Inflammatory Bowel Disease - Science topic

hello Yuning Hou , I have tested NO production through Griess reagent assay, also qPCR for IL6, IL1B, TNF-alpha but all showed no difference compared to untreated. I ordered LPS based on what has been used by some papers but still I see no response. I will try conditioned medium this is a good suggestion. thank you!

thanx for details and your answer

Hello

LPS is not a good way to induce inflammation in Caco-2 cell line. TNFa + IFN 50ng/ml each at basal side is a better way

OK!

Thanks!

Many synthetic drugs are currently in use to treat IBD such as 5-aminosalicylic acid corticosteroids. However, they all have some drawbacks as long-term use result in many complications. These problems encourage us to look out for alternative medicine. Numerous in vitro and in vivo experiments showed that the plant-derived secondary metabolites including phenolic compounds, glucosinolates, alkaloids, terpenoids, oligosaccharides, and quinones could reduce permeability, ameliorate-related dysfunctions with promising results. In addition, many of them could modulate enzymatic activity, suppress the inflammatory transcriptional factors, ease oxidative stress, and reduce pro-inflammatory cytokines secretion.

Zaim Gashi thank you very much sir for the insight.

Hello Zhiheng Shi

I would like you to refer to the article below. It may be helpful.

After the colitis induction the animals develop several manifestations of acute colitis. Those include inconsistent stool formation and occult or even bloody diarrhea. Intracolonic administration of TNBS/ethanol to rats induces a severe illness characterized by bloody diarrhea and a dramatic loss of body weight during the first week. Then the body weight increases but diarrhea still persists for about two weeks. Introduction of TNBS, for example, led to significant weight loss (mean 10% loss in body weight) and development of liquid bloody stools in all exposed animals, whereas control (saline) animals remained healthy and gained weight (mean 30% gain over 12 days). Body weight loss is caused in part by the marked effects of TNBS itself on the gut (diarrhea and perhaps reduced fluid absorption additionally), but systemic inflammatory response may also play a role. Further non-specific signs that point to a general deterioration include piloerection of fur and decreased movements of the animals. The onset and severity of the aforementioned symptoms is variable and depends on the specific animal species, strain and the dosing of ethanol and TNBS that are administered. The significance of assessing those symptoms is reflected by the development of multiple scores that quantify acute intestinal inflammation.

Best,

Wolfgang Schechinger Thank you for your answer.

Yes, I for the protein nice layer but don't know what happened the after steps because I couldn't see any proteins bands in the gel.

Of course, searching the " detection of PLA2 in stool" was my 1st thing.

Hi, I was just wondering if you ever came up with anything regarding the rumen fluid. I am trying to use it in media for bacterial culture and I am having a hard time filtering mine. Any suggestions?

Hello Sara

IBD is a result of a defective immune system. IBD patients have significantly elevated serum levels of IgG and IgM.

Azathioprine is an immunosuppressant that decreases synthesis of IgG and IgM. But it does not have any effect on serum immunoglobulin concentrations. So even if the production of immunoglobulins

show a decrease there is no significant decrease in the serum immunoglobulin levels when treated with azathioprine.

You can refer to the research article given below for more information.

J Clin Invest. 1972

Best Wishes.

I am also having the same problem. Its great honor if you could share with me.

Simon N Young thank you for your response.

1. Yes, they cannot cross the BBB but kynurenine and tryptophan can. Trp can be converted by microglia and astrocytes using IDO to kynurenine. From thereon, they can be metabolized into these acids. Is this not a mechanism which kynurenine metabolites exert negative effects on the brain?

2. Thank you for this. I , too, am more familiar with the low serotonin associated with depression. I just asked about high serotonin and IBS because it conflicts this explanation. Perhaps inflammation and other factors caused by IBS influence increased depression incidence found in patients.

And yes, MDD is indeed multifactorial. I am interested in how gut microbiota possibly exert influence in the etiology of depression via Trp metabolism.

Thank you so much Jose Alberto Lopez-Aleman for this input. I will surely try those. I was using IBD application. Thanks, again.

What's the prognosis of UC and crohn's disease in patients with coagulation problems and what are best way of management

@Maria Jara Montibeller

Thank you for your suggestions.

I found a good website for German animal testing numbers

For every animal project you find a ntp, which gives you the planned animals for this study. You can also specify your search with keywords.

Inulin is fermented by bacteria that normalize the colon and is considered a prebiotic. Prebiotics may improve gastrointestinal health as well as potentially enhance calcium absorption. Inulin is classified as a prebiotic because of its ability to stimulate the growth of beneficial bacteria such as Bifidobacteria

Becker, C., Watson, A. J., & Neurath, M. F. (2013). Complex roles of caspases in the pathogenesis of inflammatory bowel disease. Gastroenterology, 144(2), 283-293.

This is the ResearchGate link:

(see under paragraph: 'The Intrinsic Apoptosis Pathway'

Peppelenbosch, M. P., & Van Deventer, S. J. H. (2004). T cell apoptosis and inflammatory bowel disease. Gut, 53(11), 1556-1558.

@ Ayman Tawbe

PLEASE CHECK IT

https://toxnet.nlm.nih.gov › cgi-bin › sis › search

With inmunochemical studies about the lymphocytes subtipes and several interleukines perhaps

Hi Rasmus,

Thanks for your answer. We have tried some of the inital pipeline for 16s sequencing using the Omnigut kit from DNAgenotek and the Power fecal kit from Qigen/MoBio. We also tried (only once though) to run a total bacterial PCR for the V3-V4 region and quality checked it on the bioanalyzer and it looked acceptable.

Do you think that working out the qPCR will give us an idea of how well the samples will work for sequencing and a general idea of what to expect from the run or should I spend my time making sure all the samples get past the first PCR and clean-up stage?

Dear collegue

we have tried to test different models of colitis and made the conclusion that DSS colitis is the most easy, cheap and pathogenesis - related model.

But if you decided to use TNBS, you should be sure that all mice (or rats) have the similar microbiome. They should be SPF and parazites -free too. In our practice the presence of the ectromelia virus meets much more often than we think and it induce a very heavy colitis with the total death of the group. I recommend tp pay the attention to this side of the process

What are the differences between liver and colon mitochondria may be the first question to answer? Some inflammatory products influence some pathways but not others due to their multiple, or single targetted effects.

Logic, not current expertise.

Glycine prevent colitis by inhibiting induction of inflammatory cytokines and chemokines. It is postulated that glycine may be useful for the treatment of inflammatory bowel diseases as an immunomodulating nutrient.

Hi

Cannot see how you could replicate what happens in colonic epithelium in IBD using cells lines. Maintaining normal human colonic epithelial cells in culture is very difficult, and doing the same with inflamed cells from IBD patients likely to be even harder. The dextran sulphate model of acute colitis in rats produces the same histological features and ion transporter/channel abnormalities as seen in human ulcerative colitis. Hope that helps.

Regards

Professor Geoffrey Sandle

I think your question is very well answered in the following study recently published .

Dear Michael after reading your answer i did look up to the internet and started reaading bout flouride in water.

I am now able to get some insight about the big problem the humans are facing.

Lets hope that the council for water management will take up the issue in a more seroius manner.

It also has made a difference to my understnading of the disease patterns of autoimmunity.

Thank you for the insights.

Thanks for your answers.

Now I'm trying TNBS model on rat,but there is a problem that rat's distal colon is easily wounded during clysis and rat died of ileus. Could you give me some suggestions to solve the problem?@ Maria Zizzo

genetic risk for IBD and microbiota or entero*

No , laparoscopy is not the gold standard but endoscopy is.

thank you for your response .

thank you so much. and I have read the guideline of 2014 ECCO ,which said "when there is latent TB and active IBD,anti-TNF should be at least 3 weeks after starting  

chemotherapy".we now had a patient who is in active IBD,but had a history of TB 5years ago which had been totally cured. so the question is where did you get the contents of "isoniazid 600 mg/day during 6 month. I am looking forward to your answer.

Inflammatory bowel disease is not a contraindication to PD unless the patient has had multiple complicated bowel surgeries. However, patients with IBD are prone to adhesions due to either peritoneal inflammation or bowel surgeries. With this in mind, laparoscopic PD catheter insertion (which allows for direct visualization and adhesiolysis if necessary) would be the preferred catheter insertion method. The impact of IBD on the peritoneal membrane is unknown, but the majority of patients would likely achieve adequate clearance and UF.

These papers that may be of interest to you. The rise of obesity and the use of anti-biotics and proton-pump inhibitors have greatly reduced the diversity of the microbiome. The use of emulsifiers in the food supply compromises the gut barrier. 

Just found an answer...  https://www.researchgate.net/publication/309335976_a-Defensin_5_gene_expression_is_regulated_by_gut_microbial_metabolites

Dear Douglas,

Thanks for your input. Yes there is concern that my mice might not be "dirty" enough. That is why I started breeding in non-SPF condition. Also I am considering having them co-housing with a hamster or something. I heard that co-housing Rag1 KO with inflammasome-defective mice in SPF condition also helps, but I dont have those strains.

Dear Akshat,

Thanks for your input. It is routine dose for inducing colitis. I heard more than that would actually inhibit colitis but don't know why exactly.

The article by Woodford is useful, I already had this one. The side effects of BCM7 seem to be serious and would require a RCT to ascertain the safe doses for patients with asthma and those without.

HI Dear 

it is suitably to use liver biopsy to used them in IHC. the best marker is 8-OHDG. Also you can check them using ELISA kits but the collection and preservation time of serum must be not more than 6 hours.

BR

Hi Jakob,

  Thanks for your message.

  Do you know why IgG2a specifically increased in intestine of KO mice, I mean which bacteria or  antigen can specifically stimulate the epithelial cell and then further stimulate the B cells to secrete the IgG2a? 

Xin

Dear Xin,

The statistical problem you are stating , and I interpreted as, you need to compare two groups based on the some quantitative variable. Try applying the Binomial test of proportion to check the statistical difference. If you find the sample size is large enough then you can think of applying Z-test too.

The best treatment  for recurrent multiple oral ulcers undoubtedly is the gluten-free diet, followed strictly and for the whole life, because most of these patients are celiacs and they don´t know it

You could try treating with serum-free LPS media for a few hours, then replacing with fresh LPS-free media.

A greater concern is that Caco-2 cells have been reported to be unresponsive to LPS treatment (lack of TLR4 expression):

Inflammatory parameters in Caco-2 cells: Effect of stimuli nature, concentration, combination and cell differentiation.

Responsiveness of intestinal epithelial cell lines to lipopolysaccharide is correlated with Toll-like receptor 4 but not Toll-like receptor 2 or CD14 expression.

Oxazolone can also be used (Schiechl et al 2011; Gerlach et al 2014)

Overall, mixed results have been reported regarding the efficacy of haematopoyetic and mesenchymal stem cells in refractory luminal Crohn's disease in phase I/II trials.

Concerning perianal fistulae secondary to Crohn's disease, results have been in general optimistics and several reports suggest an improved healing in patients subject to stem cell therapy (Dis Colon Rectum 2009, 52:79; J Colorectal Dis 2013, 28:313; Int J Colorectal Dis. 2013;28:313; Gastroenterology. 2015;149:918)

Great thanks for answering my question.I,am really very grateful to you for the wonderful and reaching out your Scientific answers.

Dear Giuseppe,

I would suggest you to have a look to this paper: http://www.hindawi.com/journals/bmri/2012/718617/

I found it quite helpful when I wrote the animal protocol for the DSS-induced colitis in mice. Hope this will help you!

Cheers,

Alessia

You can have a look in this review. They have nicely explained different targets in colitis with references. 

Nat Rev Immunol. 2014 May;14(5):329-42. doi: 10.1038/nri3661. Epub 2014 Apr 22.

Cytokines in inflammatory bowel disease.

Neurath MF1.

In case of suspicion Crohn , we prefer, the first one, US study of the last ileal loop (SICUS). The RMN secondarily . If your service does not have a sonographer delicate, resonanc, with the study of the loops, is indicated .

You are right. Ampicillin and amoxicillin are more polar / hydrophilic than benzylpenicillin ( Pen G) because of the presence of a polar group (NH₂) in the amide side chain  and (OH) in amoxicillin at para position. It is therefore because of this hydrophilic property  that makes them to have high affinity to the Gram negative cell wall.  hence these two compounds are indicated for both Gram (+) and (-) infection while Pen G is useful only for Gram (+) infection.

Thank you for responding

Hello, Dr. Nylund, you are right, thank you. It was my first opinion, also, but the child is hospitalized since last month and NBT test is in favor of CGD. Other tests are in work now (DHR?) and I wait  a call from the clinician...

1..Loddo I, Romano C.Inflammatory Bowel Disease: Genetics, Epigenetics, and Pathogenesis. Front Immunol. 2015 Nov 2;6:551. eCollection 2015. Review. Free article

2. Beaudet AL. Epigenetics and complex human disease: is there a role in IBD? J Pediatr Gastroenterol Nutr. 2008 Apr;46 Suppl 1:E2. doi: 10.1097/01.mpg.0000313815.73649.37. Review. No abstract available.

3. Jenke AC, Zilbauer M. Epigenetics in inflammatory bowel disease. Curr Opin Gastroenterol. 2012 Nov;28(6):577-84. doi: 10.1097/MOG.0b013e328357336b. Review.

inflammatory bowel disease AND epigenetics[TW]

I would say NO "most promising" lab marker for IBD till now.

We need multiple approaches to make the diagnosis of IBD, even to differentiate the other diagnosis such as tuberculosis, esp in developing countries.

You can please follow the links below to obtain certain valuable informations:-

1. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4277955/

2. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3773507/

3. http://www.nature.com/nri/journal/v14/n5/abs/nri3661.html

4. http://www.nature.com/mi/journal/v7/n1/full/mi201331a.html

Dear Morgane, 

here is a review of :

Current methodologies used for evaluation of intestinal permeability and absorption

Praveen V Balimane, Saeho Chong, Richard A Morrison

Journal of Pharmacological and Toxicological Methods

Volume 44, Issue 1, July–August 2000, Pages 301–312

Current Directions in Drug Discovery:A Review of Modern Techniques

Serum Pepsinogen has been proposed as a marker of injury in GORD, but with non satisfactory results (Serum gastrin and pepsinogens do not correlate with the different grades of severity of gastro-oesophageal reflux disease: a matched case-control study. Aliment Pharmacol Ther, 2008). Instead, measurement of pepsinogen in the salivary secretion (Peptest (R)) has yielded better results.

OMG... I suddenly realized I've seen your report in DDW about your IL-33 research this year. I do infuse very slowly and agreed, there are lot variability with enema method. So here comes the question, is there's any standard to minimize the effect of the the variability. Say, if you want to compare a drug with this model, you got to make it reliable. Any experience on that? 

Thanks a lot.

IBD is a complex disease, rather than using a single cell line, I'd suggest to use at least a co-colture model as proposed in Leonard 2010 (http://www.ncbi.nlm.nih.gov/pubmed/20809575)

Thanks Michal.

Its very useful review paper. i will study it, However i m still searching any recent reported colinic delivery system loaded with BDP in the therapy of IBD, which i could not find.

Thanks James. I have tried using younger mice to no avail and in each trial I have used mice that fit into the weight category you specify. 

The strain was made by a colleague so I will go back and make sure it is as it should be, thank you very much for your suggestions.

I thought you planned something like that, and I guess it would work. May depend on the genetic background of the Rag2-/- mice: are they also 129SvEv? I think these experiments were done in the early days of adoptive transfer colitis in Powrie's lab.

thank you very much.but at day3,it is very hard to collect the fecal sample because of the TNBS administration.The cecum content may be more avaliable.but in that way.the microbiome is signficantly different to the day -14 and day0(natural excrement ).I am looking forward for your advices!

Hi,

 If you want to measure the impact of a mobile health web application, then you have to conduct a research on it and find out the impact percentage from it.

1) To access and controlled sharing of health data and information

2) The health and lifestyle tracking

3) The management of chronic disease and outcomes

4) to share the decision making

5) The social networks and learning health systems

6) The management of behavioral healthcare.

* GUIDELINES - PREPARATION OF KETAMINE/XYLAZINE COCKTAIL FOR MICE

Use of a sterile injection vial is required (e.g. redtop blood collection tube; commercial injection vial)

Mixing instructions:Verify the concentration of your drugs prior to mixing

For a 10mL vial using ketamine 100 mg/mL and xylazine 100 mg/mL add:1.75mL ketamine (100 mg/mL)

0.25 mL xylazine (100 mg/mL)

8 mL saline or sterile water for injection

Use of the following template for a label is recommended:Mouse Anesthetic Mix: Ketamine/Xylazine

Dosage: 0.1 ml/ 20gm IP  

Delivers: 87.5 mg/kg Ketamine/12.5 mg/kg Xylazine

Concentration:  17.5 mg/mL Ketamine/2.5 mg/mL Xylazine

or 

Ketamine/Xylazine

The Ketamine/Xylazine combination is considered to be a very reliable anesthetic for mouse surgery. The recommended dose varies: It is 100-200 mg/kg body weight for Ketamine and 5-16 mg/kg body weight for Xylazine injected intraperitoneally. Although some labs use Ketamine at a dose of 35-50 mg/kg body weight, Ketamine at 50 mg/kg combined with 10 mg/kg Xylazine does not produce a consistent reliable level of immobilization or anes¬thesia in Syrian hamsters, and a dose of 150 mg/kg Ketamine combined with 10 mg/kg Xylazine has been suggested).

The most widely used dose of Ketamine/Xylazine for mouse surgery is 100 mg/kg and 10 mg/kg body weight, respectively (Flecknell 1993). Duration of the effect may be extended by increasing the proportion of Xylazine or by an additional dose of Ketamine.

Premedication with anticholinergic drugs such as atropine (0.02-0.05 mg/kg body weight) is often used to prevent bradycardia (slow heart rate) caused by Xylazine, and to control excessive bronchial and salivary secretions caused by Ketamine (Nowrouzian 1981; Magoon et al. 1988).

The recipe below provides an onset of 3-5 minutes with 30-40 minutes dura

tion of surgical anesthesia.

Ketamine (50 mg/ml) (Vetalar, Ketaset, Ketalar) 2 ml (100 mg)

Xylazine (20 mg/ml) (Rompun) 0.8 ml (16 mg)

Water (sterile) to 10 ml

Store at 4°C for a maximum of 2 weeks. Inject 0.1 ml per 10 grams of body weight (100 mg/kg Ketamine, 16 mg/kg Xylazine).

Jun,

The randomization group is very important in vivo studies. Please do not put small mice or bigger mice into control group.

The "perfect" ratio depends on many factors. Usually it is driven by cost, since it is often easier and cheaper to obtain controls than cases. As the ratio deviates from 1:1 the incremental value per person in the more common group (sometimes one can get more cases than controls), decreases. This gives rise to the general rule of thumb that there is little benefit to be had for rations more extreme than 1:5. 

A very important consideration is that with extreme ratios one should guard against potential stratification; that is assembling a very large cohort from different sources can introduce interesting occult (hidden) stratification, which could generate spurious results.

See : F.T.Tirol; Congenital Hyperrotated Colon, Abdominal Surgery, Winter/Spring, 2015; 11-17

At UEGW last year and ECCO this year treatment results with TSO were presented for patients with Crohn's disease. It doesn't work. Several reasons, some of the authors blamed the inadequacy of the CDAI for such studies. Anti-TNF therapy is also heavily debated in my country and with again recent data that the rate of surgery might not decrease and surely the cost savings of fewer hospital admissions is offset by the cost of biologicals, maybe the answer is surgery!

I would like to consider the possibility of a necrotic granuloma and do a Acid-fast staining to rule-out a Mycobacterium infection and a GMS staining to explore for yeast and fungi. I would also suggest a staining for anti-mouse CD68 to study the presence of epithelioid macrophages which may look like the fibrosis described by some colleagues above.

When dealing with mice with IL10 deficiency, we need to remember that they show increased susceptibility to some infections and this should be a diagnosis to be included in the differential.

Reference: http://jaxmice.jax.org/strain/002251.html 

Regards.

Francisco G. La Rosa, MD

Pathologist - Associate Professor

University of Colorado Anschutz Campus

Department of Pathology

303-724-3782

I think it depends on your hypothesis. A better experiment may be a pilot where you look at various places throughout the colon, then for future experiments focus in on the area(s) where you saw the most DSS-induced change.  I know that isn't an ideal answer and it will take a lot more work, but I also see it as a potential reviewer question in the future once you try to publish. This would kill two birds with one stone, so to speak

 i am really intersted as i am suffering from IBS

Dr.Rodrigo,

Thank you for the reply!

May I ask as to have you any statistical data on the glandular metaplasia comparing the mucosal linings' alterations in the acute cholecystitis and in the chronic one?

Hi 

there is a considerable body of work suggestion that vitamin D has anti-inflammatory effects in inflammatory bowel disease (IBD), particularly from animal models of IBD (Cantrona  and others).  The human translation is less clear at this point, but emerging evidence suggests that vitamin D supplementation may prevent relapse in RCTs in IBD.  As well as anti-inflammatory effects, newer mechanisms such as effects on autophagy, gut barrier functional and on microbiota have been suggested for vitamin D. 

The immuno modulatory effect is  1,25OH2 related, so it would make sense to measure 1,25 OH2. However, measuring 1,25OH2 gives you the blood / plasma level, when you want to know also the tissue level.

The mechanism of immunomodulatory effect of 1,25OH is clearly presented in a paper of van Etten and Mathieu in J Steroid Biochem Mol Biol 2005.

Another point to take into account:  the level of 1,25OH2 is maintained in a narrow range even over a wide range of 25OH concentrations, including severe deficiency.

the literature "Advances in IBD genetics" has suggested that there are more than 160 loci containing IBD susceptibility genes

Thanks for the lead, shall do that. 

Excellent question! It depends on what your method to assess the inflammasome is. If you are measuring IL1b or IL18 readout by ELISA or cell death by LDH then you can use complete medium for all steps. If, on the other hand, you are doing a Western Blot then it is generally recommended to use serum-free medium because detection of activated caspase-1 and processed IL1b and IL18 in the supernatant have to be concentrated (by your favorite method) and serum will overload your concentrated sample. I usually wash once with serum-free medium and then add serum-free medium with my stimulus.

Be aware, however, that some stimuli take many hours to take effect and serum starvation can cause off target responses. In these cases it is sometimes effective to use a very low concentration of serum (1% or less) simply to keep your cells alive. You might still run the risk of overloading the concentration process, so you will have to optimize it according to your protocol.

For ATP, since that's the example you used, you should be OK with serum-free conditions because the effect is quite fast (30-60 minutes for very robust response).

Thank you for your answer

1. you need to stage his colitis - is it severe ? moderate? - staging is important to further management - clinical staging (Trulove Witts), endoscopic staging (Mayo or UCEIS).

2. if the patient has severe colitis, he needs hospitalization, iv steroids, enteral nutrition, sometimes antibiotics iv, if it fails after 3-5 days there is a seroous argument for colectomy or antiTNF or cyclosprin iv

3. if colitis is moderate there is room for azatioprine therapy since it takes weeks to months for this treatment to have an effect

4. important thing is to rule out other etiology of colitis - bacterial infection, parasites, CMV colitis, drugs, cancer .....

As you describe, seems to have no mechanical ileus, but we do not know if the patient has not eliminated gas and the passing stool, and if so then I think that colonoscopic assessment of anastamosis and the part above its .

Yes there have been some reports of Balbc mice being resistant to Citrobacter infection.  You could increase the dose but the best way to determine if your mice are infected is to plate homogenized feces onto selective media plate to determine the shedding kinetics.  Also the microflora of the mouse colony in that particular mouse room could be very competitive with Citrobacter.

I am agreed with Prof.Enzo entirely in that it may become a novel investigation: to compare the ADA and FC levels in a certain group of patients with Crohn's disease.

Some attention may be paid as to the sources of ADA and FC to explain that discrepancy. What is your opinion, are they produced by the same or by the different cells?

I hópe I am doing it the right way!

Of course they can cause bowel obstruction. However, maybe the more correct term is “phytobezoar”. I had a cause with multiple "stones", that even in the presence of bowel stenosis due to cancer, do not caused ileus: