ABSTRACT
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.
Subject(s)
Endothelium, Vascular/pathology , Pulmonary Artery/pathology , Pulmonary Disease, Chronic Obstructive/physiopathology , Disease Progression , Endothelium, Vascular/diagnostic imaging , Female , Humans , Longitudinal Studies , Male , Middle Aged , Pulmonary Artery/diagnostic imaging , Pulmonary Disease, Chronic Obstructive/diagnostic imaging , Pulmonary Disease, Chronic Obstructive/genetics , Respiratory Function Tests , Smokers , Tomography, X-Ray ComputedABSTRACT
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.
Subject(s)
Cell Culture Techniques/methods , Intestines/cytology , Organoids/cytology , Animals , Cell Differentiation , Cell Lineage , Cells, Cultured , Electric Impedance , Intestines/ultrastructure , Organoids/ultrastructure , Paneth Cells/cytology , Sus scrofa , Tight Junctions/metabolism , Tight Junctions/ultrastructureABSTRACT
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.
Subject(s)
B-Lymphocytes/immunology , Cholera Vaccines/immunology , Cholera/immunology , Intestine, Large/immunology , Respiratory System/immunology , T-Lymphocytes/immunology , Vibrio cholerae/physiology , Administration, Oral , Adolescent , Adult , Cell Movement , Cell Proliferation , Cells, Cultured , Female , Humans , Immunoglobulin A/metabolism , Immunologic Memory , Intestine, Large/microbiology , Lymphocyte Activation , Male , Pregnancy , Respiratory System/microbiology , Vaccination , Young AdultABSTRACT
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.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Differentiation/genetics , Gene Expression Regulation, Developmental/genetics , Islets of Langerhans/cytology , Nerve Tissue Proteins/genetics , Animals , Antibodies, Monoclonal , Basic Helix-Loop-Helix Transcription Factors/metabolism , Basic Helix-Loop-Helix Transcription Factors/physiology , Cellular Reprogramming , Glucagon-Secreting Cells/cytology , Glucagon-Secreting Cells/metabolism , Humans , Immunohistochemistry , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Islets of Langerhans/metabolism , Mice , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/physiology , Pancreatic Polypeptide-Secreting Cells/cytology , Pancreatic Polypeptide-Secreting Cells/metabolism , Somatostatin-Secreting Cells/cytology , Somatostatin-Secreting Cells/metabolismABSTRACT
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.
Subject(s)
Chemokines, CC/immunology , Eosinophilic Esophagitis/diagnosis , Eosinophilic Esophagitis/pathology , Gastroesophageal Reflux/pathology , Adult , Biomarkers , Chemokine CCL26 , Diagnosis, Differential , Eosinophilic Esophagitis/drug therapy , Female , Gastroesophageal Reflux/diagnosis , Humans , Immunohistochemistry , Male , Middle Aged , Proton Pump Inhibitors/therapeutic use , Retrospective StudiesABSTRACT
REG3γ is considered to have a protective role against infection with Gram-positive bacteria due to its bactericidal activity, but evidence from in vivo studies is lacking. We generated a REG3γ(-/-) mouse, and investigated the effect of lack of REG3γ on intestinal mucus distribution, spatial compartmentalization of bacteria, and expression of innate immunity genes. Infection studies were also performed with Gram-positive and Gram-negative pathogens to investigate the antimicrobial role of REG3γ. REG3γ(-/-) mice display altered mucus distribution, increased bacterial contact with the epithelium, and elevated inflammatory markers in the ileum without histological evidence of pathology. Infection response pathway genes were differentially expressed in both Listeria monocytogenes and Salmonella enteritidis infected REG3γ(-/-) and wild-type (wt) mice. Higher amounts of myeloperoxidase and interleukin-22 transcripts were present in the ileal mucosa of REG3γ(-/-) than wt mice, but translocation to the organs was unaffected. We concluded that REG3γ has a protective role against mucosal infection with pathogenic Listeria and Salmonella in vivo. REG3γ is equally distributed throughout the mucus and its absence results in increased epithelial contact with the microbiota resulting in low-grade inflammation. REG3γ can bind to Gram-negative and Gram-positive bacteria and influence mucus distribution in the ileum, properties which may contribute to mucosal protection.
Subject(s)
Ileum/immunology , Ileum/metabolism , Inflammation/genetics , Inflammation/immunology , Intestinal Mucosa/immunology , Intestinal Mucosa/metabolism , Mucus/metabolism , Proteins/genetics , Animals , Disease Models, Animal , Gene Expression Profiling , Ileum/microbiology , Immunity, Innate , Inflammation/microbiology , Interferon Regulatory Factors/metabolism , Interferons/metabolism , Interleukin-1beta/metabolism , Intestinal Mucosa/microbiology , Listeria monocytogenes/physiology , Mice , Mice, Knockout , Microbiota , Myeloid Differentiation Factor 88/metabolism , Pancreatitis-Associated Proteins , STAT1 Transcription Factor/metabolism , STAT3 Transcription Factor/metabolism , Salmonella enteritidis/physiology , Signal Transduction , Toll-Like Receptor 3/metabolismABSTRACT
Faecalibacterium prausnitzii is the most abundant bacterium in the human intestinal microbiota of healthy adults, representing more than 5% of the total bacterial population. Over the past five years, an increasing number of studies have clearly described the importance of this highly metabolically active commensal bacterium as a component of the healthy human microbiota. Changes in the abundance of F. prausnitzii have been linked to dysbiosis in several human disorders. Administration of F. prausnitzii strain A2-165 and its culture supernatant have been shown to protect against 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice. Here, we discuss the role of F. prausnitzii in balancing immunity in the intestine and the mechanisms involved.
Subject(s)
Gastrointestinal Tract/microbiology , Gastrointestinal Tract/physiology , Gram-Positive Bacteria/immunology , Gram-Positive Bacteria/physiology , Symbiosis , Animals , Humans , MiceABSTRACT
Beneficial effects of certain probiotic strains have been established in the treatment and prevention of various immune and intestinal disorders in humans, including allergic diseases, chronic inflammatory diseases and diarrhoea. The proposed mechanisms underlying the immunomodulatory effects of probiotics in humans are not understood in precise detail but include enhancement of intestinal barrier function, altered epithelial signalling, competition with pathogens and effects on immune cells and immunity depending on the probiotic strain. The publication of controversial or inconclusive probiotic studies in humans highlights the need for a better understanding of the mechanisms and improved strain selection criteria. This review focuses on the immunomodulatory properties of lactobacilli and bifidobacteria in vitro and in vivo, current knowledge concerning the mechanisms in vivo and challenges in translational research on probiotics. A better understanding of the molecular mechanisms of probiotics, the effect of probiotic mixtures versus single strains, the effect of formulation of probiotics and the fate of ingested probiotics should help to clarify the value of immune assays as selection criteria for probiotics.
Subject(s)
Biological Products/administration & dosage , Lactobacillus/physiology , Probiotics/administration & dosage , Translational Research, Biomedical/methods , Translational Research, Biomedical/trends , Biological Products/pharmacology , Humans , Immunologic Factors/administration & dosage , Immunologic Factors/pharmacology , Lactobacillus/immunology , Probiotics/pharmacologyABSTRACT
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.
Subject(s)
Bacteriophages/growth & development , Campylobacter Infections/complications , Campylobacter jejuni/pathogenicity , Gangliosides/metabolism , Guillain-Barre Syndrome/microbiology , Virulence Factors/metabolism , Campylobacter Infections/immunology , Campylobacter Infections/microbiology , Campylobacter jejuni/genetics , Campylobacter jejuni/immunology , Campylobacter jejuni/virology , Computational Biology , DNA, Bacterial/genetics , Gangliosides/immunology , Humans , Virulence Factors/immunologyABSTRACT
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.
Subject(s)
Butyrates/metabolism , Clostridium tyrobutyricum/physiology , Colitis, Ulcerative/microbiology , Interleukin-18/biosynthesis , Probiotics/therapeutic use , Tumor Necrosis Factor-alpha/biosynthesis , Acute Disease , Administration, Rectal , Animals , Bacterial Translocation , CD11b Antigen/biosynthesis , CD11b Antigen/genetics , Colitis, Ulcerative/chemically induced , Colitis, Ulcerative/genetics , Colitis, Ulcerative/immunology , Colitis, Ulcerative/pathology , Colon/metabolism , Colon/microbiology , Colon/pathology , Dextran Sulfate/toxicity , Fatty Acids/metabolism , Immunocompetence , Interleukin-18/genetics , Membrane Proteins/biosynthesis , Membrane Proteins/genetics , Mice , Mice, Inbred BALB C , Mice, SCID , Mucin-2/biosynthesis , Mucin-2/genetics , Mucins/biosynthesis , Phosphoproteins/biosynthesis , Phosphoproteins/genetics , Severe Combined Immunodeficiency/genetics , Severe Combined Immunodeficiency/immunology , Specific Pathogen-Free Organisms , Tumor Necrosis Factor-alpha/genetics , Zonula Occludens-1 ProteinABSTRACT
BACKGROUND/PURPOSE: Urinomas have been thought to protect renal function in boys with posterior urethral valves (PUVs), although recent reports have disputed this. This study tested the hypothesis that urinomas protect global renal function in boys with PUV. METHODS: A retrospective analysis of all boys with PUV presenting to a tertiary unit derived from a region with an estimated population of 5.5 million was performed. Comparisons of the initial nadir creatinine, current creatinine, and renal status score (RSS) were made between those with and without urinomas. The RSS was derived from nephrology assessment of current renal status (0 = normal to 4 = end-stage renal failure or transplantation). Results were given as median (range), except for RSS, which was given as mean +/- SEM. P < or = .05 was regarded as significant. RESULTS: During 1989-2009, 9 of 89 PUV boys were diagnosed with urinomas. Initial nadir creatinine was statistically lower in boys with urinomas (31 [18-44] vs 45 [20-574] mumol/L, P < .01). Length of follow-up was similar (5.1 [2.2-17.3] vs 5.9 [1.8-19.7] years, P = .59). Follow-up creatinine was significantly lower in urinoma boys (44 [25-77] vs 61 [29-1227] micromol/L, P < .05), as was the RSS (0.14 +/- 0.14 vs 0.91 +/- 0.14, P < .01). No urinoma boys progressed to end-stage renal failure or required transplant. CONCLUSION: This population-based study of PUV boys demonstrates that urinomas reduce nadir creatinine and significantly protect long-term global renal function.
Subject(s)
Kidney Failure, Chronic/epidemiology , Kidney Function Tests/statistics & numerical data , Urethra/abnormalities , Urethra/surgery , Urinoma/epidemiology , Urinoma/surgery , Adolescent , Child , Child, Preschool , Creatinine/blood , Follow-Up Studies , Functional Laterality , Humans , Incidence , Infant , Kidney Transplantation , Longitudinal Studies , Male , Nephrostomy, Percutaneous/methods , Prognosis , Treatment Outcome , Urinary Bladder Neck Obstruction/surgery , Urinoma/congenitalABSTRACT
Over the past decade it has become clear that probiotic and commensal interactions with mucosal dendritic cells in the lamina propria or epithelial cells lining the mucosa can modulate specific functions of the mucosal immune system. Innate pattern-recognition receptors such as TLRs, NLRs and CLRs play a crucial role in the host recognition of probiotics and other microorganism. Signalling via these receptors directly influences the chemokine and cytokine response of dendritic cells as well as the crosstalk between the epithelium and the immune cells in the lamina propria. This can influence the population of effector and regulatory T cell subsets in the mucosa. Immune assays with probiotics have shown that the in vitro immune response is both species and strain-specific. Such assays may be useful for the selection of probiotic strains that have beneficial effects on the regulation of intestinal inflammation but more comparative studies are needed to confirm recent findings. A better understanding of the molecular mechanisms of probiotics, the effect of dose, and frequency of administration on microbial sampling by mucosal APC will also help to clarify the value of immune assays as selection criteria for probiotics.
Subject(s)
Dendritic Cells/immunology , Intestines/immunology , Probiotics/pharmacology , T-Lymphocytes/immunology , Animals , Bacteria/immunology , Cytokines/immunology , Dendritic Cells/drug effects , Humans , Intestines/drug effects , Intestines/microbiology , T-Lymphocytes/drug effectsABSTRACT
We evaluated the safety, reactogenicity and immunogenicity of escalating doses of a new Francisella tularensis Live Vaccine Strain (LVS) lot by scarification (SCAR) or subcutaneously (SQ) in humans. Subjects (N=10/group) received one dose of LVS via SCAR at 10(5),10(7) or 10(9)cfu/ml or SQ at 10(2), 10(3),10(4) or 10(5)cfu/ml; 14 subjects received placebo. All doses/routes were well tolerated. When compared to placebo, vaccination with 10(7) SCAR and 10(9) SCAR resulted in significantly higher serologic response frequencies, as measured by ELISA for IgG, IgM, IgA and microagglutination; whereas vaccination with 10(5) SCAR, 10(7) SCAR 10(9) SCAR and 10(5) SQ elicited a significantly higher interferon-gamma response frequency.
Subject(s)
Bacterial Vaccines/adverse effects , Bacterial Vaccines/immunology , Francisella tularensis/immunology , Adolescent , Adult , Antibodies, Bacterial/blood , Bacterial Vaccines/administration & dosage , Enzyme-Linked Immunosorbent Assay/methods , Female , Humans , Immunoglobulin A/blood , Immunoglobulin G/blood , Immunoglobulin M/blood , Injections, Subcutaneous , Interferon-gamma/blood , Male , Placebos/administration & dosage , Vaccines, Attenuated/administration & dosage , Vaccines, Attenuated/adverse effects , Vaccines, Attenuated/immunology , Young AdultABSTRACT
Volatile organic compounds released from the biosphere are known to have a large impact on atmospheric chemistry. Field instruments for the detection of these trace gases are often limited by the lack of instrument portability and the inability to distinguish compounds of interest from background or other interfering compounds. We have developed an automated sampling and preconcentration system, coupled to a lightweight, low-power cylindrical ion trap mass spectrometer. The instrument was evaluated by measuring isoprene concentrations during a field campaign at the University of Michigan Biological Station PROPHET lab. Isoprene was preconcentrated by sampling directly into a short capillary column precooled without the aid of cryogens. The capillary column was then rapidly heated by moving the column to a preheated region to obtain fast separation of isoprene from other components, followed by detection with a cylindrical ion trap. This combination yielded a detection limit of approximately 80 ppt (parts per trillion) for isoprene with a measurement frequency of one sample every 11 min. The data obtained by the automated sampling and preconcentration system during the PROPHET 2005 campaign were compared to those of other field instruments measuring isoprene at this site in an intercomparison exercise. The intercomparisons suggest the new inlet system, when coupled with this ion trap detector, provides a viable field instrument for the fast, precise, and quantitative determination of isoprene and other trace gases over a variety of atmospheric conditions.
Subject(s)
Air Pollutants/analysis , Automation , Mass Spectrometry/methods , Organic Chemicals/analysis , Butadienes/analysis , Hemiterpenes/analysis , Humidity , Mass Spectrometry/instrumentation , Pentanes/analysis , Time Factors , VolatilizationABSTRACT
Genomic sequencing projects are beginning to reveal regions of extensive DNA homology between bacterial genera. Public fears of the spread of genetically modified organisms into the food chain and the increasing prevalence of multi-drug resistant disease in humans highlight the implications of horizontal gene transfer. The striking DNA sequence similarity between the two uniquely identified tetracycline resistant (Tc(R)) Campylobacter plasmids, pCC31 and pTet, suggests their conserved acquisition and maintenance within Campylobacter [Batchelor, R.A., Pearson, B.M., Friis, L.M., Guerry, P., Wells, J.M. 2004. Nucleotide sequences and comparison of two large conjugative plasmids from different Campylobacter species. Microbiology 150, 3507-3517]. It is thus likely that these and other conjugative plasmids are highly prevalent and broadly distributed across several continents. Microarray technology is now enabling fast and extensive genomic comparisons to be made and allows us to investigate intra- and inter-genetic conservation and variability. This study details the development of a microarray specific for genes from Campylobacter plasmids pCC31, pTet and pVir and its application to the analysis of Campylobacter plasmid gene presence and preservation throughout environmental and clinical isolates. Application of the iterative algorithm GENCOM (freely available at ) is used as a rapid and effective way of comparing the content and conservation of plasmids in bacteria and provides details of the Campylobacter flexible gene pool and its contribution to genomic plasticity.
Subject(s)
Campylobacter/genetics , DNA, Bacterial/physiology , Gene Transfer, Horizontal , Plasmids/physiology , Algorithms , Campylobacter/isolation & purification , Campylobacter/physiology , DNA, Bacterial/genetics , Oligonucleotide Array Sequence Analysis , Plasmids/genetics , Tetracycline ResistanceSubject(s)
Military Personnel , Physicians, Family , Career Choice , Career Mobility , Education, Medical , HumansABSTRACT
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.
Subject(s)
Bacteriological Techniques/methods , Campylobacter Infections/microbiology , Campylobacter/pathogenicity , Epithelial Cells/microbiology , Models, Biological , Animals , Caco-2 Cells , Campylobacter/drug effects , Cell Culture Techniques , Drug Resistance, Bacterial , Gentamicins/pharmacology , HumansABSTRACT
The mucosal and cellular responses of mice were studied, following mucosal-route administration of recombinant Lactococcus lactis expressing tetanus toxin fragment C (TTFC), which is a known immunogen protective against tetanus. A TTFC-specific T-cell response with a mixed profile of T-helper (Th) subset-associated cytokines was elicited in the intestine, with a Th2 bias characteristic of a mucosal response. These results correlated with the humoral response, where equivalent titers of anti-TTFC immunoglobulin G1 (IgG1) and IgG2a in serum were accompanied by an elevated IgA-specific response at more than one mucosal site. The route of vaccination had an important role in determining the immune response phenotype, as evidenced by the fact that an IgG1-biased subclass profile was obtained when lactococci were administered parenterally. Stimulation of splenic or mesenteric lymph node cells with lactococci resulted in their proliferation and the secretion of gamma interferon via antigen-specific and innate immune mechanisms. The data therefore provide further evidence of the potential of recombinant lactococcal vaccines for inducing systemic and mucosal immune responses.