Rapid removal of lead and cadmium from water by specific lactic acid bacteria.

Cadmium and lead are highly toxic metals. People are exposed to them primarily through food and water. Available conventional methods (precipitation, flocculation, ion exchange, and membrane filtration) for removal of these metals from water at low concentrations are claimed to be expensive and inefficient. Different microbes have been proposed to be an efficient and economical alternative in heavy metal removal from water. In this work, specific lactic acid bacteria (LAB) were assessed for their ability to remove cadmium and lead from water. Significant removal was observed, and it was found to be metal and bacterial strain specific. Removal was a fast, metabolism-independent surface process. It was also strongly influenced by pH, indicating that ion exchange mechanisms could be involved. The most effective metal removers were Bifidobacterium longum 46, Lactobacillus fermentum ME3 and Bifidobacterium lactis Bb12. The highest maximum cadmium and lead removal capacities of 54.7 mg metal/g and 175.7 mg/g dry biomass, respectively, were obtained with B. longum 46.

In vivo targeting of intestinal and extraintestinal transglutaminase 2 by coeliac autoantibodies.

BACKGROUND: IgA class serum autoantibodies against type 2 (tissue) transglutaminase (TG2) bind to both intestinal and extraintestinal normal tissue sections in vitro, eliciting endomysial, reticulin, and jejunal antibody reactions. It is not known whether similar binding also occurs in coeliac patients in vivo, and may thereby contribute to disease manifestations. AIMS: To investigate intestinal and extraintestinal coeliac tissues for the presence of in vivo bound TG2 specific IgA and its relation to small intestinal mucosal atrophy. PATIENTS: We investigated jejunal samples with normal villous morphology from 10 patients with developing coeliac disease who subsequently progressed to a flat lesion, from 11 patients with dermatitis herpetiformis, and from 12 non-coeliac controls. Six extrajejunal biopsy samples (liver, lymph node, muscle, appendix), obtained based on independent clinical indications from patients with active coeliac disease, were also studied. METHODS: Double colour immunofluorescent studies for in situ IgA, TG2, and laminin were performed. IgA was eluted from tissue sections and tested for TG2 specificity by enzyme linked immunosorbent assay and indirect immunofluorescence. RESULTS: IgA (in one IgA deficient case IgG) deposition on extracellularly located TG2 was detected in jejunal and extrajejunal specimens of all coeliac patients, and also in seven of 11 dermatitis herpetiformis patients, of whom two had no circulating endomysial antibodies. IgA eluted from extraintestinal coeliac tissues was targeted against TG2. CONCLUSIONS: Coeliac IgA targets jejunal TG2 early in disease development even when endomysial antibodies are not present in the circulation. Extraintestinal target sites of coeliac IgA further indicate that humoral immunity may have a pathogenetic role.

Missing endomysial and reticulin binding of coeliac antibodies in transglutaminase 2 knockout tissues.

BACKGROUND: Autoantibodies against transglutaminase 2 (TG2) are thought to be responsible for the endomysial (EMA), reticulin (ARA), and jejunal antibody (JEA) tissue binding of serum samples from coeliac patients but the exclusive role of TG2 in these staining patterns has not yet been established. AIMS: To evaluate whether antigens other than TG2 contribute to EMA/ARA/JEA reactions. PATIENTS: Serum samples from 61 EMA/ARA/JEA positive untreated patients with coeliac disease, 40 dermatitis herpetiformis patients, and 34 EMA/ARA/JEA negative non-coeliac controls were tested. METHODS: TG2 knockout (TG2-/-) and wild-type mouse oesophagus, jejunum, liver, and kidney sections, and TG2-/- sections coated with human recombinant TG2 were used as substrates in single and double immunofluorescent studies for patient IgA binding and tissue localisation of TG2, fibronectin, actin, and calreticulin. RESULTS: None of the patient serum samples elicited EMA, ARA, or JEA binding in TG2-/- morphologically normal tissues. In contrast, 96 of 101 gluten sensitive patient samples (95%) reacted with wild-type mouse tissues and all 101 reacted in EMA/ARA/JEA patterns with TG2-/- mouse tissues coated with human TG2. Serum IgA binding to TG2-/- smooth muscle cells was observed in low titres in 31.1%, 27.5%, and 20.5%, and to TG2-/- epithelium in 26.3%, 5.0%, and 8.8% of coeliac, dermatitis herpetiformis, and control samples, respectively. These positivities partly colocalised with actin and calreticulin but not with TG2 or fibronectin. CONCLUSIONS: EMA/ARA/JEA antibody binding patterns are exclusively TG2 dependent both in coeliac and dermatitis herpetiformis patients. Actin antibodies are responsible for some positivities which are not part of the EMA/ARA/JEA reactions.

Blisters in the small intestinal mucosa of coeliac patients contain T cells positive for cyclooxygenase 2.

BACKGROUND AND AIMS: Coeliac disease is characterised by atrophy of the villi and hyperplasia of the crypts in the mucosa of the small intestine. It is caused by an environmental trigger, cereal gluten, which induces infiltration of the mucosa by inflammatory cells. We hypothesised that these inflammatory cells express cyclooxygenase 2 (COX-2), an enzyme that contributes to the synthesis of pro and anti-inflammatory prostaglandins and is known to be expressed at sites of inflammation in the stomach and colon. We have investigated expression of COX-2 in the coeliac disease affected small intestinal mucosa where it may be an indicator of either disease induction or mucosal restoration processes. PATIENTS AND METHODS: Small intestinal biopsy samples from 15 coeliac patients and 15 non-coeliac individuals were stained immunohistochemically for COX-2. Samples from 10 of the patients were also stained after these patients had been on a gluten free diet for 6-24 months. Various cell type marker antigens were used for immunohistochemical identification of the type of cell that expressed COX-2. To further verify colocalisation of the cell type marker and COX-2, double immunoperoxidase and immunofluorescence methods were employed. Immunoelectron microscopy was used to investigate the subcellular location of COX-2. RESULTS: In all samples taken from coeliac patients, clusters of cells with strong immunoreactivity for COX-2 were found in those areas of the lamina propria where the epithelium seemed to blister or was totally detached from the basement membrane. These clusters were reduced in number or totally absent in samples taken after a gluten free diet. No such clusters were seen in any control samples. The density of COX-2 positive cells lining the differentiated epithelium decreased significantly from 13.5 (5.1) cells/10(5) microm(2) (mean (SD)) in the untreated patient samples to 6.5 (2.0) cells/10(5) microm(2) after a gluten free diet (p<0.001), and was 3.3 (1.9) cells/10(5) microm(2) in control samples (p<0.001 compared with untreated or diet treated coeliac samples). Staining for COX-2 was localised to CD3+ T cells and CD68+ macrophages in the mucosal lesions but not all of these cells were positive for COX-2. Immunoelectron microscopy revealed that the ultrastructure of the COX-2 positive cells resembled that of lymphocytes, and the immunoreaction was localised to the rough endoplasmic reticulum and the nuclear envelope. CONCLUSIONS: Our results show that in coeliac disease, blistering of small intestinal epithelial cells is associated with accumulation of COX-2 positive T cells, and the number of these cells decreases after a gluten free diet. These observations suggest that COX-2 mediated prostanoid synthesis contributes to healing of the coeliac mucosa and may be involved in maintenance of intestinal integrity.

Differentially expressed CC3/TIP30 and rab11 along in vivo and in vitro intestinal epithelial cell crypt-villus axis.

We have previously shown that transforming growth factor-beta1 (TGF-beta1) is involved in the fibroblast-induced organization and differentiation of transformed phenotypically crypt-like T84 intestinal epithelial cells into absorptive enterocyte-like cells, when cultured within a three-dimensional collagen gel. We have used differential display polymerase chain reaction to find genes that are either up- or downregulated by TGF-beta in the T84 cells cultured in three-dimensional collagen gel and then studied how these in vitro differentially expressed genes are expressed in vivo in the small intestinal crypt-villus axis. We found that the TGF-beta1-treated T84 cells, like the villus tip enterocytes, expressed increased levels of CC3/TIP30 when compared to the undifferentiated cells. Furthermore, the expression of rab11 showed the opposite pattern, being higher in the undifferentiated cells both in vivo and in vitro. We conclude that the three-dimensional cell culture model where TGF-beta induces organization and differentiation of secretory T84 epithelial cells makes it possible to find up- and downregulated transcripts that also play a role in the human small intestinal crypt-villus axis.

Identification of novel transcription factor-like gene from human intestinal cells.

Intestinal crypt epithelial T84 cells form luminal structures and differentiate to intestinal enterocyte-like cells in response to IMR-90 fibroblast-secreted transforming growth factor-beta when grown within three-dimensional collagen gel. In search of TGF-beta regulated genes involved in this differentiation process, we isolated a TGF-beta downregulated cDNA, human homologue of rat apoptosis antagonising transcription factor that codes for a 560-amino-acid protein. Human AATF-mRNA was expressed at high levels in human brain, heart, thymus, kidney, and placenta while in skeletal muscle and colon the expression was lower. The gene was mapped to chromosome 17q11.2-q12.

Tissue transglutaminase is the target in both rodent and primate tissues for celiac disease-specific autoantibodies.

BACKGROUND: Endomysial antibodies have recently been shown to react with tissue transglutaminase. This study was undertaken to investigate whether the tissue distribution of transglutaminase is also compatible with reticulin, jejunal, and fibroblast autoantibody binding patterns. METHODS: Sera from patients with and without celiac disease, monoclonal tissue transglutaminase antibodies, and sera from mice parenterally immunized against commercially available tissue transglutaminase, transglutaminase complexed with gliadin, or gliadin were used in indirect immunofluorescence and double-staining studies using both rodent and primate tissues as substrates. Also, antibody competition, affinity chromatography, and potassium thiocyanate extraction studies were undertaken. RESULTS: Tissue transglutaminase antibody binding patterns were identical with the extracellular binding patterns seen with celiac patient sera. Human umbilical cord-derived fibroblasts exhibited both cytoplasmic and extracellular matrix staining. Double staining with patients' sera and tissue transglutaminase antibodies showed complete overlapping. Tissue transglutaminase effectively absorbed reticulin-endomysial antibodies from celiac sera, and patients' sera blocked the staining of the monoclonal tissue transglutaminase antibodies. Potassium thiocyanate extraction abolished the staining patterns, but they were elicited again after readdition of tissue transglutaminase. CONCLUSIONS: Reticulin, endomysial, and jejunal antibodies detect transglutaminase in both rodent and primate tissues, indicating that these tissue autoantibodies are identical.

The role of transforming growth factor-beta on retarded osteoblastic differentiation in vitro.

Various matrix growth factors play important roles in the development and growth of cartilage and bone. Among them transforming growth factor-beta superfamily and especially bone morphogenetic proteins are known to be important factors, since they induce bone and cartilage formation in ectopic sites in vivo. We have previously shown that the human osteosarcoma cell line Saos-2 expresses molecules that in vivo induce new bone formation with asymmetric bone maturation. In this study we examined the role of Saos-2-conditioned medium in prolonged cultures of mesenchymal C3H/10T1/2 cells. The C3H/10T1/2 cells were cultured with Saos-2-conditioned medium for 28 days. We show that Saos-2-treated C3H/10T1/2 cells performed retarded osteoblastic differentiation when compared to recombinant BMP-2 and -4 induced differentiation. We further show that this retardation is due to excessive amounts of transforming growth factor-beta in Saos-2-conditioned medium. Our results also suggest that this model can well be used to study additional cofactors involved in retarded osteogenesis.

Serum immunoglobulin A from patients with celiac disease inhibits human T84 intestinal crypt epithelial cell differentiation.

BACKGROUND & AIMS: Celiac disease is characterized by disturbed jejunal crypt-villus axis biology with immunoglobulin (Ig) A deposits underlining the epithelium. The aim of this study was to test whether celiac disease serum IgA (reticulin/endomysial autoantibodies) interferes with the mesenchymal-epithelial cell cross-talk. METHODS: Differentiation of T84 epithelial cells was induced with IMR-90 fibroblasts or transforming growth factor beta in three-dimensional collagen gel cultures. The effects of purified celiac IgA and monoclonal tissue transglutaminase antibodies (CUB7402) were studied by adding the antibodies to the cocultures. RESULTS: Active celiac disease IgA, reactive for tissue transglutaminase, significantly inhibited T84 epithelial cell differentiation (P < 0.001) and increased epithelial cell proliferation (P = 0.024). Similar effects were obtained with antibodies against tissue transglutaminase. CONCLUSIONS: Celiac disease-associated IgA class antibodies disturb transforming growth factor beta-mediated fibroblast-epithelial cell cross-talk in this in vitro crypt-villus axis model. This primary finding indicates that celiac disease-specific autoantibodies may also contribute to the formation of the gluten-triggered jejunal mucosal lesion in celiac disease.

Tissue transglutaminase autoantibody enzyme-linked immunosorbent assay in detecting celiac disease.

BACKGROUND & AIMS: Tissue transglutaminase has been reported to be the target for endomysial antibodies in celiac disease. We sought to establish whether immunoglobulin (Ig) A class tissue transglutaminase autoantibodies can be considered specific for celiac disease. METHODS: Serum samples from 136 patients with untreated celiac disease (diagnosed according to the criteria of the European Society for Pediatric Gastroenterology, Hepatology and Nutrition) and 207 disease controls were studied. Enzyme-linked immunosorbent assay (ELISA) and Western blots were performed using calcium-treated and untreated tissue transglutaminase as antigen. Reticulin, endomysial, and mouse monoclonal tissue transglutaminase antibodies were studied by an indirect immunofluorescence method and gliadin antibodies with ELISA. RESULTS: The calcium-activated tissue transglutaminase autoantibody ELISA was highly sensitive (129 of 136) and specific (194 of 207) in detecting celiac disease. The new autoantibody ELISA test correlated well with the endomysial antibody test. Tissue transglutaminase autoantibody ELISA showed a clearly better predictive potential than the IgA class gliadin antibody ELISA. Immunoblots and ELISA blocking studies showed that calcium is needed for the specific antigen-antibody reaction to occur. Double immunofluorescence staining in human umbilical cord with sera from patients with celiac disease and with monoclonal tissue transglutaminase antibodies showed complete overlap. CONCLUSIONS: Calcium-activated tissue transglutaminase autoantibody ELISA is highly accurate in detecting untreated celiac disease. Tissue transglutaminase seems to be the target self-antigen for endomysial antibodies.

Autoantibodies in celiac disease: importance of fibroblasts.

BACKGROUND: Serum reticulin and endomysium autoantibodies are highly celiac disease-specific, and the autoantigens have been shown to be derived from human fibroblasts. Among human tissues, the umbilical cord also expresses these antigens. This study was conducted to compare different autoantibody tests and especially to elucidate whether human umbilical cord is a suitable substrate in tests and whether the cord jelly-derived fibroblasts express the antigens. METHODS: The indirect immunofluorescence method was used to detect the tissue and Wharton's jelly-derived fibroblast antibodies in 334 celiac disease and control sera samples. Affinity chromatography studies were used to show the correlation between human fibroblast-derived autoantigens and tissue and gliadin antibodies. The jelly-derived fibroblasts were used as antigen in a whole-cell enzyme-linked immunosorbent assay. RESULTS: Celiac disease patient sera showed IgA-class human umbilical cord antibody with high sensitivity (100%) and specificity (99%). All celiac disease patient sera tested showed in indirect immunofluorescence the molecules expressed by Wharton's jelly-derived fibroblasts. The whole-cell fibroblast autoantibody enzyme-linked immunosorbent assay had a sensitivity of 100% and a specificity of 81%. Human fibroblast-derived celiac disease autoantigens absorbed most of the IgA responsible for human umbilical cord antibodies but not the IgA responsible for gliadin antibodies in the same sera. CONCLUSIONS: Wharton's jelly-derived fibroblast autoantibodies tested in a novel whole-cell enzyme-linked immunosorbent assay correlated well with the human umbilical cord but not with gliadin antibodies.

Measurement of total and local bone morphogenetic protein concentration in bone tumours.

Bone morphogenetic protein (BMP) has been shown to be one of the significant factors in the prognosis of bone tumours. In normal development BMP induces new bone formation and later takes part in fracture healing, but its function in malignant tumours is not known. In this study the concentration of bone morphogenetic protein was measured in primary bone tumours by two methods. Local staining intensity was detected immunohistologically by the avidin-biotin-peroxidase method determining the highest dilution of anti-serum against bovine bone morphogenetic protein. The total amount of BMP in a tumour sample was measured by an enzyme-linked immunosorbent assay technique after digesting the tissue with collagenase to remove proteins from the connective tissue. Immunohistochemical staining showed that bone morphogenetic protein was present in the cytoplasm and in reactive bone formed by malignant cells. The local concentration was highest in the tissue of giant cell tumours compared to chondrosarcoma, osteosarcoma and benign bone tumours. The total amount in malignant bone tumours was 2.4 times higher compared to benign bone tumours.

Fibroblasts and transforming growth factor beta induce organization and differentiation of T84 human epithelial cells.

BACKGROUND & AIMS: The gut epithelium in the crypt-villus axis represents a continuous developmental system in which the role of fibroblast-epithelial interactions is obvious. The aim of this study was to establish an in vitro method whereby fibroblast-guided differentiation of crypt-like gut epithelial cells can be studied. METHODS: Intestinal epithelial cells (T84 and HT-29) were cultured within type I collagen gel together were fibroblasts without cell-to-cell contact. T84 cells were also grown in the presence of transforming growth factor beta and hepatocyte growth factor. The gels were studied using light and electron microscopy and histochemical and immunohistochemical methods. RESULTS: The epithelial cells formed unorganized cell clusters within the gels, but when given fibroblast support, 76% of the T84 cell colonies (not HT-29) organized into luminal formations, and basement membranes including laminin were well deposited. The cells in the columnar single cell-layer luminal formations (49% of all colonies) were differentiated, showing microvilli, up-regulated alkaline phosphatase brush border activity, and mucin profiles typical for small intestine. This fibroblast-induced organization and differentiation was induced by transforming growth factor beta. CONCLUSIONS: Crypt-like T84 epithelial cells are able to differentiate when grown three-dimensionally together with fibroblasts or transforming growth factor beta. This method may be used for mesenchymal-epithelial cell cross-talk studies.