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• Re: Intestinal inflammation in cystic fibrosis
  Rosalind L Smyth
  Published on: 22 March 2016
• Intestinal inflammation in CF - An alternative hypothesis
  Larry Eisenberg
  Published on: 22 March 2016
• Intestinal inflammation in cystic fibrosis
  G L Briars
  Published on: 22 March 2016

• Published on: 22 March 2016

  Re: Intestinal inflammation in cystic fibrosis

  • Rosalind L Smyth, Professor of Paediatric Medicine

  Dear Editor:

  We thank Dr Briars for his comments and are aware of his opinions regarding the potential source of the intestinal cytokines which we have discussed in the paper including reference to his previous paper.[1] We do not agree that our data is dependant upon IL-8 alone. We have shown statistically significant differences for a whole range of proteins and types of assays.

  Due to the large number of pr...

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  Dear Editor:

  We thank Dr Briars for his comments and are aware of his opinions regarding the potential source of the intestinal cytokines which we have discussed in the paper including reference to his previous paper.[1] We do not agree that our data is dependant upon IL-8 alone. We have shown statistically significant differences for a whole range of proteins and types of assays.

  Due to the large number of proteins and types of assays that we have performed we have not the performed the extensive experiments as reported by Dr Briars for IL-8. We do know that the polyethylene glycol, a key constituent of the lavage fluid, does not affect the IL-8 assay. There are two reasons why variable recovery is unlikely to be a major factor in our results. First by collecting whole gut lavage (WGL) any intestinal secretions present, including bile, are extremely dilute. Substances found in faeces such as stercobilin are effectively absent. Secondly WGL is a perfusion system found to be equivalent to balloon perfusion systems.[2,3] Thus the dilution of any interfering factors would be very similar between the subjects and controls. Using WGL minimises any interference from intestinal material as much as feasible in vivo.

  Assuming the worst case scenario from Dr Briars' data (i.e. a two fold overestimate of IL-8 in the cystic fibrosis (CF) patients which is not found in the controls) still shows very significantly increased IL-8 output in the CF patients (p<_0.0001 and="and" unfeasible="unfeasible" volumes="volumes" of="of" sputum="sputum" would="would" still="still" be="be" required.="required." p="p"/> For these and reasons detailed in our paper and previous correspondence[4] we do not believe that sputum is the primary cause of the abnormalities found. Our observations concerning the increase in intestinal inflammatory markers in the WGL of CF patients have now been supported by a study which investigates intestinal inflammation within mucosal biopsy samples.[5] This provides additional support to the hypothesis that the basic defect of CFTR can be proinflammatory.

  Dr Eisenberg correctly points out the potential influence of pancreatic enzymes and degradation. The results we found for alpha-1- antitrypsin were unexpected given differences for albumin and IgG. Some discordance in data has been found previously in WGL from subjects with active inflammatory bowel disease[6] who are pancreatic sufficient and also can have raised intestinal permeability.[7]

  However our data demonstrating raised albumin and IgG are consistent with well established data showing raised intestinal permeability in children with CF.[8] As we discussed, it has been found that protein outputs from balloon perfusion experiments (which exclude upper intestinal secretions) are similar to those found in whole gut lavage suggesting that any potential effect of degradation from pancreatic enzymes is minimal.[2,3] We also showed ECP to be raised in CF children. Like alpha-1- antitrypsin this is relatively stable in faeces at room temperature (approx 21% loss over 24 hours).[10] This loss would be considerably lower during WGL. Thus degradation would be unlikely to explain this difference.

  Nicholas M Croft
  Department of Paediatric Gastroenterology
  St Bartholomew’s and the Royal London School of Medicine and Dentistry, Queen Mary and Westfield College
  London, UK

  Rosalind L Smyth
  Una O’Hea
  University Institute of Child Health
  Royal Liverpool Children’s Hospital
  Liverpool, UK

  Tom G Marshall
  Royal Hospital for Sick Children
  Edinburgh UK

  References

  (1) Briars GL, Dean TP, Murphy JL, Rolles CJ, Warner JO. Faecal interleukin-8 and tumour necrosis factor-alpha concentrations in cystic fibrosis. Arch Dis Child 1995;73:74-6.

  (2) Ferguson A, Sallam J, McLintock L, Croft NM, Poxton I. The gut as an immune organ: intestinal antiendotoxin antibodies. In: Kinney JM, Tucker HN, eds. Organ Metabolism and Nutrition: Ides for Future Critical Care. New York: Raven Press, Ltd, 1994:231-44.

  (3) Croft NM, Ferguson A. High intraluminal fluid flow increases intestinal IgA output. Scand J Gastroenterol 2000;35:726-31.

  (4) Croft NM, Marshall TG, Ferguson A. Gut inflammation in children with cystic fibrosis on high dose enzyme supplements [letter]. Lancet 1996;347:327.

  (5) Raia V, Maiuri L, de Ritis G, et al. Evidence of chronic inflammation in morphologically normal small intestine of cystic fibrosis patients. Pediatr Res 2000;47:344-50.

  6) Choudari CP, O’Mahony S, Brydon G, Mwantembe O, Ferguson A. Gut lavage fluid protein concentrations; objective measures of disease activity in inflammatory bowel disease. Gastroenterology 1993;104:1064-71.

  (7) Tibble JA, Sigthorssen G, Bridger S, Fagerhol MK, Bjarnason I. Surrogate markers of intestinal inflammation are predictive of relapse in patients with inflammatory bowel disease. Gastroenterology 2000;119:15-22.

  (8) Flick JA, Perman JA. Increased intestinal permeability in cystic fibrosis correlates with the degree of pancreatic exocrine dysfunction. Pediatr Pulmonol 1990;(suppl):97-8.

  (9) Berstad A, Borkje B, Riedel B, Elsayed S. Increased fecal eosinophilic cationic protein in inflammatory bowel disease. Hepatol Gastroenterol 1993;40:276-8.

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  Conflict of Interest:
  None declared.

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• Published on: 22 March 2016

  Intestinal inflammation in CF - An alternative hypothesis

  • Larry Eisenberg, Pediatric Gastroenterologist

  Dear Editor:

  I was interested by the report of Smyth et al regarding the finding of markers of intestinal inflammation in whole gut lavage in patients with cystic fibrosis.[1] As the alpha 1 antitrypsin levels were not increased compared to controls, perhaps another hypothesis needs to be considered.

  Conceivably the inflammatory markers are not actually increased within the bowel, but rather, they are...

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  Dear Editor:

  I was interested by the report of Smyth et al regarding the finding of markers of intestinal inflammation in whole gut lavage in patients with cystic fibrosis.[1] As the alpha 1 antitrypsin levels were not increased compared to controls, perhaps another hypothesis needs to be considered.

  Conceivably the inflammatory markers are not actually increased within the bowel, but rather, they are not degraded due to the lack of intestinal enzymes. Alpha 1 antitrypsin, which is resistent to proteolytic enzyme activity, would not be affected by such a phenomenon and therefore, would be the same in patients with cystic fibrosis and controls. Perhaps the authors would need to resort to the somewhat dated technique of radiolabeled albumin to definitively answer this question.

  Larry Eisenberg

  Reference

  (1) Smyth RL, Croft NM, O'Hea U, Marshall TG, Ferguson A. Intestinal inflammation in cystic fibrosis. Arch Dis Child 2000;82:394-9.

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  Conflict of Interest:
  None declared.

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• Published on: 22 March 2016

  Intestinal inflammation in cystic fibrosis

  • G L Briars, Consultant in Paediatrics & Paediatric Gastroenterology

  Dear Editor

  Following their studies of whole gut lavage fluid Smyth et al have suggested that a non-idiopathic intestinal inflammation occurs constituitively in CF patients, as a consequence of a proinflammatory effect of the patient's CFTR mutations.[1] They reported marginally elevated excretion of IgG, IgM, IL-1 , neutrophil elastase and eosinophil cationic protein, and much more significant increase in excreti...

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  Dear Editor

  Following their studies of whole gut lavage fluid Smyth et al have suggested that a non-idiopathic intestinal inflammation occurs constituitively in CF patients, as a consequence of a proinflammatory effect of the patient's CFTR mutations.[1] They reported marginally elevated excretion of IgG, IgM, IL-1 , neutrophil elastase and eosinophil cationic protein, and much more significant increase in excretion of IL-8 and albumin, but no increase in excretion of alpha 1-antitrypsin or IgA. In this study where lavage fluid was administered continuously, and intestinal effluent was collected in discrete samples, pooling of the effluent before analysis would have allowed small differences in calculated inflammatory marker outputs to be interpreted as representative of gastroentestinal output. Of all the inflammatory markers presented only interleukin-8 shows a range of cytokine outputs in CF patients with or without fibrosing colonopathy that does not extend into the range seen in controls, in these non parametric datasets. The author's evidence for intestinal inflammation therefore relies heavily on the validity of their IL-8 Quantikine assay (R&D Minneapolis) protocol.

  I have explored the validity of this assay for use in supernatants of faecal homogenates in children with cystic fibrosis and found it wanting. Recovery of a 500 pg/ml spike of IL-8 progressively increased from 41% in samples which were a 12-fold dilution of faeces to 189% in samples which were a 120,000-fold dilution of faeces, when used according to manufacturers instructions. Pre-diluting the samples 50:50 in newborn calf serum, and using calf serum for further dilutions gave this assay (R&D catalogue no D8000) mean (sd) spike recovery of 92.1 (12.5)% and coefficients of variation of 3.46% (intra assay) and 6.85% (inter assay).[2]

  Without knowledge of Smyth et al's IL-8 ELISA validation data, I assume that this assay returns similarly spuriously high IL-8 concentrations in polyethylene glycol based wholegut lavage fluid to my 120,000-fold dilution faecal supernatant. The absence of a significant difference between CF patients and controls in their alpha 1-antitrypsin outputs suggests that intestinal inflammation was not presentin the CF patients. Overestimation of the WGLF IL-8 concentration would explain the apparently implausably large volumes of swallowed sputum that the authors estimate would be required to account for their results. In this study which could not turn off the mucociliary escalator, but did dramatically increase the rate of intestinal transit and exclude exogenous pancreatic enzymes, swallowed sputum is the most likely explanation for the results.

  References

  1. Smyth RL, Croft NM, O'Hea U, Marshall T G, Ferguson A. Intestinal inflammation in cystic fibrosis. Arch Dis Child 2000;82:394-9.

  2. Briars GL, Shepherd RW. Faecal interleukin-8 and TNF ELISA: The first validated assay protocol. Immunology and Cell Biology 1997;suppl 1:A114

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  Conflict of Interest:
  None declared.

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