Interpretation of Research Article
1. The aim of this study was to prove that Clostridium difficile toxin B is an inflammatory enterotoxin in humans.
2. The study was carried out as a group of people all from different organisations. Tor C. Savidge, Wei-Hua Pan and Paul Newman from Laboratory of Developmental Gastroenterology, Combine Department of Pediatric Gastroenterology & Nutrition, Massachusetts General Hospital and Harvard Medical School, Charlestown, Massachusetts; Michael O’Brien from Department of Anatomic Pathology, Boston University School of Medicine, Boston, Massachusetts and Pauline M. Anton and Charalabos Pothoulakis from the Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts.
3. This study was funded by two organisations called the Crohn’s and Colitis Foundation and the National Institutes of Health.
4. The study found that not only is C.difficile toxin A an enterotoxin but toxin B is too. Current therapeutic drugs only target toxin A which are not effective in preventing the spread of C.difficile due to high relapse rates. Strains of C.difficile could potentially change due to these drugs, causing antibiotic resistance. The researchers therefore discovered that there must be improved drugs or even vaccines, aimed at C.difficile infection which are targeting both toxins. Without the research, treatment will solely target toxin A alone and this will create a serious problem in the future as the number of incidences will increase.
5. There were 8 methods used in this study to achieve the aim of proving that toxin B is an enterotoxin in humans. Purification of C.difficile toxin A and toxin B, mouse illeal loops, inoculation of human intestinal xenografts with toxins, histology and in situ hybridization, a myeloperoxidase assay, an Interleukin 8 Enzyme-Linked Immunosorbent Assay (ELISA), Real-Time Quantitative RT-PCR and a laser capture microdissection of tissue.
6. Purification of toxins A and B was for ensuring both were C.difficle toxins and for their cytotoxic properties. Toxin masses were measured (kilodaltons) for confirmation of their correct molecular masses. Severe combined immunodeficiency mice were the chosen organism for tissue inoculation of the contaminated human intestine. Some mice had toxin A, others with toxin B and some used as a control during the experiment. The mice were chosen for ethical reasons as a chimeric model for the inoculation with human intestine as it was the toxic effects on the human that the research was interested in. The principle in this method was for justifying the aim of the research to prove that toxin B has enterotoxic properties in humans as well as toxin A. Once the mice had died after 6 hours, the tissue was removed and stained using h&e staining for examining the necrotic factors both toxins had on the structure of the intestinal tissue. In Situ Hybridization was carried out using specific staining for fixing the cells. Fluorescent staining such as dapi blue was used to bind to the cells DNA for showing what was happening within the cell. In the myeloperoxidase assay was used for measuring the amount of myeloperoxidase in the tissue segments. The amount of enzyme produced was used to help determine how much toxin was degraded by the neutrophils and how increased the immune response was. A rate of absorbance was determined to measure neutrophil activity in the tissue and allow for comparison with known numbers of human blood-derived neutrophils. Using myeloperoxidase, the activity of the enzyme was determined by measuring the rate of conversion of the substrates to the products produced in the reaction. An ELISA was carried out on the tissue. This is through the use of antibodies to detect for antigens on the cells. The secondary antibody used binds to the antigen-antibody complex and produces a colour change. This was useful for determining the uptake of IL-8 protein in cells treated with either toxin A or B. Real-time PCR was used to determine the induced IL-8 response. By observing the amplification of the gene sequence throughout, any mutations could be identified as a result of IL-8 expression.
7. The methods used were to justify the main aim of the study – to examine the potential of C.difficile toxin B as an enterotoxin. In situ hybridization using species specific human and mouse DNA probes proved that the epithelium of the intestinal tissue used for the xenograft was well differentiated and exclusively of human origin. This was useful to know as the effects of toxin B on human intestinal tissue was being examined, and not on mice. Both toxin A and B grafts showed signs of mucosal inflammation and damage to epithelial cells. H&E staining was used as an indicator for this, as it was found there was a reduced intensity of stained cells, visible congestion and edema of cells, the presence of sub-epithelial blebs and lifting of cells from the basal lamina. It was also found that few intestinal cells apoptosed on exposure to both toxins due to the clear nuclear fragmentation and condensation of the chromatin in the histopathologic analysis. However, it was evident that most cells only had necrotic damage with ruptured membranes and stable nuclei. A raised myeloperoxidase activity with toxin B was examined in graft tissue due to a high infiltration of granulocytes in the mucosal lining and evidence of edema. A higher myeloperoxidase activity in toxin B grafts than in toxin A, suggests that toxin B is a potent enterotoxin. For ruling out the possibility of a toxin A contamination causing the enterotoxic effects on the toxin B xenografts, the content of toxin A was measured. This was carried out using a toxin A specific ELISA. It was found that no detectable toxin A was measured in dilutions up to 14 µg/mL. As the toxin was inserted into both CD-1 and C.B.-17 mice, there was no response to toxin B by CD-1 mice and only showed secretory responses to toxin A. This backs up the findings of no toxin contamination in the tissues. Both types of mice showed an increase in fluid secreted in response to toxin A, proving that the enterotoxic effects produced by toxin B was not caused by a contaminant. An increase in granulocytes were produced in response to an increased synthesis of IL-8 in the mucosa. Real-time PCR and ELISAs were used for evaluating the levels of IL-8 mRNA and protein expression in the xenografts of both toxins and compared with a control tissue. It was found that there was a significant increase in epithelial-derived IL-8 expression after exposure to toxin A and B. This proved that epithelial cells were solely responsible for the recruitment of granulocytes to the site of infection.
8. The study has proved partly what was set out in the aim by providing evidence that toxin B is a potent enterotoxin in the human intestine. However, the animal intestine used failed to respond to toxin B due to absence of toxin B receptors on the epithelial cells. Due to rise of drug-resistant strains to commonly used metronidazole and vancomycin, the development of non-antibiotic treatments strategies for the future was recommended from the study. The study has suggested that stronger and better approaches need to be developed to target C.difficile infection more invasively. They suggest that antitoxin antibodies, toxin binding polymers and toxin receptor decoys would be best used and must target both toxins to be effective. A possible vaccine could be developed. With the use of these drugs, the spread of resistant strains would be decreased and better treat the infection, being beneficial for the patient.
9. The study discovered new information which could be used for targeting future pathological studies on C.difficile toxin B. Neural precursors were discovered in the foetal intestine which could be used for the regeneration of a functional enteric nervous system. Therefore, this allows for future research to focus on whether the effects of toxin B are dependent on enteric neurons. The study has also recommended that a vaccine could be developed for limiting the amount of cases. As toxin B is a potent inflammatory enterotoxin, the focus of treatment would be to target both toxins instead of solely toxin A.