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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Liver Enzymes

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The liver is the largest internal organ. It is incredibly functionally complex. It carries out more than 500 different functions that include detoxification, protein synthesis and chemical synthesis to aid in digestion. Some of the livers main functions will be talked about. Bile produced by the liver aids in digestion in the small intestine. It breaks down and absorbs fats, cholesterol, and occasionally some vitamins. Bile is produced by the liver and is stored in the gallbladder. The liver also absorbs and metabolizes bilirubin. Bilirubin is caused by the breakdown of hemoglobin in red cells. This can either be extravascular or intravascular hemolysis and can either be normal breakdown as red cells only live for 120 days before being recycled or it can be not normal. The iron that is released by the bilirubin is stored in the liver or the bone marrow that is used to make new red cells. Bilirubin is metabolized to its direct conjugated form where it is excreted by the urine and feces. The liver produces clotting factors. Vitamin K is necessary for some clotting factors to be produced and in order for vitamin K to be absorbed from the diet the liver needs to produce bile. Without the bile clotting factors would not be produced. The liver aids in filtering the blood. It filters and removes different waste material produced from the body as well as exogenous compounds like alcohol and other drugs. The liver also has a immunological function in that it contains Kupffer cells. These Kupffer cells are the macrophages of the liver and are part of the mononuclear phagocyte system that destroy any foreign antigen that enters the liver. The liver produces albumin which is arguably the most important protein in the body. Albumin is used as a transport protein and maintains colloid pressure in the blood vessels. Angiotensinogen is produced by the liver that is used in the angiotensinogen-Renin system in the kidneys that raises blood pressure by vasoconstriction.

Regeneration is a unique feature of the liver. Its important to the body is unmatched and it has evolved the ability of regeneration. Regeneration is the ability for the organ to regrow rapidly as long as it is kept healthy. During this process of regeneration the function of the liver is not compromised. If needed there are a number of compounds that can aid in the regeneration process like hepatocyte growth factor, insulin, epidermal growth factor, IL-6, and even norepinephrine.

Analysis of bilirubin is based on the reaction of bilirubin with a diazotized sulfanilic acid. Three fractions of bilirubin is measured. Conjugated, unconjugated, and delta bilirubin. Delta bilirubin is bilirubin bound to albumin. A fasting sample is preferred and hemolyzed samples should be avoided. Bilirubin is sensitive to light so care should be taken to shield the sample from the light.

Liver enzymes are relatively nonspecific indicators that can indicate tissue destruction in several organs. Alkaline phosphatase is a group of isoenzymes that are found on the membranes of cells in almost every tissue. Alkaline phosphatase can become elevated in many different conditions including bone diseases, puberty, and even in late pregnancy. One important feature of alkaline phosphatase is that the isoenzyme found in bone is the most heat labile. Differentiation in the laboratory includes heating the sample and then remeasuring the alkaline phosphatase and measuring the difference.

Gamma-glutamyltransferase (GGT) is a membrane enzyme that is important in glutathione metabolism. It transfers glutamate to the amino acid peptide chain. GGT is among one of the first enzymes to become elevated in acute liver diseases such as hepatitis. Its important to note that GGT is normal in patients with bone diseases making it one of the most clinically specific of all the liver enzymes. Lactate dehydrogenase is a cytosolic enzyme that interconverts pyruvate and lactate. There are five isoenzymes. Isoenzymes 4 and 5 are increased in viral or toxic hepatitis, biliary obstructions and cirrhosis. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) convert aspartate and alanine to oxaloacetate and pyruvate respectively. ALT is very specific for liver pathology. AST is found in liver tissues, but also present in heart and muscle tissue. The AST/ALT ratio is an important factor to look at. A ratio less than 1.0 indicates viral hepatitis. This is an increase in ALT greater than the increase in AST. An AST/ALT ratio greater than 1.0 usually means that the ALT is elevated to a lesser degree than the AST and this can be found in cirrhosis, bile duct obstruction, or metastatic cancer of the liver.

-Caleb

Viral Hepatitis

Viral hepatitis is acute or chronic symptomatic or even sometimes asymptomatic inflammation of the liver. Usually the infection is self-limiting and clears within 12 weeks, but in immunocompromised patients it can pose serious complications and development of systemic problems. All viral hepatitities can lead to liver cirrhosis if untreated. There are important serological markers involving different proteins of the virus that can tell us whether or not the infection is acute or chronic and whether or not the virus is actively replicating or not. The viruses replicate one week before detection and two weeks post-detectable levels. The most common types of hepatitis are A, B and C. Although Hep A and B are on the decline as vaccination campaigns have been more successful. Hep C is gradually declining, but as of recently there has been an increase in incidence. It should be noted that as of now there is no currently FDA approved vaccination for Hep C. There are some more less common Hep virus like E, and D, but the incidence is low and clinically not very relevant. One important distinction to point out is that a person much have Hep B to acquire Hep D. Hep D requires the viral envelope protein from Hep B to survive.

Hep A is a +ssRNA picornavirus. It is non-enveloped which means when it is shedding and replicating it causes lysis of the hepatocytes in the liver. The disease is spread by fecal/oral route and the virus is exceptionally hardy. It is very hard to inactivate and can survive in water or on surfaces for an extended period of time. The incubation period is 20-40 days and the disease itself is mild in nature. Clinical presentation includes variable prodromal symptoms usually fever, malaise, weakness, nausea, loss of appetite, and myalgia. Jaundice may or may not be present. The virus usually presents as asymptomatic, especially at a younger age as the immune system is able to hold it in check. Symptoms can actually be a good thing as it means that the immune system is recognizing the virus and trying to rid of it. Risk groups include those who have direct contact with persons who have Hep A such as a healthcare worker, travelers to endemic countries are at high risk even when good hygiene and sanitation measures are being applied. Less commonly users of injection and non-injection drugs, and people with clotting factor disorders. Diagnostic workout includes serologically workup and liver enzyme panel as well as bilirubin levels.

Hep B is a dsDNA-RT enveloped hepadnavirus. The Hep B genome is unique in that it virally replicates using an RNA intermediate and uses its reverse transcriptase mechanism to complete its DNA replication making the life cycle very complex. Transmission is through body fluids or sexually and is considered a sexually transmitted disease (STD). Activities involving percutaneous or mucosal contact such as heterosexual contact, injection drug-use with sharing of needles, birth to an infected mother or contact with blood or sores of an infected person. Medical professionals should take care to avoid any needle sticks or other exposures to those infected. Its estimated that 400 million people worldwide are infected with Hep B. The clinical presentation often presents with very generalized symptoms similar to Hep A with the addition of joint pain, jaundice, dark urine or clay-colored bowel movements. Chronic Hep B infection is not commonly talked about as Hep C is primarily dubbed as the chronic hepatitis infection, but 5% of adults become chronically infected with Hep B. They can live asymptomatically or they can display a spectrum of disease ranging from chronic hepatitis flares to cirrhosis or hepatocellular carcinoma.

The Hep B diagnosis is largely reliant on serology. The Hep B surface antigen (HBsAg) is a protein on the surface of HBV. It can be detected in high levels in the serum during acute or chronic HBV infection. It should be noted that individuals typically lose the expression of the HBsAg after 12 weeks when chronically infected. The presence of HBsAg means that person is infectious. Hep B surface antibody (Anti-HBs) present means that the person is recovering from an infection and that they now have immunity to the HBV. Anti-HBs also is developed when an individual is vaccinated against Hep B. IgM antibody to Hep B core antigen (IgM-Anti-HBc) indicates that an individual has an acute Hep B infection or has had an infection within the last 6 months. IgG-Anti-HBc indicates previous infection with Hep B and immunity towards the virus. The core antibody persists for life. Its important to note that when you run labs looking for hepatitis if the Anti-HBs antibody is present, but the Anti-HBc antibody is NOT present this indicates the individual has been vaccinated, not previously infected. The core antibody will not develop during vaccination. The Hep B (e) antigen is a secreted product of the nucleocapsid of HBV that is found in the serum during actively replicating infection. This indicates that the individual has an incredibly high viral burden. Anti-HBe antibody produced is an indicator of seroconversion and is used as a predictor of clearance of the Hep B virus for individuals undergoing treatment. Acute Hep B is usually not treated and the individual receives supportive therapy if needed. Chronic Hep B is treated using interferon or five different antiviral nucleoside analogs. The treatment is not curative, and only has a 20-50% response rate, but improves prognostic markers. Hep B is HIGHLY infective, 50-100x more infectious than HIV and 10x more infectious as HCV.

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Hep C is a +ssRNA enveloped flavivirus. Typical transmission is through exposure to infectious blood. This can either be through injection drug use of shared needles or through recipient of donated blood, or organs, or neonatal birth to an HCV infected mother. Sexual transmission can still occur, but not at the rate that Hep B is transmitted. Acute infection usually is asymptomatic or presents with only mild symptoms similar to that of HAV and HBV. The incubation period is 4-12 weeks. Chronic HCV is generally asymptomatic while some individuals present with mild chronic liver disease and others with cirrhosis or hepatocellular carcinoma. 15-25% of individuals who develop Hep C will be able to clear the disease. Of the 75-85% that develop chronic HCV 60-70% will develop chronic liver disease, 5-20% will develop cirrhosis, and 1-5% will develop HCC. Hep D diagnosis is dependent on screening tests for the HCV antibody. These screening methods include enzyme immunoassay (EIA) or enhanced chemiluminescence immunoassay (CIA). There are PCR tests that detect the HCV RNA and also that quantify the viral RNA to determine the disease burden. A diagnosis based solely on elevation of liver enzymes AST, ALT is not acceptable. There is no currently accepted vaccine, but there is research ongoing. Previous infection does not grant immunity for subsequent infections like one would think likely due to high rte of viral mutation. There are 3 characteristic classes of drugs for treatment of Hep C and one novel advancement of one. HCV protease, polymerase inhibitors and interferon are most commonly used. A new novel treatment called direct acting antivirals (DAA) is a combination therapy that is shown to be highly effective at suppressing viral replication and can actually result in a cure in as short as 12 weeks of treatment. The cure rate is 95%. This novel treatment protocol is showing unprecedented success, but it comes as cost. A cost that no insurance company wants to pay for and that most people can’t afford.

Chronic HBV and HCV are associated with an increased incidence of HCC. This is due to the continuous cycles of viral replication, the immune-mediated killing of the hepatocytes and inflammation. Inflammation induces oxidative damage and altered cellular metabolism. This creates an unhealthy environment in the liver.

-Caleb