1. IMMUNODEFICIIENCY DISEASES
Dr. Nehad Mohammad Sayed
Professor of Microbiology&Immunology
Ain Shams University

2. Define primary and secondary immune deficiencies.
Explain the immunologic defect and presentation of clinical conditions resulting from primary immunodeficiency disorders.
Solve problems concerning defects resulting from primary immunodeficiency disorders

3. A diverse spectrum of illnesses due to various abnormalities of the immune system .
Patients with immunodeficiency diseases are more susceptible to infections and cancers.
The type of infection may give an idea about the site of defect in immune system e.g . ????

5. Clinical manifestations
Increased susceptibility to infections
Diagnosis of ID is considered when infections are:
1. Frequent and severe..
2. Unusually resistant to therapy ..
3. Caused by unusual ( opportunistic ) organisms .
It is critical to maintain an index of suspicion
for diagnosis of I.D.
Early diagnosis reduce morbidity..

6. CLASSIFICATION A. PRIIMARY ( CONGENIITAL) DEFECTS. Heritable disorders
B. SECONDARY (ACQUIIRED) DEFECTS.
The incidence of primary immunodeficiency is lower than secondary immunodeficiency

7. Primary (congenital) or Secondary (acquired)
lesions may affect :
1. Natural immune components:
Phagocytic cells.
Complement system. OR
2. Acquired immune components:
T- lymphocytes.
B- lymphocytes.

8. A-Phagocyte deficiencies QUANTITATIVE OR QUALITATIVE
Quantitative defects:
1. Congenital agranulocytosis:
Defect in the gene inducing G-CSF
Respond to G-CSF therapy.

10. phagocytic function defects
Defect in response to chemotactic agents.
1- Leukocyte adhesion deficiencies: Decreased expression of adhesion molecules on the surface of leukocyte causes failure of leukocyte migration to site of infection.
Leukocytes are trapped in the circulation.
Leukocyte count can reach 100,000
cells per mm3.
Abscesses do not suppurate

11. 2-Defect in intracellular killing:
a- Defect in lysozymes (Chediak Higashi syndrome):
. Defective lysosomal function in neutrophils, macrophages, and dendritic cells, and defective granule function in natural killer cells. It is characterized by the presence of giant lysosomes with defective phagosome lysosome fusion leading to diminished intracellular killing. Associated with: Abnormal platelet function.
Partial albinism .

12. B.Defective production of reactive oxygen species (chronic granulomatous disease):
X-LIINKED.. (75%) or AUTOSOMAL RECESSIIVE ..(25%)..
Defective production of reactive oxygen species (e.g NADPH oxidase deficiency) results in failure to kill phagocytosed microbes with recurrent bacterial and fungal infections.
Granulomatous inflammation (chronic T-cell stimulation)

13. Chronic antigenic stimulation of macrophages by surviving intracellular bacteria invites T lymphocytes. Continuous release of cytokines from sensitized T lymphocytes, leads to accumulation of large number of activated macrophages with formation of granuloma hence the name chronic granulomatous disease.

14. CGD patient with skin infections due to Serratia marcescens

15. Complement deficiencies
Deficiency of all complement components have been described C1-C9.

17. 1- Deficiencies of Early Complement Components
1- Deficiencies of Early Complement Components. The early components of complement are particularly important in generating the opsonin, C3b. Patients with deficiencies of C1, 4, or 2 from the classical pathway or with deficiency in C3 itself have increased infections with encapsulated organisms (S. pneumoniae, Streptococcus pyogenes, H. influenza) and increased incidence of immune complex diseases. The reason for the latter is the improper clearance of immune complexes as a consequence of low C3b generation, and often the autoimmune disease is most striking.

18. 2- C1 inhibitor (C1 INH) deficiency (hereditary angioneurotic oedema):
Without this inhibitor, uncontrolled activation of classical pathway with generation of large amounts of vasoactive mediators (kinins) that increase blood vessel permeability. Patients suffer from localized edema at mucosal surfaces which becomes life threatening when it occurs in the larynx obstructing the airway passages.

19. C1 - inhibitor deficiency: hereditary angioedema
Laboratory findings revealed that serum levels of the early components of classical pathway of the complement (C1, C2, C4) are decreased due to their overuse.

20. 3- Deficiencies of the late complement components (C5–C9) interfere with the generation of the membrane attack complex (MAC). The MAC is directly lytic and responsible for the primary defense against Gram-negative bacteria, particularly Neisseria meningitides.

23. B- cell (humoral ) deficiencies
1. Diverse spectrum of diseases ranging from:
Complete absence of B-cells , Plasma cells and Immunoglobulin 
Selective absence of certain immunoglobulin classes.
2. X- linked disease :
Female carriers normal .
Males manifest the disease .
3. Severity of the disorder parallels the degree of the deficiency .

24. Patients with B-cell defects are subject to:
Recurrent bacterial infections.
but
Display normal immunity to most viral & fungal infections because-cells are unaffected.

25. 1.X- linked agammaglobulinaemia (XLA). Bruton’s Disease
Failure of bone marrow to generate mature B-cells. The disease is recognized in boys in the late first year of life (after maternal immunoglobulins decreased). Patients suffer from recurrent pyogenic infections.
The disease is diagnosed by:
 Reduced or absent B cells in blood and lymph nodes.
 Reduced or absent immunoglobulins of all classes.

26. Management
Periodic intravenous immunoglobulin (IVIG) reduces infectious complications.

27. 2-Selective immunoglobulin deficiencies
A. IgA deficiency
Most are asymptomatic. ( but have
increased rate of (R.T.I.)
Some have recurrent R.T.I. and G.I.T. symptoms .
Increased incidence of allergic manifestations.
low levels of serum IgA with normal levels of IgM and IgG

28. 3-X-linked hyper- IgM syndrome.. (HXM)
Defect in T cell dependant B cell activation
Mutations in gene encoding the CD 40L in T- cells which is required for class switching from IgM to IgG and other immunoglobulin classes to occur
* No co-stimulatory signal for B-cells.
* No class – switching.
* No memory cells
Characterized by :
Low IgG, IgA & IgE.
Markedly elevated IgM.
Recurrent infections .

29. Characterized by :
Low IgG.
Markedly elevated IgM.
Patients will also show defect in cell mediated immunity.
Recurrent infections .

30. 4-Common variable immunodeficiency (CVID): is a heterogeneous group of disorders characterized by poor antibody responses to infections and reduced serum levels of IgG, IgA, and often IgM. The underlying causes of CVID include defects in various genes involved in B cell maturation and activation. Patients have recurrent infections, and lymphomas.

31. T-cell deficiency Di George Syndrome (congenital thymic aplasia )
Characterized by :
Absence of the Thymus gland .
Hypoparathyroidism..
Cardiovascullar abnormalities.
Characteristic facial features .

33. Defect in Di GEORGE syndrome
Failure of the third & fourth pharyngeal pouches to develop .
Features :
Extreme susceptibility to mycobacterial,
viral , protozoal, and fungal infections.
Profound depression of T-cell numbers
Absence of T-cell responses.Give negative delayed skin hypersensitivity tests.

34. Di George, (cont.)
B cells function is altered due to absence of T cell help but the phagocyte function is normal.
Treatment is by thymic transplant

35. B Cell Deficiency
Pyogenic bacteria (otitis, pneumonia, meningitis, osteomyelitis), enteric bacteria and viruses, some parasites
Serum Ig levels Reduced
DTH reactions to common antigens
Normal
Absent or reduced follicles and germinal centers (B cell zones)

36. T Cell Deficiency
Many viruses, atypical mycobacteria, fungi
Diagnosis
Serum Ig levels Normal or reduced
DTH reactions to common antigens
Reduced
Usually normal follicles, may be reduced para-follicular cortical regions (T cell zones)

37. Combined B cell and T cell deficiency:
Severe combined immunodeficiency (SCID)
 Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders characterized by profound abnormalities in T and B lymphocytes, natural killer cell development and function.

38. 2. Severe combined immunodeficiency (SCID).
Children characteristically present with failure to thrive, recurrent infections, and increased susceptibility to opportunistic infection.
The age of presentation is variable but occurs typically between 3 and 6 months when the protective effect of maternally transmitted Ab decreased.
Lymphocytes fail to develop .
Markedly depressed T-cells & B-cells.

39. Vaccination with attenuated live virus could prove fatal in infants with SCID.
Without bone marrow transplantation, most of these conditions are incompatible with long term survival.

40. Adenosine deaminase deficiency (ADA) is one of the most common forms of severe combined immunodeficiency (SCID). Deficiency in expression of the ADA enzyme which leads to the accumulation of toxic purine metabolites in cells . Since developing lymphocytes are particularly sensitive to ADA deficiency B- and T-cell numbers are impacted

41. Clinical approach to suspected immunodeficiency
1.History.
* Infections of unusual frequency,
Chronicity or severity .
* Family history of infectious problems..
Consanguinity should be investigated
(inter-family marriages ).

42. 2.Physical examination.
* Absence of tonsils.
* Partial albinism.
* Telangiectasia ( bleeding capillaries ).

43. 3.Radiologic evaluation ..
* Absence of thymic shadow ..

44. Therapy of immunodeficiency
1. IVIG .( IV infusion of immunoglobulin)
For : a. agammaglobulinaemia ..b.. WAS..
2. Periodic antibiotic treatment.
3. Bone marrow transplantation .
For : a. SCID .
4. Enzyme replacement .
For : ADA deficiency.

45. 5. G-CSF.(colony stimulating factor )
For : neutropenia .
6. Thymus transplantation ;For : DiGeorge syndrome.
7. IFN – gamma; For : CGD.

46. Secondary immunodeficiency
One is born with normal immune responses but immunosuppression may occur later on as a complication of another disease process or drug intake.
Causes:
1- Malnutrition.
2- Bone marrow tumors.
3- Infections e.g. HIV and HTLV-1. Both infect CD4+T helper cells which may be the base for immunosuppression.

47. 4- Chronic diseases: as diabetes mellitus and renal failure
5- Iatrogenic immunosuppression :
Immunosuppressive drugs as corticosteroides and cyclosporines.
Cytotoxic drugs used in cancer chemotherapy cause a decreased production of white blood cells precursors in the bone marrow.
Irradiation: exposure to X-rays and gamma rays causes a decrease in production of lymphocyte precursors in the bone marrow.
Spleenectomy

48. Immunological changes in AIDS:
There is progressive decrease in number and function of CD4+ T helper lymphocytes. The function of other cells of immune system may be also altered

49. A-Decrease in CD4 +T helper number is attributed to:
1- Direct lysis of infected cells by the virus
2- Antibodies may bind to HIV infected cells inducing antibody dependant cell mediated cytotoxicity
3- CTL may directly kill CD4+ T cells
4- Chronic activation of T cells (uninfected with HIV) by infections common in HIV patients may predispose to apoptosis

50. B- Impaired CD4+ T helper function:
HIV virus binding to CD4 receptor delivers a signal that down regulates T cell function via:
 Decreasing secretion of IL-2 and interferon γ.
 Loss of the ability of CD4+ cells to interact with MHC molecules on APC.

51. C- Impaired function of macrophage and dendritic cells:
 HIV virus can infect other cells carrying CD4 as macrophages and dendritic cells. These cells are not generally killed by the virus but they become a reservoir for the virus with impaired antigen presentation and cytokine secretion.

52. D- Other affected components of the immune system are B lymphocytes (produce excessive inappropriate functioning antibodies) and natural killer cells (defective killing abilities)

53. Evaluations of Immunodeficiency
Lymphocytes (T & B):
Quantitative:
-MoAb : immunofluorescence or flow cytometry
-Total & differential leucocytic count
-For immunoglobulins : RID / ELISA

54. Tests for Phagocytic cells:
Phagocytic cells in blood are (neutrophils and monocytes)
A- Tests for the number
1- Total and differential leukocytic count.
2- Number of monocytes can be assayed by flowcytometry using fluorescein labeled monoclonal antibodies (MABs) directed against a surface marker CD14.

55. PRINCIPLE OF FLOW CYTOMETRY

56. B- Tests for the function
I- Tests for random motility and chemotaxis:
The modified Boyden chamber assay is based on a chamber of two compartments separated by a microporous membrane.
 Tested cells are placed in the upper compartment and a chemotactic chemical substance is put in the lower compartment.
The number of cells that have migrated to the lower side of the membrane is determined

57. Modified Boyden chamber assay

58. II-Tests for ingestion:
Incubate white blood cells with heat killed yeast or Staph aureus and count the ingested particles by microscope.
III- Tests for intracellular killing:
Defects in oxygen dependent intracellular killing as in congenital NADPH oxidase deficiency is detected by Nitroblue tetrazolium test.

59. In this test a blood sample is taken from the patient
In this test a blood sample is taken from the patient. Neutrophils are isolated from blood and stimulated with LPS to produce superoxide, then NBT dye (yellow) is added.
 Cells with normal phagocytic function produce superoxide with reduction of NBT to blue insoluble crystals that are readily detected within the phagocyte microscopically

60. Nitroblue tetrazolium dye reduction test:

61. 2- Tests for complement:
A- Measurement of the amounts of individual complement factors by radial immunodiffusion or ELISA.
B- Functional assay for the complement:
The hemolytic activity of the complement is the only assay of complement activity available in the clinical laboratory

62. 3- Tests for B lymphocytes:
A- Tests for the number
Counting the number of B cells in peripheral blood and lymph node biopsy by flowcytometry using fluorescein labeled MABs directed against B cell surface markers (anti-CD19 & anti-CD 20).
B- Tests for the function
Serum levels of antibody are evaluated by ELISA, radial immunodiffusion and immunoelectrophoresis. (after vaccination)

63. 4- Tests for T lymphocytes
A- Tests for the number
Lymphocytic count (T cells are the major population of circulating blood lymphocytes).
Flowcytometry using fluorescein labeled MAbs directed against T cell surface markers (anti-CD3).
Counting T cell subsets using fluorescein labeled anti-CD4 and antiCD8.
Lymph node biopsy .

64. B- Tests for the function
1- Delayed hypersensitivity skin testing using candidin antigen or tuberculin. Absence of reaction suggests impairment of cell mediated immunity.
2- Assessment of circulating cytokines levels by ELISA.
3- Lymphocyte proliferation and blast transformation assays.

65. B- Tuberculin test:

66. Lymphocyte proliferation assays
In this technique, lymphocytes are isolated from peripheral blood and placed with mitogen for 48 to 72 hours.
A radionucleotide (such as H-thymidine) is added.
Proliferation is measured by detection of radioactive cells (dividing cells use more radionucleotide).

67. Tests for Cytotoxic T-lymphocyte function
CD 8+ T cells of the patient are incubated with foreign cells labeled with radioactive sodium chromate (a substance that readily crosses the cell membrane of live cells and binds to cytoplasmic proteins in the cell).
o Within 4 hours, CD8+ T cells specifically destroy these labeled cells causing the release of radioactive chromate into the culture medium.

68. 2-A 4 years old boy was brought to you because of repeated boil lesions arising in his body in different occasions and resolved after antibiotic intake. His Investigations revealed normal immunoglobulin levels, normal B and T cell count and function, normal complement level and negative nitoblue tetrazolium test. What do these findings mean?
a- Chronic granulomatous disease
b- Defective neutrophil chemotaxis
c- Digeorge syndrome
d- Normal finding
e- X linked infantile agammaglobulinaemia

69. Which of the following finding in the serum support the diagnosis of hereditary angioneurotic oedema?
a-Elevated C6
b-Depressed C4
c-Depressed C6
d-Elevated C1
e-Elevated C4

70. A newborn was evaluated for immunologic function
A newborn was evaluated for immunologic function. he has distorted shape of mouth, malformed ears and widely spaced eyes. Radiologically there is evidence of cardiac malformation and absence of thymic shadow. Which of the following parameters would be normal in this child?
a-Antibody dependent cell mediated cytotoxicity
b-Presence of T cells in peripheral lymphoid tissues
c-Generation of oxygen metabolite in phagocytic cell
d-Killing of viral infected cell by cytotoxic T cell
e-Reaction of cytotoxic T cell to cells displaying foreign MHC molecule

71. Thank You
and
Any Question