Download Case #1 At 3 years old, Daisy Miller was admitted to the Boston

Case #1
At 3 years old, Daisy Miller was admitted to the Boston Children’s Hospital with pneumonia.
Her mother had taken her to Dr. James, a pediatrician, because she had a fever and was
breathing fast. Her temperature was high, at 40.1°C (104.2°F), her respiratory rate was 40 per
minute (normal 20), and her blood oxygen saturation was 88% (normal >98%). Dr. James also
noticed that lymph nodes in Daisy’s neck and armpits (axillae) were enlarged. A chest X-ray
was ordered. It revealed diffuse consolidation (whitened areas of lung due to inflammation,
indicating pneumonia) of the lower lobe of her left lung, and she was admitted to the
hospital.
Daisy had had pneumonia once before, at 25 months of age, as well as 10 episodes of
middle-ear infection (otitis media) that had required antibiotic therapy. Tubes had been
placed in her ears to provide adequate drainage and ventilation of the ear infections.
In the hospital a blood sample was taken and was found to contain 13,500 white blood
cells/ml, of which 81% were neutrophils and 14% were lymphocytes. A blood culture grew
the bacterium Streptococcus pneumoniae.
Because of Daisy’s repeated infections, Dr. James consulted an immunologist. She tested
Daisy’s immunoglobulin levels and found that her serum contained 470 mg/dl of IgM (normal
40-240), undetectable IgA (normal 70-312), and 40 mg/dl of IgG (normal 639-1344). Although
Daisy had been vaccinated against tetanus and Haemophilus influenzae, she had no
specific IgG antibodies against tetanus toxoid or to the polyribosyl phosphate (PRP)
polysaccharide antigen of H. influenzae. Because her blood type was A, she was tested for
anti-B antibodies. Her IgM titer of anti-B antibodies was positive at 1:320 (upper limit of
normal), whereas her IgG titer was undetectable.
Daisy was started on intravenous antibiotics. She improved rapidly and was sent home on a
course of oral antibiotics. Intravenous immunoglobulin (IVIG) therapy was started, which
resulted in a marked decrease in the frequency of infections.
Analysis of Daisy’s peripheral blood lymphocytes revealed normal expression of CD40L on T
cells activated by anti-CD3 antibodies, and normal expression of CD40 on B cells.
Nevertheless, her blood cells completely failed to secrete IgG and IgE after stimulation with
anti-CD40 antibody (to mimic the effects of engagement of CD40L) and IL-4, a cytokine that
also helps to stimulate class switching, although the blood cells proliferated normally in
response to these stimuli. cDNAs for CD40 and for the enzyme activation-induced cytidine
deaminase (AID) were made and amplified by RT-PCR on mRNA isolated from blood
lymphocytes activated by anti-CD40 and IL-4. Sequencing of the cDNAs revealed a point
mutation in the AID gene that introduced a stop codon into exon 5, leading to the formation
of truncated and defective protein. The CD40 sequence was normal.
1) What is the name of the syndrome that Daisy has?
Hyper IgM syndrome. This is a group of gene disorder which is characterized by defects in
CD40 signaling process.
2) How does an AID deficiency contribute to this syndrome?
AID deficiency contribute to the syndrome by defects caused during gene encoding process. Deficiency
in AID lead to no conversion of cytidine to uridine hence no DNA breakage and repair process.
3) What is causing her to have so many bacterial infections?
Daisy is having so many infections because the body is incapable of producing enough
antibodies to counter the infections making him susceptible to bacteria diseases.
4) Why is Daisy placed on IVIG? What is that and why would it help her?
IVIG is a drug that is administered to treat variety of disorders resulting from deficiency in
antibody production it is a blood product which is made up of immunoglobulins refer to us antibodies
which helps in protecting Daisy against germs, virus and bacterial infections. Daisy was placed under
the drug because he lacked enough antibodies in her blood
5) What other role does AID play in generating antibody diversity?
Aid plays and important role in somatic cell mutation and class switch combination.
The Function of AID in Somatic Mutation and Class Switch Recombination
Case #2
At birth, Ricardo seemed to be a normal, healthy baby. He gained weight normally and
cried vigorously. Soon after birth, however, his mother noticed that he had 10 loose bowel
movements a day. On the 17th day after birth, he developed a rash on his legs, which over
the next 7 days spread over his entire body. His parents brought him to the ER at Children’s
Hospital, and also reported that he had a dry cough for the past week.
On physical exam, Ricardo’s weight, length, and head circumference were normal. The
diffuse popular scaly rash was worst on his face, but also covered his trunk and extremities.
Small blisters were present on his palms and the soles of his feet, which were red. He had
purulent conjunctivitis (yellow discharge from his eyes), his eardrums were normal, no lymph
nodes could be felt, and his heart and lungs were normal. The liver and spleen were not
enlarged.
Ricardo’s parents had three normal children, but had had two other children, a boy and a
girl, who had died soon after the onset of a similar rash at 1 month old. The parents were first
cousins of Portuguese extraction.
Ricardo was admitted to the hospital. Blood tests showed that his hemoglobin was 8.4 g/dl
(low), his platelet count was 460,00 (slightly elevated), and his white blood cell count was
8000/µl (normal), of which 56% were eosinophils (normal <5%), 23% monocytes (normal 10%),
15% neutrophils, and 6% lymphocytes (normal 50%). An examination of his bone marrow
revealed a preponderance of eosinophil precursors. Ricardo’s serum IgG level was 55mg/dl
(normal 400 mg/dl), IgA and IgM were undetectable, and IgE was 7200 IU/ml (normal <50
IU/ml). A skin biopsy showed that the dermis was infiltrated with large numbers of eosinophils,
lymphocytes, and macrophages. Large numbers of cells surrounded the blood vessels. An Xray of Ricardo’s chest showed clear lungs and a normal cardiac shadow; there was no
thymic shadow.
In the hospital, Ricardo’s condition rapidly worsened. He developed enlarged lymph nodes
in the neck and groin, and pus accumulated in the skin behind his ear. This was drained, and
Staphyloccus aureus and Candida albicans were cultured from the drainage fluid. Thrush (C.
albicans) was noticed in his mouth. An immunologist was consulted. He ordered blood tests
that revealed an absence of B cells and a paucity of T cells. Ricardo’s peripheral blood
lymphocytes responded poorly to stimulation with phytohemagglutinin and with anti-CD3
monoclonal antibodies. On FACS (flow cytometry) analysis, no cells were found that reacted
with anti-CD19, which detects B cells. All the lymphocytes were CD3+, of which 90%
coexpressed the activation marker CD45R0, and 65% expressed MHC II molecules, another
marker of T cell activation. Eighty percent of the lymphocytes were CD4+, and 15% were
CD8+. Flow-cytometry analysis of Ricardo’s peripheral T cells, using monoclonal antibodies
directed against various families of T-cell receptor sequences showed that only a few of
them were expressed, indicating an oligoclonal T-cell receptor repertoire. The RAG1 and
RAG2 genes were sequenced, and homozygosity for the Arg222Gln mutation was found in
the RAG2 gene. The T cells were definitively identified as Ricardo’s (and not as transferred
maternal T cells) by HLA typing.
While these studies were being carried out, Ricardo developed Pneumocystis jirovecii
pneumonia and died of respiratory failure. 
1) Summarize Ricardo’s symptoms below. What is(are) the overall defect(s) in the immune
response?
Ricardo heard lose bowel in a day and developed rush in his legs which spread to the entire body
within a week. He also heard dry cough. Small blisters were noticed in his arm and soles on his feet
which were red in color. The child had some discharge in his eyes which were yellow. Dermis layer of
the skin was infiltrated with eosinophils, macrophages, and lymphocytes. The patient has missense gene
mutation in the RAG genes leading to partial enzyme expression. The missense mutation in the RAG
genes allowing development of few T cells and B lymphocytes. Low production of T cells in the thymus
leads to impaired of growth and maturation of and epithelial cells and delayed AIRE expression. This
expression then impinges deletion of reactive T cells leading to decreased body immunity. Due to
reduced T cells generation by Ricardo immune system, there is high peripheral expansion, high
cytokines, and inflammatory which led to formation of bowels and sores.
2) What does it mean to have an Arg222Gln mutation in the RAG2 gene? What does Arg222Gln
specifically refer to? How would a mutation in a RAG gene contribute to Ricardo’s symptoms?
Arg222Gln is missense mutation in the in the RAG2 gene that help in determining gen evolution
and presence of T cells. To have Arg222Gln mutation in the RAG2 genes mean easy identification of T
cells in the child blood. The partial disappearance of RAG genes functions leads to severe immune
deficiency syndrome which made Ricardo prone to opportunistic infections. RAG genes are important
in gene restructuring and recombination of the T cells and B cell receptors. If the genes losses these
abilities, the immune system will not be capable of recognizing any attacking pathogens.
3) What is the name given to this syndrome?
This is Omenn syndrome. The disease is characterized by red skin, hair loss, failure grow and
thrive, swollen lymph nodes, sore in the body, and elevated serum. The patients suffering from this
immune disease are susceptible to diseases or infections and may develop both fungal, viral and bacteria
related diseases. The virus is caused by RAG genes mutation. There are elevated number of T cells with
their role impaired.
4) Is there a treatment for this syndrome?
Omenn syndrome can only be treated through bone marrow transplant. The patient should be put
under nutritional care and meticulous skin care supplemented by better hygiene practices before the
bone marrow transplant is done. The process must involve matching of the sibling with the maternal
5) How does Ricardo’s family history help you determine the mode of inheritance of this
syndrome?
Ricardo’s had no had no Omenn syndrome. Ricardo brothers had a brother and a sister who experienced
the same problem and died when they were young. Presence of the syndrome the children shows Mendelian
autosomal recessive transfer of the defects. This means if the defects was connected to X- linked genes, Ricardo
sister would not have been affected. In the case of dominant genes, one of the parents would have suffered from
the syndrome.
Case #3
Helen Burns was the second child born to her parents. She thrived until 6 months of age
when she developed pneumonia in both lungs, accompanied by a severe cough and fever.
Blood and sputum cultures for bacteria were negative, but a tracheal aspirate revealed the
presence of abundant Pneumocystis jirovecii. She was treated successfully with the antiPneumocystis drug pentamidine and seemed to recover fully.
As her pneumonia was caused by the opportunistic pathogen, P. jirovecii, Helen was
suspected to have severe combined immunodeficiency. A blood sample was taken and her
peripheral blood mononuclear cells were stimulated with phytohemagglutinin (PHA) to test
for T-cell function by 3H-thymidine incorporation into DNA. A normal T cell proliferative
response was obtained, with her T cells incorporating 114,050 counts/mi of 3H-thymidine
(normal control 75,000 counts/min). Helen had received routine immunizations with orally
administered polio vaccine and DPT (diphtheria, pertussis, and tetanus) vaccine at 2 months
old. However, in further tests her T cells failed to respond to tetanus toxid in vitro, although
they responded normally in the 3H-thymidine incorporation assay when stimulated with
allogeneic B cells (6730 counts/min incorporated, in contrast with 783 counts/min for
unstimulated cells).
When it was found that Helen’s T cells could not respond to a specific antigenic stimulus, her
serum immunoglobulins were measured and found to be very low. IgG levels were 96 mg/dl
(normal 600-1400), IgA was 6 mg/dl (normal 60-380), and IgM 30 mg/dl (normal 40-345).
Helen’s white blood cell count was elevated at 20,000 cells/µl (normal range 4000-7000). Of
these, 82% were neutrophils, 10% lymphocytes, 6% monocytes, and 2% eosinophils. The
calculated number of 2000 lymphocytes/µl was low for her age (normal >3000/µl). Of her
lymphocytes, 27% were B cells as determined by an antibody against CD20 (normal 10-12%),
and 47% reacted with antibody to the T-cell marker CD3. In particular, 34% of Helen’s
lymphocytes were positive for CD8, and 10% were positive for CD4. Thus, at 680 cells/µl her
number of CD8 T cells was within the normal range, but the number of CD4 T cells (200/µl)
was much lower than normal (her CD4 T cell count would be expected to be twice her CD8
T cell count). The presence of substantial numbers of T cells, and thus a normal response to
PHA, ruled out a diagnosis of severe combined immunodeficiency.
Helen’s pediatrician referred her to the Children’s Hospital for consideration for a bone
marrow transplant, despite the lack of a diagnosis. When an attempt was made to HLA-type
Helen, her parents, and her healthy 4-year-old brother by serology, a DR type could not be
obtained from Helen’s white blood cells. Her circulating B lymphocytes were transformed
with Epstein-Barr virus (EBV) to establish a B-cell line, which was then analyzed by flow
cytometry. The EBV-transformed B lymphocytes did not express HLA-DQ or HLA-DR molecules.
Hence, a diagnosis of MHC class II deficiency was established.
Her brother was found to have the same HLA type as Helen, and therefore was chosen as a
bone marrow donor. Helen was given 1mg/kg of body weight of the cytotoxic drug busulfan
every 6 hours for 4 days and then 50mg/kg cyclophosphamide each day for 4 days to
ablate her bone marrow. The brother’s bone marrow was administered to Helen by
transfusion without any in vitro manipulation. The graft was successful and immune function
was restored
1) Why is a deficiency in MHC II a problem? What is the role of MHC II in the normal immune
response?
This problem is inherited by autosomal recessive feature. It health complications show up in
early childhood development period. The deficiency creates a problem because it weakens the immune
system making the child susceptible to both pathogens and opportunistic infections. In a normal person,
MHC II help in determining histocompatibility between two people. In human cell, they are responsible
for presenting pathogen defined peptides to T cell
2) Why did Helen lack CD4 T cells in her blood?
Helen lacked CD4 T cells in her blood because there was no MHC II class was presented for
selection in the thymus region hence no CD4+ cells managed to pass this point or stage.
3) Why did Helen have a low level of immunoglobulins in her blood?
The low level of immunoglobulins was as result of very few IgA molecule productions caused
by lack of CD4 T cells.
4) What are HLA-DR and HLA-DQ molecules?
HLA-DR is a receptor of MHC II which is encoded by leukocyte antigens complex on
chromosome 6. The molecule gives instructions for making proteins that play a major function in body
immune system. HLA-DQ this gene encodes a section of protein receptor. They are of two types
designated A and B which bind together forming gluten
5) Why would a bone marrow transplant cure Helen?
Helen was cured by the bone marrow transplant. Hematopoietic stem cell transplantation is the only
solution patient with the deficiency have.
6) Why was Helen placed on busulfan and cyclophosphamide prior to bone marrow transplant?
What does each of these drugs do?
Busulfan is used to sulfonate groups that are linked to the N7 of guanines to form two strands DNA and
then cross link them. The drug is used before transplant case of Helen to held in preconditioning bone
marrow transport because of suppression. Cyclophosphamide is used to increase DNA repair hence
increasing glutathione