How does measuring expression reveal anything about the Immune response?

How does measuring expression reveal anything about the Immune response?

How does measuring expression reveal anything about the Immune response? 150 150 Nyagu

EXPERIMENTAL DESIGN F E E D B AC K ( E X P T. D E S I G N PA RT A ) C A S E S T U DY – E S T R A D I O L T R E AT M E N T T R I A L A S S E S S M E N T PA RT B FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #1 – Text: Toxoplasma Gondii – Feedback: Incorrect nomenclature – Correction: Toxoplasma gondii (a few still missed this ) FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #2 – Text: The aim of this study was to determine whether DNA vaccines engineered to express the T. gondii ROP13 protein or the cytokine IL-18 can induce long term immunity – Feedback: Is the immunity targeted against ROP13 or IL-18? Consider the purpose of IL-18 as described in the paper – Comments: • pVAX/IL-18 is an adjuvant, not a vaccine – the mice are not “immunised” with IL-18, even if the paper expresses it this way • Do you actually want immunity against IL-18? FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #3 – Text: immune response was evaluated by measuring expression of ROP13 – Feedback: How does measuring expression reveal anything about the Immune response? – Comments: • Not an immune response – this experiment was used to assess whether pVAX-ROP13 was functioning as expected • Can Marc-145 cells demonstrate an immune response? • Demonstrate expression of ROP13 from pVAX-ROP13 FEEDBACK: EXPERIMENTAL DESIGN PART A • Two transfected cell populations (as per the figure legend below) – IFA detection of T. gondii ROP13 on Marc-145 cells previously transfected with pVAX-ROP13 (A) or the empty vector pVAX-1(B) – What technique(s) could be used to verify this result? FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #4 – Text: In each experiment, there is a dependent and independent variable. – Feedback: Doesn’t every well-designed experiment have this? – Comments: • Most removed this, but some replaced it with other overly obvious statements… • Interrogate every statement in your submissions – Are these words necessary to demonstrate my understanding? – Or are they simply extras that don’t address the point of the exercise? – Be careful with “filler” statements… FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #5 – Text: the positive control was the presence of pVAX-ROP13 in one group of cells and the negative control was pVAX-1 in a second group – Feedback: If the experiment is testing the effect of pVAX-ROP13, how can that same construct also be a positive control? – Comments: • This was the construct being tested • Cannot be a test sample and a positive control! • But can be a test sample and then a positive control in a subsequent experiment FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #6 – This experiment looked for specific green fluorescence (dependant variable) as a measure of ROP13 expression Text: and was dependent on whether or not the cells had been transfected. – Feedback: While transfection needs to occur to get a result, is it possible to have transfection but find there is no fluorescence? What would this say about the construct? – Comments: • Cells may be transfected but not express ROP13 • Construct is defective? Cells not capable of expressing? FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #7 – Text: experimental vaccines pVAX-ROP13 and pVAX/IL-18 – Feedback: Again, check the purpose of IL-18 – it is appropriate to call it a vaccine? – Comments: • pVAX/IL-18 is not a vaccine – immune modulator • What would happen if you developed an immune response to IL-18? FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #8 – Text: The dependent variable was the average amount of antibodies – Feedback: Which antibodies? Be specific – Comments: • Need to be specific – There are going to be lots of different antibodies present • Measurement of anti-ROP13 specific IgG – ELISA using “plates coated overnight with 1g of STAg (soluble tachyzoite Ag)” – i.e. ROP13 FEEDBACK: EXPERIMENTAL DESIGN PART A HRP anti Mouse IgG (secondary antibody) anti-T. gondii IgG (primary antibody, from mouse serum) T. gondii ROP13 (STAg) anti Mouse IgG (secondary antibody) Sample containing specific T. gondii protein of interest separated by polyacrylamide gel and transferred to a membrane ELISA HRP Western Blot anti-T. gondii IgG (primary antibody) Direction of separation FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #9 – Text: independent variable was the time frame (i.e. 6 weeks) – Feedback: Really? Does that mean the experiments used different amount of time? Or was time fixed (i.e. a controlled variable)? – Comments: • Time – controlled variable – Each group examined over the same time period – Independent variable was vaccination type – A few (unnecessarily) repeated this multiple times • Dependant variable – OD(450nm) which is representative of the anti-ROP13 level – Changes based on the independent variable FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #10 – Text: immunizations containing pVAX-ROP13 or pVAX-ROP13 in combination with pVAX/IL-18 produced specific IgG levels greater than the baseline observed in the controls, with the combination producing a faster and stronger response – Feedback: In what sense? Stronger antigen binding (i.e. affinity)? Or is it a “greater” antibody response i.e. more antibody? – Comments: • The ELISA assay suggests the latter i.e. more antibody • Avoid using descriptive words that are actually misleading FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #11 – Text: Mice immunised with pVAX-ROP13 or pVAX/IL-18 showed increased proliferation over the controls. – Feedback: Are the mice really proliferating? Care must be taken to provide the correct experimental details. – Comments: • It’s not the mice proliferating, it’s the splenocytes! • Alternative: add the words “Lymphocytes from” to the sentence and it makes perfect sense • i.e. Lymphocytes from mice immunised with pVAX-ROP13 or pVAX/IL18 showed increased proliferation over the controls. FEEDBACK: EXPERIMENTAL DESIGN PART A • Lymphoproliferation assay – In this assay used to measure splenocyte proliferation – Harvest splenocytes from 5 mice groups • Culture with STAg (soluble tachyzoite antigen), ConA (positive control) or medium (negative control) – Expected outcomes? • G1 & G2 – immunised with PBS and pVAX-I respectively • G3 – immunised with pVAX/IL-18 • G4 – immunised with pVAX/ROP13 • G5 – immunised with pVAX/ROP13 + pVAX/IL-18 FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #12 – Text: immunization of the mice and challenge measured the survival times. – Feedback: This sentence is very unclear. How can challenge ‘measure’ survival times? – Comments: • Misleading grammar – “challenge” doesn’t measure anything; the measurement (actually an observation) is POST-challenge. • Suggested alternative – The immunised mice were challenged with a lethal dose of T. gondii and the survival time was monitored. FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #13 – Text: it would have been better experimental design to test all 5 mouse groups for fluorescence. – Feedback: What does fluorescence have to do with the mice experiments? – Comments: • To look for what exactly? And where? – What additional information would this provide beyond what Figure 1 already tells you? • Mice were NOT tested for fluorescence at all. • Fluorescence was observed in Marc-145 (monkey kidney cells) FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #14 – Text: additional controls could have been used to provide more contrast in the results. – Feedback: Such as? This statement adds nothing if specific examples are not provided. Is it likely that the experiment would have been accepted for publication if a necessary control was missing? – Comments: • A statement commonly made for “padding” – Meaningless without suggestions for what the additional controls might be • In any case, no extra controls are warranted in this study FEEDBACK: EXPERIMENTAL DESIGN PART A • Error #15 – Text: developing a vaccine for the future treatment of toxoplasmosis. – Feedback: Is a vaccine meant to “treat” disease? – Comments: • Vaccines prevent infection, they don’t treat disease!!! FINAL WORDS ON THE PAPER • Figure 4/Table 3 – Immune responses tested before this challenge – Used 6 of the 40 mice/grp to harvest serum/spleens • Challenge 17 mice/grp – Intraperitoneally or Orally (i.e. 34 mice total per group) – Asking the question – “Does the treatment (independent variable) change survival time or number of brain cysts (dependent variables)?” EXPERIMENTAL DESIGN CAS E S TUDY E S T R A D I O L T R E AT M E N T TRIAL K A R AG H L O R I N G – P O S T D O C TO R A L S C I E N T I S T Estradiol treatment trial – study design E2/V injections P0 P3 P10 Male WT and mutant mice (n = ~10 each) P21 Seizure monitoring Behaviour testing P56 P70 P42 P35 RNAseq 40ng/g daily Sesame oil Blinded 100% Behaviour testing RNAseq Proportions of seizures over lifetime 50% 0% 0 10 20 30 Untreated PA1 40 Untreated PA2 50 60 70 BREAK TIME! RETURN IN 5 MINS ASSESSMENT #3 – EXPT. DESIGN PART B • Aim of this assessment – Understand how observational details translate into a hypotheses – Utilise prior learning about experimental design to design experiments that correctly address the supplied hypotheses • Learning outcomes – By successfully completing this assessment you will demonstrate the ability to: • Select appropriate techniques to address given hypotheses • Design properly controlled experiments and explain how they address the given hypotheses EXPERIMENTAL DESIGN PART B • Scenario – 37 patients in their early 20’s – Present with symptoms described as sudden onset dementia – Pattern of presentation consistent with a localised outbreak – Likely a chemical or infectious cause • Findings/Assumptions – No common chemicals causes detected – All patients attended common event • Uni BBQ for Biomed Students, all ate the beetroot salad! • Further testing revealed presence of a previously uncharacterised gastrointestinal bacteria EXPERIMENTAL DESIGN PART B • Discussion – What questions would you ask about this disease? – These questions will be translated into hypotheses • Consider pathogen characteristics e.g. – Route of infection – in this case? – Ability to colonise host in one or more locations – Mechanisms for causing disease • Toxins, secretion systems, host cell invasion, etc. – Use this additional information to formulate three relevant hypotheses to investigate this case… EXPERIMENTAL DESIGN PART B • Recall the following – Controls – Dependant variables – Independent variables • All these things are relevant – Your turn to design three experiments – You’ll need to research techniques and experimental approaches relevant to hypotheses – This is meant to be a straightforward exercise and is not designed to “trip you up”! EXPERIMENTAL DESIGN PART B • What is the process we need to consider? 1. What exactly is being tested? (i.e. the sample) 2. How/where will the sample(s) be acquired? 3. What technique(s) are suitable to test these sample(s)? 4. What are you hoping/expecting to find using these techniques? 5. How does this outcome address the hypothesis? EXPERIMENTAL DESIGN PART B • Supplied Hypotheses 1. The pathogen-secreted effector molecule is secreted after the pathogen binds to (colonise) gastrointestinal epithelial cells 2. The pathogen-secreted effector molecule localises in the cytoplasm of neurons 3. Expression of the mouse gene xyzA (which is already known to induce dementia-like symptoms when overexpressed) is upregulated in neurons exposed to the pathogen-secreted effector molecule. EXPERIMENTAL DESIGN PART B • Some suitable techniques – Western/Northern blots, ELISA, Immunohistochemistry – Other techniques can be used if you wish • You may need to make some assumptions – E.g. hypothesis that toxin affects gene expression • Don’t need to know exact gene(s) to design an experiment that would measure changes in gene expression • Assessment mechanics? – Template and rubric available now – Start thinking about techniques you want to use – Collaborate if you wish, but do not collude/plagiarise! (******) – Submission: Monday 1st June, 11:59pm (Week 12) THE END ☺ Assessment #3 – Experimental Design Part B Delete pages 1-3 before submitting your work! Aim and Learning objectives This assessment provides an opportunity to utilise what you have learned in the course so far about experimental design. You will need to design three experiments that correctly address the hypotheses shown in the template on page 4 of this document. By successfully completing this assessment you will demonstrate the ability to: 1. Select appropriate techniques to address given hypotheses 2. Design properly controlled experiments and explain how they address the given hypotheses Scenario and Assessment Notes 37 patients in their early 20’s have presented with symptoms described by medical professionals as “sudden onset dementia” The timing and pattern of presentation is consistent with a localised outbreak of disease, suggesting either a chemical or infectious cause rather than a common genetic defect. After extensive testing, no common chemical causes were able to be identified, suggesting an infectious cause for the disease. Concurrent epidemiological investigations found that all the patients attended a university barbeque for Biomedical science students several days before the outbreak. Further to this, it was discovered the only common food all of the patients had eaten was a beetroot salad. Bacteriological testing discovered a previously uncharacterised bacteria in stool samples from all of the affected individuals. Before you start thinking about experiments, you should consider what information you can extract from the scenario. For example, if the beetroot salad was in fact the source of the bacteria, then how did the patients get infected? It would have to be self-inoculation by ingestion. If the ingested bacteria were in fact the ultimate cause of the observed dementia like symptoms, then you should be asking “How did a suspected gut pathogen somehow affect the brain?” This type of thinking is crucial to good experimental design; you always need to be considering the implications of the information you have available, and how that information can suggest interesting areas to research. Remember that this is an artificial scenario, and we don’t have or need to know every single step in the process between infection and disease. In fact, you need to make some assumptions (e.g. you have access to a specific reagent or mutant strain) in order to properly draft your experiments. The hypotheses you are basing your designed experiments on have been chosen as they highlight important steps in what might happen in real life, and they lend themselves to some key experimental techniques that are common in most biomedical research. For example, in the first hypothesis (see the last page of this document) you could consider making a mutant in the either the bacteria or the host cells to investigate the hypothesised binding activity. Exemplar for the hypothesis “Bacterial Toxin X binds to T lymphocytes” Experimental Design (240 words, inclusive of in-text references) To test the hypothesis “Bacterial Toxin X binds T-lymphocytes”, a flow cytometry experiment will be performed. First, a culture of mouse T-lymphocytes will be grown in AIM V™ Media (ThermoFisher Scientific, 2020) and split into 6 separate samples. Each sample will receive buffer solution with or without added toxin. Samples 1 & 3 will receive toxin buffer alone (negative control samples). Sample 2 will receive 1g/mL of purified Toxic shock syndrome toxin-1 (TSST; in toxin buffer), which is known to bind to T-lymphocytes (technique control). Samples 4, 5 and 6 will receive 0.1g/mL, 1g/mL and 10g/mL of purified Toxin X (in toxin buffer) respectively. As the binding affinity of toxin X for T-cells is unknown, this range of concentrations should allow for toxin binding detection where the binding affinity is higher or lower than that observed for TSST. Following toxin binding the T-lymphocytes will be washed (with AIM V™ Media) and incubated with either mouse anti-TSST (for cultures 1 & 2) or mouse anti-toxin X (for cultures 3-6). After incubation, all cultures will be washed to remove unbound primary antibody, then incubated with secondary antibody (fluorescently tagged sheep antimouse immunoglobulin). Following a final wash to remove unbound secondary antibody, all samples will be fixed with 3% paraformaldehyde (Sigma-Aldrich; 2014). After fixing the samples will be analysed using a flow cytometer to determine both the proportion of cells that have bound Toxin X, and the relative amount of Toxin X binding. Conclusion (66 words) The buffer-only negative controls establish the background autofluorescence in the presence of each primary antibody; values above this level in the experimental samples can be correctly attributed to the binding of toxin to the cells rather than to binding of antibody directly to the cells. The TT control ensures the technique is functioning as expected, although it does not control directly for the anti-Toxin X antibody. Exemplar notes This exemplar contains the level of detail expected. You DO NOT need to discuss statistical analysis; for the purposes of this assessment it is assumed each experiment has a statistically relevant number of replicates. Nor should you describe how individual reagents were generated; purified Toxin X was used in the exemplar, but nothing was said about how it was obtained, since that relates to preparatory work not covered in this assessment. Providing specific details about aspects such as reagent concentrations, times for incubation, volumes, reagent suppliers etc. is somewhat flexible. For example, the exemplar describes toxin concentrations but not antibody concentrations. This should make sense – the antibody concentration would be fixed at a level appropriate for this particular assay (i.e. it would be kept the same across all the samples tested) and as such the actual concentration isn’t critical for the experimental design. Given that we don’t yet know whether toxin X binds, specifying a range of concentrations makes good sense from an experimental design perspective – if you selected a single concentration, a lack of fluorescence might be due to the concentration being too low, rather than because it doesn’t actually bind to the T-lymphocytes. You are not expected to go into excruciating detail for every single element of the experiment – this would take far too many words! Inclusion of some specific details adds a level of authenticity to your experimental design i.e. it shows that you have bothered to properly research how these experiments might be done! Keep your focus on the design aspect – getting the controls correct and using an appropriate technique to address each hypothesis is far more important than specifying what supplier a particular buffer came from. Use the exemplar as a guide and make sure you reference correctly! type=grid ninc=1 ndpm=1 ndpt=1 Experimental design 1 (clearly explains the process for addressing hypothesis #1) Experimental design 2 (clearly explains the process for addressing hypothesis #2) Experimental design 3 (clearly explains the process for addressing hypothesis #3) Conclusions (Clearly indicates how the experiments address the hypotheses) Clarity (Grammar, Spelling and References) Penalties (late or over word limit) Total Percentage Relative Semester Mark (as …
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