1. Treatment of Chronic Granulomatous Disease
PHM Fall 2018
Instructor: Ms. Maya Latif
Coordinator: Dr. J. Henderson
Treatment of Chronic Granulomatous Disease
Ingrid Gan Jin Kong Simran Sharma Aniko Yeats

2. Introduction Chronic granulomatous disease (CGD)
Primary immunodeficiency disorder of phagocyte oxidative metabolism
Defect in NADPH oxidase complex of phagocytes
Difficulty forming ROS to destroy invading pathogens
Increased risk of severe bacterial & fungal infections, autoimmunity and inflammatory complications leading to granuloma formation
Incidence
1:200,000 to 1: (US and Europe)
Prevalence varies worldwide: from 1:1 million in Italy to 1:70,000 in the Israeli Arab population
Introduction: Chronic Granulomatous Disease (CGD) is an inherited immunodeficiency disorder of phagocyte oxidative metabolism. It is caused by a defect in any of the peptide subunits of the NADPH oxidase complex in phagocytes. Without a functioning NADPH oxidase complex, phagocytes have difficulty forming the ROS needed to destroy invading pathogens and this leaves patients at an increased risk of recurrent life-threatening infections,autoimmunity, inflammatory complications and granuloma formation. CGD may present at any age from infancy to late adulthood; however, the vast majority of patients are diagnosed at less than 5 years of age. The incidence of CGD is about 1: to 1: in the US and Europe, but the prevalence fluctuates worldwide.

3. Inheritance Inherited in 2 manners: X-linked recessive - most severe
CYBB gene defect (encodes gp91-phox subunit)
> 2/3 of cases
Autosomal recessive
NCF1 (encodes p47phox ) - 20%
NCF2 (encodes p67phox) - 5%
CYBA (encodes p22phox) - 5%
Inheritance: CGD can be inherited in 2 manners: X-linked or autosomal. Patients with X-linked CGD generally have a more severe disease. More than two- thirds of all cases are X-linked recessive and result from defects in the CYBB gene that encodes the gp91-phox subunit; the remaining cases are autosomal recessive and caused by defects in NCF-1, CYBA, and NCF-2.
Image: Stasia et al, 2008

4. Signs & Symptoms Recurrent bacterial and fungal infections:
Lymph nodes, liver, lungs, skin, and bones
Most commonly by Staphylococcus aureus and Aspergillus species
Granuloma formation: GIT and Genitourinary system
Poor quality of life with advancing age
Death from infectious complications within first years of life
Signs and symptoms: Patients with CGD often have bacterial and fungal infections involving the lymph nodes, liver, lungs, skin, and bones. Excessive granuloma formation occurs most commonly in the GUT or the genitourinary system. Patients tend to become increasingly debilitated with poor quality of life with advancing age and most die of infectious complications within the first years of life.
Image: symptoms
Image: Horizon Pharma, 2018

5. Mechanism gp91-phox and p22-phox form flavocytochrome b558
Cytosolic complex (p67+p47+p40) translocates and binds to b558
Binding activates b558
Catalyzes transfer of e- from NADPH to O2 → superoxide
‘Respiratory Burst’
CGD is caused by mutations in any of the genes that encode these four subunits
Imag: (in works cited)
Text
works cited)
(inworks cited)
Image: Heyworth et al, 2002

6. Diagnosis
Detect absence of respiratory burst → production of superoxide/hydrogen peroxide
Nitroblue Tetrazolium (NBT) Reduction Test
Oldest and most recognizable
Superoxide reduces NBT to formazan
quantify;light microscope; subjective;
Healthy: blue
CGD: Yellow
X-linked carriers → both
Dihydrorhodamine (DHR) Test
Replaces NBT test, objective and more reliable
Neutrophils can be stimulated by Phorbol-12- Myristate-13 Acetate (PMA) → Hydrogen peroxide
Hydrogen peroxide reduces DHR to rhodamine (fluorescent)
flow cytometer; a shift of curves
Healthy: increased fluorescence → shifts right
CGD: no shift
X-linked carriers: mosaic forms

7. Treatment Overview
Early diagnosis and treatment can be instrumental in improving patient outcomes
Therapeutic options for CGD include:
Prolonged treatment with:
Antimicrobials
Antifungals
Surgical intervention
Interferon Gamma-1b
Curative: Hematopoietic Stem Cell Transplantation
Challenges: Inflammatory complications }
Prophylaxis or treatment of acute infections as they occur
Over almost 60 years, CGD has evolved from a disease of early fatality to one of effective management with high survival, allowing patients to live well into adulthood. This has been made possible by the evolution of the different treatments listed here, which include prophylaxis and aggressive treatment of acute infections, surgery as well as stem cell transplants. I’d like to emphasize early diagnosis as being crucial for improving patient outcomes.
Treatment for inflammatory complications does still remain challenging. Steroids are sometimes used for inflammatory bowel disease in CGD patients but they come with their own subset of adverse effects like heightened infection risk but such immunosuppression is perhaps the last thing CGD patients need.

8. Treatment: Antifungals
Fungal infection is a main cause of death in chronic granulomatous patients with severe immune deficiencies
Fungal infections typically involve the pathogen Aspergillus spp which targets the lung, bone, and the brain
Mechanism of action: Activity against a variety of clinically significant yeasts and molds
Azole antifungal agents (five-membered heterocyclic compounds containing N)
Voriconazole
Broad-spectrum triazole
Can be administered orally or through IV
Posaconazole
Extended spectrum triazole
Isavuconazol
So I’ll start us off with antifungal treatment. Fungal infections are the leading cause of death for CGD patients that have severe immune deficiences. These infections often involve Aspergillus species which target the lung, bone and brain. Patients diagnosed before the advent of antifungal azole agents had different outcomes, often with poor survival into their 30s and 40s.
Unfortunately, incidence of triazole-resistant Aspergillus is increasing. So as mentioned, aggressive treatment of fungal infections when they appear is imperative. Azoles are the primary agents used for this purpose. They have antifungal activity against a variety of clinically significant yeasts and molds. Three different agents with varied spectrum activity are VoriCONazole, PosaCONazole and IsavuCONazole.

9. Treatment: Antimicrobials
Chronic granulomatous patients also experience frequent bacterial infections from species of Staphylococcus, Klebsiella, and Pseudomonas.
Mechanism of action: Synergistic bactericidal activity through preventing bacterial synthesis of thymidine (used in bacterial DNA synthesis)
Procedure: Antibacterial prophylaxis is recommended using:
Trimethoprim(TMP)/Sulfamethoxazole(SMX)
Long term use of TMX has been used for patients since the 1970s
TMP-SMX treatment decreases bacterial infections without increasing fungal infections
TMP-SMX is the treatment of choice due to its broad spectrum of microbicidal activity that does not compromise gut flora activity
CGD patients also experience frequent bacterial infections from species of Staphylococcus, Klebsiella, and Pseudomonas.These bacteria break down hydrogen peroxide, which is utilized by the host to fight against infection. So an important agent on the market critical for antimicrobial prophylaxis against this bacterial action is Trimethoprim/Sulfamathoazole or TMP/SMX.
These agents act on sequential steps in the enzymatic pathway of bacterial synthesis of tetrahydrofolic acid. The net result is a decrease in the bacteria’s ability to synthesize thymidine which is needed for DNA synthesis. TMP-SMX is the treatment of choice because it has a broad spectrum of microbicidal activity and does not compromise gut flora activity. Studies also show that this treatment does not inadvertently increase fungal infections while treating bacterial infections.

10. Treatment: Surgical Intervention
Necessary when antimicrobial agents alone are unable to remove infections
Procedure involves:
Drainage of abscesses (in skin, lymph node, liver and rectum wall)
Relief of obstruction from granulomas
Excision of established granulomatous lesions (in lung and liver)
However:
Operative sites can become infected and heal slowly
Postoperative treatment of these patients is demanding
May require prolonged use of antibiotics
This is a tool for CGD patients which is necessary when antimicrobial agents alone are unable to remove infections. It’s used in a variety of ways, such as drainage of abscesses in body regions such as skin and lymph nodes. Liver abscesses also occur in approximately 35% of patients. Surgery is also used to remove consolidated granulomatous lesions in the body and relieving obstructions caused by granulomas.
The drawbacks unfortunately are that the operative sites do have the potential to become severely infected and require prolonged use of antibiotics for the patient while they heal. Studies do however show a significantly reduced rate of relapse in those treated with a combination of medical and surgical therapy vs. medical therapy alone.

11. Treatment: Interferon Gamma-1b
Properties: A proinflammatory cytokine secreted by T cells, macrophages, and NK cells
Indication: Reduces the frequency and severity of serious infections associated CGD
Mechanism of action: INF-gamma stimulates superoxide release and improves microbicidal function
Administration: Administered subcutaneously with dose based on body surface area
Adverse reactions: Fever, headache, chills and fatigue along with the potential for less common but severe adverse reactions (including cardiovascular, lymphatic, pulmonary and renal disorders)
While antibiotics, fungal, and surgical interventions help to treat existing infections caused by CGD, preventing such infections is just as crucial to help alleviate serious infections.
One clinical treatment is Interferon Gamma 1beta, which is a proinflammatory cytokine secreted by cells of the immune system, particularly T cells, macrophages, and NK cells. It has been proved to reduce the frequency and severity of serious infections associated with CGD. Its mechanism of action is to increase the production of nitric oxide as well as the bactericidal properties of neutrophils
This treatment is comprised of daily injections under the skin, and clinical results has demonstrated benefit in a majority of patients and reduced the risk of infection in 70% of the cases. Of course like all treatments it can lead to adverse reactions in patients such as fever, headaches, chills, and fatigue.

12. Curative Treatment: Hematopoietic Stem Cell Transplantation (HSCT)
Procedure: Patient receives blood forming cells from a genetically similar donor
HSCT from a human leukocyte antigen (HLA) compatible donor can cure CGD
It provides patients with donor neutrophils with functional NADPH and superoxide anion production
Traditionally a high risk procedure but advances in stem cell transplantation has evolved, resulting in improvement in survival
Recent HSCT trial for high risk patients results in survival in 12 out of 13 adult males
Challenges:
There is no general consensus on best source of stem cells
No optimal conditioning regimen has yet been identified
Hematopoietic stem cell transplantation treatment is currently the only known cure for CGD, and this treatment works by providing patients with donor neutrophils with functional NADPH and superoxide anion production
It is a traditionally a high risk procedure where most infections are due to complications of infection and graft-versus-host disease (GVHD). In recent years, however, advances in stem cell transplantation has evolved and studies have shown efficacy and improvement in survival.
Nevertheless, many challenges still remain for this technique, such as no general consensus on the best source of stem cells, and that no optimal conditioning regimens have yet been identified
The complexities of the clinical phenotype of CGD + the perceived and real risks associated with HSCT have prevented many patients from getting this curative treatment
Image: Creative Biolabs Vaccine

13. Current Clinical Research & Future Therapy
Development of target therapy: drugs/therapies that interfere with specific molecules responsible for the pathogenesis of CGD
Progression from HSCT to modification of germline DNA to correct mutated genes
Currently used for patients that lack an HLA-matched donor for HSCT
Procedure: Initial gene addition trials using gamma-retroviral vectors to treat X-linked CGD
Limited gene transduction = low levels of functional protein
Results are not promising
However: Patients still able to clear persistent life-threatening fungal infection & receive significant clinical benefit
Continued study will be beneficial in finding effective gene therapy
The difficulty in treating patients with CGD is that currently there is no targeted treatment, which refers to drugs or therapies that interfere with specific molecules responsible for the pathogenesis of this disease. Current gene therapy trials such germline DNA modification to correct mutated genes, using gamma retroviral vectors to limit gene transduction and CRISPR CAS9 gene editing are all under investigation. Although results are not efficient as of yet due to limited transfection and selection efficiency, continued study in targeted gene therapy are promising techniques for the treatment and maybe even cure for chronic granulomatous disease
(in works cited)

14. Summary Chronic Granulomatous Disease:
Inherited disorder of phagocyte oxidative metabolism
Increased risk of infections & inflammation → granuloma formation
Mechanism: defect in one of the NADPH oxidase complex subunits of phagocytes
Difficulty forming ROS to destroy invading pathogens
Life expectancy increased three-fold over the last few decades → improved management of infectious and inflammatory complications
Treatment allows survival of most patients into adulthood:
Antibacterial, azole antifungals, surgery, interferon gamma 1b, HSCT
Gene therapy for patients lacking a suitable stem cell donor
CGD is an immunodeficiency disorder where phagocytes have difficulty forming the ROS required to destroy pathogens. As a result, pateints at left with an increased risk of infections, inflamation and gramuloma formation. Life expenctacy has increased significantly over the years due to improved treament options. These include antibacterials, azole antifungals, surgery, interferon gamma 1b, HSCT, and gene therpy.

15. Works Cited
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van den Berg, J.M., et al., Chronic granulomatous disease: the European experience. PLoS One, (4): p. e5234.
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