Tutor Guide : Yr 1 Wk 15 "Astro Boy" Case Summary

This weeks work is about the basic functions of the immune system, triggered by a patient with compromised defences who has repeated infections. In addition to its obvious role in defence against bacterial infection, the immune system mediates the killing of body cells infected with viruses, cancer cells expressing abnormal proteins on their surfaces, and, in autoimmune disease, even normal cells. However, to begin at the beginning, defence against infection involves more than a functional immune system. The epithelial barriers of the skin, gut, airways, and urogenital system give defence against invasion. This is supported by specific adaptations such as the cilia of the airway sweeping debris away, the antibacterial substances secreted by glands (e.g. lysozyme in saliva), and mucus layers. Normal bacterial flora provides an environment hostile to pathogens: e.g. the acidity of the skin and female genital tract. Nonspecific immune defences is a term for processes which do not require specific recognition of the invader: such as inflammation, and phagocytosis by neutrophils and macrophages Specific immune defences involve recognition of antigen by immunoglobulin molecules and are mediated by lymphocytes. Additionally, the nonspecific process of phagocytosis is greatly enhanced by coating the bacterium with specific antibody (circulating immunoglobulin). Lymphocytes come in several varieties, but the most basic division is between B and T cells. The labels are historical, referring to early understanding of the immune system based on studies in birds. B stands for Bursa of Fabricius, a lymphoid organ associated with the hindgut: bursectomised chicks cannot make antibodies against injected bacterial antigens. In mammals, B cells originate in the bone marrow and circulate to the secondary lymphoid organs: the spleen, lymph nodes, tonsils, and lymphoid cells of the gut wall. B cells differentiate into plasma cells, which secrete immunoglobulins, and memory cells, which are responsible for the rapid response to a repeat dose of antigen. T cells in mammals leave the bone marrow to mature in the thymus before migrating to the secondary lymphoid organs. They come in a number of types. Cytotoxic T cells kill virus-infected body cells and some cancer cells without needing circulating antibody molecules to direct them. Helper T cells stimulate B cells by secretion of cytokines: without T cells, B cells cannot be activated properly. All patriotic Australians should be aware of the clonal selection theory which replaced the earlier instructional or template theory of antibody generation. The essence is that every possible antibody we can make (say up to 100 million) exists BEFORE exposure to an antigen, as a cell surface immunoglobulin molecule on a few cells which constitute one clone of B lymphocytes. If they never meet a complementary antigen, they just continue to lie in wait. But, on exposure to antigen, a process is triggered which results in multiplication of the cells of the clone, from a handful up to millions in number, and their differentiation into antibody-secreting plasma cells and memory cells. It is the 20 or so generations of cell division needed to bring the numbers up which accounts for the delay of about a week in developing a useful antibody response to a new antigen. Antigen presentation is an essential function in the generation of an immune response. When a macrophage engulfs a bacterium, it digests the proteins down to peptides and presents these on its surface, complexed with Class II MHC protein. The complex binds to the helper T cell receptor. Helper T cells produce cytokines which are needed to trigger proliferation of B cells as well as T cells. It is this step, the production of the cytokine Interleukin-2, which is blocked by the famously successful immunosuppressant cyclosporin that is used in transplant patients (cytotoxic T cells are the mediators of graft rejection). There is an elegant story about the generation of this huge number of possible antibody sequences: so called generation of diversity. The variable regions of the genes for immunoglobulin light and heavy chains

are assembled from 2 or 3 segments chosen out of many alternatives coded in the genome: a dozen D segments, a hundred or more VH segments Somatic point mutations also occur rapidly in the early development of B cell clones. Finally, there is the double-whammy of immunoglobulin structure: with say 1,000 heavy chains and 1,000 light chains one could assemble 1,000,000 different antibody specificities. All this occurs early in embryonic life, so we are born with the potential for every antibody we can ever make. The sharp paradox, which this case brings into focus, is this: a patient may have B cells, but be unable to produce antibodies because of a lack of functional T cells. This highlights the critical function of interactions between the B and T lines in mounting an immune response.

Triggers and Tutor Notes

Tutorial 1 : Trigger 1
PRESENTATION Timothy J. aged 8 months is brought to the Emergency Department with a two-week history of gradually increasing shortness of breath such that he becomes distressed on minimal exertion such as suckling.

Discussion Checklist
What are the important features of Timothys presentation? Why? Outline possible causes for his symptoms and explain the mechanisms underlying them. What information do you need to refine your hypotheses?

Tutor Notes
This is a good general brainstorm to start the tutorial. What does it take to cope with exertion? Respiratory and cardiac conditions are common causes of shortness of breath. The time course is interesting here. He was probably relatively well until recently, the way the history is presented. Thus, congenital conditions are less likely, and an infection or acquired process is probable (of course in this case we have the congenital immunodeficiency predisposing to an infection). The time course is also a bit long for a typical infection but in this case the patients inflammatory response to the infection is reduced. The students should be able to come up with a focussed enquiry strategy. Was he well at birth? Was there any congenital disorder detected? Is there any family history of genetic diseases: cystic fibrosis, immotile cilia syndrome, immunodeficiency? Any early deaths in the familly? In recent history: respiratory infections, URTI and ENT especially.

Learning Objective Questions

What are common causes of shortness of breath?

Tutorial 1 : Trigger 2
HISTORY

Timothy is the youngest of three children, and has a 10-year-old sister and 8 year old brother. His father, William is a commercial lawyer and his mother, Bernice, works part-time as an accountant. In response to your questions, Bernice tells you that Timothys birth was normal. His birth weight was 3.5 Kg and his APGAR score was 10. Despite being well initially, for most of the time since his birth, he has had a persistent nasal discharge. He has also failed to gain weight despite a good appetite for his mother's breast milk. At 5 months of age, Timothy had an upper respiratory tract infection, which proceeded to bronchitis but responded to antibiotics. At 6 months, he had otitis media, which also responded to antibiotics. The ear infection recurred two weeks later but responded again to antibiotics. Over the last 6 weeks, he has developed moderate diarrhoea of between 8 and 20 times/day. He has had five admissions to hospital since he was born, the most recent 3 weeks ago for IV fluid replacement.

Discussion Checklist
Which aspects of this history are relevant to Timothys current problem? Why? How does this information help you refine your hypotheses? Which ones are now the most likely? Explain. What information from the physical examination would help you? Are there any special features you would look for? Predict the findings for each of your hypotheses and explain your reasoning.

Tutor Notes
There are some solid clues here. The child has been seriously ill many times, and has had a number of episodes of infection. His diarrhoea is apparently quite significant since he has become dehydrated and needed IV fluids. These are pointers to more than one body system being involved: multiple infections in different systems might be the earliest clue to this week's theme of immunodeficiency. What factors could predispose to infections? He also has failure to thrive: this path has been well trodden in recent cases, so the students should be able to come up with several hypotheses including repeated or chronic infection. What features should be sought on examination? This will be limited by their clinical insight, but a good answer could include: 1. Temperature, pulse rate, respiratory rate, blood pressure, weight 2. Full Respiratory examination 3. Full CVS examination 4. Features of cystic fibrosis eg chronic liver disease, mucus plugging, steatorrhoea 5. Full ENT Examination 6. Abdominal Examination including PR and faeces examination 7. Nutritional assessment

Learning Objective Questions

What are the critical issues in taking a history (from carer) about an ill infant? What are the major causes of impaired immune defences?

Tutorial 1 : Trigger 3
EXAMINATION On examination, Timothys vital signs were: Respiratory rate 40 /min BP 100/60

Heart rate 160/min (Age-adjusted average:115/min) Temp 37.8 C Weight 6.5 Kg His weight was less than the first centile for his age. He was noted to be a wasted, irritable, cyanosed male infant with an excoriated red raised genital rash extending posteriorly to the anus. He had white plaques on his tongue and buccal mucosa and a muco-purulent discharge from his nose. There was no visible tonsillar tissue.

Auscultation confirmed an audible wheeze with widespread crackles over the lungfields. He had intercostal recession and a tracheal tug. His tympanic membranes were bilaterally inflamed. Abdominal examination was difficult and during the examination he was incontinent of 50mls of yellow liquid faeces. There were no signs of chronic liver disease. Timothy was admitted to intensive care. Samples were taken for lab investigations.

Discussion Checklist
Which of the above findings are most pertinent to your hypotheses? Why? Can you prioritise your list of hypotheses? What investigations would help you decide what is wrong with Timothy? What results would you expect if your principal hypothesis is correct? Why?

Tutor Notes
This is a sick distressed child. He has poor gas exchange as shown by cyanosis. The essence of his respiratory situation is that he has a severe problem with mechanics of moving air and with gas exchange, probably due to infection causing swelling of the airway tissues and increased secretions. The alveoli or the interstitial tissues of the lungs are involved as he shows crackling noises. He is severely ill and needs urgent treatment. The failure to thrive is a story they will handle well, as it has occurred in earlier cases. Here we see a chronic illness with an acute episode. Multiple infections could be the key, especially involving the gastrointestinal system. Other thoughts might include respiratory illness with poor feeding, diabetes, malignant disease and some haemopoietic diseases. Our leading hypotheses at this point must have the child with multiple organs involved in infective processes. This leads to the puzzle of what type of process could give rise to infections in multiple organs? It would be useful to get the students to think, for each impaired organ, what type of defences normally exist to protect against foreign insult? And then what general processes might impair several organs' defences. The skin rash plus white flecks on the tongue suggest colonisation by Candida - an opportunistic pathogen, striking where the environment, or the defences, are altered. They might pick up on the lack of tonsils. After they have argued it out, you can tell them that they were NOT surgically removed. The point comes up in a later trigger, where the thymus is missing. Interestingly, his fever is very modest for such a severe clinical situation with a pneumonia. It is possible his responses to the invaders are reduced, or that the organism is an unusual one, which does not trigger massive responses. The answer comes later. There are a huge number of investigations, both specific and general, which are performed in this situation

by experienced intensivists and paediatricians. The students should be able to identify some principles. We will need microbiology for sputum, nasal discharge, faeces, skin scrapings and oral scrapings. A chest X-ray is basic for assessment of the situation in the lungs, plus numerous (positive or negative) findings about the heart and great vessels, the skeleton, and the thymus, which is prominent in infants. We need electrolytes and blood gases. We need haematology for basic cell counts, and also subclasses of lymphocytes, immunoglobulin levels, complement components and neutrophil functions. They might also think of CF screen, or a glucose tolerance test.

Learning Objective Questions

What are the priorities in assessing an ill Infant? What are the signs of impaired defences?

Tutorial 1 : Trigger 4
INVESTIGATIONS Timothy's chest radiology showed bilateral diffuse opacities throughout both lung fields in an interstitial pattern. The radiologist commented that the pattern of interstitial pneumonitis was consistent with infection by Pneumocystis carinii. There was no thymic shadow seen on the chest film. Laboratory results showed: Microbiology: Nasopharyngeal aspirate: gram-negative bacilli on gram stain. Culture subsequently grew Pseudomonas aeruginosa. Faecal examination: No parasites seen. Latex agglutination for Rotavirus - positive. Oral, groin scrapings: Candida albicans

Dihydrorhodamine test 95% (>90%) DNA testing for cystic fibrosis: . negative Electron Microscopy of Cilia: . normal HIV antibody : . negative Serum biochemistry and blood gases were consistent with severe dehydration and respiratory failure requiring mechanical ventilation.

Discussion Checklist
How do these results affect your thinking about Timothys condition? What principles of treatment would underlie your acute management of this patient?

What further information (if any) do you need to fully understand Timothys condition? Justify your answer.

Tutor Notes
The onslaught of data contained in this and the next trigger might induce severe distress in the students, if not in us, the tutors! Firstly, summarizing the clinical situation, the child is very ill, with both upper and lower respiratory infection producing respiratory failure. As the students have not yet tackled the respiratory system, or gas exchange, this is downplayed. The focus is on these severe, multiple infections and why they might be occurring. The pattern of infections includes superficial fungal infections and enteric infection with rotavirus, which are typical in severe T cell immunodeficiencies. In passing, we discount CF, which they have recently studied, and would be a good starter, plus the immotile cilia syndrome. In addition to failure to thrive and multiple infections, infection with strange or otherwise non-pathogenic organisms is one of the clinical features of immunodeficiencies. Pneumocystis carinii is a fungus which has become known through its high incidence in AIDS, the most famous of the immunodeficiencies. The clinical picture for AIDS would be indistinguishable from SCID at this stage. Infants acquire HIV from infected mothers. HIV infects helper T cells by binding to the CD4 protein on the cell surface. Without the helper function an effective immune response cannot be mounted through either antibody or cell mediated functions.

Learning Objective Questions

What are the clinical features of immune dysfunction?

Tutorial 1 : Trigger 5
INVESTIGATIONS (cont'd) Timothy's haematology results were: Hb . . . . . 8.0 (10.0-14.0 g/dl) WCC . . . 4.8 (4.0-11.0) x10e9/l Lymphocytes . 0.76 (3.0-9.0) x 10e8/l Immunoglobulins: IgG . . 0.3 (3-8) g/l IgA . . 0.1 (0.2-1.0) g/l IgM . . 0.1 (0.3-1.3) g/l Lymphocyte subsets: CD3 . . . 0.0 . (3.4-4.6) x10e9/l . . (all T cells) CD4 . . . 0.0 . (2.6-3.5) x10e9/l . . (helper-inducer T cells) CD8 . . . 0.0 . (0.11-0.96) x10e9/l . . (cytotoxic/suppressor T cells) CD16 . . 0.7 . (0.07-0.53) x10e9/l . . (natural killer cells) CD19 . . 0.75 . (0.02-0.43) x10e9/l . . (all B cells) Complement activity/titre

C3 . . . 1.6 (1.1-2.1) g/l C4 . . . 0.31 (0.15-0.45) g/l CH50 . . 578 (520-660) U/ml

Discussion Checklist
How do these results affect your thinking about Timothys condition? What further information (if any) do you need to fully understand Timothys condition? Justify your answer.

Tutor Notes
Here we have solid evidence of the cause of Timothy's multiple-system problems. He is deficient in lymphocytes. The thymus, normally prominent on chest X-ray in infants, and the site of maturation of T lymphocytes, is missing or reduced in size. While this sign occurs in many severe illnesses, in this case it is a good clue for the students to the role of the thymus in lymphocyte function. He is severely deficient in immunoglobulins. As many of the students will know, antibodies are produced by the B cell line. However, the CD markers present a sharp puzzle, and this is the clue to the deepest understanding they will derive from this case. CD3 is a marker for all T cells; CD4 for helper-inducer T cells; CD8 for suppressor-cytotoxic T cells; CD16 for natural killer cells; and CD19 marks all B cells. This patient has plenty of B cells but their progeny are not producing antibody. The deficiency is in T cells, and the B cells are failing to respond to antigenic challenge because of a lack of helper T cell function. His neutrophils are able to produce the normal oxidative burst, which kills phagocytosed bacteria (dihydrorhodamine test), but in the absence of antibodies coating the bacteria (opsonisation), the phagocytic activity will be reduced. Complement, the multi-enzyme effector system responsible for punching holes in cell membranes (the membrane attack complex of five activated complement components) and triggering a number of aspects of the inflammatory response, is normal in this patient. CH50 is a biological assay that picks up defects in any of C1 through C9. This is the sharp end of the PBL. Here they need to formulate good LOs on the functions of T cells and B cells, helper cell function and antigen presentation, and the effector limbs of the immune system including the phagocytes and the complement system.

Learning Objective Questions

How do B cells proliferate and differentiate to manufacture antibody? What are the functions of the subsets of T cells? Why are helper T cells essential for an antibody response? How does antibody facilitate the phagocytic response? How is complement activated and what does it do?

Tutorial 2 : Trigger 1
PROGRESS Timothys results were consistent with Severe Combined Immunodeficiency Disease (SCID). Timothy required mechanical ventilation and treatment with multiple intravenous antibiotics and a topical antifungal. Intravenous feeding was instituted in an effort to improve his nutrition. He was transfused with a unit of cross-matched CMV-negative red cells and treated with 50 ml fresh frozen

plasma daily for four days. He made slow but steady progress on this regimen and came off the ventilator after seven days. Siblings and parents were HLA typed.

Discussion Checklist

Tutor Notes
Prompts: What is SCID? What immunological principles underlie its treatment? What do these tests indicate about Timothys prognosis and management? What is the likely impact on Timothys family of his condition? What community support services are available to help? The close relatives will be HLA typed since bone marrow transplantation is the only available treatment. By now the students will have met the HLA system, including the Class I expressed on all nucleated cells, and the Class II expressed on macrophages and other cells capable of antigen presentation (macrophage-like cells and B cells). Here the question is tissue compatibility for transplantation. The genetic map of the HLA system on chromosome 6 shows loci adjacent in each class and Class II separated from Class I only by the genes for Tumour Necrosis Factor and components of the complement system. Therefore, it is usual (98%) to find that the HLA complex is inherited intact as two haplotypes (one from each parent), so that siblings have a 1 in 4 chance of being HLA identical. In contrast, the chances of finding a match in unrelated individuals is many thousands to one against. Since the patient is immunoincompetent, donor lymphocytes in transfused blood could set up a graft versus host reaction. Ionizing radiation kills the lymphocytes (or renders them incapable of proliferation) while the red cells which have no nucleic acid suffer only minimal damage. Cytomegalovirus will infect the patient with a defective T system if present in the transfused blood. Molecular biology may prove useful in the detailed workup. It may identify the exact defect causing the SCID and thereby be useful for establishing whether the condition is inherited or a new mutation. A family history of early infant loss is also important in this regard. It is most likely that the patient has X-linked SCID because he had normal numbers of B cells and an absence of T cells. Thus it was likely the B cells were present but non-functioning as evidenced by the very low immunoglobulins. At this stage of the week, the group will be trying to synthesise a coherent picture of the immune system, without losing themselves in the infinity of detail. That is quite a challenge.

Learning Objective Questions

What are the avenues of treatment for immunodeficiencies? Which are the essential functions to try to improve or replace? What are the HLA antigens? What is the difference between Class I and Class II MHC?

Tutorial 2 : Trigger 2
PROGRESS (contd)

Timothy continued to respond well to treatment. One month following admission, he was healthy enough to have a bone marrow transplant. His 10 year old sister, Jodie, was found to have the closest match in HLA antigens, but her mother expressed concerns about the donation. "Can't I donate instead, rather than putting her through it?" she asked.

Discussion Checklist
How would you answer Mrs. J's question?

Tutor Notes
Prompts: Will Timothy require ongoing long-term therapy? If so, what and why? What is the likelihood that Timothy will be able to lead a normal life? There is a lot of technical detail in the assessment and management of a patient through a bone marrow transplant. As with any transplant, if the match is imperfect, rejection is a possible problem. With bone marrow, the Graft vs. Host response is a major risk. If the donor were a perfect match then no preconditioning would be required. Since the donor is haploidentical, preconditioning with cyclophosphamide will be necessary followed by cyclosporin posttransplant to prevent rejection. Timothy should have CMV prophylaxis with acyclovir at the time of transplant if either he or his donor sister are positive for CMV. CMV surveillance should occur with antigen testing and CMV-PCR testing. He is treated prophylactically with itraconazole and then post-transplant with nilstat and chlorhexidine mouth washes (fungi), norflox whilst neutropaenic, and once engraftment occurs with cotrimoxazole (pneumocystis). The long-term management involves regular review, however, the prognosis after a successful transplant is excellent.

Learning Objective Questions

What determines whether the BMT graft will attack the host cells? Which parts of the immune system are reconstituted by BMT?

Tutorial 3 : Trigger 1
CLOSURE By three weeks there was definite evidence of engraftment based on the rising lymphocyte count. By three months there was complete engraftment so that all the immunology tests had returned to normal. Timothy was now at the 10th centile for height and weight.

Discussion Checklist
nil

Tutor Notes

Learning Objective Questions