Case 6

Published on 18/02/2015 by admin

Filed under Allergy and Immunology

Last modified 22/04/2025

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CASE 6

Jason, a 6-year-old boy who lives on a farm, has convinced his parents that he should be allowed to take care of the chicks that are in the hay loft area of the barn. He is intrigued with this brood of chicks and spends hours just lying down in the hay to watch them or dragging hay over to their nest. When he is not in the barn, Jason is “helping” his mother with the new garden. After several weeks of this regimen, Jason started having difficulty breathing and seemed to “always be coughing.” The parents took him to their family physician, who had already treated Jason for a severe diaper rash (Candida albicans) when he was an infant and over the past few years for numerous severe bacterial infections.

The physician ordered several tests, including a complete blood cell count and differential, total serum immunoglobulins, immunization antigen titer, T cell function test, and a bronchoalveolar lavage to use for culture. The white blood cell count showed a mild leukocytosis, but the differential indicated a normal percentage of each cell type. Total IgG was normal for the pediatric population (see Appendix), as was the antibody titer to immunization antigens. T cell function was normal. An inquiry into Jason’s family revealed that he had three older sisters and two brothers, none of whom had presented with clinical symptoms analogous to those for which Jason’s parents had sought medical attention. How would you proceed?

QUESTIONS FOR GROUP DISCUSSION

1. Fungal infections (e.g., Candida albicans) occur frequently in patients with T cell disorders. However, laboratory tests indicated that this patient’s T cell number and function were normal. What other cell type plays an important role in the elimination of fungal infections?
2. This patient has a history of both fungal and bacterial infections. Therefore, which cell type should you consider investigating with respect to cell number and function?
3. The phagocyte plays an important role in the innate immune system using as its armamentarium lysosomal enzymes, reactive oxygen intermediates (ROIs), and nitric oxide. ROIs are generated during the respiratory burst. Which enzyme complex is required to trigger the sequence of events that generates the ROIs?
4. Describe the laboratory tests used to determine if the NADPH oxidase enzymatic complex is functional.
5. Culture of the bronchoalveolar lavage fluid revealed that Jason was infected with Aspergillus fumigatus, a fungus that is widely distributed in nature, including garden soil and hay lofts. Despite this, aspergillosis is not common. Explain.
6. In a normally functioning immune system, T cells enhance the phagocytic ability of phagocytes. Explain.
7. Recently, prophylactic administration of the cytokine interferon gamma (IFNg) has gained popularity as a therapeutic intervention to treat patients with a defect in a component of the NADPH oxidase complex. Explain. What is the source of IFNγ in an intact immune system?
8. Describe some of the effects of IFNγ, other than effects described in question 7.
9. Describe the rationale for bone marrow transplantation and gene therapy.
10. Explain the role of NRAMP-1 in host defense.

RECOMMENDED APPROACH

Implications/Analysis of Family History

An inquiry into Jason’s family revealed that he had three older sisters and two brothers, none of whom had presented with clinical symptoms analogous to those for which Jason’s parents had sought medical attention.

Implications/Analysis of Clinical History

Whereas bacterial infections can be commonly seen in complement and cell-trafficking abnormalities, susceptibility to fungal infections should trigger a concern to investigate a defect in phagocyte or T cell function.

Implications/Results of Laboratory Investigation

Laboratory tests revealed a mild elevation in white blood cell count, normal differential, normal serum immunoglobulins, and normal B cell and T cell function tests. These tests rule out obvious B cell and T cell development disorders (see Cases 2 and 3). It is reasonable to conclude that Jason’s infections are not due to defects in adaptive immunity. As well, because the complete blood cell count and differential were normal, a deficiency in development of cell population(s) in the innate immune system can also be ruled out.

Culture indicated an infection with Aspergillus fumigatus. Infection is generally acquired from breathing in Aspergillus conidia (spores), which are widely distributed in nature, including hay lofts and garden soil. In immunocompromised individuals these infections can give rise to aspergillosis (Fig. 6-1). Individuals with normal numbers and function of phagocytes and CD4+ T cells can usually overcome these infections

image

FIGURE 6-1 Aspergillosis in lung of patient with chronic granulomatous disease. Slide is viewed by fluorescence microscopy, showing yellow branching hyphae typical of Aspergillus. (Note: unlike Candida, Aspergillus lacks budding yeasts.) (Acridine orange fluorescence, ×400.)

Given that T cell numbers and function are normal, as are the myeloid cell numbers, we need to consider the possibility that a functional defect in the innate immune system is the cause of the infections.

Additional Laboratory Tests

Destruction of pathogens by the phagocyte is mediated by the armamentarium in the phagosome, and this includes reactive oxygen intermediates, lysosomal enzymes, and nitric oxide. Although any one of these can be defective, the most prominent defects that alter intracellular killing of pathogens by phagocytes are those that result in mutations of the proteins that compose the NADPH oxidase system. Activation of this enzyme complex induces a respiratory burst (an increase in oxygen consumption), so named because oxygen is used by the cell to generate reactive oxygen intermediates (ROIs: e.g., superoxide anion, hydroxyl ion, hydroxyl radical, and hydrogen peroxide).

Nitroblue Tetrazolium Test

Classically, the overall function of the NADPH oxidase complex has been determined using the nitroblue tetrazolium (NBT) dye reduction test. NBT is a yellow dye that becomes insoluble and turns purple (chemical reaction) resulting from the presence of NADPH oxidase activity. In this test, a droplet of the patient’s blood is placed onto a slide along with a neutrophil activator and NBT. The patient’s cells take up the dye, and if NADPH oxidase is functioning normally the dye is reduced, causing it to change to dark blue/purple.

Flow Cytometry

More recently, flow cytometry has been used to assess the respiratory burst. In this approach, phagocytes take up dihydro-rhodamine-123, a dye that does not fluoresce in resting cells. On activation of NADPH oxidase, the dye emits a fluorescence that is detected using flow cytometry. One of the advantages of this more recent approach is that it has been easier to quantitate this assay than the NBT assay. Analysis of NADPH oxidase in Jason’s phagocytes indicated that the NADPH oxidase system was defective.

DIAGNOSIS

Jason was diagnosed with chronic granulomatous disease. Because some NADPH oxidase defects are X linked, the flow cytometry test for NADPH oxidase activity was extended to Jason’s mother, sisters, and brothers. Only his 12-year-old brother showed evidence for defective NADPH oxidase function. Interestingly, blood cells from his mother and one sister showed mixed populations of neutrophils, with about 50% exhibiting a normal respiratory burst and 50% not doing so. Females carry two X chromosomes, one of which is randomly inactivated in all cells. Therefore, females who are carriers but do not express the disease have a mutated gene on only one X chromosome. A normal gene is expressed in about half the cells and a defective gene in the other half. Thus “functionally normal” females (carriers) are, in fact, genetic mosaics.

THERAPY

Administration of antibiotics to combat bacterial infections and potent antifungal agents (e.g., oral itraconazole) represent the mainstay of treatment. Prophylactic administration of IFNγ has gained popularity as a therapeutic intervention.

Interferon γ

In one study of chronic granulomatous disease (CGD) patients with a defect in p47phox, 70% of patients treated with IFNγ had enhanced production of ROIs. However, this response itself has not been seen uniformly. Such conflicting reports may represent patient populations that have different subunit defects or different mutations in any particular subunit. Some mutations might cause total inactivation of NADPH oxidase activity, whereas other mutations may cause a decrease in activity that can be rescued with high concentrations of IFNγ. Despite the conflicting reports regarding increases in ROIs after IFNγ treatment, a reduction in the number of bacterial infections is generally observed consistently, suggesting that IFNγ is also stimulating other intracellular antimicrobial defense mechanisms. In normal individuals, IFNγ induces transcription of gp91 PHOX and enhances production of ROIs.

Other Effects of IFNγ Treatment

IFNγ induces transcription of inducible nitric oxide synthase, an enzyme that catalyzes the conversion of l-arginine (+O2) to l-citrulline and nitric oxide. Nitric oxide is toxic to many pathogens. Other systems activated by IFNγ include, but are not limited to, IDO (indoleamine 2,3 dioxygenase oxidase) and NRAMP (natural resistance associated macrophage protein).

IDO is a key enzyme required for the catabolism of tryptophan, an amino acid that is required for the protein synthesis. NRAMP1 is a membrane protein and, as such, is present on the phagosome membrane because the phagosome is formed when membrane pseudopods extend around the pathogen. This strategic location of NRAMP1 allows it to transport metal ions from the phagosome to the cytosol. The removal of metal ions from the phagosome is a host defense mechanism because the phagosome milieu contains metal ions required by some bacteria present in that vacuole. These bacteria have metal ion transporters that take up metal ions from this milieu and incorporate them into metalloenzymes. This host defense mechanism is believed to be particularly important in patients infected with Mycobacterium species because these metal ions are essential for their survival.

Bone Marrow Transplant

In more severe cases of CGD, bone marrow transplantation to replace hematopoietic cells has been performed when a suitable donor has been available. However, bone marrow transplantation carries a high mortality risk.

Gene Therapy

In the future, treatment may rely on gene therapy in which a corrected version of the defective NADPH oxidase subunit is cloned into the patient’s cells. As an example, one recent clinical trial performed in association with the NIAID’s Laboratory of Host Defenses, described five adult patients with a defect in p47phox who were treated using a gene therapy approach.

These patients were first treated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF), a growth factor for CD34+ stem cells (precursors of hematopoietic cells), in order to expand the CD34+ stem cell pool. CD34+ peripheral blood stem cells were then isolated from the patients and transduced ex vivo with a retroviral vector containing a DNA encoding the NADPH oxidase subunit, p47phox. The transduced cells were then infused back into the patients. In all five patients, functionally active (NADPH+) neutrophils were detected in circulation, with activity peaking at about 1 month. In two patients, the transduced cells were still detectable 6 months later. No toxicity of treatment was observed.

ETIOLOGY: CHRONIC GRANULOMATOUS DISEASE

Chronic granulomatous disease (CGD), one of the more common immunodeficiencies that affects innate immunity, is characterized by the absence of a respiratory burst and associated ROIs due to a defective subunit in the multi-subunit NADPH oxidase enzyme complex. In the resting phagocyte, some of the subunits are distributed in the plasma membrane; others are in the cytosol. Phagocytosis triggers subunit assembly on the phagocytic vacuole, a respiratory burst, and production of ROIs (see Fig. 4-1).

Some of the genes encoding the NADPH oxidase subunits that assemble to form a complex are encoded on autosomal chromosomes. However, others (e.g., gp91 PHOX) map to the X chromosome. The gp91 PHOX is the most common mutation in patients presenting with CGD and so the majority (>80%) of affected individuals are male. This is consistent with the observation that Jason’s 12-year-old brother was the only family member who had a defective NADPH oxidase test. Although a defective NADPH oxidase leaves individuals susceptible to recurrent bacterial infections, the most frequent cause of death is chronic infection with Aspergillus species. Chronic infections are often complicated by the end result of chronic inflammatory processes and tissue remodeling, namely, excessive scar tissue contributing to noncompliant lung function.

When patients with a defective NADPH oxidase are infected with bacteria that are hydrogen peroxide producing but catalase negative, some bacterial destruction can still occur. Here the hydrogen peroxide produced by the bacteria in the phagocytic vacuole acts as a substrate for myeloperoxidase (a lysosomal enzyme) to generate bactericidal hypochlorite when chloride ion is present.

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