Immunodeficiencies: Phagocyte and complement dysfunction: Pathology review

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Case study0:00–1:34

Alyssa is a 3 week old newborn baby girl that’s brought to the clinic by her parents. They’re a bit concerned because they’ve noticed that Alyssa’s umbilical cord stump hasn’t fallen off yet.
On physical examination, you notice that the stump looks red and swollen, but there’s no pus. You decide to run a blood test, which reveals an increased level of neutrophils.
Finally, you perform flow cytometry, which shows that these neutrophils have reduced expression of CD18. Next comes Eddie, a 2 year old boy who has a fever that won’t go away after 2 weeks.
His parents also mention that he has frequent infections involving the respiratory tract, and he once also had an infection of the knee joint.
Upon physical examination, the first thing you notice is that Eddie has extremely light skin, hair, and eyes. Then, you find swollen lymph nodes all around the body, and you palpate an enlarged liver and spleen.
So again you run some blood tests, but now you find decreased white blood cells, especially neutrophils, and a prolonged bleeding time.
Finally, you do a peripheral and bone marrow smear, which shows abnormally large granules within the white blood cells and platelets.
Based on the initial presentation, both cases seem to have some form of immunodeficiency, meaning that their immune system's ability to fight pathogens is compromised.

Pathology1:34–2:04

Immunodeficiencies can be classified according to the component of the immune system that is defective. In this video, we’ll be focusing on phagocyte dysfunction and complement disorders.
Okay, let’s start with phagocyte dysfunction. First we have leukocyte adhesion deficiency, which is an autosomal recessive disorder, meaning that an individual needs to inherit two copies of the mutated gene, one from each parent, to develop the condition.

Leukocyte adhesion deficiency2:04–6:30

Normally, when there’s an infection or inflammatory process, as well as for wound healing, chemical signals are released by cells in the affected area, to attract leukocytes such as phagocytes that are circulating in the blood, and this is called chemotaxis.
But to actually get to the affected area, they first have to squeeze and pass through the endothelial cells that line the blood vessel wall.
To do this, what’s important to know is that there’s a tight interaction between cellular adhesion molecules on the surface of endothelial cells, and the integrins on the surface of the phagocytes.
Once at the infected site, phagocytes start phagocytosing or eating invading pathogens and damaged cells, and then undergo apoptosis or programmed cell death, destroying themselves and all of the pathogens they’ve taken in.
This may form a collection of pus, which can accumulate in closed tissue spaces and develop into an abscess. Now, there are many types of leukocyte adhesion deficiency, but the most common and high yield one is type 1.
So type 1 leukocyte adhesion deficiency is caused by a mutation in the gene coding for CD18, which is a subunit of integrin molecules.
Without integrins, phagocytes in the circulation can’t make their way to the infected or damaged tissues. This allows pathogens, like bacteria and fungi, to spread uncontrollably, causing recurrent bacterial or fungal infections of the skin or mucosal membranes.
A high yield fact is that there’s never pus or abscess formation since the neutrophils never make it to the pathogens. Another important thing to keep in mind for your exams is that these patients are also at risk for much more serious infections such as pneumonia or peritonitis.
Unfortunately, because of this, life expectancy can be severely shortened, and many babies don’t survive past infancy. At the same time, without the help of phagocytes, damage cells and tissue debris cannot be removed.
As a consequence, wounds are slow to heal, leading to poorly formed, thin, and bluish scars. Now, a very high yield fact is that phagocytes are also required to help the umbilical cord stump separate or fall off from the baby’s belly button.
For your exams, remember that this normally takes 1 to 2 weeks, while with leukocyte adhesion deficiency, it may take longer than a month, and it can often get inflamed and infected, but again there’s no pus.
Diagnosis is based on the elevated number of phagocytes, especially neutrophils, in the blood. This is because they simply don’t move into pathogen infected tissue.
For this reason they’re also absent at the infection sites. Diagnosis can be confirmed with flow cytometry looking for the reduced expression of CD18 on the membrane of phagocytes.
For treatment, prophylactic antibiotics are often given to help prevent serious infections, while the only cure is a hematopoietic stem cell transplant that can replace all types of blood cells, including new leukocytes that are able to extravasate normally.
Another high yield phagocyte dysfunction is Chediak-Higashi syndrome, which is also autosomal recessive. The mutated gene here is the LYST gene, which codes for the LYSosomal Trafficking regulator, or LYST for short.

Chediak-Higashi syndrome6:30–11:13

LYST is a vesicular transport protein that’s particularly important for the transport of substances into lysosomes. Normally, when a phagocyte detects a pathogen, it wraps around it and engulfs it, forming a vesicle inside the phagocyte called a phagosome.
Then, the phagosome fuses with a lysosome, forming a phagolysosome, and lysosomal enzymes destroy the pathogen. In Chediak-Higashi syndrome, there’s defective transport into lysosomes, which results in an impaired phagolysosome formation.
Affected phagocytes produce giant granules, but are unable to kill engulfed pathogens. Platelets are also affected in Chediak-Higashi syndrome.
That’s because, normally, platelets have intracellular vesicles or granules that contain clotting and platelet-activating factors, but in Chediak-Higashi syndrome, these granules can’t be released, so they become giant and there’s impaired platelet aggregation.
Another type of cells affected in Chediak-Higashi syndrome are melanocytes, which produce a protein pigment called melanin.
Melanin is stored in vesicles called melanosomes, which then carry it to the surrounding tissue cells, and it contributes to the color of our skin, hair, and eyes.
In Chediak-Higashi syndrome, melanosomes fail to transport melanin to the surrounding cells. Finally, neurons also rely on vesicular transport to release neurotransmitters and communicate with other cells.
As a consequence, Chediak-Higashi syndrome can cause damage to neurons. Because of all this, Chediak-Higashi syndrome usually presents in infancy or early childhood with a classic combination of recurrent infections and abscesses; mild coagulation defects; albinism, and neurologic symptoms, including progressive neurodegeneration and peripheral neuropathy, with loss of sensation in the arms and legs.
For your exams, remember that infections are typically severe, are caused by bacteria or fungi and can involve the skin, soft tissues, respiratory tract, bones, and joints.
Ultimately, many individuals with Chediak-Higashi syndrome reach the so-called accelerated phase, in which lymphocytes start proliferating uncontrollably, and can invade and damage various organs, including the liver, spleen, and the bone marrow.
This is known as lymphohistiocytosis, and can manifest with fever, lymphadenopathy or swollen lymph nodes, hepatosplenomegaly or an enlarged liver and spleen, and pancytopenia or low counts of red blood cells, white blood cells, and platelets.
Diagnosis of Chediak-Higashi syndrome begins with blood tests which show pancytopenia or a decrease in all types of blood cells, especially neutrophils, and a prolonged bleeding time.
Confirmation comes with a peripheral and bone marrow smear, showing giant clumped up granules within granulocytes and platelets.
Finally, genetic tests can also be done to look for mutations in the LYST gene. For treatment, antibiotics can be used to treat infections, and individuals in the accelerated phase may get chemotherapy, but the only cure for Chediak-Higashi syndrome is a bone marrow transplant.
The last high yield phagocyte dysfunction is chronic granulomatous disease, which is caused by a mutation in the genes that code for the enzyme complex NADPH oxidase.

Chronic granulomatous disease11:13–16:44

There are many ways to inherit these mutations, but the most important for your exams is an X-linked recessive mutation, and since men only have one X chromosome, they get the disease, whereas because women have two X chromosomes, they only get the disease if both of their X chromosomes are affected.
Remember the phagolysosome? Great!
So if we zoom into its membrane, we’ll find this enzyme complex called NADPH oxidase. And inside the phagolysosome we have the lysosomal enzymes that can destroy a pathogen.
The lysosomal enzymes also activate NADPH oxidase, which causes NADPH to undergo oxidation and lose two electrons. Nearby oxygen molecules can grab these electrons to form superoxide ions, or O2- ions.
Another enzyme called superoxide dismutase can then take these superoxide ions and combine them with hydrogen ions, forming hydrogen peroxide, or H2O2.
Finally, superoxide ions and hydrogen peroxide destroy pathogens by breaking down their cell membranes and damaging their proteins.
This process is called the respiratory burst, and it’s very high yield. Now, in chronic granulomatous disease, there’s a decrease in the amount of functioning NADPH oxidase, so there’s less superoxide ions and hydrogen peroxide, resulting in a weaker respiratory burst.
This makes it difficult to kill certain pathogens, specifically fungi and bacteria that have an enzyme called catalase, and are therefore called catalase-positive pathogens.
To help you remember these pathogens, remember the mnemonic “CATs have BeeN PLACESS" which stands for Burkholderia, Nocardia, Pasteurella, Listeria, Aspergillus, Candida, E.
coli, Staphylococcus, and Serratia. Now, what catalase does is break down hydrogen peroxide, into water and oxygen, rendering it harmless.
Normally, there's enough hydrogen peroxide being produced, so catalase positive pathogens can only break down some of it, but in the case of chronic granulomatous disease, there's already so little hydrogen peroxide, that catalase-positive pathogens can break down all of it.
Over time, phagocytes gather up to stop the pathogen from spreading, forming little clusters called granulomas throughout the body.
So they’re not killing the pathogens, but at least they’re keeping them sort of under control. And that’s where chronic granulomatous disease gets its name from!
In terms of symptoms, individuals can develop recurrent pneumonia, or recurrent skin and soft tissue infections like cellulitis and abscesses, as well as bone and joint infections like osteomyelitis and septic arthritis.
It's also common to see bacteremia or fungemia, which is when the pathogens that don't get killed find their way into the bloodstream.
Diagnosis of chronic granulomatous disease involves testing neutrophil function in a blood sample. A very old test is the nitroblue tetrazolium test, which is when a colorless dye called nitroblue tetrazolium is added to the blood sample.
If the respiratory burst is working properly, superoxide ions are produced and react with the dye, changing its color to a deep blue, while in chronic granulomatous disease the sample fails to turn blue.
A newer test is the dihydrorhodamine 123 test, which is where dihydrorhodamine 123 is added to the blood sample. If levels of NADPH oxidase are normal, then it will oxidize dihydrorhodamine 123, causing it to shine with a green color or fluorescence, but in chronic granulomatous disease, there’s reduced green fluorescence.
Treatment of chronic granulomatous disease involves prophylactic antibiotics and antifungals. In addition, interferon gamma can be given to stimulate the production of superoxide in phagocytes.
In some cases, a hematopoietic stem cell transplantation can be done. Okay now!

Complement deficiencies16:44–21:22

We’ve reached our final topic, which is complement deficiencies! Here, there’s a deficiency in one or more of the complement proteins.
Let’s start with the complement proteins that make up the complement system, going from C1 all the way to C9. Generally speaking, each complement protein is normally inactive, and it becomes activated when it’s cleaved, meaning that a part or fragment of it is removed.
Normally, the complement system helps with the immune response through three mechanisms. First, it enhances the function of phagocytes by opsonization, meaning that complement proteins bind to and coat the pathogen’s or damaged cell’s membranes as well as immune complexes made up of antibodies bound to pathogenic antigens, so that they can be more easily picked up by phagocytes.
The main opsonin complement protein is C3b. This is an active fragment of C3, and its cleavage involves complement proteins C1, 2, and 4.
And so C1 to C4 are called the early components of the complement system. Second, the complement promotes inflammation and chemotaxis by recruiting immune cells into the affected area, which is mainly carried out by C3a, that’s a fragment of C3, as well as C5a, which is a fragment of C5.
And third, proteins C5b and C6 to C9 cluster in the pathogen’s membrane and form the membrane attack complex, or MAC, which creates a hole in the membrane and ultimately kills the pathogen.
And so C5 to C9 are called the terminal components of the complement system. For your tests, keep in mind that the formation of the MAC complex is especially useful against Neisseria meningitidis and gonorrhoeae.
Now, many cases of complement deficiency are inherited, and most are autosomal recessive. However, remember that it's also possible to develop an acquired complement deficiency, for example, when a severe infection or autoimmune condition causes complement proteins to get used up.
For your exams, a high-yield example is systemic lupus erythematosus, which causes both C3 and C4 protein deficiency. So complement deficiencies can be classified as early complement deficiencies when they involve C1 to C4, and terminal complement deficiencies when they involve C5 to C9.
Early complement deficiencies are associated with an increased risk of severe, recurrent pyogenic infections, mainly involving the sinuses and respiratory tract.
In addition, due to the decreased clearance of damaged cells and immune complexes, these individuals often develop autoimmune manifestations, the most high yield one being again systemic lupus erythematosus.
On the other hand, terminal complement deficiencies impair the formation of the MAC, and bear in mind that individuals are particularly susceptible to recurrent Neisseria infections, so they’re at higher risk for developing meningitis or gonorrhea.
Diagnosis of complement deficiencies can be done through blood analysis, by testing overall complement activity, denoted by "CH50" or "CH100".
In addition, it's possible to test for specific protein levels, like C3 or C4. Treatment of complement deficiencies depends on the cases.
It's important to treat harmful infections, and to take precautions like prophylactic antibiotics. In addition, individuals that develop autoimmune manifestations can be given immunosuppressors.
All right, as a quick recap… Leukocyte adhesion deficiency type 1 is caused by an autosomal recessive mutation in the CD18 subunit of the LFA-1 integrin, which is necessary for extravasation of phagocytes.

Review21:22–23:28

This results in recurrent bacterial and fungal infections of the skin and mucosal membranes, impaired wound healing, and delayed separation of the umbilical cord.
Chediak-Higashi syndrome is an autosomal recessive disease caused by a mutated LYST gene, which impairs vesicular transport, particularly to lysosomes, and is characterized by recurrent bacterial infections as well as defects in blood clotting, pigmentation, and neurologic function.
Chronic granulomatous disease is caused by an X-linked recessive mutation in NADPH oxidase, resulting in an inability of phagocytes to create superoxide and hydrogen peroxide.
This causes them to struggle to kill catalase-positive pathogens, which can be remembered with the mnemonic “CATs have BeeN PLACESS".
Complement deficiencies are a group of disorders, all of which have a deficiency in one or more complement proteins, and can be inherited or acquired.
Early complement deficiencies involve C1 to C4, and are associated with an increased risk of severe, recurrent pyogenic infections of the sinuses and respiratory tract, as well as autoimmune manifestations, like systemic lupus erythematosus.
On the other hand, terminal complement deficiencies involve C5 to C9, and individuals are particularly susceptible to recurrent Neisseria infections.

Summary23:28–25:08

Okay, back to our cases. Alyssa is a 3 week old newborn girl that’s taken to the clinic because her umbilical cord stump still hasn’t separated from her belly button.
This already should make you think of leukocyte adhesion deficiency. Another very important fact here is that her stump is inflamed, but there’s no pus, which would indicate that there are no neutrophils fighting the infection.
However, her blood tests show high levels of neutrophils which means they are somehow defective. The final clue is given by flow cytometry, which reveals that these neutrophils lack expression of CD18 on their membranes, which confirms the diagnosis of leukocyte adhesion deficiency type 1.
Next comes Eddie, a 2 year old albino boy who has frequent infections involving a variety of organs and tissues. He presents with a history of fever, lymphadenopathy, and hepatosplenomegaly.
This may suggest lymphohistiocytosis, which is typical of the accelerated phase of Chediak-Higashi syndrome. This is further supported by Eddie’s blood tests, which show a combination of pancytopenia and a prolonged bleeding time.
Finally, a peripheral and bone marrow smear reveals giant clumped up granules within granulocytes and platelets, which definitely confirms the diagnosis of Chediak-Higashi syndrome.