Interleukin 4 induces rapid mucin transport, increases mucus thickness and quality and decreases colitis and Citrobacter rodentium in contact with epithelial cells.

Citrobacter rodentium infection is a murine model for pathogenic intestinal Escherichia coli infection. C. rodentium infection causes an initial decrease in mucus layer thickness, followed by an increase during clearance. We aimed to identify the cause of these changes and to utilize this naturally occurring mucus stimulus to decrease pathogen impact and inflammation. We identified that mucin production and speed of transport from Golgi to secretory vesicles at the apical surface increased concomitantly with increased mucus thickness. Of the cytokines differentially expressed during increased mucus thickness, IFN-γ and TNF-α decreased the mucin production and transport speed, whereas IL-4, IL-13, C. rodentium and E. coli enhanced these aspects. IFN-γ and TNF-α treatment in combination with C. rodentium and pathogenic E. coli infection negatively affected mucus parameters in vitro, which was relieved by IL-4 treatment. The effect of IL-4 was more pronounced than that of IL-13, and in wild type mice, only IL-4 was present. Increased expression of Il-4, Il-4-receptor α, Stat6 and Spdef during clearance indicate that this pathway contributes to the increase in mucin production. In vivo IL-4 administration initiated 10 days after infection increased mucus thickness and quality and decreased colitis and pathogen contact with the epithelium. Thus, during clearance of infection, the concomitant increase in IL-4 protects and maintains goblet cell function against the increasing levels of TNF-α and IFN-γ. Furthermore, IL-4 affects intestinal mucus production, pathogen contact with the epithelium and colitis. IL-4 treatment may thus have therapeutic benefits for mucosal healing.

Core 1- and 3-derived O-glycans collectively maintain the colonic mucus barrier and protect against spontaneous colitis in mice.

Core 1- and 3-derived mucin-type O-glycans are primary components of the mucus layer in the colon. Reduced mucus thickness and impaired O-glycosylation are observed in human ulcerative colitis. However, how both types of O-glycans maintain mucus barrier function in the colon is unclear. We found that C1galt1 expression, which synthesizes core 1 O-glycans, was detected throughout the colon, whereas C3GnT, which controls core 3 O-glycan formation, was most highly expressed in the proximal colon. Consistent with this, mice lacking intestinal core 1-derived O-glycans (IEC C1galt1-/-) developed spontaneous colitis primarily in the distal colon, whereas mice lacking both intestinal core 1- and 3-derived O-glycans (DKO) developed spontaneous colitis in both the distal and proximal colon. DKO mice showed an early onset and more severe colitis than IEC C1galt1-/- mice. Antibiotic treatment restored the mucus layer and attenuated colitis in DKO mice. Mucins from DKO mice were more susceptible to proteolysis than wild-type mucins. This study indicates that core 1- and 3-derived O-glycans collectively contribute to the mucus barrier by protecting it from bacterial protease degradation and suggests new therapeutic targets to promote mucus barrier function in colitis patients.

Species diversity regarding the presence of proximal tubular progenitor cells of the kidney.

The cellular source for tubular regeneration following kidney injury is a matter of dispute, with reports suggesting a stem or progenitor cells as the regeneration source while linage tracing studies in mice seemingly favor the classical theory, where regeneration is performed by randomly surviving cells. We, and others have previously described a scattered cell population localized to the tubules of human kidney, which increases in number following injury. Here we have characterized the species distribution of these proximal tubular progenitor cells (PTPCs) in kidney tissue from chimpanzee, pig, rat and mouse using a set of human PTPC markers. We detected PTPCs in chimpanzee and pig kidneys, but not in mouse tissue. Also, subjecting mice to the unilateral urethral obstruction model, caused clear signs of tubular injury, but failed to induce the PTPC phenotype in renal tubules.

New developments in goblet cell mucus secretion and function.

Goblet cells and their main secretory product, mucus, have long been poorly appreciated; however, recent discoveries have changed this and placed these cells at the center stage of our understanding of mucosal biology and the immunology of the intestinal tract. The mucus system differs substantially between the small and large intestine, although it is built around MUC2 mucin polymers in both cases. Furthermore, that goblet cells and the regulation of their secretion also differ between these two parts of the intestine is of fundamental importance for a better understanding of mucosal immunology. There are several types of goblet cell that can be delineated based on their location and function. The surface colonic goblet cells secrete continuously to maintain the inner mucus layer, whereas goblet cells of the colonic and small intestinal crypts secrete upon stimulation, for example, after endocytosis or in response to acetyl choline. However, despite much progress in recent years, our understanding of goblet cell function and regulation is still in its infancy.

Primary clear cell renal carcinoma cells display minimal mitochondrial respiratory capacity resulting in pronounced sensitivity to glycolytic inhibition by 3-Bromopyruvate.

Changes of cellular metabolism are an integral property of the malignant potential of most cancer cells. Already in the 1930s, Otto Warburg observed that tumor cells preferably utilize glycolysis and lactate fermentation for energy production, rather than the mitochondrial oxidative phosphorylation dominating in normal cells, a phenomenon today known as the Warburg effect. Even though many tumor types display a high degree of aerobic glycolysis, they still retain the activity of other energy-producing metabolic pathways. One exception seems to be the clear cell variant of renal cell carcinoma, ccRCC, where the activity of most other pathways than that of glycolysis has been shown to be reduced. This makes ccRCC a promising candidate for the use of glycolytic inhibitors in treatment of the disease. However, few studies have so far addressed this issue. In this report, we show a strikingly reduced mitochondrial respiratory capacity of primary human ccRCC cells, resulting in enhanced sensitivity to glycolytic inhibition by 3-Bromopyruvate (3BrPA). This effect was largely absent in established ccRCC cell lines, a finding that highlights the importance of using biologically relevant models in the search for new candidate cancer therapies. 3BrPA markedly reduced ATP production in primary ccRCC cells, followed by cell death. Our data suggest that glycolytic inhibitors such as 3BrPA, that has been shown to be well tolerated in vivo, should be further analyzed for the possible development of selective treatment strategies for patients with ccRCC.

Slc26a3 deficiency is associated with loss of colonic HCO3 (-) secretion, absence of a firm mucus layer and barrier impairment in mice.

AIM: Downregulated in adenoma (DRA, Slc26a3) is a member of the solute carrier family 26 (SLC26), family of anion transporters, which is mutated in familial chloride-losing diarrhoea (CLD). Besides Cl(-) -rich diarrhoea, CLD patients also have a higher-than-average incidence of intestinal inflammation. In a search for potential explanations for this clinical finding, we investigated colonic electrolyte transport, the mucus layer and susceptibility against dextran sodium sulphate (DSS)-induced colitis in Slc26a3(-/-) mice. METHODS: HCO3 (-) secretory (JHCO3 (-) ) and fluid absorptive rates were measured by single-pass perfusion in vivo and in isolated mid-distal colonic mucosa in Ussing chambers in vitro. Colonocyte intracellular pH (pHi ) was assessed fluorometrically, the mucus layer by immunohistochemistry and colitis susceptibility by the addition of DSS to the drinking water. RESULTS: HCO3 (-) secretory (JHCO3- ) and fluid absorptive rates were strongly reduced in Slc26a3(-/-) mice compared to wild-type (WT) littermates. Despite an increase in sodium/hydrogen exchanger 3 (NHE3) mRNA and protein expression, and intact acid-activation of NHE3, the high colonocyte pH in Slc26a3(-/-) mice prevented Na(+) /H(+) exchange-mediated fluid absorption in vivo. Mucin 2 (MUC2) immunohistochemistry revealed the absence of a firm mucus layer, implying that alkaline secretion and/or an absorptive flux may be necessary for optimal mucus gel formation. Slc26a3(-/-) mice were highly susceptible to DSS damage. CONCLUSIONS: Deletion of DRA results in severely reduced colonic HCO3 (-) secretory rate, a loss of colonic fluid absorption, a lack of a firmly adherent mucus layer and a severely reduced colonic mucosal resistance to DSS damage. These data provide potential pathophysiological explanations for the increased susceptibility of CLD patients to intestinal inflammation.

Overexpression of podocalyxin-like protein is an independent factor of poor prognosis in colorectal cancer.

BACKGROUND: Podocalyxin-like 1 (PODXL) is a cell-adhesion glycoprotein and stem cell marker that has been associated with an aggressive tumour phenotype and poor prognosis in several forms of cancer. In this study, we investigated the prognostic impact of PODXL expression in colorectal cancer (CRC). METHODS: Using tissue microarrays and immunohistochemistry, PODXL expression was evaluated in 536 incident CRC cases from a prospective, population-based cohort study. Kaplan-Meier analysis and Cox proportional hazards modelling were used to assess the impact of PODXL expression on cancer-specific survival (CSS) and overall survival (OS). RESULTS: High PODXL expression was significantly associated with unfavourable clinicopathological characteristics, a shorter CSS (hazard ratio (HR)=1.98; 95% confidence interval (CI) 1.38-2.84, P<0.001) and 5-year OS (HR=1.85; 95% CI 1.29-2.64, P=0.001); the latter remaining significant in multivariate analysis (HR=1.52; 95% CI 1.03-2.25, P=0.036). In addition, in curatively resected stage III (T1-4, N1-2, M0) patients (n=122) with tumours with high PODXL expression, a significant benefit from adjuvant chemotherapy was demonstrated (p(interaction) =0.004 for CSS and 0.015 for 5-year OS in multivariate analysis). CONCLUSION: Podocalyxin-like 1 expression is an independent factor of poor prognosis in CRC. Our results also suggest that PODXL may be a useful marker to stratify patients for adjuvant chemotherapy.

Lactobacillus and Bifidobacterium species do not secrete protease that cleaves the MUC2 mucin which organises the colon mucus.

The colon epithelium is covered by two layers of mucus built around the MUC2 mucin. An inner dense and attached mucus layer does not allow bacteria to penetrate, thus keeping the epithelial cell surface free from bacteria. An outer loose mucus layer is the habitat for the commensal bacterial microbiota. The inner mucus layer is renewed from the epithelial side and gets converted into the outer layer due to proteolytic cleavages by host proteases. We have now analysed if potential probiotic bacteria, namely Lactobacillus brevis, Lactobacillus plantarum, Lactobacillus bulgaricus and Bifidobacterium lactis, can secrete protease that cleaves the MUC2 mucin. We found that none of the potential probiotic bacteria Lactobacillus and Bifidobacterium could cleave the MUC2 core protein in the form of recombinant MUC2 N and C-termini although they secreted active proteases. This was in contrast to crude mixtures of oral and faecal bacteria that cleaved the MUC2 mucin. This observation further supports the view that these potential probiotic bacteria are of no harm to the host, as these bacteria cannot disrupt the mucin organised mucus as long as they are covered by glycans.

The glycosylation of rat intestinal Muc2 mucin varies between rat strains and the small and large intestine. A study of O-linked oligosaccharides by a mass spectrometric approach.

The large glycosylated domains obtained from the rat intestinal mucin Muc2 were isolated from the large and small intestine of the inbred rat strains GOT-W and GOT-BW. The expression of the rat Muc2 in the large intestine was confirmed immunochemically and by Northern blotting. Released oligosaccharides were structurally characterized by gas chromatography-mass spectrometry (neutral and sialylated species) or by tandem mass spectrometry (sulfated species), and a total of 63 structures was assigned. The large intestinal oligosaccharides were found to be identical between the strains, while the small intestinal glycosylation differed. Until now, detailed structural analysis of oligosaccharides isolated from a single mucin core or mucin domain with different origin have not been performed, and the information of different mucin glycoforms has been limited to immunochemistry. Blood group A-determinants (GalNAcalpha1-3(Fucalpha1-2)Galbeta1-, and structures related to the blood group Sda/Cad-related epitope NeuAc/NeuGcalpha1-3(GalNAcbeta1-4)Galbeta1-, were found in GOT-BW small intestine, and also in both large intestines. Blood group H-determinants and NeuAc/NeuGcalpha1-3Galbeta1- were found in all samples. Core 1 (Galbeta1-3GalNAcalpha1-), core 2 (Galbeta1-3(GlcNAcbeta1-6)GalNAcalpha1-), core 3 (GlcNAcbeta1-3GalNAcalpha1-), and core 4 (GlcNAcbeta1-3(GlcNAcbeta1-6)GalNAcalpha1- were also found in all the samples. The large intestine were enriched in sulfated oligosaccharides and the small intestine contained higher amounts of sialylated species. Sulfation were found exclusively on C-6 of GlcNAc.

Molecular characterization of the large heavily glycosylated domain glycopeptide from the rat small intestinal Muc2 mucin.

The largest high-glycosylated domain, glycopeptide A, of the "insoluble' mucin complex of the rat small intestine has earlier been purified and characterized (Carlstedt et al., 1993, J Biol Chem 268: 18771-81). A rabbit antiserum raised against deglycosylated glycopeptide A was used to clone part of a mucin showing homology to the human MUC2 mucin (Hansson et al., 1994, Biochem Biophys Res Commun 198. 181-90). This serum specifically stained goblet cells (paranuclear) in the mouse small intestine. The size of the coding sequence of glycopeptide A was estimated by using reversed transcriptase PCR of mRNA from an inbred rat strain (GOT-W) using primers in the unique central and C-terminal parts of the proposed rat Muc2 sequences. The PCR and Southern blot of the PCR products showed a fragment of about 5.5 kb corresponding to about 1700 amino acids when the known Cys-rich sequences used for the primers were subtracted. This is slightly larger than the size estimated earlier by biochemical studies. The mRNA encoding the rat Muc2 was slightly smaller than the mRNA encoding the human MUC2 in a colorectal cell line. Although the size of glycopeptide A estimated from biochemical results and by PCR is not identical, the results obtained here further support that the "insoluble' mucin of the rat small intestine is encoded by the Muc2 gene. Most of the oligosaccharides in glycopeptide A were either neutral (40%) or sialylated (40%). The remaining ones were sulfated with the sulfate group attached to C-6 of N-acetylglucosamine linked to C-6 of the N-acetylgalactosaminitol as revealed by tandem mass spectrometry of the perdeuteroacetylated oligosaccharides. Eighteen oligosaccharides were found of which fourteen were characterized and found to be mostly novel. Our findings thus expand the current knowledge of the core peptide of the rat intestinal goblet cell mucin and provide a relatively complete picture of the glycosylation of a defined mucin domain.

Adenoviruses isolated in the Stockholm area during 1987-1992: restriction endonuclease analysis and molecular epidemiology.

Adenoviruses recovered from the northern Stockholm area during 1987-1992 have been subjected to restriction endonuclease analysis. Adenoviruses of all subgenera (A-F) were represented and a considerable degree of serotype variation was seen, e.g. the rarely encountered subgenus A viruses were frequently isolated in the present study. Of 16 subgenus A isolates, Ad31 predominated with 12 strains which were equally distributed into the DNA-variants D2 and D7. Analysis of 38 Ad3 isolates revealed four DNA-variants: D1, D3, D10, and "Sto1". The ten Ad7 isolates belonged all to the DNA-variant D5 of Ad7. Of 27 Ad1 strains, 11 belonged to D10, followed by the DNA-variants D4 and D7 with four strains each. Among Ad2 isolates, D2 or D2-like strains prevailed (23/28). Of six Ad5 strains, three belonged to the DNA-variant D2. The most notable subgenus D event was a nosocomial outbreak of keratokonjunctivitis due ot Ad19a. In addition, a collection of heterogenous subgenus D strains was detected, most of which untypable by RE-analysis. Among the six Ad4 isolates of subgenus E, both genomic clusters (p and a, respectively) of Ad4 were recognized. Concerning the clinical important subgenus F adenoviruses, only two strains of Ad40 were detected as compared to 12 strains of Ad41, all of which ascribed to the DNA-variant D12 of Ad41.

Molecular epidemiology of adenoviruses isolated in Iceland during 1988-1990.

All human adenoviruses isolated in Iceland during 1988-1990 have been subjected to restriction endonuclease analysis. Of 55 isolates altogether, subgenus C (Ad1, Ad2, and Ad5) predominated with 42 isolates followed by subgenus B (Ad3 and Ad7) with 12. Analysis of the 9 Ad1 isolates revealed 6 DNA-variants. Among these the established DNA-variants D4, D7, and D10 were recognized. The remaining 3 DNA-variants were primarily found in Iceland. Among the 22 Ad2 isolates, 7 DNA-variants could be distinguished. D2 predominated with 15 isolates whereas the prototype was isolated only once. The novel 5 DNA-variants of Ad2 were all closely related to D2. Analysis of the 11 Ad5 isolates revealed 6 DNA-variants, 2 of which (D2 and D5) were already established. Ice2 and D3 were the most common occurring DNA-variants of Ad5. Ad5 showed the highest degree of genomic variability within subgenus C, both in terms of the low degree of pair-wise comigration of restriction fragments and the number of principal variants of RE-patterns. Analysis of the 9 Ad3 isolates revealed 3 DNA-variants: D3, D10, and Ice1 (a novel DNA-variant that resembles D10). The DNA-variants D3 and D10 were each represented by 4 isolates. The three Ad7 isolates belonged all to the DNA-variant D5.

Characterization of two divergent adenovirus 31 strains.

Two divergent strains of adenovirus type 31 were analyzed by neutralization test and restriction endonuclease (RE) patterns in an effort to find the basis for their genetic variability. One strain, isolated from the throat of a child in Maryland during an upper respiratory illness in 1968, was partially neutralized by Ad 31 antisera (to 16-fold lower than homologous titer) while its own antiserum fully neutralized prototype Ad 31 virus, but shared only 9% of comigrating RE fragments with Ad 31 prototype (vs. 30% with Ad 18 prototype); however, PCR tests specific for the inverted terminal repeat (ITR) sequence of Ads 12 and 18 were negative. The other strain, recovered from a stool sample from an infant with diarrhea in Georgia in 1979, was inhibited by Ad 31 antiserum to within 4-fold homologous titer, but shared only 15% of comigrating fragments with Ad 31 prototype (vs. 91% with Ad 18 prototype); ITR-specific PCR tests with this virus were positive for Ad 12/Ad 18. These data suggest that both strains are from separate evolutionary lines of Ad 31 unrelated to all other isolates studied to date by RE analysis, and that the partial neutralization by prototype Ad 31 antisera might represent small mutations in the hexon gene.

Restriction endonuclease patterns of adenovirus type 12 and 18.

Restriction endonuclease analysis using 10 restriction enzymes was performed on six and three wild isolates of adenovirus (Ad) type 12 and 18, respectively. Among the Ad12 strains, five DNA variants could be identified. The degree of pairwise comigration of restriction fragments suggests a high degree of genomic diversity within Ad12. The wild isolates of Ad18, on the other hand, displayed a low degree of genetic variability and comprised one DNA variant closely related to the prototype strain. The BglII, BstEII, and HindIII restriction endonuclease patterns of Ad18 were inconsistent with those originally presented. Identical RE-patterns among Ad18 prototype strains (DC) obtained from four different sources, including directly from the American Type Culture Collection, verify that the genuine Ad18 prototype was used in the present study. Using the revised restriction patterns of BglII, BstEII, and HindIII, a proper identification of Ad18 will be facilitated.

Intrauterine fetal death due to echovirus 11.

In a case of intrauterine fetal death in the 29th week of gestation, echovirus 11 could be isolated from the umbilical cord of the fetus. The mother had no apparent signs of infection but serological evidence of current echovirus 11 infection. Enterovirus PCR performed on paraffin-embedded specimens of various tissues (myocardium, lung, liver and placenta) from the fetus yielded positive results in all cases. These findings, together with supporting serological and epidemiological findings--e.g. proven echovirus 11 infection 3 weeks before in the 18-month-old son of the woman--constituted strong evidence that echovirus 11 infection was responsible for the fetal death.

Genome analysis of adenovirus type 31 strains from immunocompromised and immunocompetent patients.

Adenovirus type 31 (Ad31) was isolated from 15 immunocompromised patients in 12 of whom seroconversion was also recorded. Ad31 infection has a substantial clinical relevance since 8 of 10 with lower respiratory tract infection and 4 of 4 with hepatitis died. Therefore, Ad31 isolates from immunocompetent and immunodeficient hosts were compared by restriction endonuclease analysis. Nine genome types were identified among the 79 Ad31 isolates. Pairwise comparison of comigrating restriction fragments indicated that the genome types could be divided into three genomic clusters. Several Ad31 genome types were isolated from immunocompromised patients, but no highly virulent genome type could be found. A genome type was identified in a child with severe combined immunodeficiency who originally was infected with another genome type. This observation is suggested to have evolutionary implications.

Characterization of two genome types of adenovirus type 31 isolated in Stockholm during 1987.

The authors isolated during 1987 seven adenovirus type 31 (Ad31) within a 9-month period. The isolates were obtained from urine, throat, and feces, implying a systemic spread of the infection. Most patients displayed gastrointestinal symptoms, but some had respiratory symptoms and fever. All of the strains differed by restriction endonuclease analysis from the prototype strain (1315) by an additional Bgl II restriction site. Ad31 isolates 1-6 could be divided into two groups by the enzymes Bam HI, Msp I, and Xho I. Each enzyme gave rise to the same group distribution: isolates 1-3 and 4-6, respectively. Digestion with Bst EII, Hind III, Kpn I, and Sma I resulted in identical patterns for isolates 1-6. Isolate 7, however, demonstrated a DNA deletion of approximately 0.8 kbp, but it was otherwise identical to isolates 4-6. In conclusion, two separate genome types of Ad31 were isolated, one of which included a DNA deletion mutant. The increased isolation rate may reflect an epidemiological situation, as the same isolation procedure had been used both before and after this period.

Optimization of a fluid bed spray coating process using reduced factorial design.

A sequential approach to the optimization of a fluid bed coating process of pellets for controlled release using organic solvents and ethylcellulose has been applied using reduced factorial experiments. The optimization was started by applying a 2(4-1) experiment then, based on the results from that study, further experiments were carried out where only the variables which had the most significant effect on the film yield were used. Finally, a 2(3-1) experiment was performed. A relationship was found between the film yield and degree of agglomeration, indicating a limiting value of the film yield (75 per cent) below which only little agglomeration takes place. Above this value, the degree of agglomeration increases dramatically. The limit value was found to change only to a minor extent as a function of experimental conditions.