B-Cells and T-Cells

These specialized cells are a critical part of the bodies humoral immune system. They recognize foreign antigens or invaders and mount a quick response. B-cells act quickly by developing antibodies to the antigen epitopes. T-cells react based on what serological class they are in. If it is a CD8 T-cell, its cytotoxic and can quickly fight and phagocytize the antigen, if it is a CD4 T-cell, it works in conjunction with B-cells and other T-cell subclasses to defend the host. This article will dive into B-cells, and every subclass of T-cells and how they work together to form the humoral branch of the immune system.

B-Cells

B-cells also known as B lymphocytes are a type of lymphocyte that functions as part of the humoral component of the adaptive immune system. It’s role is to secrete antibodies, but it also functions as an antigen-presenting cell (APC) that secretes cytokines. It possesses a B-cell receptor (BCR) on its surface that allows it to bind to a specific target antigen and initiate an immune response. B-cells develop from hematopoietic stem cells (HSCs) that originate within the bone marrow. They then develop into multipotent progenitor cells (MPP), which further differentiates into the common lymphoid progenitor (CLP). Development further progresses through several stages through various gene expression patterns and arrangements. Before maturation occurs, positive selection takes place to make sure that the pre-BCR and BCR can recognize and bind to specific ligands through antigen-independent signaling. If the cells are unable to bind, these B-cells cease to develop. Negative selection occurs through binding of self-antigen with the BCR. If the BCR is able to bind self-antigen it undergoes four fates; clonal deletion, receptor editing, anergy, or ignorance. Clonal deletion is the destruction of the B-cell through programmed cell death, in other words known as apoptosis. This is only for those B-cells that have expressed receptors for self-antigens. Receptor editing is exactly what the name suggests; editing of the BCR during the maturation process in an attempt to change the specificity the receptor to not recognize self-antigens. Anergy is used to describe lack of reaction by the bodies immune system. Its a way of saying that the B-cells that express BCRs for self-antigen will simply not be used. The last fate; ignorance means that the B-cell ignores the signal and continues through natural development. When negative selection is complete, the B-cells are now in a state of central tolerance. These mature B-cells do not bind with self antigens. From the bone marrow, B-cells migrate to the spleen as transitional B-cells. Within the spleen they become Follicular B-cells or Marginal zone B-cells depending on the signal received through the BCR. Once completely differentiated, they are now called naive B-cells.

B cell

B-Cell Activation

Activation usually occurs within the secondary lymphoid organs, such as the spleen and the lymph nodes. This is where naive B-cells are positioned once mature. When these naive immunocompetent B-cells encounter an antigen through its BCR, the antigen is internalized by receptor-mediated endocytosis, digested, and positioned on MHC II molecules on the B-cell surface. This allows the B-cell to act as an antigen-presenting cell to T-cells. T-cell dependent activation requires a T-cell helper, most commonly a follicular T-helper cell, to bind to the antigen-complexed MHC II molecule on the B-cell surface through its T-cell receptor (TCR) which drives T-cell activation. These T-cells express the surface protein CD40L and secrete cytokines IL-4, and IL-21 which bind to CD40 on the B-cell surface and act as co-stimulatory factors for B-cell activation. The co-stimulatory factors promote proliferation, immunoglobulin class switching, and somatic hypermutation. Activated T-cells then provide a secondary wave of activation that cause the B-cells to proliferate and form germinal centers. During the production of these germinal centers, activated B-cells may differentiate into plasma blasts, which can produce weak IgM antibodies. Within the germinal centers, B-cells differentiate into high affinity memory B-cells or long-lived plasma cells. The primary function of plasma cells is the secretion of clone-specific antibodies. There are very few antigens that can directly provide T-cell independent B-cell activation. Some components of bacterial cell walls (lipopolysaccharide), and bacterial flagellin are some to name a few. One other mechanism through which B-cell activation is enhanced is through the activity of CD21, CD19, and CD81; all three are surface proteins that form a complex. When the BCR binds to an antigen that is tagged with the complement protein C3, CD21 binds to C3, and downstream signaling lowers the activation threshold of the cell.

Memory B-cell Activation

Activation begins through detection and binding of the target antigen. When the antigen binds, it is taken up by the B-cell through receptor-mediated endocytosis, degraded, and presented onto the MHC II molecule within the B-cell surface. The memory B-cell then acts as an antigen-presenting cell that presents the antigen:MHC II complex to T-cells. Most commonly memory follicular T-helper cells that bind through their TCR. The memory B-cell is then activated and differentiates into either plasmablasts and plasma cells or generate germinal centers.

T-Cells

A T-cell is another lymphocyte, which is a subset of white blood cells. They are called T-cells because they mature in the thymus from thymocytes. There are several subsets of T-cells, each with a specific role in the immune system. These T-cells, just like B-cells originate from hematopoietic stem cells in the bone marrow. These lymphoid progenitor cells populate the thymus and expand by cell division to immature thymocytes. The earliest thymocytes do not express either CD4+ or CD8+ and are classified as double negative cells. Through progression they become double positive and then eventually differentiate into single positive cells, either becoming CD8+, or CD4+. Its interesting to note that there is a small population of double positive T-cells within the peripheral circulation, although their function is unknown. About 98% of thymocytes undergo apoptosis during the development process by failing either positive selection or negative selection. The 2% that survive leave the thymus and become mature immunocompetent T-cells. Lets review positive and negative selection again. Positive selection selects for T-cells that are capable of interacting with MHC molecules. During positive selection signals by double positive precursors express either MHC class I or II receptors. A thymocytes fate is determined during positive selection. Double positive CD4+/CD8+ cells that interact with MHC class II molecules eventually become CD4+ cells, and on the contrary thymocytes that interact well with MHC class I molecules mature into CD8+ cells. Negative selection removes thymocytes that are capable of strongly binding with self MHC peptides.

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T-Helper Cells

T-helper cells do just what their name suggests, they help other cells in immunological processes. This is evident in the activation of B-cells talked about previously. These cells are also most well known as CD4+ T-cells because the highly express CD4 glycoprotein on their surfaces. These T-cells become activated when they are presented with peptide antigens or epitopes by MHC class II molecules, usually present on antigen-presenting cells. Once activated, these cells proliferate rapidly and secrete multiple cytokines. T-helper cells differentiate into several subtypes; TH1, TH2, TH3, TH17, TH9, and THF, each secreting different cytokines to facilitate different pathways of the immune response. This is an article for another time.

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Cytotoxic T-Cells

These killer T-cells destroy virus-infected cells and tumor cells. These cells are known as CD8+ T-cells since they express the CD8 glycoprotein on their surface. These cells recognize targets by binding to antigen epitopes that are associated with MHC class I molecules. Cytotoxic T-cells are highly regulated by Regulatory T-cells through IL-10, adenosine, and other molecules. They can be inactivated to an anergic state, which prevents autoimmune diseases.

T-cell CD8

Memory T-Cells

These memory T-cells are long-lived and when presented with an antigen that is recognized they can quickly expand and differentiate into large numbers of effector T-cells. These memory T-cells can either be CD4+ or CD8+ T-cells. There are four subtypes of memory T-cells that will be discussed below.

Central memory T-cells express CD45RO, C-C chemokine receptor type 7 (CCR7) and L-selectin which are all surface protein markers. They have high expression of CD44, and is commonly found within the lymph nodes.

Effector memory T-cells express CD45RO, but lack expression of CCR7 and L-selectin. These T-cells also have high expression of CD44, but are not found in the lymph nodes. These T-cells are found in the peripheral circulation and tissues.

Tissue resident memory T-cells occupy tissues without recirculating. The one specific surface marker that is associated with these cells is integral aeB7.

Virtual memory T-cells differ from all other memory subsets in that they do not originate from a clonal expansion event. These cells reside at low frequencies.

Natural Killer T-cells (NK)

First off, it should be mentioned that these cells should not be confused with natural killer cells of the innate immune system. Unlike conventional T-cells that recognize antigen epitopes presented on MHC I/II molecules, NKT cells recognize glycolipid antigens presented by a molecule called CD1d. When these cells are activated, these cells perform functions from both T-helper cells and cytotoxic T-cells. These cells specialize in recognizing tumor cells and cells infected with herpes viruses.

 

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Enzyme-Linked Immunosorbent Assays (ELISA)

The first step in any ELISA assay is the immobilization of the antigen within the sample to the wall of the wells within a microtiter plate. These microtiter plates are usually 96-wells. This is by direct adsorption to the plates surface or by using a capture antibody. The capture antibody has to be specific to the  target antigen. After immobilization, another antibody is added called the detection antibody. This detection antibody binds to the adsorbed antigen which forms an antigen:antibody complex. This detection antibody is either directly conjugated to an enzyme, such as horseradish peroxidase (HRP), or provides an antibody-binding site for a secondary labeled antibody. There are four different types of ELISAs which will all be discussed below. ELISAs take advantage of an enzymatic label to produce a signal that can be quantified and correlated to the binding of an antibody to an antigen. The final assay signal is measured using spectophotometry.

Direct ELISA

In the direct ELISA, the detection antibody is conjugated with either alkaline phosphatase (AP) or horseradish peroxidase (HRP). These substrates produce a colorimetric output that is then measured. The advantages of a direct ELISA is that it is a short protocol which saves time and reagent, and money. There is no cross-reactivity from a secondary antibody that can cause interference. The disadvantages are that there is no signal amplification, so the primary antibody must be conjugated for it to work.

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Indirect ELISA

In the indirect ELISA, antibodies can be conjugated to biotin, which is then followed by a streptavidin-conjugated enzyme step. This is becoming more common place within the clinical laboratory. Alternatively, the detection antibody is typically a human IgG antibody that binds to the antigen within the wells. This primary antibody has multiple antibody-binding sites on it. A secondary rabbit anti-human IgG antibody conjugated with an enzymatic substrate is added. This secondary antibody binds to the first antibody and gives off a colorimetric signal which can be quantified by spectrophotometry. There are advantages over the direct ELISA, mainly that there is signal amplification by using several antibodies, allowing for high flexibility. This also creates a longer protocol, and increases the chances for cross-reactivity, which can be deemed disadvantages.

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Sandwich ELISA

The sandwich ELISA is less common, but is highly efficient in antigen detection. It quantifies antigens using multiple polyclonal or monoclonal antibodies. Monoclonal antibodies recognize a single epitope, while a polyclonal antibody recognizes multiple antigen epitopes. The antigen that is to be measured must contain at least two antigenic epitopes capable of binding to an antibody for this reason. The first step is to coat the microtiter plate wells with the capture antibody within a carbonate/bicarbonate buffer (pH 9.6). Proceed to incubate the plate overnight at 4 degrees Celsius. Wash the plate twice using PBS. Incubate the plate again for at least 2 hours at room temperature. Wash the plate again using PBS. The next step is to add diluted unknown samples to each well. Its important to run unknown samples against those of a standard curve by running standards in duplicates or triplicates. Incubate for 90 minutes at 37 degrees Celsius. then remove the sample and wash with PBS again. Next, add diluted detection antibody to each well. Its important to make sure that the detection antibody recognizes a different epitope on the target antigen than the capture antibody. The prevents interference with antibody binding. To maximize specificity and efficiency, use a tested matched pair. Once the detection antibody has been added, incubate for 2 hours at room temperature. Wash once again with PBS. After washing, add conjugated secondary antibody to each well. Incubate once again at room temperature, then proceed to wash. Once again, horseradish peroxidase and alkaline phosphatase are used as enzymes conjugated to the secondary antibody. The substrates for HRP are called HRP chromogens. Cleavage of hydrogen peroxide is coupled to an oxidation reaction which changes color. Another common substrate used is ABTS. The end product is green.

Sandwich-ELISA

The sandwich ELISA employs high specificity, even when using complex samples. Within the sandwich ELISA, both direct and indirect methods can be used. It can be challenging to find two different antibodies against the same target the recognize different epitopes.

Competitive ELISA

The competitive ELISA is exactly what its name suggests; it is a competitive binding process which is produced by the sample antigen, and an add-in known concentration of antigen. A primary unlabeled antibody is incubated with the unknown sample antigen. This creates antigen:antibody complexes, which are then conjugated to a microtiter plate which is pre-coated with the same antigen. Any free antibody binds to the same antigen on the well. Unbound antibody is removed by washing the microtiter plate. The more antigen within the unknown sample means that less antibody will be able to bind to the antigens within the wells, hence the assay gets its name. Its a competition. A secondary conjugated antibody that is specific for the primary antibody bound to the antigen on the pre-coated on the wells is added. When a substrate is added, the reaction elicits a chromogenic or fluorescent signal. The higher the sample antigen concentration, the weaker the eventual signal.

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References

https://www.bio-rad-antibodies.com/elisa-procedure.html

https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/pierce-protein-methods/overview-elisa.html

Donath-Landsteiner Antibodies

The history of the DL antibody goes back to the 1900’s. It was one of the first recognized forms of immune mediated hemolysis and responsible for inducing Paroxysmal Cold Hemoglobinuria (PCH). PCH is a transient condition, meaning that it comes on when immunoglobulins (Antibodies) are formed in response to a viral, bacterial, or spirochete infection. Its history will suggest that there is an association between PCH and syphilis. In over 90% of the cases of PCH in early history, the patient was co-diagnosed with syphilis. Throughout the 1900’s the condition began to evolve and is now seen most commonly in children following some sort of infection. Although it should be noted that PCH is not limited to those of adolescent age. So what really is the Donath-Landsteiner antibody and how does it contribute to PCH?

Clinical Presentation

Paroxysmal Cold Hemoglobinuria (PCH) is an autoimmune hemolytic anemia (AIHA). Autoimmune meaning that they are antibodies that have cross-reacted to attack the individuals own cells. Hemoglobinuria means that there will be hemoglobin present in the blood, which suggests intravascular hemolysis. PCH is one of the more common intravascular hemolytic anemias. Typical patients present with fever, chills, abdominal and back pain, and pronounced hemoglobinuria. PCH typically presents in children following and upper respiratory infection or immunization. These patients often have a rapidly progressing anemia with hemoglobins that can fall as low as 2.5 g/dL. Peripheral blood smears show significant red blood cell agglutination and anisocytosis and poikilocytosis. Anisocytosis indicating variance in size of the red blood cells and poikilocytosis indicating variance in structure to the red blood cells. Schistocytes, spherocytes, and polychromasia are common findings. The spherocytes and polychromasia are indicative of the bone marrow trying to replenish the red cell population as best it can so it forces out immature erythrocytes into the peripheral blood. Its an effort to sustain the hemoglobin as best it can. One distinguishing peripheral blood smear finding in patients with PCH is erythrophagocytosis. Lets break this word down. Erythro- short for erythrocyte meaning red blood cells. Phagocytosis is mediated by neutrophils and monocytes as a way to kill foreign pathogens. In the case of erythrophagocytosis in PCH, neutrophils are characteristically seen engulfing red blood cells, which is diagnostic for AIHA.

The Donath-Landsteiner Antibody

The DL antibody, although being recognized as an cold autoantibody, is an IgG antibody that has developed P antigen specificity and it is a biphasic hemolysin. What that means is that when someone has the DL antibody and is exposed to cold temperatures, it will bind to the individuals red blood cells through the P antigen, but does not cause hemolysis until the coated red blood cells are heated to 37 degrees Celsius as they (RBC:antibody complex) travel from the peripheral fingertips and toes to the core of the human body.   At cold temperatures, the IgG molecule is able to recruit complement (C3), and at the higher temperatures, activates the membrane attack complex (C5-C9) and lyses the red blood cells. One very interesting piece of information regarding the difference between Cold Agglutinin Syndrome (CAS), another autoimmune hemolytic anemia caused by Anti-I, is that the hemolysis from PCH is stronger and more severe because of the DL antibodies ability to detach from lysed red blood cells and reattaching to other cells. 

Laboratory Diagnosis

There are a few different ways to pinpoint PCH in the blood bank. One is by use of a Direct Coombs test (DAT). This test provides information regarding the type of hemolysis, whether it be acquired or inherited. It also tests for antibodies that have are bound in vivo. The most common DAT result in PCH is red blood cells coated with C3d causing a positive reaction. This is sensitive in 94-99% of cases. The other way to diagnosis DLAIHA (Donath-Landsteiner Autoimmune Hemolytic Anemia) is by the indirect DL test. This process involves collection of a fresh serum specimen that is strictly maintained at 37 degrees Celsius from collection all the way through to testing. If the sample is allowed to cool or is refrigerated, there could potentially be autoadsorption of the DL anti-P antibodies onto the patients autologous red blood cells. This could cause a false negative result. Upon testing, the patients serum is mixed with P antigen positive, group O red blood cells, and fresh donor serum. The fresh donor serum is added because the complement level within the patients may be low due to consumption. The patient and donor serum mixture is incubated in a melting ice bath (O degrees Celsius) for 30 minutes, then warmed to 37 degrees Celsius for one hour. The specimen is then centrifuged and examined for hemolysis. If hemolysis is present then this constitutes a positive result for DL antibody.

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Indirect DL test: As you can see in tubes 1 and 4, the presence of hemolysis indicates a positive test result for the DL antibody.

Treatment

There is unfortunately no cure for PCH, and very little reliable treatment options for those with the DL antibody. It is recommended to avoid cold climates as much as possible and when inside to have the temperature at 30 degrees Celsius to keep the hemoglobinuria low. This doesn’t treat the PCH, but it will minimize the recurrence and induced anemia. Steroids have been through extensive trials for treatment of PCH and there are mixed results. Theory is that steroids are better at clearing red blood cells coated with IgG, and less effective at clearing red blood cells that are coated with complement. More aggressive treatment such as splenectomy and Rituximab, which is an monoclonal antibody that targets the transmembrane protein CD20 present on B cells has been found effective for those patients with refractory PCH.

Adrenal 101

The adrenal glands also known as the suprarenal glands. Supra meaning above, and renal meaning kidneys. So these glands are situated on top of the kidneys. These are endocrine glands that produce a variety of hormones, but most notable adrenaline, and the steroids aldosterone and cortisol. Each gland has an outer cortex which is divided into three different zones and an inner medulla. The three zones of the cortex are the zone glomerulosa, zone fasciculate, and zone reticularis.

This article will go briefly touch on the structure of the adrenal gland, including each zone of the cortex. Then it will dive into the function of the adrenal gland and the hormones it produces along with their specific cellular target. Finally the article will conclude with an overview of adrenal insufficiency and cortisol overproduction and diseases that illustrate those two conditions.

Structure

adrenal gland sections

As mentioned earlier, the gland is composed of an outer cortex, and an inner medulla. The outer cortex can be further divided into three zones that each have a specific function.

Zona Fasciculata

The zona fasciculata sits between the other two zones (zona glomerulosa, and zona reticularis) and consists of cells responsible for producing glucocorticoids such as cortisol. Its the largest of the three zones consisting of about 80% of the space in the cortex.

Zona Glomerulosa

The zona glomerulosa is the outermost zone of the adrenal cortex. The cells that are situated in this zone are responsible for the production of mineralocorticoids such as aldosterone. Aldosterone is an important regulator of blood pressure. Review the article covering the Renin-Aldosterone system.

Zona Reticularis

The zona reticular is the innermost cortical layer which is primarily responsible for producing androgens. Its main component synthesized is dehydroepiandrosterone (DHEA), and androstenedione, which is the precursor to testosterone.

Medulla

The medulla is in the centre of each adrenal gland with the cortex around the entire periphery. The chromatin cells within the medulla are the bodies main source of catecholamines. Catecholamines produced in the medulla are adrenaline (epinephrine), and noradrenaline (norepinephrine). Regulation of the synthesis of these catecholamines is driven by the sympathetic nervous system via the preganglionic nerve fibers stemming from the thoracic spinal cord (T5-T11) to the adrenal glands. When the medulla gets stimulated to produce these hormones it secretes them directly into the cardiovascular circulation system, which is unusual of sympathetic innervation as they usually have distinct synapses on specialized cells.

Mineralocorticoids

Mineralocorticoids such as aldosterone are named according to its function. They regulate minerals, such as salt and regulate blood volume (blood pressure). Aldosterone, the most prominent mineralocorticoid acts on the distal convoluted tubules and the collecting ducts by increasing the reabsorption of sodium and the excretion of both potassium and hydrogen ions. The amount of salt present in the body affects the extracellular volume, which influences the blood pressure.

Glucocorticoids

Glucocorticoids are also named due to its function. Cortisol is a prominent glucocorticoid that regulates the metabolism of proteins, fats and sugars (glucose). Cortisol increases the circulating level of glucose. They cause protein catabolism into amino acids and the synthesis of glucose from the amino acids in the liver. They also increase the concentration of fatty acids by increasing lipolysis (fat breakdown) which cells can use as an alternative energy source in situations of glucose absence. Glucocorticoids also play a role in suppression of the immune system. They induce a potent anti-inflammatory effect.

Cortisol

Cortisol is the prominent glucocorticoid produced by the adrenal gland. The adrenal gland secretes a basal level of cortisol depending on the time of day it is. Cortisol concentrations in the blood are highest in the early morning and lowest in the evening as part of the circadian rhythm of adrenalcorticotropic hormone (ACTH) secretion. The article on general endocrinology explains what ACTH is and how it affects the adrenal gland. Basically what happens is the hypothalamus secretes corticotropin releasing hormone that acts on the pituitary to produce ACTH that acts on the adrenal gland cortex to produce cortisol.

Androgens and Catecholamines

The primary androgen produced by the adrenal gland is DHEA, which is converted to more potent androgens such as testosterone, DHT, and estrogen in the gonads. DHEA acts as a precursor. Androgens drive sexual maturation.

Catecholamines are produced by the chromaffin cells from tyrosine. The enzyme tyrosine hydroxyls converts tyrosine to L-DOPA. L-DOPA is then converted to dopamine before it can be turned into norepinephrine. Norepinephrine is then converted to epinephrine by the enzyme phenylethanolamine N-methyltransferase (PNMT). Epinephrine and norepinephrine act as adrenoreceptors throughout the body, whose primary effect is to increase the blood pressure and cardiac output by way of vasoconstriction. Catecholamines play a huge role in the fight-or-flight response.

Corticosteroid Overproduction

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The normal function of the adrenal gland can be impaired from infections, tumors, autoimmune diseases, or from previous medical therapy such as radiation and chemotherapy. Cushing’s syndrome is the manifestation of glucocorticoid excess. Symptoms and sign are a direct result of chronic exposure to glucocorticoids. Diagnosis is difficult because the symptoms are often nonspecific and pathognomonic of the syndrome in isolation. Symptoms include proximal (distant) muscle weakness, wasting of the extremities, increased fat in the abdomen and face often leading to a moon face, bruising without trauma, and a buffalo hump. A buffalo hump is fat on the back of the neck and supraclavicular pads. In women, menstrual irregularities are common such as oligomenorrhea (infrequent menstrual periods), amenorrhea (absence of menstrual periods), and variable menses. Hyperpigmentation can occur by increased secretion of cortisol. Cortisol acts on the melanocyte-stimulating hormone receptors.

Glucose intolerance is common in Cushing’s syndrome. Primarily due to stimulation of gluconeogenesis by cortisol and insulin resistance caused by the obesity. This leads to hyperglycemia, which can exacerbate any diabetic patient.

Bone loss and osteoporosis is common in patients with Cushing’s syndrome because there is less intestinal calcium absorption. Calcium is vital to bone health and growth. The decrease in bone formation is coupled with an increased rate of bone reabsorption which can lead to more pathological fractures.

Adrenal Insufficiency

Addison’s disease is considered primary hypoadrenalism. There is an inherent deficiency of glucocorticoids and mineralocorticoids. Most commonly caused by an autoimmune condition. Autoimmune means that the body is attacking itself by production of antibodies against cells of the adrenal cortex. In cases of adrenal crisis due to autoimmune primary adrenal insufficiency clinical presentation is usually the patient presenting in a state of shock. Abdominal tenderness upon deep palpation is common. Patients present with hyperpigmentation due to chronic ACTH release by the pituitary. Proopiomelanocortin is overproduced which is a pro hormone that is cleaved into its biologically active hormones corticotropin and melanocyte-stimulating hormone (MSH). This causes increased melanin synthesis, causing the hyperpigmentation. Other non-specific symptoms such as lethargy, fatigue, weakness, confusion, anorexia, nausea, vomiting, or even coma can occur. One of the most commonly presented symptoms is fever and infection, which can be exaggerated by the hypocortisolemia.

Its important to take this article slowly. There a lot of different parts, but the aim was to look at the hormones themselves and how they physiologically act on the body, then take what was learned about those and apply them to two scenarios, hypo/hyperadrenalism and how it affects the body. Cushing’s syndrome is where there is hyperproduction of cortisol primarily leading to many disastrous effects on the body. Addisons disease is an autoimmune disease where the body produces antibodies against the cells of the adrenal cortex, causing destruction of the gland itself, again leading to detrimental effects on the body.

Iron Absorption and Metabolism

Iron is an essential element for almost all forms of life, but most important as an oxygen transporter. When iron is in its ferrous state, oxygen binds to it within the hemoglobin molecule allowing erythrocytes to circulate and deliver oxygen to all the human bodies cells and tissues. The human body also requires iron in order to obtain ATP from cellular respiration (Oxidative phosphorylation). Although iron is an essential element to the body, like anything in nature, too much of it can be toxic. Its ability to donate and accept electrons readily means that it can spontaneously catalyze the conversion of hydrogen peroxide into free radicals. Free radicals cause a wide array of damage to cellular structures and tissues. To minimize the chances of toxicity, almost every iron atom is bound to protein structures, an example being hemoglobin. The iron is bound to the globin protein. To learn more about the structure of hemoglobin, review the previous article written. There is tight regulation of iron metabolism that allows the body to remain in homeostasis. Understanding iron metabolism is important for understanding multiple diseases of iron overload, and iron deficiency.

Iron Absorption

Most of the bodies iron comes from dietary uptake. There is continuous iron recycling occurring within the body from the sequela of hemoglobin metabolism by the spleen. The macrophages of the reticuloendothelial system store iron from the process of breaking down engulfed red blood cells. Its stored as hemosiderin. Hemosiderin is just a defined deposit of protein and iron that occurs as a result of iron overload, either systemically or locally. The metabolic functions of iron depend on the ability to change its valence state from reduced ferrous state (Fe2+) to the oxidized ferric state (Fe3+). Ferrous iron in the lumen of the duodenum is transported across the luminal side of the enterocyte by a protein called divalent metal transporter-1 (DMT1). Once iron has been absorbed across the cell membrane of the enterocyte, it can either be stored by binding to apoferritin or the cell can release the iron through the help of another transporter called ferroportin. Ferroportin is the only know protein that exports iron across cell membranes. One of the ways that the human body manages iron homeostasis is by the production of hepcidin. When iron stores are adequate, the liver will produce hepcidin, which competitively binds to ferroportin and inactivates it. When iron stores begin to drop, suppression of synthesis of hepcidin allows ferroportin to transport iron again. Before iron is taken by ferroportin across the membrane, it must be converted to its ferric form. Hephaestin, another protein on the enterocyte cell membrane oxidizes iron as it exits to its ferric form (Fe3+). Once oxidized and in its ferric state, the iron binds to apotransferrin (ApoTf). This iron:apotransferrin complex is known as transferrin (Tf). Its important to note that two molecules of ferric iron can bind to one molecule of apotransferrin.

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Iron Uptake into Cells

Individual cells regulate the amount of iron they absorb to avoid adverse toxicity. Cells possess a receptor for transferrin (Tf), called transferrin receptor-1 (TfR1). The physiological pH of the plasma and extracellular fluid allow for a strong affinity to transferrin for TfR1. Through receptor mediated endocytosis transferrin saturates the TfR1 and once a critical mass has accumulated, endocytosis begins. The iron is passed into the cell into an endosome vesicle. Hydrogen ions are then pumped into the endosome and as a result the pH drops causing dissociation of the iron from the transferrin. Almost simultaneously the affinity for TfR1 to apotransferrin increases so it remains bound to the receptor while the iron remains free. The iron is then exported from the endosome vesicle into the cytoplasm by divalent metal transporter 1 (DMT1). The molecules of iron are then either stored, or transported into the mitochondria where they are incorporated into cytochromes or heme for the production of hemoglobin. While the iron is transported in the cytoplasm, the endosome fuses again with the cell membrane and in the extracellular space pH, TfR1 has a low affinity for apotransferrin so it dissociates and begins circulating again in the plasma for free transferrin. Again transferrin being a diiron:apotransferrin complex. Cells are able to store iron so they have a reserve if needed. Ferric iron (Fe3+) is stored in a protein called apoferritin. When iron binds to it it known as ferritin. Ferritin can be used at anytime during iron depletion by lysosomal degradation of the protein.

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Regulation

Just like hepcidin, there are other ways that the body maintains iron homeostasis. Transcription of TfR1 on the surface of the cells can either decrease or increase depending on iron stores within the cell. When iron stores are sufficient, production of TFR1 decreases, and vice versa. This is also useful in diagnosis of iron deficiency. Turns out there is a truncated form of TfR1 that circulates in serum as soluble transferrin receptors (sTfR). These sTfRs reflect the amount of tFR1 in the body. So in iron depletion there will be more circulating sTfRs indicating more production of TfR1 on the cells surface. A useful tool in the diagnosis of iron deficiency anemia.

Iron Recycling

When cells die, they are sequestered by the spleen and liver in which mechanisms salvage iron. These mechanisms are often referred to as the haptoglobin-hemopexin-methemalbumin system. Free hemoglobin in the plasma is quickly complexed with haptoglobin. By binding haptoglobin, the hemoglobin, and consequently, the iron avoid filtration by the glomerulus in the kidneys. This complex is taken up by macrophages, primarily those in the liver, spleen, bone marrow and even in the lungs. These macrophages express CD163, which is the haptoglobin scavenger receptor. The entire complex is internalized into the macrophage within a lysosome. Inside this lysosome, the iron is salvaged, the globin is catabolized as any protein would be, and the protoporphyrin is converted to unconjugated bilirubin. To learn more about the process of bilirubin metabolism, review the previous article. The haptoglobin is also degraded by the lysosome. The iron in free hemoglobin becomes oxidized to its ferric state (Fe3+), and as a result, forms methemoglobin. The heme (metheme) molecule of the free hemoglobin binds to hemopexin, preventing oxidative injury to the cells and tissues, as well as prevents loss of iron through glomerulus filtration. Albumin acts as a carrier for many proteins, including metheme. So albumin acts as a carrier for metheme to find hemopexin, which has a much higher affinity for the metheme itself. This allows for more rapid degradation of the toxic metheme.

There was a lot to learn in this article. Read carefully and go back and refer. I will try to highlight certain areas that I think are more important to the bigger picture. The next step is what happens in certain physiological disease states that leads to either iron overload or iron deficiency.