(a) (i) State the therapeutic indications, drug interactions and side effects of Methotrexate. (5 marks) (ii) State the…

(a)(i) Methotrexate (5 marks)

Methotrexate is an antimetabolite that inhibits dihydrofolate reductase, so tetrahydrofolate is depleted and the synthesis of thymidylate and purines (DNA synthesis, S phase) is blocked.

Therapeutic indications.

• Malignant: acute lymphoblastic leukaemia (including intrathecal prophylaxis), choriocarcinoma and gestational trophoblastic disease, osteosarcoma (high dose with leucovorin rescue), non-Hodgkin lymphoma including CNS lymphoma, and cancers of the breast, head and neck, and bladder.
• Non-malignant: rheumatoid arthritis (weekly low dose, the standard DMARD), psoriasis and psoriatic arthritis, ectopic pregnancy, medical termination of pregnancy (with misoprostol), Crohn's disease, and vasculitis or lupus as an immunosuppressant.

Drug interactions.

• NSAIDs, salicylates, penicillins and probenecid reduce renal tubular secretion of methotrexate, so its level and toxicity rise.
• Co-trimoxazole and trimethoprim add antifolate action and cause pancytopenia.
• Proton-pump inhibitors reduce its renal clearance; nephrotoxic drugs such as cisplatin reduce its excretion.
• Alcohol, leflunomide and other hepatotoxic drugs add to liver damage. Live vaccines should not be given.

Side effects. Bone marrow suppression (megaloblastic anaemia, leucopenia, thrombocytopenia), mucositis and stomatitis, nausea and diarrhoea, hepatotoxicity (fibrosis and cirrhosis), pneumonitis, nephrotoxicity (high dose), neurotoxicity (arachnoiditis with the intrathecal route), alopecia and skin rash, and teratogenicity (contraindicated in pregnancy). The toxicity is reduced by folic acid supplements, and severe toxicity is treated with leucovorin (folinic acid).

(a)(ii) Furosemide (5 marks)

Furosemide is a high-ceiling loop diuretic. It inhibits the Na⁺–K⁺–2Cl⁻ cotransporter (NKCC2) in the thick ascending limb of the loop of Henle, blocking reabsorption of about 25% of the filtered sodium.

Therapeutic indications. Acute pulmonary oedema and left ventricular failure, congestive heart failure, cirrhotic ascites, nephrotic and renal oedema, oliguria in acute and chronic kidney disease, hypertension with renal impairment, hypercalcaemia and hyperkalaemia, and forced diuresis in poisoning. It is also used with other measures in cerebral oedema.

Drug interactions.

• Aminoglycosides and cisplatin add to ototoxicity and nephrotoxicity.
• NSAIDs blunt its diuretic effect (prostaglandin inhibition) and add to nephrotoxicity.
• Digoxin toxicity is provoked by the hypokalaemia it causes. Corticosteroids, amphotericin B and laxatives add to potassium loss.
• It raises lithium levels, and adds to the effect of antihypertensives (with ACE inhibitors, first-dose hypotension). Probenecid reduces its tubular secretion and action.
• Thiazides give a synergistic sequential nephron block, with severe electrolyte loss.

Side effects. Hypovolaemia and hypotension, hypokalaemia, hyponatraemia, hypomagnesaemia and hypocalcaemia, metabolic alkalosis, ototoxicity (tinnitus and deafness, with high dose or rapid intravenous injection), hyperuricaemia (gout), hyperglycaemia, allergic reactions (it is a sulphonamide), interstitial nephritis and pancreatitis.

(b)(i) Antigen-presenting cells and antigen processing pathways (5 marks)

Antigen-presenting cells. Dendritic cells (the most potent professional APC), macrophages, B lymphocytes and Langerhans cells of the skin (a dendritic cell type), and Kupffer cells (macrophages). All express MHC class II and costimulatory molecules.

Antigen processing pathways.

• Endogenous (cytosolic) pathway, MHC class I: endogenous antigens (viral or tumour proteins made inside any nucleated cell) are tagged with ubiquitin and cut into peptides by the proteasome. The peptides are carried into the endoplasmic reticulum by the transporter TAP, loaded on MHC class I with β₂-microglobulin (with tapasin and calreticulin), and the complex reaches the surface through the Golgi. It is recognised by CD8⁺ cytotoxic T cells.
• Exogenous (endocytic) pathway, MHC class II: extracellular antigen is taken up by phagocytosis or endocytosis into endosomes and lysosomes and is broken into peptides by proteases. MHC class II, made in the endoplasmic reticulum with the invariant chain, reaches the endosome, where the invariant chain is cut to CLIP and HLA-DM exchanges CLIP for the antigen peptide. The complex goes to the surface, and is recognised by CD4⁺ helper T cells.
• Cross-presentation allows dendritic cells to present exogenous antigen on MHC class I as well.

(b)(ii) Effector T cells and Treg cells (5 marks)

Effector T cells (any two).

• CD4⁺ Th1 cells (transcription factor T-bet) secrete IFN-γ, activate macrophages against intracellular microbes and mediate delayed hypersensitivity.
• CD4⁺ Th2 cells (GATA3) secrete IL-4, IL-5 and IL-13, and defend against helminths and mediate allergy.
• CD4⁺ Th17 cells (RORγt) secrete IL-17, and recruit neutrophils against extracellular bacteria and fungi.
• CD8⁺ cytotoxic T lymphocytes kill virus-infected and tumour cells with perforin and granzymes and Fas ligand.

Treg cells. Regulatory T cells are CD4⁺ CD25⁺ cells that carry the transcription factor FOXP3. They arise in the thymus (natural Tregs), or in the periphery under TGF-β and IL-2 (induced Tregs). They suppress immune responses by secreting IL-10, TGF-β and IL-35, by CTLA-4 blocking the costimulatory signal, and by consuming IL-2 through CD25. They maintain self-tolerance and prevent autoimmunity, and FOXP3 mutation causes the IPEX syndrome.

(c) Sixteen-year-old boy with fever and a lytic bone lesion with sclerosis (10 marks)

(i) Diagnosis. The most probable diagnosis is haematogenous pyogenic osteomyelitis of the lower femoral (or upper tibial) metaphysis, presenting as a subacute Brodie's abscess (Staphylococcus aureus), seen as a lytic focus with a sclerotic rim, with fever and chills.

(ii) Pathogenesis.

• Bacteria (most often Staphylococcus aureus) reach the bone by the blood from a distant focus, and they settle in the metaphysis of the long bones near the knee in children. There the terminal vessels form sluggish hairpin loops and the phagocytic cover is poor.
• Suppurative inflammation and pus form. Since bone is rigid, the pressure of pus rises and causes severe throbbing pain, and pus spreads through the Haversian and Volkmann canals to the medulla and under the periosteum.
• The subperiosteal abscess strips the periosteum, cutting off the blood supply, and a piece of bone dies (sequestrum). The periosteum lays down new bone around it (involucrum), and pus may discharge through openings (cloacae) and skin sinuses.
• If the organism has low virulence or host resistance is good, the abscess is walled off by reactive sclerotic bone (Brodie's abscess), which explains the lytic focus with a sclerotic rim.

(iii) Complications (any four). Septicaemia with metastatic abscesses, septic arthritis (through the epiphysis), chronic osteomyelitis with sequestrum, involucrum and discharging sinus, pathological fracture, growth disturbance of the limb, secondary amyloidosis, and squamous carcinoma in a long-standing sinus (Marjolin's ulcer).

(d) Acute lymphoblastic leukaemia: laboratory diagnosis and immunophenotypic classification (10 marks)

Laboratory diagnosis.

• Blood count: anaemia (normocytic, normochromic), thrombocytopenia, and a total white count that may be raised, normal or low, with neutropenia.
• Peripheral smear: circulating lymphoblasts, small to medium cells with a high nuclear-cytoplasmic ratio, scant basophilic agranular cytoplasm, fine chromatin and inconspicuous nucleoli. In the FAB system L1 (small, uniform), L2 (larger, heterogeneous) and L3 (vacuolated, deeply basophilic, Burkitt type) are recognised.
• Bone marrow aspirate and biopsy: hypercellular, replaced by lymphoblasts, with 20% or more lymphoblasts required for diagnosis (WHO), and reduced normal haematopoiesis.
• Cytochemistry: myeloperoxidase and Sudan black B are negative (which separates it from AML), PAS gives coarse block positivity, and TdT is positive. Acid phosphatase is focally positive in T-ALL.
• Ancillary tests: flow cytometry (immunophenotyping), cytogenetics and molecular tests (t(12;21) ETV6-RUNX1 and hyperdiploidy are favourable, t(9;22) BCR-ABL1, t(4;11) KMT2A rearrangement and hypodiploidy are unfavourable), CSF examination for blasts, chest X-ray for a mediastinal mass (T-ALL), and biochemistry (raised LDH, uric acid).

Immunophenotypic classification.

• B-lineage ALL (about 85% of childhood cases): TdT⁺, CD19⁺, CD22⁺, CD79a⁺, HLA-DR⁺. The subtypes are pro-B (CD10⁻), common (CD10⁺, CALLA), pre-B (cytoplasmic μ chain⁺) and mature B (surface immunoglobulin⁺, Burkitt type).
• T-lineage ALL: TdT⁺, cytoplasmic CD3⁺, CD7⁺, CD2⁺ and CD5⁺. The subtypes are pro-T (CD7 and cytoplasmic CD3 only), pre-T (CD2, CD5, CD8), cortical T (CD1a⁺, CD4⁺ CD8⁺) and mature T (surface CD3⁺, CD4 or CD8).

(e) Thermal injuries and burns (10 marks)

Classification of thermal injuries (3 marks).

• By agent: injuries due to heat (dry heat burns from flame, radiant heat and hot metal, and moist heat scalds from hot liquids and steam) and injuries due to cold (frostbite, trench foot and hypothermia). Systemic heat effects (heat stroke, heat exhaustion, heat cramps) also belong here.
• By depth (Dupuytren's six degrees): first, erythema; second, vesicles (blisters); third, destruction of the epidermis and the superficial part of the dermis; fourth, destruction of the whole thickness of the skin; fifth, involvement of the deep fascia and muscle; sixth, charring of bone. In the modern scheme these are superficial, partial-thickness and full-thickness burns.

Estimating the body surface area burnt in an adult (4 marks). Wallace's rule of nines is used. The area burnt is the sum of the regions affected:

• Head and neck 9%.
• Each upper limb 9% (18% for both).
• Front of the trunk 18% and back of the trunk 18%.
• Each lower limb 18% (36% for both).
• Perineum and genitalia 1%.

The total is 100%. Only second-degree and deeper burns are counted. For small or scattered burns the patient's palm (about 1%) is used, and the Lund and Browder chart is used in children, because their head is proportionately larger.

Causes of death in burns (3 marks).

• Immediate (first 24 hours): neurogenic shock from severe pain and fright, asphyxia from smoke and carbon monoxide poisoning or laryngeal oedema in inhalation burns, and hypovolaemic (burn) shock from loss of plasma into the burn.
• Early (2 to 7 days): continued burn shock, acute renal failure, toxaemia and early infection, acute respiratory distress syndrome, and stress (Curling's) ulcer with gastrointestinal bleeding.
• Delayed (after a week): septicaemia (often Pseudomonas or staphylococcal), pneumonia, renal failure, pulmonary embolism, tetanus, and malnutrition and exhaustion, with contractures and Marjolin's ulcer as late complications.