John Kevin Collins

Double blind, placebo controlled trial of two probiotic strains in interleukin 10 knockout mice and mechanistic link with cytokine balance

Background: Prophylactic efficacy against colitis following lactobacillus consumption in interleukin 10 (IL-10) knockout (KO) mice has been reported. Whether this applies equally to other probiotic strains is unknown, and the mechanism is unclear. Aims: (1) To compare the effect of feeding Lactobacillus salivarius subspecies salivarius 433118 and Bifidobacterium infantis 35624 against placebo on enterocolitis, the intestinal microflora, and (2) to compare the systemic immunological response to in vitro microbial challenge in probiotic fed and control IL-10 KO mice. Methods: Three groups of 10 IL-10 KO mice were fed fermented milk products containing Lb salivarius 433118 at 109 CFU/ml, B infantis 35624 at 108 CFU/ml, and unmodified milk, respectively, for 19 weeks. Faecal samples were taken at regular intervals to confirm gut transit, recovery of fed probiotics, and to assess the impact on the microflora. At sacrifice, the bowels were histologically scored. Cytokine production from Peyers’ patches and splenocytes was measured in vitro by ELISA. Results: Faecal recovery of probiotics was confirmed in all probiotic fed mice but not in controls. Colonic and caecal inflammatory scores were significantly decreased in both groups of probiotic fed mice (p<0.05). Proinflammatory cytokine production by Peyers’ patches and splenocytes was significantly reduced in probiotic fed animals whereas transforming growth factor β (TGF-β) levels were maintained. Conclusion: Both Lactobacillus salivarius 433118 and Bifidobacterium infantis 35624 significantly attenuate colitis in this murine model. Attenuation of colitis is associated with a reduced ability to produce Th1-type cytokines systemically and mucosally, while levels of TGF-β are maintained.

Comparative survival rates of human-derived probiotic Lactobacillus paracasei and L. salivarius strains during heat treatment and spray drying.

ABSTRACT Spray drying of skim milk was evaluated as a means of preservingLactobacillus paracasei NFBC 338 and Lactobacillus salivarius UCC 118, which are human-derived strains with probiotic potential. Our initial experiments revealed that NFBC 338 is considerably more heat resistant in 20% (wt/vol) skim milk than UCC 118 is; the comparable decimal reduction times were 11.1 and 1.1 min, respectively, at 59°C. An air outlet temperature of 80 to 85°C was optimal for spray drying; these conditions resulted in powders with moisture contents of 4.1 to 4.2% and viable counts of 3.2 × 109 CFU/g for NFBC 338 and 5.2 × 107 CFU/g for UCC 118. Thus, L. paracasei NFBC 338 survived better than L. salivarius UCC 118 during spray drying; similar results were obtained when we used confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining. In addition, confocal scanning laser microscopy revealed that the probiotic lactobacilli were located primarily in the powder particles. Although both spray-dried cultures appeared to be stressed, as shown by increased sensitivity to NaCl, bacteriocin production by UCC 118 was not affected by the process, nor was the activity of the bacteriocin peptide. The level of survival of NFBC 338 remained constant at ∼1 × 109 CFU/g during 2 months of powder storage at 4°C, while a decline in the level of survival of approximately 1 log (from 7.2 × 107 to 9.5 × 106 CFU/g) was observed for UCC 118 stored under the same conditions. However, survival of both Lactobacillus strains during powder storage was inversely related to the storage temperature. Our data demonstrate that spray drying may be a cost-effective way to produce large quantities of some probiotic cultures.

Probiotic impact on microbial flora, inflammation and tumour development in IL‐10 knockout mice

The enteric bacterial flora has been implicated in the pathogenesis of enterocolitis and colon cancer in C57BL/6 IL‐10 knockout mice. Probiotic Lactobacilli modify the enteric flora and are thought to have a beneficial effect on enterocolitis. We conducted a controlled feeding trial in IL‐10 knockout mice using the probiotic Lactobacillus salivarius ssp. salivarius UCC118.

Is the mucosal route of administration essential for probiotic function? Subcutaneous administration is associated with attenuation of murine colitis and arthritis

Background: We and others have reported the prophylactic efficacy of oral consumption of probiotic lactobacilli in the interleukin 10 knockout (IL-10 KO) model of colitis. It has not been demonstrated that the oral route is essential for probiotic efficacy. Aims: (i) To determine the effect of parenteral administration (subcutaneous) of Lactobacillus salivarius 118 on colitis of IL-10 KO mice; and (ii) to determine if observed responses are disease specific. Methods: (i) IL-10 KO mice were injected subcutaneously with L salivarius 118 or saline over 19 weeks. At sacrifice, the bowels were histologically scored. Isolated splenocytes were cultured in vitro and cytokine levels measured. (ii) In the collagen induced arthritis model, DBA/1 mice were injected subcutaneously with the probiotic or saline. At sacrifice, paw thickness was measured and joints were histologically scored. Results: (i) Colonic inflammatory scores were significantly decreased in IL-10 KO mice injected with L salivarius 118 compared with controls (p<0.05). Proinflammatory cytokine production from stimulated splenocytes was significantly lower for the probiotic group whereas stimulated transforming growth factor β (TGF-β) levels were significantly increased (p<0.05). (ii) Scoring of arthritis and paw thickness were significantly improved in the group of mice injected with L salivarius 118 compared with controls. Conclusions: (1) Subcutaneous administration of L salivarius 118 significantly attenuated colitis in the IL-10 KO model and suppressed collagen induced arthritis, suggesting that the oral route may not be essential for probiotic anti-inflammatory effects and that responses are not disease specific. (2) The probiotic effect was associated with reduced production of proinflammatory (T helper 1) cytokines and maintained production of anti-TGF-β.

Probiotic bacteria: myth or reality?

With the emergence of a more health-conscious society, the role of probiotic bacteria in human health has gained considerable attention from both the consumer and the producer. Considerable controversy has been associated with the lack of scientific evidence for the probiotic concept. However, current scientifically based research efforts worldwide are now focusing on the development of high-quality probiotic products containing microorganisms preselected for specific probiotic characteristics.

Culture‐independent analysis of the gut microbiota in colorectal cancer and polyposis

A role for the intestinal microbiota is routinely cited as a potential aetiological factor in colorectal cancer initiation and progression. As the majority of bacteria in the gut are refractory to culture we investigated this ecosystem in subjects with colorectal cancer and with adenomatous polyposis who are at high risk of developing colorectal cancer, using culture-independent methods. Twenty colorectal cancer and 20 polypectomized volunteers were chosen for this analysis. An exploration of the diversity and temporal stability of the dominant bacteria and several bacterial subgroups was undertaken using 16S rRNA gene denaturing gradient gel electrophoresis and ribosomal intergenic spacer analysis (RISA). Metabonomic analysis of the distal gut microbiotas environment was also undertaken. A significantly reduced temporal stability and increased diversity for the microbiota of subjects with colorectal cancer and polyposis was evident. A significantly increased diversity of the Clostridium leptum and C. coccoides subgroups was also noted for both disease groups. A clear division in the metabonome was observed for the colorectal cancer and polypectomized subjects compared with control volunteers. The intestinal microbiota and their metabolites are significantly altered in both colorectal cancer and polypectomized subjects compared with controls.

Direct In Situ Viability Assessment of Bacteria in Probiotic Dairy Products Using Viability Staining in Conjunction with Confocal Scanning Laser Microscopy

ABSTRACT The viability of the human probiotic strains Lactobacillus paracasei NFBC 338 and Bifidobacterium sp. strain UCC 35612 in reconstituted skim milk was assessed by confocal scanning laser microscopy using the LIVE/DEAD BacLight viability stain. The technique was rapid (<30 min) and clearly differentiated live from heat-killed bacteria. The microscopic enumeration of various proportions of viable to heat-killed bacteria was then compared with conventional plating on nutrient agar. Direct microscopic enumeration of bacteria indicated that plate counting led to an underestimation of bacterial numbers, which was most likely related to clumping. Similarly, LIVE/DEAD BacLight staining yielded bacterial counts that were higher than cell numbers obtained by plate counting (CFU) in milk and fermented milk. These results indicate the value of the microscopic approach for rapid viability testing of such probiotic products. In contrast, the numbers obtained by direct microscopic counting for Cheddar cheese and spray-dried probiotic milk powder were lower than those obtained by plate counting. These results highlight the limitations of LIVE/DEAD BacLight staining and the need to optimize the technique for different strain-product combinations. The minimum detection limit for in situ viability staining in conjunction with confocal scanning laser microscopy enumeration was ∼108 bacteria/ml (equivalent to ∼107 CFU/ml), based on Bifidobacterium sp. strain UCC 35612 counts in maximum-recovery diluent.

A DNA diagnostic biosensor: development, characterisation and performance

Abstract The aim of this work is to develop a sensor for specific DNA sequences, using non-complex synthetic single-stranded oligonucleotides as a model system. A capacitance-based sensor for the direct detection of DNA sequences is described. Hybridisation of analyte DNA with immobilised DNA on the silicon surface induces charge effects, altering the dielectric properties of the biolayer, and can be detected by the associated change in the measured capacitance. DNA has been immobilised on a silicon electrode either by passive adsorption or covalent coupling via 4-aminobutyldimethylmethoxysilane (4-ABDMMS). The work presented here introduces a colourimetric immunodetection technique for the evaluation of the immobilisation process and describes the electrical characterisation and performance of three silicon-based sequence-specific DNA sensors. These sensors consisted of a standard electrolyte-insulator-semiconductor (EIS) structure with covalently bound probe DNA, a mechanically degraded structure with passively adsorbed probe DNA and a mechanically degraded structure with covalently bound probe DNA. The last device had an improved signal to noise ratio and was, therefore, used to construct a standard curve, revealing a detection limit of 100 pmol DNA. On addition of analyte DNA, there was a decrease in measured capacitance. This response was fast, specific and required no addition of mediators to enhance or amplify the signal. This device can be optimised for the detection of complex sequences.

PCR detection of Mycobacterium paratuberculosis in Crohn’s disease granulomas isolated by laser capture microdissection

Background and aims: The uncertainty surrounding the role of Mycobacterium avium subsp paratuberculosis (Map) in Crohn’s disease has been compounded by possible contamination from Map present in the lumen microflora. This study used laser capture microdissection (LCM) and polymerase chain reaction (PCR) to detect Map DNA in subepithelial granulomas, isolated from 15 surgically resected, formalin fixed specimens of granulomatous Crohn’s disease and from 12 granulomatous disease controls (10 bowel, 2 non-bowel). Methods: The effect of amplicon size on reliability of PCR from formalin fixed samples was examined by amplifying 435 bp and 133 bp sequences of the human APC gene. After this, nested primers were designed to detect a small fragment (155 bp) of the Map specific IS900 gene in Crohn’s granulomas. LCM isolated granulomas from Map culture positive bovine intestine was used as positive control. PCR product specificity was confirmed by direct DNA sequencing. Results: The smaller, but not the larger, fragment of the APC gene amplified reliably in all samples. Amplification of the 155 bp fragment of the IS900 gene detected Map DNA in microdissected Crohn’s granulomas in 6 of 15 cases, and in 0 of 12 disease control granulomas. Conclusions: LCM can be used to detect Map DNA in granulomas in a proportion of patients with Crohn’s disease. However, formalin fixation requires that comparatively short DNA fragments of the Map specific IS900 gene be targeted, to permit consistent detection. Detection of Map DNA within granulomas might suggest an infectious aetiology in a subset of patients; alternatively, a transmissible agent may not be involved but mycobacterial DNA may influence pathogenesis by modifying the local cytokine responses.

Expression of Fas ligand by human gastric adenocarcinomas: a potential mechanism of immune escape in stomach cancer

Background Despite being immunogenic, gastric cancers overcome antitumour immune responses by mechanisms that have yet to be fully elucidated. Fas ligand (FasL) is a molecule that induces Fas receptor mediated apoptosis of activated immunocytes, thereby mediating normal immune downregulatory roles including immune response termination, tolerance acquisition, and immune privilege. Colon cancer cell lines have previously been shown to express FasL and kill lymphoid cells by Fas mediated apoptosis in vitro. Many diverse tumours have since been found to express FasL suggesting that a “Fas counterattack” against antitumour immune effector cells may contribute to tumour immune escape. Aim To ascertain if human gastric tumours express FasL in vivo, as a potential mediator of immune escape in stomach cancer. Specimens Thirty paraffin wax embedded human gastric adenocarcinomas. Methods FasL protein was detected in gastric tumours using immunohistochemistry; FasL mRNA was detected in the tumours using in situ hybridisation. Cell death was detected in situ in tumour infiltrating lymphocytes using terminal deoxynucleotidyl transferase mediated dUTP nick end labelling (TUNEL). Results Prevalent expression of FasL was detected in all 30 resected gastric adenocarcinomas examined. In the tumours, FasL protein and mRNA were co-localised to neoplastic gastric epithelial cells, confirming expression by the tumour cells. FasL expression was independent of tumour stage, suggesting that it may be expressed throughout gastric cancer progression. TUNEL staining disclosed a high level of cell death among lymphocytes infiltrating FasL positive areas of tumour. Conclusions Human gastric adenocarcinomas express the immune downregulatory molecule, FasL. The results suggest that FasL is a prevalent mediator of immune privilege in stomach cancer.