Practical 3: Analysis of Markers of Inflammation P15179888GROUP 4By Nooria Attia  Class results foreffect of drugs (salbutamol) on mast cell degranulation (Experiment 1)  EXPERIMENT 1 – MAST CELL DEGRANULATION RESULTS TABLES ResultsTable 1. The Effect of Drugs on Mast Cell Degranulation (expressed aspercentage) ResultsTable 2. The Effect of Drugs on Degranulation (expressed as mean +/- sem)  Group Degranulation Score (mean +/- sem)   O                       +                               ++   1. Control 128.90 +/- 7.

81 56.90 +/- 5.83 18.

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50 +/- 4.07 2. 48/80 200ng/ml 22.80 +/- 3.60 96.60 +/- 11.16 82.10 +/- 13.

38 5. Salbutamol, 2ng/ml 97.50 +/-15.30 82.10 +/- 13.47 23.20 +/- 5.

90 6. 48/80 200ng/ml + Salbutamol 2ng/ml 42.50 +/- 10.90 81.80 +/- 10.12 76.20 +/- 13.

11    Experiment 1questions 1.    –         Group1 (control) had no degranulator (48/80)/inhibitor (salbutamol) present, sominimal ‘++’ state observed.-         Group2 mast cells had a degranulator so a high percentage of ‘++’ was expected whencompared to group 3 (no degranulator) and control group. This is supported byclass results; group 2 has highest ‘++’ percentage (40.74%) compared to control(9.02%) & group 3 (11.44%). The ‘++’ and ‘+47.

04%’ are close together-could be due to difficulty distinguishing between differentstates/characteristics of cells. –         Group3 had inhibitor so I expected a higher % of cells in ‘0’ state as evident byclass results (48.08%) compared to group 2 (11.32%). –         Group4 contained both degranulator and inhibitor so one would expect similar %’sacross all states. Unfortunately, the class outcomes show a higherdegranulation ‘40.80%’ in the ‘+’ state which is close to ‘++38%’ than ‘O21.

20%’. This could be because a weak inhibitor was used compared to morepotent degranulator (Schemann.M et al, 2012). 2.   Salbutamolstimulates ?2 adrenergic receptors in bronchial s.

m leading to activation ofadenylyl cyclase forming AMP from ATP. AMP inhibits release of mediators frommast cells (emc, 2016) : imitated by theresults in groups 2 & 3 in ‘O’ & ‘++’ states.  3.   Angioedema,headache, bronchospasm & tremor (BNF, 2018)Word count: 214References BNF.(2018).

British National Formulary. London: BMJ Group and pharmaceuticalpress.emc. (2016, 08 1). easyhalersalbutamol 100mcg. Retrieved January 10, 2018, from Medicines complete: https://www.

medicines.org.uk/emc/medicine/21084Schemann.M et al.

(2012, December 18). NCBI. Retrieved January 10, 2018, from The mastcell degranulator compound 48/80 directly activates neurons:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525567/   Experiment 2 Results table 3. Theresults of ELISA assay for detection of faecal calprotectin in twelve knownconcentrations, two control and four patients.

sample Concentration (µg/g) Absorbance (A) 1 1000 0.700 2 500 0.651 3 250 0.844 4 125 0.520 5 63 0.454 6 31 0.

296 7 16 0.157 8 8 0.154 9 4 0.103 10 2 0.057 11 1 0.081 12 0 0.043 + unknown 0.

782 – 2 0.057 Patient A 45 0.334 Patient B 1.4 0.046 Patient C 1.

6 0.050 Patient D 1.4 0.046                 Experiment2 questions4.

     –         PatientA – 45 µg/g-         PatientB – 1.4 µg/g-         PatientC – 1.6 µg/g-         PatientD – 1.4 µg/gTheconcentration threshold of faecal calprotectin is ?50 µg/g and therefore anyvalue greater is classified as abnormal (Lamb, 2010).My results show that none of the patients have an abnormal level, indicatingthat inflammation is unlikely to be present.5.    Erythrocyte sedimentation rate and C -reactive protein tests can identify inflammation; but can be influenced bynon-intestinal diseases (so cannot localise it to the bowel) and can lackdiagnostic accuracy. Faecal calprotectin test (recommended by NICE)distinguishes between inflammatory bowel diseases (Crohn’s, ulcerative colitis)and non-inflammatory bowel diseases (IBS).

It’s high specificity (95%) andsensitivity (100%) gives a quick reliable diagnosis without furtherinvestigations (i.e. endoscopies) (BMJ, 2014). Raised calprotectinlevel indicates active inflammation in the intestines.

If the level is belowthe cut-off value (50 µg/g in some brands), its considered negative/normal,above is positive/abnormal (Marechal, 2017).Wordcount: 160ReferencesLamb, J. M. (2010).Measurement of Faecal calprotectin and lactoferrin in inflammatory boweldisease. BMJ, 13-18.BMJ.

(2014). What is the faecal calprotectin test? BMJ, 102-104.Marechal, e.

a. (2017).compliance with faecal calprotectin test in patients with inflammatory boweldisease. SAGE journals, 702-707.                     Abs Concentration (µ/g) Control(+) 1.260 Unknown Control(-) 0.236 2.0 Patient 1 0.

237 2.2 Patient 2 1.233 80 Patient 3 0.

434 4 Patient 4 0.393 3 Patient 5 0.225 1.4 Patient 6 1.035 25  Experiment3                     Question6Giventhe Antidrug antibody (ADAs) detection limit is 5 µg/g, it indicates thatpatients 2 (80 µg/g) & 6 (25 µg/g) are above the ADA limit, compared topatients 1 (2.2 µg/g), 3 (4 µg/g), 4 (3 µg/g) and 5 (1.4 µg/g) withconcentrations below the limit. This suggests that there is a presence of ADAsin patients 2 & 6 which explains why they’re not responding to Adalimumabas they neutralise the biologics ability to bind to its target protein.

Howeverwe must bear in mind that patients 1, 3, 4 and 5 did not have ADAs present, yetthey too were unresponsive. The reason for their non-responsiveness totreatment should be investigated further. NICE guidelines states thatEtanercept can be used as a suitable monotherapy for all patients as they areeither intolerant/contraindicated to DMARDs (e.g.

methotrexate) and haveinadequate/failed response to at least one TNF-alpha inhibitor (alternativetreatment) which is Adalimumab in our experiment. However, Toclizumab which isanother biologic can also be used if the first TNF-alpha inhibitor is stoppedwithin 6 months of treatment. Further alternative biologic treatment for thesepatients can include Abatacept. Patient 2 & 6 could try another biologic -Certrolizumabpegol which works similar to Adalimumab (ADAs present were specific againstAdalimumab) (NCBI, 2004)Question7RheumatoidArthritis (RA) is caused by both TNF-alpha and other cytokines which contributeto the disease, for instance IL-6.

 Adalimumabis a TNF-alpha inhibitor and so if a patients’ disease is not triggered byTNF-alpha, then use pf Adalimumab would be unsuccessful and lack efficacy inthese patients. A more targeted medicine for example Toclizumab which targetsIL-6 should be used in such cases (nordqvist, 2017). Additionally, Adalimumabis administered intravenously and this may pose problems such as injectionssite reactions and fear of needle leading to non-adherence.

Furthermore, theseverity of RA can mean that the drug will simply just not be enough to providea therapeutic effect (Pubmed, 2005). Wordcount: 323 References NCBI. (2004). Efficayand safety of Adalimumab as monotherapy in patients with rheumatoid arthritisfor whom previous disease modifying antirheumatic drug treatment has failed. Pubmed,508-516.

nordqvist, c. (2017,January 6). Humira (adalimumab) and its uses. Retrieved from Medicalnews today: https://www.

medicalnewstoday.com/articles/248215.phpPubmed. (2005).Adalimumab: a review of side effects. NCBI, 637-41.

  

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