Practical 3: Analysis of Markers of Inflammation

 

P15179888

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GROUP 4

By Nooria Attia

 

 

Class results for
effect of drugs (salbutamol) on mast cell degranulation (Experiment 1)

  
EXPERIMENT 1 – MAST CELL DEGRANULATION RESULTS TABLES

 

Results
Table 1. The Effect of Drugs on Mast Cell Degranulation (expressed as
percentage)

 

Results
Table 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

18.50 +/- 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 1
questions

1.   
 

–         
Group
1 (control) had no degranulator (48/80)/inhibitor (salbutamol) present, so
minimal ‘++’ state observed.

–         
Group
2 mast cells had a degranulator so a high percentage of ‘++’ was expected when
compared to group 3 (no degranulator) and control group. This is supported by
class 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 different
states/characteristics of cells.

–         
Group
3 had inhibitor so I expected a higher % of cells in ‘0’ state as evident by
class results (48.08%) compared to group 2 (11.32%).

–         
Group
4 contained both degranulator and inhibitor so one would expect similar %’s
across all states. Unfortunately, the class outcomes show a higher
degranulation ‘40.80%’ in the ‘+’ state which is close to ‘++38%’ than ‘O
21.20%’. This could be because a weak inhibitor was used compared to more
potent degranulator (Schemann.M et al, 2012).

 

2.   
Salbutamol
stimulates ?2 adrenergic receptors in bronchial s.m leading to activation of
adenylyl cyclase forming AMP from ATP. AMP inhibits release of mediators from
mast cells (emc, 2016) : imitated by the
results in groups 2 & 3 in ‘O’ & ‘++’ states.

 

3.   
Angioedema,
headache, bronchospasm & tremor (BNF, 2018)

Word count: 214

References

BNF.
(2018). British National Formulary. London: BMJ Group and pharmaceutical
press.

emc. (2016, 08 1). easyhaler
salbutamol 100mcg. Retrieved January 10, 2018, from Medicines complete: https://www.medicines.org.uk/emc/medicine/21084

Schemann.M et al.
(2012, December 18). NCBI. Retrieved January 10, 2018, from The mast
cell degranulator compound 48/80 directly activates neurons:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3525567/

 

 

 

Experiment 2

Results table 3. The
results of ELISA assay for detection of faecal calprotectin in twelve known
concentrations, 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

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Experiment
2 questions

4.     

–         
Patient
A – 45 µg/g

–         
Patient
B – 1.4 µg/g

–         
Patient
C – 1.6 µg/g

–         
Patient
D – 1.4 µg/g

The
concentration threshold of faecal calprotectin is ?50 µg/g and therefore any
value greater is classified as abnormal (Lamb, 2010).
My results show that none of the patients have an abnormal level, indicating
that inflammation is unlikely to be present.

5.    Erythrocyte sedimentation rate and C –
reactive protein tests can identify inflammation; but can be influenced by
non-intestinal diseases (so cannot localise it to the bowel) and can lack
diagnostic 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%) and
sensitivity (100%) gives a quick reliable diagnosis without further
investigations (i.e. endoscopies) (BMJ, 2014). Raised calprotectin
level indicates active inflammation in the intestines. If the level is below
the cut-off value (50 µg/g in some brands), its considered negative/normal,
above is positive/abnormal (Marechal, 2017).

Word
count: 160

References

Lamb, J. M. (2010).
Measurement of Faecal calprotectin and lactoferrin in inflammatory bowel
disease. 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 bowel
disease. 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

 Experiment
3

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Question
6

Given
the Antidrug antibody (ADAs) detection limit is 5 µg/g, it indicates that
patients 2 (80 µg/g) & 6 (25 µg/g) are above the ADA limit, compared to
patients 1 (2.2 µg/g), 3 (4 µg/g), 4 (3 µg/g) and 5 (1.4 µg/g) with
concentrations below the limit. This suggests that there is a presence of ADAs
in patients 2 & 6 which explains why they’re not responding to Adalimumab
as they neutralise the biologics ability to bind to its target protein. However
we must bear in mind that patients 1, 3, 4 and 5 did not have ADAs present, yet
they too were unresponsive. The reason for their non-responsiveness to
treatment should be investigated further. NICE guidelines states that
Etanercept can be used as a suitable monotherapy for all patients as they are
either intolerant/contraindicated to DMARDs (e.g. methotrexate) and have
inadequate/failed response to at least one TNF-alpha inhibitor (alternative
treatment) which is Adalimumab in our experiment. However, Toclizumab which is
another biologic can also be used if the first TNF-alpha inhibitor is stopped
within 6 months of treatment. Further alternative biologic treatment for these
patients can include Abatacept. Patient 2 & 6 could try another biologic -Certrolizumab
pegol which works similar to Adalimumab (ADAs present were specific against
Adalimumab) (NCBI, 2004)

Question
7

Rheumatoid
Arthritis (RA) is caused by both TNF-alpha and other cytokines which contribute
to the disease, for instance IL-6.  Adalimumab
is a TNF-alpha inhibitor and so if a patients’ disease is not triggered by
TNF-alpha, then use pf Adalimumab would be unsuccessful and lack efficacy in
these patients. A more targeted medicine for example Toclizumab which targets
IL-6 should be used in such cases (nordqvist, 2017). Additionally, Adalimumab
is administered intravenously and this may pose problems such as injections
site reactions and fear of needle leading to non-adherence. Furthermore, the
severity of RA can mean that the drug will simply just not be enough to provide
a therapeutic effect (Pubmed, 2005).

Word
count: 323

References

NCBI. (2004). Efficay
and safety of Adalimumab as monotherapy in patients with rheumatoid arthritis
for whom previous disease modifying antirheumatic drug treatment has failed. Pubmed,
508-516.

nordqvist, c. (2017,
January 6). Humira (adalimumab) and its uses. Retrieved from Medical
news today: https://www.medicalnewstoday.com/articles/248215.php

Pubmed. (2005).
Adalimumab: a review of side effects. NCBI, 637-41.

 

 

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