Longer Questions

Question 1

Describe the stages of spermatogenesis

Answer 1

Spermatogenesis has three stages: mitotic proliferation, meiotic division, and spermiogenesis.

Mitotic Proliferation:
Spermatogenesis begins with spermatagonia/ gonocytes (the term for primordial germ cells once they have entered the testis) which are found on the basement membrane in the testis. Primordial germ cells remain arrested at the gonocyte stage in the foetus all the way up until puberty. They undergo several rounds of mitosis to form multiple type A spermatogonia. In rodents there are Asingle, Apair, Aaligned, A1, A2, A3, A4, and B stages. in primates there are Adark, Apale, Atransition (controversial), and B stages. B spermatogonia undergo one more mitotic division to form primary spermatocytes.

Meiotic Division
Primary spermatocytes undergo two rounds of meiosis (first slow, second quick) to form secondary spermatocytes, and then spermatids. Primary spermatocytes at the leptotene or zygotene stage of prophase can cross the blood-testis barrier into the adluminal compertment

Spermiogenesis
Divided into 4 main stages in mammals: Golgi, Capping, Acrosomal, and Maturation.
Golgi: the Golgi apparatus migrates to the pole where the acrosome will form. At the opposite pole form the axoneme begins to grow from the centrioles.
Capping: The acrosomal vesicle touches the nuclear envelope and flattens into a cap.
Acrosome: The nucleus begins to elongate, and mitochondria migrates to the caudal end
Maturation: Outer dense fibres grow around the axoneme. Mitochondria condense and form a mitochondrial sheath around the outer dense fibres. Most of the cytoplasm is discarded as the residual body.

The sperm is then release from its Sertoli cell - spermiation

Question 2

Describe the mechanism by which GnRH affects the synthesis and secretion of LH and FSH, and how this leads to a carefully controlled menstrual cycle.

Answer 2

GnRH is released in a pulsatile fashion from neurons in the medio-basal hypothalamus (pulse generator) and acts on its G-protein coupled receptor in the pituitary. This triggers a MAPK cascade via PKA and PKC activation, leading to transcription of FSH and LH genes. GnRH activation also raises intraceullar calcium whch leads to exocytosis of stored gonadotrophins. The frequency of pulses determines which gondadotrophin is released, and constant release of GnRH prevents gonadotrophin release.

In men, GnRH pulses are released roughly every 2 hours. In women pulses are released at different frequencies depending on the stage of the menstrual cycle. In the early follicular phase pulses are released every 90-120 minutes, however in the mid-late follicular phase this frequency increases to ~ 1 pulse every hour. After ovulation GnRH pulses slow to once every 3-5 hours which favours FSH production, though high oestrogen prevents secretion. Towards the end of the luteal phase GnRH pulse frequency increases and FSH is secreted

Question 3

HPV infection and how it causes cancer (50%) primary and secondary prevention. (25%) and management (25%)

Answer 3

HPV is a dsDNA virus which is spread by skin-skin contact (often through sexual contact) and infects keratinocytes in the basal layer of the epidermis. Infection is often asymptomatic and ~80% of cases will be cleared or go into spontaneous remission within 6-12 months. However in cases of chronic infection the virus may cause warts or cancer.

HPV infection is considered a necessary prerequisite for cervical cancer, though it is not necessarily sufficient. The most common subtypes are 16 and 18, which account for 75% of cervical cancer, and there are 7 subtypes which account for 90% of cervical cancer worldwide. The most vulnerable region of the cervix to cancer is the squamo-columnar junction/ transformation zone. HPV causes cancer via the actions of its E6 and E7 oncogenes: E6 ubiquitinates and degrades p53, and E7 inactivates and degrades pRb.

Vaccination uses virus-like particles and is a relatively successful prophylaxis, but does not work therapeutically. Treatment of carcinoma in situ is with resection and follow-up monitoring. Invasive cancer is treated with radical hysterectomy and lymph node dissection or radical chemoradiation therapy (depends on stage).

Question 4

Describe the main functions of Sertoli and Leydig cells in spermatogenesis

Answer 4

Sertoli cells respond to FSH, and produce androgen-binding proteins, oestrogen, Anti-Mullerian hormone, and inhibin. They also regulate the inernal environment of the seminiferous tubules

Leydig cells respond to LH and produce tesosterone and DHT

Question 5

Name an endocrine cause of secondary amenorrhoea, discuss the investigations and management

Answer 5

Hypothalamic:
FSH and LH will be low, as will gonadal steroids. Management will depend on cause: potentially increase weight and decrease exercise, but pulsatile replacement GnRH may be needed. Prolactinoma may be the cause, in which case prolactin will be high, and FSH and LH will be low. An MRI of the head may be needed, and treatment is with dopamine agonists, and occasionally surgery. Hyperthyroidism may also lead to secondary amenorrhea so TFTs should be done

HPG axis dysfunction:
Usually PCOS: Normal FSH and oestrogen, LH may be raised, advise weight loss if they have BMI>30. Address associated obesity and insulin resistance with diet and medication. Oral contraceptive pill helps regulates periods. Letrozole or clomiphene or FSH will help fertility

Primary ovarian insufficiency:
FSH will be high, oestrogen will be low, Management is with HRT

Question 6

How do obesity and insulin resistance increase risk of endometrial cancer?

Answer 6

Obesity is a very important factor in the development of endometrial cancer, with 50% of UK cases being considered attributable to obesity. Obesity predominantly increases the risk of type I cancer, but also increases the risk of type II.

Pre-menopausally, obesity can lead to anovulatory cycles, which leads to high, unopposed oestrogen production. Post-menopausally, obesity mainly influences cancer risk by conversion of androstenedione to oestrone by aromatase in adipocytes.

Insulin resistance results from chronically high insulin levels, which lead to high IGF-1. IGF-1 mediates action of steroid hormones on the endometrium and is associated with proliferation, hence chronically raised levels increase the risk of endometrial cancer.

Furthermore, adipocytes produce adipokines which may stimulate endometrial proliferation, and obese people often display chronic low-level inflammation which may increase cancer risk

Question 7

Extrinsic and intrinsic factors for meiotic regulation and how this differs in males and females

Answer 7

DAZL is crucial for a primordial germ cell to be meiosis-competent in both males and females

Retinoic acid is expressed in gametes of both males and females, but in males is degraded by Cyp26b1

Stra8 expression is triggered by retinoic acid and is required for pre-meiotic DNA replication

Question 8

Discuss an example of how maternal lifestyle and diet affects the fetus

a. Dutch hunger winter
b. Monozygotic twins
c. Agouti mouse/ BPA

Answer 8

a) The Dutch Hunger Winter refers to the winter of 1944-45 when food to the west of the Netherlands was restricted by the Nazis. As a result there was a cohort of children exposed to famine in utero (mothers having 400-800 calories per day), but were then well nourished throughout childhood. These children has a higher risk of CVS disease (early gestation starvation), and obesity/ insulin resistance (prenatal starvation). The neonates had high head: birth weight ratio, suggesting brain sparing.
b) A study examined normal metaphase chromosomes of monozygotic twins. 3-year-old twins had vritually identical chromosomes, but 50 year-old twins had very different patterns of methylation. This supports the idea that methylation is due to environment
c) The Agouti gene is normally expressed in the mous hair growth cycle, but when hypomethylated becomes continuously active and ectopically expressed, and leads to a yellow, obese phenotype. Agouti is theorised to have target sites in adipocytes and the hypothalamus, which lead to its metabolic effects. Pregnant Agouti mice were exposed to BPA, which increased the number of offspring with yellow coats. BPA was found to demethylate the Agouti gene, resulting in over-activation and continuous expression - causing the yellow-coat phenotype

Question 9

Describe the role of Sertoli cells in testis development

Answer 9

Sertoli cell precursors arise from the coelomic epithelium at the genital ridges. Differentiation of coloemic epithelial cells into Sertoli cells is driven by expression of SRY, which leads to expression of Sox9.

Sox9 initiates two pathways: release of Fibroblast Growth Factor-9 (FGF-9) which induces Sertoli cell differentiation in coloemic epithelium; and activation of PgD synthase which causes PgD to be released and trigger Sox9 expression in neighbouring cells via paracrine signalling, even if those cells don’t express SRY (PgD also has autocrine action to further boost Sox9 expression)

After differentiation, Sertoli cells surround primordial germ cells and form the cord structures. Sertoli cells are critical in the differentiation of the testis: a threshold number are needed to initiate testis development.

Additionally, Sertoli cells release Desert hedgehog factor (DHH) which facilitates differentiation of Leydig cells.

Question 10

Discuss causes of recurrent miscarriage (RM), investigations, and treatments

Answer 10

Causes
Thrombophillic: Antiphospholipid syndrome is the most important and treatable cause of RM. Antibodies bind to platelets and endothelial cells, activating them and triggering clotting and infarction in placental vessels. Investigations are ELISA for anti-cardiolipin antibodies, and dilute Russel’s viper venom time test with platelet neutralisation test for lupus anticoagulant. Treatment with aspirin and unfractionated heparin has been shown to significantly reduce RM rates. RM patients should also be screened for factor V leiden, protein C&S deficiency, and antithrombin and prothrombin mutations.

Karyotypic disorders
Parents can be carriers of balanced translocations which lead to unbalanced translocations in the foetus, leading to miscarriage. Screening and IVF with PGD can be done to only select euploid, normal embryos, but has been shown to not affect healthy birth rate.

Uterine malformation
There is a wide variety of possible uterine malformations that can increase RM rates. These can often be detected using ultrasound, but the gold standard is using hysteroscopy, which also allows the malformation to be fixed as it is examined. Laparascopy will sometimes be necessary (usually in the case of didelphus - two uteri).

Metabolic and Endocrine disease
PCOS, elevated basal LH, thyroid disease, and insulin resistance are all associated with RM. TFTs, thyroid antibody tests, glycated haemoglobin, ovarian USS, and gondaorophins could all be done to investigate. Control of thyroid disease and diabetes improves live birth rate, as does metformin in PCOS, but LH suppression does not

Sperm fragmentation
Sperm samples from RM men have revealed higher incidence of sperm fragmentation. There are multiple tests to assess sperm, but the alkaline COMET assay results in better prediction for male infertility

Question 11

Describe investigations and management for an infertile couple (not IVF)

Answer 11

Tests for ovarian reserve would include: ultrasound antral follicle count, early follicular FSH, AMH, and day 21 serum progesterone. An HSG (contrast x-ray) would be used to establish whether the fallopian tubes were patent.

Semen analysis would be carried out to establish: volume, pH, number and concentration of sperm, morphology, and motility. If oligospermia/ azoospermia is found, further analysis can be done with karyoptyping, cystic fibrosis screen, hromonal tests (FSH, LH, tesosterone).

If the cause is hypothalamic dysfunction in the woman, increasing BMI>19, or decreasing exercise levels if they are high may help. If not, pulsatile administration of GnRH or gonadotrophins should induce ovulation.

HPG axis dysfunction is usually a result of PCOS, which can be helped by reducing BMI below 30. Clomiphene or letrozole can be given to help, with or without metformin. Exogenous FSH may be used.

If there is an issue with sperm, aspiration or microTESE may be beneficial

Question 12

Describe the process of IVF

Answer 12

Pituitary down-regulation:
Either a GnRH agonist or an antagonist is given. The agonist produces an initial 2-week ‘flare-up’ of the HPG axis, but afterwards the pituitary down-regulates its GnRH receptors and becomes de-sensitised/ Alternatively, an antagonist can be given which blocks pituitary GnRH receptors immediately. Agonists have a better live birth rate, but antagonists are safer.

Ovarian stimulation with hCG (if GnRH agonist was used) or GnRH agonist (if antagonist was used) to produce multiple follicles.

Ovulation is triggered with an LH surge, and oocytes are collected.

Sperm are placed in a dish with oocytes (or injected - ICSI) and the oocytes are fertilised

Embryos are transplanted back into the woman

Question 13

Describe three reprogramming technologies

Answer 13

Somatic cell nuclear transfer:
This is how Dolly the sheep was cloned. An oocyte has its nucleus removed, then a whole somatic cell is inserted into the oocyte. The new cell is activated with an electric shock and cultured. The cells can then be used for reproductive cloning (e.g. Dolly) or therapeutic cloning (cells are taken and differentiated to somatic cells). Somatic cell nuclear transfer could allow creation of tissue that could be transplanted without being rejected, and could produce all cell types. However there are ethical issues RE where to get the oocytes. Furthermore the process is currently inefficient (Dolly took 400 nucleated oocytes)

Fusion with embryonic stem cells:
Somatic and embryonic cells are fused to create a pluripotent tetraploid cell. This cell is unstable and used for research, but not regenerative medicine

Reprogramming with defined factors:
The theory was that the factors responsible for maintaining the pluripotency of embryonic stem cells could also be used to reprogram differentiated cells. Oct4 and Sox2 were found to be essential - they form a complex which binds to the nanog promoter and are sufficient to activate reprogramming of somatic to pluripotent cells

Question 14

Describe the characteristics of induced pluripotent stem cells, their pros and cons, and their uses in regenerative medicine

Answer 14

Characteristics:
iPSC are pluripotent cells re-programmed from somatic cells and form ESC-like colonies, express ESC genetic markers, and have similar epigenetic signatures to ESCs. They also form teratomas and will form chimeras when injected into a blastocyst. iPSCs have similar differentiation potency to ECs and can be amplified significantly in vitro.

Pros:
There are no associated ethical issues regarding obtaining oocytes, as for somatic cell reprogramming
They can be patient-matched to overcome immune rejection
They can be used to produce any required cell type

Cons:
They are inefficient
It may be tricky to culture the exact cell type needed
Reprogramming does not totally erase epigenetic marks, so they may still have some differentiation preference
There is a risk of tumourigenesis due to residual undifferentiated cells

Uses:
Modelling of diseased tissue through gene knockouts
Testing of drug toxicity on models
Cell therapy/ regenerative medicine
e.g. cardiac repair where they are a good option because they can differentiate into authentic mature cardiomyocytes (as opposed to other stem cell options like bone-marrow derived stem cells). However the cardiomyocytes produced are immature, and there may be mixed phenotypes which contribute to arrhythmia, and the process of reprogramming of somatic cells may cause genetic/ epigenetic changes in the cells. Overall iPSCs have been shown to be effective in improving ejection fraction in heart failure

Question 15

Describe the roles of progesterone and cAMP in decidualisation

Answer 15

Progesterone:
Progesterone is release from the corpus luteum in response to hCG from the blastocyst. It induces myometrial quiescence and inhibits the proliferative effects of oestrogen on endometrial epithelial cells. Progesterone also acts to induce immune tolerance of the conceptus, and regulates the window of receptvity to the blastocyst. During decidualisation the inhibitory PRA is the dominant subtype of receptor, whilst PRB is down-regulated.

cAMP:
Progesterone on its own is insufficient to induce decidualisation. cAMP is created by activation of GPCR (e.g. by oestradiol, prostaglandins) and initiates the process, but is insufficient to sustain it. cAMP is key in initiating differentiation of endometrial stromal cells through the PKA pathway, and sensitises the cells to the effects of progesterone. In addition, cAMP acts as an intracellular messenger for the effects of hormones triggering progesterone release from the corpus luteum

Question 16

Describe the 2 well-established diagnostic methods for TB

Answer 16

Tuberculin skin test
Involves subcutaneous injection of TB antigens and monitoring for sensitivity response. It has multiple issues: it requires some technical skill, it has low specificity (infection with other mycobacteria can return a false positive, as can BCG vaccination, and latent disease), it has low sensitivity (particularly poor for detecting early disease, disease in children, disease in immunocompromised patients e.g. malnourished or HIV)

Microscopy
Staining with Ziehl-Neeson reveals acid-fast bacili (consequence of hydrophobic mycolic acid cell wall). However it takes 4-6 weeks to culture samples, requires a prohibitive amount of resources, and sufficient samples of sputum or gastric washings are hard to get, and in children the bacterial load is usually low, making this test unsuitable

Question 17

Describe 5 newer diagnostic methods for TB

Answer 17

Xpert
The Xpert machine purifies and concentrates TB samples and amplifies Mtb DNA. Within 2 hours the machine can give a diagnosis, and detect resistance. Though the machine is expensive, each separate test is relatively cheap

IGRA
Interferon gamma release assays measure TB infection by analysing the host immune response. It is essentially the same principle as a tuberculin test, but is more specific. However it is technically challenging and expensive, and carries ethical issues RE venepuncture in children

MODS
Microscopic observation drug susceptibility testing is microscopy of sputum samples cultured in broth for 8-15 days, which is faster than using solid media. Mtb forms cords in culture which can be observed. This method allows for diagnosis and testing for drug resistance. It is cheap, fast, sensitive, and specific.

PCR
PCR can be used to amplify Mtb-specific regions of DNA. It is specific, but sensitivity varies

LAM urine test
A point of care, 30 minute, cheap test which is useful for HIV+ adults with suspected TB, as its sensitivity increases the lower the CD4 count is

Question 18

Briefly summarise pre-implantation development

Answer 18

After fertilisation, the zygote undergoes four rounds of mitosis, but no growth. This results in a ball of eight cells with the same volume as the original zygote. These cells then compact very closely together through cadherin interactions to form a mass known as the morula. The zona pellucida is still intact and important: it stops the morula from sticking to the fallopian tube wall, and prevents disintegration by leucocytes.

Position of cells within the morula determines differentiation: outer cells differentiate and develop polarity to become the trophectoderm (express Cdx2) and form tight junctions between each other. Inner cells retain pluirpotency and so express Sox2, Oct4, and Nanog.

A cavity appears in the morula, and it enlarges to form a blastocyst. The cavity is formed by the action of Na+ transporters which draws water into the morula.

Question 19

Describe examples of first, second, and third generation sequencing technologies

Answer 19

1st - Sanger Sequencing:
Involves fragmenting and amplifying DNA, then mixing it with ddNTPs which don’t have a free 3’ OH, and so terminate polymerisation. The different fragments formed are then separated by electrophoresis. This technique is quick and very accurate, but is far more expensive than other methods.

2nd - Pacific Bio
A real time technique that is fast and cheap with a good read length, but is inaccurate

3rd - Nanopore:
Hollow transmembrane proteins are inserted into a membrane of high electrical resistance with a current applied across it. As a single strand of DNA passes through the pore, the different bases cause distinct changes in the current, which are detected in real time. The issue with this technology is its comparatively low accuracy: 80-85%

Question 20

Give the advantages and disadvantages (with examples) of using the following in infectious disease research:

Animals
In vitro/ explant surrogates
Alternative hosts
Humans

Answer 20

x

In vitro:
Most research is carried out on cultured cell lines (e.g. epithelium, endothelium). The issues are that the in vivo environment and phenotype is not accurately replicated (e.g. it’s not dynamic, doesn’t account for human genetic variation). This can be partially addressed with additions to culture media (e.g. add EGF or use collagen surface). Explants also help to simulate the in vivo enviornment, though they are difficult to obtain, and may be altered in the process of removing them

Alternative hosts:
Easy to grow/ obtain and genetically well-defined, so specific mutants are easily available. KOs can be induced in the organism as well as the pathogen. E.g. C. elegans, Drosophilia, Zebra fish

Humans:
The ideal host for modelling disease as it is obviously the most accurate. However human testing is ethically tricky and requires sophisticated facilities to contain the pathogen (e.g. total control air handling, ensuite bathroom). Furthermore the recruitment and follow-up is logistically challenging

Question 21

Give 5 uses of next generation sequencing in infectious disease with examples

Answer 21

Analysing microbiome components to determine their effect on health and disease (e.g. gut microbiome)

Detecting pathogens for diagnosis, especially those that can’t be grown on agar (e.g. S. constellatus in CF patients in 2008)

Identifying and monitoring emergence of new resistance mechanisms

Discovering new pathogens or strains in outbreaks (e.g. Arena virus in 2008)

Identification of essential genes in pathogens so that they can be targeted with antibiotics (e.g. genes for survival in bile salt - S. typhi)

Question 22

Describe the differences between IgE and non-IgE ‘allergy’

Answer 22

Presentation:
IgE-mediated allergy presents with acute. rapid-onset itchiness, rash, uriticaria, angiooedma, breathlessness, and anaphylaxis
Non-IgE allergy generally presents as a chronic issue, with delayed-onset symptoms

Question 23

Discuss 3 methods of diagnosing allergy and their pros and cons

Answer 23

Skin tests:
Either a prick test or an intradermal test can be done (the latter being trickier and more painful, but also more sensitive). Both are quick, cheap, easy (though errors with pricking can cause false negative or positive), and give fast results. They have a high negative predictive value, and a fair positive predictive value, but carry a small risk of causing a systemic allergic reaction. The skin tests also have a variable response depending on time of day (circadian rhythm) and season

Blood SpIgE Tests:
A fluorescent antibody test using the allergen absorbed onto a cellulose sponge measures which allergens the patient has produced IgE against. It’s more expensive, slower than skin tests, and has a worse negative predictive value. Blood tests are useful in cases where the patient has severe dermatitis, or recent anaphylaxis, or is taking anti-histamines

Oral food tolerance test:
40% of young adults will have a positive skin test to some common allergens, but only ~20% develop symptoms if challenged (i.e. positive predictive value is not high). An oral food challenge is a more relevant test, but is riskier than skin or blood testing, and so requires access to a properly trained clinician, and full equipment to deal with anaphylaxis.

Question 24

Describe the epigenetic regulation of chromatin in embryonic stem cells (ESCs)

Answer 24

Chromatin can be modelled into different states via epigenetic modification, which affects accessibility of gene sequences to transcription machinery. Euchromatin refers to accessible chromatin, and is characterised by H3K9 and 14 acetylation, and H3K4 methylation. Heterochromatin refers to inaccessible, tightly wound chromatin, and is characterised by H3K9 and 14 de-acetylation, and H3K27 methylation.

Chromatin in ESCs must be kept accessible in order to make the cells pluripotent, and not commit them to any one lineage. Specialised epigenetic regulation of ESC DNA allows expression of the crucial components for pluripotency: Oct4, Sox2, and Nanog. However, chromatin must also be regulated so that transcription of all genes does not occur.

A defining characteristic of embryonic stem cells is the presence of bivalent methylation markings in promoters of key developmental genes – i.e. the presence of both activating and repressing marks. This serves to maintain the cell’s pluripotency, and to prevent unregulated transcription of all genes. Polycomb repressive complexes (PRCs) aid this process: PRC 1 prevents RNA polymerase II from elongating DNA after it has been assembled by activating methylation marks, while PRC 2 makes repressive gene marks (e.g. methylation of H3K27).

As cells differentiate, they acquire more repressive epigenetic modifications, and their chromatin becomes less accessible to transcription machinery, consolidating their differentiated identity.

Question 25

Describe the development and activation of a type I hypersensitivity reaction

Answer 25

The allergen is encountered by a dendritic cell (e.g. as the gastrointestinal surface) and presented to a T-cell that recognises the antigen, which then differentiates to a Th2 cell. This Th2 cell then activates B-cells which mature to plasma cells and produce IgE. The Fc portion of the IgE docks in mast cells, and upon the second exposure, the antigen will cross-link IgE causing the mast cell to granulate and release histamine

Question 26

Describe the characteristics of somatic stem cells, and their pros and cons

Answer 26

Characteristics:
SSCs are found in very specific locations in the body, and are difficult to culture in vitro, however in vivo they are capable of life-long self-renewal. They are multipotent, and do not form teratomas.

Pros:
Autologous transplants can be used - no immune rejection issues
Cells need no further modification to make them suitable for transplant
The stem cells release cytokines and growth factors to stimulate endogenous stem cells

Cons:
Limited availability
Low plasticity - they are only multipotent, not pluripotent
They are difficult to culture in vitro

Question 27

Summarise the evidence that allergy is increasing as a result of changes in human environments

Answer 27

Incidence of asthma, hay fever, eczema, food allergy have all risen over the past 60-70 years. This is in conjunction with other autoimmune conditions, such as multiple sclerosis, Type I diabetes mellitus, and Crohn’s disease.

One theorised explanation for this is lower exposure to certain antigens which may have a de-sensitising effect, especially in childhood. This is supported by observations that in Germany before the fall of the Berlin wall, the opulent West side saw greater incidences of asthma and hayfever than the relatively poor East. The difference in wealth is theorised to have led to a less hygeinic environment in East Germany, resulting in greater exposure to de-sensitising antigens.

Further evidence is provided by the PARSIFAL study, where growing up on a farm, and engaging in a lifestyle free of pasteurised food and antibiotics was found to be protective against allergic disease.

Finally, one study examined rates of atopic disease in villagers in rural Poland at the time Poland joined the EU, and 9 years after. During this time agriculture underwent mechanisation, and fewer villagers came into contact with the farm environment. Over this time, rates of atopy increased significantly.

Question 28

Describe the development of oral tolerance

Answer 28

Oral tolerance refers to the induction of systemic hypo-reactivity to an antigen through ingesting it; it is a type of peripheral tolerance. Depending on the dose of antigen, tolerance can either be induced by anergy and clonal deletion of T-cells recognising it (high-dose), or by induction of regulatory T-cells (low-dose) which suppress immune response to the antigen using TGF-beta and IL-10 (this suppressive balance is less permanent than that caused by anergy or clonal deletion).

Gut flora are critical for inducing oral tolerance, as specific bacteria are needed to prime dendritic cells, inducer Treg cells, and promote IgA response. IgA is secreted into the gut to bind antigens in the lumen. IgE induces its receptor on enterocytes, then binds the antigen and accelerates uptake, leading to allergic sensitisation. Hence a deficiency of IgA is often seen in children who develop food allergies.

RCTs and systematic reviews have demonstrated that rates of allergy are lower in children who are fed allergens in early life.

Question 29

Describe the pathophysiology of preeclampsia

Answer 29

The spiral arteries fail to remodel properly and so remain narrow, resulting in high pressure, poor flow vessels. This promotes clotting within the arteries, restricting blood flow to the placenta. During this time the oxygen tension of the placenta decreases, and when the clot is thrombolysed and flow is restored, oxygen tension increases again, which causes production of oxygen free radicals.

The oxidative stress damages the sycitiotrophoblast layer, causing release of suncytiotrophoblast microparticles into maternal circulation. This leads to endothelial damage and dysfunction, which causes hypertension, proteinuria, oedema, and increased coagulability

Question 30

Describe the events that occur upon sperm-oocyte fusion

Answer 30

Once the sperm has undergone the acrosome reaction, it releases PLC-ζ into the oocyte, which triggers InsP3 signalling, leading to pulsatile Ca2+ release from the oocyte endoplasmic reticulum, known as calcium oscillations. Intracellular calcium release cleaves securing, releasing separase which cleaves cohesins between chromatids, allowing them to separate.

These calcium oscillations also lead to degradation of the Mature-Promoting Factor complex (MPF) which consists of cyclin B1 and CDK1. Degradation of MPF is prevented until this point by Mos, but calcium release activates Anaphase-Promoting Complex (APC) which degrades MPF and allows the cell to resume meiosis II. Upon the completion of meiosis II, the second polar body is formed.

Once the sperm and oocyte have fused, cortical granules are exocytosed from the oocyte and release their contents into the peri-vitelline space, cleaving ZP glycoproteins, and cross-linking tyrosine residues: this establishes a firm block to polyspermy. The zona pellucida remains important as the zygote moves along the fallopian tube towards the uterus, as it prevents the zygote from sticking in the tube, protects it from leucocytes

Question 31

Describe the immune therapies available for treating cancer

Answer 31

Cytokine therapy:
Mostly experimental therapies involving giving patients interferon gamma or IL-2 to try and activate and expand T-cell populations. It has shown limited success and has a significant side-effect profile

Vaccines:
Tumour cell vaccines - Tumour cells extracted from patient, then modified to make them more immunogenic, then re-injected
Dendritic cell vaccines - extracted in peripheral blood sample, expanded with IL-4 and GM-CSF, activated with tumour antigen, then re-injected
Peptide - specific protein designed to trigger anti-tumour immune response
Infection - e.g. HPV, limit the pathogen that causes the cancer

Monoclonal antibodies:
Signalling inhibition - e.g. Herceptin binds HER2 receptor causing internalisation and degradation. Herceptin is not often effective and resistance may arise through loss of HER2 expression, receptor mutation to stop Herceptin binding or to gain constitutive kinase activity, or compensatory expression of other receptors

Cytotoxicity - e.g. anti-CD20 Rituximab. These antibodies trigger direct cell lysis, complement activation, and opsonic phagocytosis

Conjugates - e.g. ADEPT. An antibody with an enzyme to convert a chemo pro-drug to its active form is given. The antibody attaches only to tumour cells, but doesn’t kill them. Once the unattached antibody has left the blood, the pro-drug is given, and is only metabolised to the active form at sites where the antibody has attached to tumour cells, limiting side-effects

Anti-immunosuppressive - Most successful new technology. Aims to block either CTLA-4 (Ipilimumab) or PD1 (Nivolumab) which would otherwise prevent T-cell co-stimulation and activation

Adaptive immunotherapy:
T-cell transfer - can be done with tumour infiltrating lymphocytes, which are extracted in tumour biopsy, then expanded, activated, and re-infused, but it is hard to get enough cells, it takes 6 weeks, and the response is disappointing. T-cell engineering is a promising therapy - extracted T-cells are modified to express a chimeric antigen receptor that is MHC independent.

Gamma delta T-cells - A new therapy involving using a rare subset of T-cells that are highly effective at killing cancer cells, have high clonal frequencies, and are differentiated pre-activation, giving them a fast response