Inter-kingdom relationships in Crohn's disease explored using a multi-omics approach.

The etiology of Crohn's disease (CD) is multifactorial. Bacterial and fungal microbiota are involved in the onset and/or progression of the disease. A bacterial dysbiosis in CD patients is accepted; however, less is known about the mycobiome and the relationships between the two communities. We investigated the interkingdom relationships, their metabolic consequences, and the changes in the fungal community during relapse and remission in CD.Two cohorts were evaluated: a British cohort (n = 63) comprising CD and ulcerative colitis patients, and controls. The fungal and bacterial communities of biopsy and fecal samples were analyzed, with the fecal volatiles; datasets were also integrated; and a Dutch cohort (n = 41) comprising CD patients and healthy controls was analyzed for stability of the gut mycobiome.A dysbiosis of the bacterial community was observed in biopsies and stool. Results suggest Bacteroides is likely key in CD and may modulate Candida colonization. A dysbiosis of the fungal community was observed only in the Dutch cohort; Malassezia and Candida were increased in patients taking immunosuppressants. Longitudinal analysis showed an increase in Cyberlindnera in relapse. Saccharomyces was dominant in all fecal samples, but not in biopsies, some of which did not yield fungal reads; amino acid degradation was the main metabolic change associated with CD and both bacteria and fungi might be implicated.We have shown that Bacteroides and yeasts may play a role in CD; understanding their role and relationship in the disease would shed new light on the development and treatment of CD.

Investigating the feasibility of ICP-MS/MS for differentiating NIST salmon reference materials through determination of Sr and S isotope ratios.

Inductively coupled plasma tandem mass spectrometry (ICP-MS/MS) was investigated for possible use in food fraud studies through the measurement of strontium and sulfur isotope ratios. Oxygen mass shift mode was applied to shift 87Sr/86Sr and 34S/32S isotope ratios to their respective oxides, 87Sr16O+/86Sr16O+ and 34S16O+/32S16O+, effecting a gas-phase chemical separation of the elements from Rb and Kr (for Sr) and molecular N and O species, along with P- and S-hydrides (for S). A total least squares regression approach was employed to generate the isotope ratio data from time-resolved analyses, and method uncertainties and accuracies were determined. The utility of the approach was shown by using the Sr and S isotope ratios together to differentiate between NIST RM 8256 Wild-Caught Coho Salmon and NIST RM 8257 Aquacultured Coho Salmon. These materials are currently under development at NIST as certified food fraud standards and method evaluation materials for comprehensive chemical analysis.

DNA extraction and amplicon production strategies deeply inf luence the outcome of gut mycobiome studies.

Microbial ecology studies are often performed through extraction of metagenomic DNA followed by amplification and sequencing of a marker. It is known that each step may bias the results. These biases have been explored for the study of bacterial communities, but rarely for fungi. Our aim was therefore to evaluate methods for the study of the gut mycobiome. We first evaluated DNA extraction methods in fungal cultures relevant to the gut. Afterwards, to assess how these methods would behave with an actual sample, stool from a donor was spiked with cells from the same cultures. We found that different extraction kits favour some species and bias against others. In terms of amplicon sequencing, we evaluated five primer sets, two for ITS2 and one for ITS1, 18S and 28S rRNA. Results showed that 18S rRNA outperformed the other markers: it was able to amplify all the species in the mock community and to discriminate among them. ITS primers showed both amplification and sequencing biases, the latter related to the variable length of the product. We identified several biases in the characterisation of the gut mycobiome and showed how crucial it is to be aware of these before drawing conclusions from the results of these studies.

Comparison of drug release from poly(lactide-co-glycolide) microspheres and novel fibre formulations.

Intraperitoneal cisplatin delivery has recently been shown to benefit ovarian cancer patients. Cisplatin-containing poly(lactide-co-glycolide) (PLGA) microspheres have been proposed for cisplatin delivery. The drug loading of cisplatin containing microspheres produced elsewhere is 3-10%w. Similar microspheres are reported here with a mean diameter of 38.8 µm, and a drug loading of 11.7%w, but using ethyl acetate as a safer solvent. In addition, novel formulations of cisplatin-containing solid and hollow PLGA 65:35 (lactide:glycolide) fibres were prepared and are reported here for the first time. PLGA hollow fibres were produced by phase inversion with a high drug loading of 27%w. Mechanistic mathematical models were applied to the cisplatin release profiles to allow quantitative comparison of microsphere, solid fibre and hollow fibre formulations. The diffusion coefficient of cisplatin eluting from a typical batch of PLGA microspheres was 4.8 × 10(-13) cm(2) s(-1); this low diffusivity of cisplatin in microspheres was caused by the low porosity of the polymer matrix. The diffusion coefficients of cisplatin eluting from a batch of PLGA solid fibres and hollow fibres were 6.1 × 10(-10) and 3.3 × 10(-10) cm(2) s(-1), respectively. These fibres allowed the controlled release of high doses of cisplatin over four days and may represent an improvement in slow release technology for treatment of ovarian cancer.

Combined oral and rectal mesalazine for the treatment of mild-to-moderately active ulcerative colitis: rapid symptom resolution and improvements in quality of life.

BACKGROUND AND AIMS: Mesalazine (5-aminosalicylic acid) is the standard first-line therapy for mild-to-moderate ulcerative colitis. In the PINCE study, remission rates were significantly greater with combined oral/enema vs. oral/placebo treatment at 8 weeks (64% vs. 43%, respectively; p=0.030). In this analysis, we explored early response, mucosal healing rates, cessation of rectal bleeding, and quality of life in PINCE. METHODS: Patients with extensive mild-to-moderately active ulcerative colitis received 8weeks of oral mesalazine 4 g/day, plus 4 weeks of daily active (1g mesalazine) or placebo enema. Early response was assessed using the abbreviated ulcerative colitis disease activity index. Mucosal healing was assessed by disease activity index endoscopic mucosal appearance score. Cessation of bleeding (patient diaries), quality of life (EQ-5D), and patient acceptability (questionnaire) were also assessed. RESULTS: Combined mesalazine oral/enema treatment achieved a significantly higher rate of improvement in abbreviated ulcerative colitis disease activity index (score decrease ≥ 2) within 2 weeks, compared with oral-only treatment (p = 0.032). Bleeding ceased significantly more quickly with combination vs. oral therapy (p = 0.003). More patients showed mucosal healing (disease activity index endoscopic mucosal appearance score 0/1) with combination vs. oral therapy, which was significantly different between groups at week 4 (p = 0.052). Both groups showed quality of life improvements, with a significant benefit for combination vs. oral therapy at week 4 in multiple domains. Most patients reported finding the treatment acceptable. CONCLUSIONS: Rapid cessation of symptoms was seen with combination therapy, which is particularly important to patients and may improve quality of life.

Objective assessment of nanoparticle disposition in mammalian skin after topical exposure.

The use of nanoparticles as formulation components of topical drug delivery systems for the skin has been widely investigated in the literature. Because of the conflicting conclusions resulting from these studies concerning the ultimate disposition of the nanoparticles employed, the research presented in this paper has been designed to evaluate objectively the fate of such structures when administered to mammalian skin. Confocal microscopy images of skin exposed to nanoparticles have therefore been assessed by quantitative statistical analysis. Sebum on the skin surface was naturally fluorescent and clearly defined the outermost part of the cutaneous barrier. Fluorescent polystyrene nanoparticles applied in aqueous suspension could infiltrate only the stratum disjunctum, i.e., skin layers in the final stages of desquamation. This minimal uptake was independent of contact time (up to 16 h) and of nanoparticle size tested (20-200 nm). When skin barrier function was modestly compromised, the nanoparticles remained incapable of penetration beyond the most superficial layers, corresponding to a depth of 2-3 µm, of the stratum corneum (the outermost, 15-20 µm skin layer). Overall, these results demonstrate objectively and semi-quantitatively that nanoparticles contacting intact, and even partially damaged, skin cannot penetrate beyond the superficial layers of the barrier, and are highly unlikely, therefore, to reach the viable cells of the epidermis or beyond. It follows that nanoparticulate-based, topical delivery systems may prove useful as skin surface reservoirs from which controlled drug release over time may be achieved.

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

BACKGROUND: Fibrosis is a serious consequence of Crohn's disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. METHODS: Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. RESULTS: In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR(+) cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR(+)CD45(+)CD56(+/-)CD3(-) were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. CONCLUSIONS: The data indicate that in fibrotic intestinal muscle of Crohn's patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1(+), KIR(+) innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Development and certification of green tea-containing standard reference materials.

A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability.

Identification and characterisation of an iron-responsive candidate probiotic.

BACKGROUND: Iron is an essential cofactor in almost all biological systems. The lactic acid bacteria (LAB), frequently employed as probiotics, are unusual in having little or no requirement for iron. Iron in the human body is sequestered by transferrins and lactoferrin, limiting bacterial growth. An increase in the availability of iron in the intestine by bleeding, surgery, or under stress leads to an increase in the growth and virulence of many pathogens. Under these high iron conditions, LAB are rapidly out-competed; for the levels of probiotic bacteria to be maintained under high iron conditions they must be able to respond by increasing growth rate to compete with the normal flora. Despite this, iron-responsive genera are poorly characterised as probiotics. METHODOLOGY/PRINCIPAL FINDINGS: Here, we show that a panel of probiotics are not able to respond to increased iron availability, and identify an isolate of Streptococcus thermophilus that can increase growth rate in response to increased iron availability. The isolate of S. thermophilus selected was able to reduce epithelial cell death as well as NF-κB signalling and IL-8 production triggered by pathogens. It was capable of crossing an epithelial cell barrier in conjunction with E. coli and downregulating Th1 and Th17 responses in primary human intestinal leukocytes. CONCLUSIONS/SIGNIFICANCE: We propose that an inability to compete with potential pathogens under conditions of high iron availability such as stress and trauma may contribute to the lack of efficacy of many LAB-based probiotics in treating disease. Therefore, we offer an alternative paradigm which considers that probiotics should be able to be competitive during periods of intestinal bleeding, trauma or stress.

MRI findings in herniation of the spinal cord.

Herniation of the spinal cord is a rare condition that causes non specific neurological deficits that are often a diagnostic challenge to clinicians. Despite several reports in the neurosurgical literature, it is only recently that the imaging appearances of this condition have come to be recognised, due mainly to the widespread adoption of spinal MRI. It is important for radiologists to recognise the telltale MRI features of this condition, as several cases have undergone initial misdiagnosis, resulting in delayed treatment We present a case with typical imaging features to familiarise radiologists with this condition, as it is likely that more cases will come to the fore, with more spinal MRIs being performed.

The imaging spectrum of peri-articular inflammatory masses following metal-on-metal hip resurfacing.

INTRODUCTION: Resurfacing metal-on-metal hip arthroplasty is increasing in popularity, especially in younger patients. To date, studies indicate that the procedure is associated with a good outcome in the medium-term. Formation of a peri-articuar mass is a rarely reported complication. In this study we analyse the imaging findings in patients with resurfacing implants presenting to our institution with peri-articular masses identified on cross sectional imaging. MATERIALS AND METHODS: All patients with documented peri-articular masses following resurfacing arthroplasty were included. The available imaging related to the masses was reviewed and the findings documented along with the patient's demographics. RESULTS: There were 10 patients (13 joints). All patients were female. Patients presented with periprosthetic anterior or posterolateral solid and cystic masses. The anterior masses involved psoas muscle and were predominately solid. The posterolateral masses were predominately cystic. In the three cases with bilateral arthroplasties, masses were detected in both hips. Histology in six cases showed features compatible with a type IV hypersensitivity reaction. CONCLUSIONS: The preponderance of females, the bilateral nature of the masses and the histological features suggest that peri-articular masses following resurfacing arthroplasty is due to the metal hypersensitivity.

Scarring occurs at a critical depth of skin injury: precise measurement in a graduated dermal scratch in human volunteers.

BACKGROUND: The association between scarring and the depth of dermal injury or burn is clinically recognized but not quantified. The authors tested the hypothesis that there is a critical depth beyond which a fibrous scar develops. METHODS: A novel jig produced a wound that was deep dermal at one end and superficial dermal at the other. Pilot studies in cadaveric and ex vivo breast skin confirmed the depth of injury. Healthy volunteers had a standardized dermal wound made on the lateral aspect of the hip. Digital photography recorded the surface appearance of wound healing and scar development. High-frequency ultrasound demonstrated the depth of the healing wound and subsequent scar in vivo. RESULTS: One hundred thirteen human subjects participated in the clinical study. Mean length of follow up was 28.6 +/- 13.2 weeks. The deep dermal end of the wound healed with a visible scar and the superficial end had no visible residual mark after week 18. The initial length of injury was 51.3 +/- 0.6 mm, which reduced to a scar of 34.9 +/- 1.0 mm at 36 weeks (corresponding areas were 196.6 +/- 7.5 mm and 92.7 +/- 9.4 mm). High-frequency ultrasound analysis showed a gradual reduction in scar thickness at the deep end and no detectable scar at the shallow end. The transition point between scar and no scar marked the threshold depth for scarring. This was calculated as 0.56 +/- 0.03 mm, or 33.1 percent of normal hip skin thickness. CONCLUSIONS: The dermal scratch provides a well-tolerated, standardized, and reproducible wound model for investigating the healing response to dermal injury of different depths. There is a threshold depth of dermal injury at which scarring develops.

Repertoire of the alpha beta T-cell receptor in the intestine.

The majority of T cells in the human and mouse intestine express the T-cell receptor (TCR) as an alphabeta heterodimer on their cell surface. As the major recognition element of antigens in the context of major histocompatibility complex-derived proteins, an examination of the structure of the alpha beta TCR in intestines has provided significant insights into the potential function of these cells and the major determinants that drive their selection. Studies in the human intestine have shown that the repertoires of intraepithelial lymphocytes (IELs), and likely lamina propria lymphocytes, are polyclonal before and shortly after birth, with the repertoire becoming oligoclonal in adults. Similarly, in adult mice the repertoire is oligoclonal, while in the newborn it is polyclonal. Investigations in mice have shown that some T cells may evade thymic selection. The population size and oligoclonality of IELs is influenced by the microbial content of the luminal microenvironment. This microenvironment probably directly determines the TCR repertoire. Studies in human inflammatory bowel disease (IBD) indicate that inflammation further skews the TCR repertoire. We speculate that dominant antigens associated with the pathogenesis of IBD are responsible for such skewing and that identifying the antigenic drivers may shed light on the environmental factors that trigger or potentiate human IBD.

A case-control study of drinking water and dairy products in Crohn's Disease--further investigation of the possible role of Mycobacterium avium paratuberculosis.

Similarities between Johne's disease in ruminants and Crohn's disease in humans have led to speculation that Mycobacterium avium paratuberculosis (MAP) might be a causative agent in Crohn's disease. However, evidence remains inconsistent. In this case-control study (1999-2004), the authors assessed the possible role of drinking water and dairy products potentially contaminated with MAP in the etiology of Crohn's disease. A total of 218 patients with Crohn's disease recruited from nine hospitals in England and 812 controls recruited from the community completed a short questionnaire for evaluation of proxy measures of potential exposure to MAP. Logistic regression showed no significant association with measures of potential contamination of water sources with MAP, water intake, or water treatment. Multivariate analysis showed that consumption of pasteurized milk (per kg/month: odds ratio (OR) = 0.82, 95% confidence interval (CI): 0.69, 0.97) was associated with a reduced risk of Crohn's disease. Meat intake (per kg/month: OR = 1.40, 95% CI: 1.17, 1.67) was associated with a significantly increased risk of Crohn's disease, whereas fruit consumption (per kg/month: OR = 0.78, 95% CI: 0.67, 0.92) was associated with reduced risk. This study does not support a role for water or dairy products potentially contaminated with MAP in the etiology of Crohn's disease. The observed association with meat and the negative association with pasteurized milk need further study.

Certification of methylmercury content in two fresh-frozen reference materials: SRM 1947 Lake Michigan fish tissue and SRM 1974b organics in mussel tissue (Mytilus edulis).

This paper describes the development of two independent analytical methods for the extraction and quantification of methylmercury from marine biota. The procedures involve microwave extraction, followed by derivatization and either headspace solid-phase microextraction (SPME) with a polydimethylsiloxane (PDMS)-coated silica fiber or back-extraction into iso-octane. The identification and quantification of the extracted compounds is carried out by capillary gas chromatography/mass spectrometric (GC/MS) and inductively coupled plasma mass spectrometric (GC/ICP-MS) detection. Both methods were validated for the determination of methylmercury (MeHg) concentrations in a variety of biological standard reference materials (SRMs) including fresh-frozen tissue homogenates of SRM 1946 Lake Superior fish tissue and SRM 1974a organics in mussel tissue (Mytilus edulis) and then applied to the certification effort of SRM 1947 Lake Michigan fish tissue and SRM 1974b organics in mussel tissue (Mytilus edulis). While past certifications of methylmercury in tissue SRMs have been based on two independent methods from the National Institute of Standards and Technology (NIST) and participating laboratories, the methods described within provide improved protocols and will allow future certification efforts to be based on at least two independent analytical methods within NIST.

The effect of weaning on the clonality of alpha beta T-cell receptor T cells in the intestine of GF and SPF mice.

In humans, intestinal antigen exposure during neonatal life influences the T-cell receptor (TCR) repertoire. To define the relative effects of bacteria and food antigens in early life, we examined TCR diversity in the intestine of SPF and GF mice. TCR repertoire was assessed at a single time point pre-, peri- and post-weaning in the small and large intestine of SPF and GF mice using spectratyping and/or TCR-beta-chain sequencing. There was good concordance of data obtained by the two techniques. In SPF mice, the repertoire was polyclonal shortly after birth in the small and large intestine. After weaning, there was a significant change towards an oligoclonal repertoire in the small intestine. There was some evidence that specific clones were shared between the small and large intestine. In contrast, in GF mice, the repertoire was oligoclonal after birth, and remained restricted. These data show: firstly, that under SPF conditions, the intestine is seeded with a diverse T-cell population that becomes oligoclonal around the time of weaning; secondly, that GF mice were oligoclonal at each time point.

Selective processing of gastrointestinal symptom-related stimuli in irritable bowel syndrome.

OBJECTIVES: We sought to determine whether irritable bowel syndrome (IBS) was associated with attentional bias toward symptom-related cues in IBS patients versus healthy controls, using a modified Stroop task to measure selective processing of gastrointestinal symptom-related cues. METHODS: Fifteen patients with a clinical diagnosis of IBS and 15 healthy controls were recruited into the study. All participants attended a single testing session, during which they completed a modified Stroop task using gastrointestinal symptom-related and neutral control words. RESULTS: Results indicated a significant main effect of word type (p = .013), with slower color-naming times for IBS-related compared with neutral words, and a significant main effect of exposure (p = .001), with slower color-naming times in the unmasked condition compared with the masked condition. The group x word type x exposure interaction was significant (p = .048). A series of post hoc tests indicated that among patients there was significant interference of symptom-related words in the masked condition but not in the unmasked condition, whereas among controls, the reverse was true. CONCLUSIONS: These results indicate that IBS patients selectively process gastrointestinal symptom-related words compared with neutral words when they are presented subliminally but not when they are presented supraliminally. In contrast, healthy controls demonstrate the opposite pattern. Implications for the cognitive mechanisms in IBS, and future research directions, are discussed.

Effects of microflora on the neonatal development of gut mucosal T cells and myeloid cells in the mouse.

Colonization with commensal flora in very early life may profoundly influence intestinal lymphoid development and bias later immune responses. We defined gut-homing T cell phenotypes and the influence of flora on intestinal immune development in mice. Intestinal T cells were phenotyped and quantified in conventional (CV), germfree (GF) and conventionalized germfree (GF/CV) neonatal mice by immunohistochemistry. Mucosal adressin cell adhesion molecule 1 (MAdCAM-1) was expressed by mucosal vessels at birth in CV and GF mice and was more prevalent in CV than GF small intestine, but was distributed similarly and did not change with age. Less MAdCAM-1 was expressed in the colon; its distribution became restricted after weaning, with no difference between CV and GF mice. CD3(+)beta(7) (+) cells were present in similar numbers in CV and GF intestine at birth. They were CD62L(-) in CV mice and were accompanied by further CD3(+)beta(7) (+)CD62L(-) T cells as development progressed, but in GF and GF/CV intestine they expressed CD62L and numbers did not change. IEL numbers increased at weaning in CV mice in both small and large intestine, but showed delayed development in GF intestine. Macrophages were present at high levels from birth in GF intestine, but dendritic cells did not develop until day 16. Thus, fetus-derived T cells seed the intestinal lamina propria before birth via beta-MadCAM interactions. Their activation status depends on the microbiological status of the dam, and without a commensal flora they remain naive. We propose that these cells regulate antigen responsiveness of the developing mucosal T cell pool.