Revisiting the immunocytochemical detection of Neurogenin 3 expression in mouse and man.

During embryonic development, endocrine cells of the pancreas are specified from multipotent progenitors. The transcription factor Neurogenin 3 (NEUROG3) is critical for this development and it has been shown that all endocrine cells of the pancreas arise from endocrine progenitors expressing NEUROG3. A thorough understanding of the role of NEUROG3 during development, directed differentiation of pluripotent stem cells and in models of cellular reprogramming, will guide future efforts directed at finding novel sources of ß-cells for cell replacement therapies. In this article, we review the expression and function of NEUROG3 in both mouse and human and present the further characterization of a monoclonal antibody directed against NEUROG3. This antibody has been previously been used for detection of both mouse and human NEUROG3. However, our results suggest that the epitope recognized by this antibody is specific to mouse NEUROG3. Thus, we have also generated a monoclonal antibody specifically recognizing human NEUROG3 and present the characterization of this antibody here. Together, these antibodies will provide useful tools for future studies of NEUROG3 expression, and the data presented in this article suggest that recently described expression patterns of NEUROG3 in human foetal and adult pancreas should be re-examined.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Protective effect of Clostridium tyrobutyricum in acute dextran sodium sulphate-induced colitis: differential regulation of tumour necrosis factor-α and interleukin-18 in BALB/c and severe combined immunodeficiency mice.

One of the promising approaches in the therapy of ulcerative colitis is administration of butyrate, an energy source for colonocytes, into the lumen of the colon. This study investigates the effect of butyrate producing bacterium Clostridium tyrobutyricum on dextran sodium sulphate (DSS)-induced colitis in mice. Immunocompetent BALB/c and immunodeficient severe combined immunodeficiency (SCID) mice reared in specific-pathogen-free (SPF) conditions were treated intrarectally with C. tyrobutyricum 1 week prior to the induction of DSS colitis and during oral DSS treatment. Administration of DSS without C. tyrobutyricum treatment led to an appearance of clinical symptoms - bleeding, rectal prolapses and colitis-induced increase in the antigen CD11b, a marker of infiltrating inflammatory cells in the lamina propria. The severity of colitis was similar in BALB/c and SCID mice as judged by the histological damage score and colon shortening after 7 days of DSS treatment. Both strains of mice also showed a similar reduction in tight junction (TJ) protein zonula occludens (ZO)-1 expression and of MUC-2 mucin depression. Highly elevated levels of cytokine tumour necrosis factor (TNF)-α in the colon of SCID mice and of interleukin (IL)-18 in BALB/c mice were observed. Intrarectal administration of C. tyrobutyricum prevented appearance of clinical symptoms of DSS-colitis, restored normal MUC-2 production, unaltered expression of TJ protein ZO-1 and decreased levels of TNF-α and IL-18 in the descending colon of SCID and BALB/c mice, respectively. Some of these features can be ascribed to the increased production of butyrate in the lumen of the colon and its role in protection of barrier functions and regulation of IL-18 expression.

Pulmonary Arterial Pruning and Longitudinal Change in Percent Emphysema and Lung Function: The Genetic Epidemiology of COPD Study.

BACKGROUND: Pulmonary endothelial damage has been shown to precede the development of emphysema in animals, and vascular changes in humans have been observed in COPD and emphysema. RESEARCH QUESTION: Is intraparenchymal vascular pruning associated with longitudinal progression of emphysema on CT imaging or decline in lung function over 5 years? STUDY DESIGN AND METHODS: The Genetic Epidemiology of COPD Study enrolled ever smokers with and without COPD from 2008 through 2011. The percentage of emphysema-like lung, or "percent emphysema," was assessed at baseline and after 5 years on noncontrast CT imaging as the percentage of lung voxels < -950 Hounsfield units. An automated CT imaging-based tool assessed and classified intrapulmonary arteries and veins. Spirometry measures are postbronchodilator. Pulmonary arterial pruning was defined as a lower ratio of small artery volume (< 5 mm2 cross-sectional area) to total lung artery volume. Mixed linear models included demographics, anthropomorphics, smoking, and COPD, with emphysema models also adjusting for CT imaging scanner and lung function models adjusting for clinical center and baseline percent emphysema. RESULTS: At baseline, the 4,227 participants were 60 ± 9 years of age, 50% were women, 28% were Black, 47% were current smokers, and 41% had COPD. Median percent emphysema was 2.1 (interquartile range, 0.6-6.3) and progressed 0.24 percentage points/y (95% CI, 0.22-0.26 percentage points/y) over 5.6 years. Mean FEV1 to FVC ratio was 68.5 ± 14.2% and declined 0.26%/y (95% CI, -0.30 to -0.23%/y). Greater pulmonary arterial pruning was associated with more rapid progression of percent emphysema (0.11 percentage points/y per 1-SD increase in arterial pruning; 95% CI, 0.09-0.16 percentage points/y), including after adjusting for baseline percent emphysema and FEV1. Arterial pruning also was associated with a faster decline in FEV1 to FVC ratio (-0.04%/y per 1-SD increase in arterial pruning; 95% CI, -0.008 to -0.001%/y). INTERPRETATION: Pulmonary arterial pruning was associated with faster progression of percent emphysema and more rapid decline in FEV1 to FVC ratio over 5 years in ever smokers, suggesting that pulmonary vascular differences may be relevant in disease progression. TRIAL REGISTRY: ClinicalTrials.gov; No.: NCT00608764; URL: www.clinicaltrials.gov.

Optimized procedures for generating an enhanced, near physiological 2D culture system from porcine intestinal organoids.

An important practical limitation of the three-dimensional geometry of stem-cell derived intestinal organoids is that it prevents easy access to the apical epithelium for testing food components, microorganisms, bioactive and toxic compounds. To this end, we here report on a new robust method for generating confluent intestinal cell monolayers from single-cell suspensions of enzymatically-dissociated porcine organoids using modified culture conditions. With this method, cell seeding densities can be standardised, overcoming problems with methods based on mechanical dissociation of organoids. Confluent monolayers formed tight junctions with high transepithelial electrical resistance in three days and could be used in experiments for up to two weeks. Multilineage differentiation of ileal stem cells was demonstrated by immunohistochemistry and RT-qPCR of cell-specific transcripts, also unequivocally confirming the controversial existence of Paneth-like cells in the porcine small intestine. The method described here is useful to standardize primary epithelial monolayer formation from intestinal organoids and allows rapid and robust studies of intestinal physiology.

Oral cholera vaccination promotes homing of IgA+ memory B cells to the large intestine and the respiratory tract.

Oral cholera vaccination is used to induce immune responses in the intestines to protect against cholera infection. However, oral vaccination may also affect immune responses in other mucosal tissues. To study this, tissue-specific homing potential and kinetics of B-cell responses were characterized after oral cholera vaccination. Healthy adult volunteers received two doses of Dukoral® and blood, saliva, nasal wash, and fecal samples were collected over time to detect vaccine-specific antibodies. Additionally, homing potential of lymphocytes to small intestine, colon, airways, skin, and periphery was measured by expression of Integrin ß1 and ß7, CCR9, CCR10, CCR7, and CLA. After vaccination, antibody responses to cholera toxin B (CTB) and Dukoral® were detected in serum and nasal wash. CTB-specific memory B cells in peripheral blood and tissue homing profiles of memory B cells peaked at day 18. IgA+ memory B cells expressed markers that enable homing to the airways and colon, while IgA- memory B cells primarily expressed small-intestine-homing markers. These data show that oral cholera vaccination has a differential effect on immune responses in various mucosal sites, including the respiratory tract.

Molecular analysis of N6-methyladenine patterns in Tetrahymena thermophila nuclear DNA.

We have cloned two DNA fragments containing 5'-GATC-3' sites at which the adenine is methylated in the macronucleus of the ciliate Tetrahymena thermophila. Using these cloned fragments as molecular probes, we analyzed the maintenance of methylation patterns at two partially and two uniformly methylated sites. Our results suggest that a semiconservative copying model for maintenance of methylation is not sufficient to account for the methylation patterns we found during somatic growth of Tetrahymena. Although we detected hemimethylated molecules in macronuclear DNA, they were present in both replicating and nonreplicating DNA. In addition, we observed that a complex methylation pattern including partially methylated sites was maintained during vegetative growth. This required the activity of a methylase capable of recognizing and modifying sites specified by something other than hemimethylation. We suggest that a eucaryotic maintenance methylase may be capable of discriminating between potential methylation sites to ensure the inheritance of methylation patterns.

A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila.

Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans. The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned. The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences. There is a long, 825-bp, inverted repeat near the micronuclear junctions. The inverted repeat contains two different 19-bp tandem repeats. The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome. Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences. Another family member was isolated. The 19-mers in that clone are also in close proximity to a rearrangement junction. We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites. We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions.

Oral vaccination of mice against tetanus with recombinant Lactococcus lactis.

To determine whether a protective immune response could be elicited by oral delivery of a recombinant bacterial vaccine, tetanus toxin fragment C (TTFC) was expressed constitutively in Lactococcus lactis and administered orally to C57 BL/6 mice. The antibody titers elicited were lower than those following intranasal immunization (a route already known to result in high-level systemic anti-TTFC immune responses) but the protective efficacy was the same order of magnitude. The serum antibody isotypes elicited were predominantly IgG1 and IgG2a. TTFC-specific fecal IgA responses could be detected following oral or intranasal immunization. Chemically killed lactococci administered via the intranasal route were also able to elicit serum antibody responses of similar levels and kinetics to those induced by live bacteria.

Curability of tumors bearing herpes thymidine kinase genes transferred by retroviral vectors.

Retroviral vectors constructed to contain the herpes simplex virus thymidine kinase (HSV-TK) gene were used for transduction of this gene into murine sarcoma and lymphoma cells to yield sublines susceptible in vitro to the cytotoxicity of ganciclovir, a drug specifically activated by HSV-TK. In vivo, ganciclovir induced complete, durable regressions in most mice bearing transplanted HSV-TK-positive sarcomas; its efficacy against lymphomas was only marginal, possibly because of their greater instability of gene expression. The results imply the potential value of an anticancer strategy entailing the prophylactic use of retroviral vectors to create tissue mosaicism for drug sensitivity.

In vitro cell culture methods for investigating Campylobacter invasion mechanisms.

Studying the mechanisms of Campylobacter pathogenesis is complicated by the lack of simple animal models that mimic the disease seen in humans. In vitro cell culture methods provide a useful alternative to investigate the interactions between Campylobacter and the host epithelium that occur during infection. In the genomics era there is an increasing use of in vitro cell culture techniques to interrogate the potential role of different genes in pathogenesis. The aim of this review was to discuss the suitability and limitations of the various experimental approaches that might be adopted. We review current knowledge concerning the influence of cell-specific as well as bacterial factors required for Campylobacter invasion such as flagella and secreted proteins. The involvement and effects of phase variation on the results of invasion studies in cell culture emphasise the need to verify observed strain variations. We present the use of a mathematical Invasion Success Model to analyse Campylobacter invasion and show that it can be used to derive three strain dependent characteristics Imax, k, and I0. Even by combining data from independent experiments the Invasion Success Model can be used to statistically compare Campylobacter strains for their invasion of epithelial cells. Recommendations are given for the adoption of standard assay parameters and analytical methods such as the Invasion Success Model in order to facilitate comparison of data generated in different laboratories.

Comparison of eotaxin-3 biomarker in patients with eosinophilic oesophagitis, proton pump inhibitor-responsive oesophageal eosinophilia and gastro-oesophageal reflux disease.

BACKGROUND: Proton pump inhibitor-responsive oesophageal eosinophilia (PPI-REE) is a recently described entity which resembles oeosinophilic oesophagitis (EoE), yet responds to acid suppressive treatment. AIM: To determine whether EoE shares similar staining features with PPI-REE or with gastro-oesophageal reflux disease (GERD). METHODS: This retrospective study consisted of patients with an established diagnosis of EoE, PPI-REE, or GERD identified from a database during a 1-year period. Immunohistochemistry (IHC) analysis was performed specifically targeting eotaxin-3 antibodies. All sections were qualitatively (intensity) and quantitatively (percentage of cells stained) assessed independently by two blinded pathologists. RESULTS: The cohort consisted of three groups of patients: EoE (n = 22), PPI-REE (n = 23) and GERD (n = 23) for a total of 68 patients. Study demographics included mean age 39 (14) years, 75% male and 77% Caucasian. There was a significant difference in the eotaxin-3 staining among EoE, PPI-REE and GERD groups [mean score (s.d.): 1.2 (1.2), 0.8 (1.0), 0.3 (0.7), P = 0.006]. Staining scores of EoE patients were significantly higher compared with GERD (P = 0.002) and a trend towards significance was seen between EoE and PPI-REE (P = 0.054). There was also a significant difference in EoE staining intensity score among the three groups (P = 0.006). Intensity scores of EoE were significantly higher compared with GERD [1.0 (0.9) vs. 0.22 (0.52), P < 0.001]. There was no significant difference between EoE and PPI-REE groups [1.0 (0.0) vs. 0.52 (0.75) P = 0.094]. CONCLUSIONS: A difference in eotaxin-3 staining was seen in the three groups of patients with oesophageal eosinophilia. Eotaxin-3 can distinguish EoE from GERD, but not from proton pump inhibitor responsive-oesophageal eosinophilia.

Lymphoma regression induced by ganciclovir in mice bearing a herpes thymidine kinase transgene.

The dose limitations imposed on cancer chemotherapeutic agents by their lack of selectivity can, in theory, be circumvented by a strategy entailing the prophylactic insertion into hosts of drug-sensitivity genes that are acquired or expressed in some but not all cells. This strategy predicts that neoplasms arising from drug-sensitive cells might be safely treatable with tumor-eradicative drug doses because the presence of a modicum of drug-insensitive stem cells will protect vital tissues from lethal depopulation. To test this prediction, lymphomas were induced with Abelson leukemia virus in mice bearing a herpes simplex virus thymidine kinase (HSV-TK) transgene selectively expressed in lymphoid cells. Of 12 transgenic mice treated with the HSV-TK-specific substrate ganciclovir (GCV), 11 exhibited complete tumor regressions; 5 of these mice remained tumor-free over observation periods that exceeded 100 days. Among the lymphomas that recurred, most appeared to represent mutant subpopulations that were GCV-insensitive because they had lost HSV-TK, implying that independent insertion of multiple HSV-TK gene copies might provide a means of preventing recurrences. The results of this study demonstrate that chemosensitivity genes can enhance the efficacy of treatment in hosts who subsequently develop a neoplasm. While the use of a germ-line gene insertion model precludes direct human application, the results also imply the merits of exploring an alternative version of the strategy in which somatic insertion of chemosensitivity genes in mosaic fashion is used prophylactically to enhance the prospect that a subsequent tumor will respond to therapy.

Moderate and advanced Alzheimer's patients exhibit platelet activation differences.

We previously reported that platelets from advanced sporadic Alzheimer's disease (AD) patients exhibit two defects: first, an aberrant signal transduction presenting as a thrombin-induced hyperacidification, which is more severe for donors with the apolipoprotein E4 allele (apoE4), and second, an AD-specific Amyloid Precursor Protein (APP) processing defect that presents as retention of APP on the activated platelets' surface and in independent of the apo E allele. This retention of membrane APP correlates with decreased release of soluble APP. To determine at what stage in the disease progression these defects appear, we performed signal transduction and secretion studies on moderate AD patients. Thrombin-activated platelets from these patients do not exhibit either hyperacidification or APP retention; their APP processing and secretion are normal by Western blotting, suggesting that the two platelet defects appear in the advanced stages of AD.

Activated Alzheimer disease platelets retain more beta amyloid precursor protein.

Upon activation, platelet alpha-granules' soluble contents are secreted and membrane-bound contents are translocated to the plasma membrane. Membrane-bound proteins include the beta-amyloid precursor protein (APP) from which the beta-amyloid (A beta) deposits found surrounding the cerebrovasculature of patients with Alzheimer's Disease (AD) may originate. We show here that activated platelets from AD patients exhibit less APP processing, retain more of the protein on their surface, and secrete less as soluble fragments than do controls. Surface labeling demonstrated that there is little APP or CD62 on the surface of resting platelets. Upon activation, control platelets exhibited more of both proteins on their surface, while advanced AD patients exhibited similar amounts of CD62 as controls, but retained significantly more surface APP. AD platelets secreted similar amounts of most soluble alpha-granule contents as controls, but less APP fragments. Together these results suggest a processing defect that may account for greater deposition of A beta-containing products in the vasculature to which activated platelets adhere.

The isolation of lactococcal promoters and their use in investigating bacterial luciferase synthesis in Lactococcus lactis.

18 different promoter elements, encompassing a 71-fold range of activity, were isolated from the chromosome of Lactococcus lactis (Ll) MG1363 and from an uncharacterised small isometric bacteriophage of Ll. The Vibrio fischeri (Vf) luciferase-encoding gene (lux) was used as a reporter in Ll, so that the promoters could be identified strictly on the basis of their activity in the homologous host. Sequence and primer extension analysis of six of the promoters has provided a new consensus sequence for the -35 and -10 hexanucleotide motifs present upstream from lactococcal transcription start points. When the nucleotide sequence of the most active promoter (P15) was compared with that of the highly expressed Ll usp45 gene, a novel 8-bp region of homology was identified which corresponded to the newly derived consensus -35 sequence element; this element may therefore be of general importance in Ll gene expression. The isolation of these promoters has also enabled us to investigate the characteristics of the Vf Lux activity in Ll under different physiological conditions using promoters of different strengths. Lux activity in Ll is critically dependent upon the phase of cell growth. Luminescence falls sharply in stationary phase, possibly due to a lack of FMNH2. In contrast to the kinetics of Lux function in Escherichia coli (Ec), Lux activity in Ll declines rapidly after addition of the substrate; the rate of decay is dependent both on the growth phase and on the strength of the promoter. It is apparent that the previously reported thermal instability of Lux is in fact a function of the host organism in which Lux is expressed.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative genome analysis of Campylobacter jejuni using whole genome DNA microarrays.

Whole genome DNA microarrays were constructed and used to investigate genomic diversity in 18 Campylobacter jejuni strains from diverse sources. New algorithms were developed that dynamically determine the boundary between the conserved and variable genes. Seven hypervariable plasticity regions (PR) were identified in the genome (PR1 to PR7) containing 136 genes (50%) of the variable gene pool. When comparisons were made with the sequenced strain NCTC11168, the number of absent or divergent genes ranged from 2.6% (40 genes) to 10.2% (163) and in total 16.3% (269) of the genes were variable. PR1 contains genes important in the utilisation of alternative electron acceptors for respiration and may confer a selective advantage to strains in restricted oxygen environments. PR2, 3 and 7 contain many outer membrane and periplasmic proteins and hypothetical proteins of unknown function that might be linked to phenotypic variation and adaptation to different ecological niches. PR4, 5 and 6 contain genes involved in the production and modification of antigenic surface structures.

Genetic evidence that metacyclic forms of Trypanosoma brucei are diploid.

The hypothesis that metacyclic trypanosomes are haploid has been tested genetically. Five cloned stocks of Trypanosoma brucei (each having four known isoenzyme markers and six known restriction fragment length polymorphisms) have been independently transmitted through tsetse flies. Fifteen individual metacyclic organisms were taken from flies with mature cyclical infections and used to establish fresh clones. All the sub-clones from all the flies proved to be identical to the starting (parental) stocks, with respect to all the markers examined, including those markers which were heterozygous in the parental stocks. We conclude that metacyclic trypanosomes are diploid, and are not the product of an obligatory meiosis.

DNA contents and molecular karyotypes of hybrid Trypanosoma brucei.

We have used restriction fragment length polymorphism markers to characterise parental and hybrid trypanosome stocks. Unexpected differences in the intensities of Southern hybridisation banding patterns led us to suspect that the hybrid organisms contained more DNA than the parental stocks. This has been confirmed using flow cytofluorimetry (FCF). Hybrids contained significantly more DNA than the parents, both as procyclic organisms (1.5 fold) and as bloodstream forms (1.5-1.6 fold). The DNA contents of both forms were stable through prolonged culture (procyclics), or serial passage (bloodstream forms), although limited data indicated that falls in DNA content could occur in bloodstream forms. FCF analysis of purified nuclei revealed that the increased DNA content of hybrids could be wholly ascribed to nuclear DNA. Our methods are able to detect hybrid organisms with elevated DNA contents in uncloned isolates following cyclical mixed transmission. We have used alternating field electrophoresis techniques to investigate whether the inheritance by the hybrids of the smaller chromosomes could account for their elevated DNA contents. Hybrids lacked the single 500 kb chromosome from one of the parents but appeared to have virtually double the amount of minichromosomes. However, this increase could only account for about 20% of the additional DNA. We are unable at present to distinguish between models for hybrid formation based on the fusion of predominantly diploid cells, and models in which the diploid chromosomes participate in conventional meiosis.

Gene exchange in African trypanosomes: characterisation of a new hybrid genotype.

We describe the isolation of a hybrid Trypanosoma brucei clone following the mixed cyclical transmission of two parental clones through tsetse flies. The characterisation of this clone reveals some new facets of the process of genetic exchange in T. brucei. The inheritance of four isoenzyme loci and restriction fragment polymorphisms at two loci was interpretable in 'mendelian' genetic terms, involving meiosis either before or after the genetic exchange event. However, at a further two isoenzyme loci a deviation from mendelian behaviour was observed. Pulse field gel analysis showed that the hybrid clone possessed a new combination of intermediate size chromosomes. Examination of kinetoplast DNA variants showed uniparental kinetoplast inheritance, and with reference to previous work we conclude that the kinetoplast can be inherited from either parent. The nuclear DNA content of the hybrid clone was measured and found to be identical to the parental nuclear DNA contents. This finding, together with the inheritance of isoenzyme and RFLP markers, is discussed with respect to possible models for genetic exchange.