Immunology

Biochemical Assessment of Nutrition Status – Immunocompetence and Hematological Assessment

August 3, 2016 Hematology, Immunology, Medical Nutrition No comments , , , , , , , , , , , ,

Immunocompetence Assessment

Historically, evaluation of immunocompetence has been included as a part of any discussion of protein and nutrition assessment. This is logical, since adequate and appropriate immune function is dependent in part on adequate protein status. Protein deficiency routinely results in increased risk of infection as well as altered immune and inflammatory responses. But in clinical practice, the use of this type of nutrition assessment is complicated by the presence of disease and infection, which of course also affect all components of the immune system.

Nutrition Care Indicator: Total Lymphocyte Count (TLC) When evaluating a complete blood count (CBC) and differential count, calculaltion for TLC can be completed as follows:

TLC = WBC X % lymphocytes / 100

Total lymphocyte count will be affected by presence of infection, trauma, stress, and diseaes such as cancer and HIV, as well as medications that influence the immune system (e.g., chemotherapy and corticosteroids).

Nutrition Care Indicators for Hematological Assessment

Evaluation of erythrocytes (red blod cells, or RBC) can be an important component of nutrition assessment and is key to diagnosis of all anemia types. A complete blood count includes measurement of the total number of blood cells in the volume of blood. Many types of anemias exist, including those caused by deficiencies of iron, folate, or vitamin B12 and those arising from chronic diseases such as renal failure and congestive heart failure. Anermias are diagnosed by evaluation of the complete blood count and by the microscopic evaluation of the size, shape, and color of erythrocytes.

Hemoglobin (Hgb) Hemoglobin is a protein found in erythrocytes that functions to deliver oxygen to cells and to pick up carbon dioxide for expiration by the lungs. Measurement of hemoglobin is common in diagnosis of anemias, particularly iron-deficiency anemia. Additionally, hemoglobin is decreased in some chronic diseases and protein-energy malnutrition. Even though it is commonly measured, it is not the most sensitive or the most specific of hematological assessments of nutritional status. For example, in iron deficiency, iron stores may be depleted before serum hemoglobin levels will be affected.

Hematocrit (Hct) Hematocrit is defined as the percentage of blood that is actually composed of red blood cells. Hematocrit, like hemoglobin, will be decreased only in the final stage of iron deficiency. Hematocrit is affected by other nutrient deficiencies as well as by hydration status.

Mean Corpuscular Volume (MCV) Mean corpuscular volume is a measure of the average size of an individual red blood cell. A variety of anemias are characterized by changes in RBC size; for example, MCV is reduced in iron an copper deficiencies and elevated in folic acid and vitamin B12 deficiencies.

Mean Corpuscular Hemoglobin (MCH) Mean corpuscular hemoglobin is an estimate of the amout of hemoglobin in each cell. Thi value can reflect total serum hemoglobin levels. In some situations, however, MCH remains normal while the number of red blood cells is low, resulting in low total Hgb. Abnormalities are generally specific to iron deficiency and other nutritional anemias.

Mean Corpuscular Hemoglobin Concentration (MCHC) Mean corpuscular hemoglobin concentration also estimates the amount of hemoglobin in each red blood cell, but it expresses the value as a percentage.

Ferritin Ferritin is a protein that serves as a storage form of iron; therefore, serum ferritin is an estimate of iron stores. Ferritin is a sensitive and specific measure of iron status and will be one of the first indices to change in iron deficiency.

Transferrin Saturation As discussed earlier under "Protein Assessment," transferrin is a serum protein responsible for transport of iron systemically. Each molecule of transferrin can carry two molecules of iron. Under normal conditions, approximately 30% of iron binding sites on the transferrin molecule are saturated (i.e., have iron attached). The body's requirement for iron and overall iron status will be reflected by changes in transferrin saturation. When iron status is low, transferrin is less saturated. Transferrin is calculated by using the ratio of serum levels to total iron biding capacity (TIBC). TIBC is the test used to measure the saturation ability for transferrin. TIBC is higher during iron deficiency and lower after repletion. There are numerous equations to calculate transferrin from TIBC, but, as mentioned earlier, transferrin is not the most reliable indicator of protein status due to the effect of iron status.

Protoporphyrin When there is inadeuate iron available for hemoglobin synthesis, zinc is substituted fro iron within hemoglobin. Consequently, zinc protoporphyrin (the protein transporter for zinc) levels rise during iron deficiency and are considered a sensitive measure of iron-deficiency anemia.

Serum Folate Coenzymes associated with folate are necessary for amino acid metabolism, including many one-carbon transfer reactions such as the conversion of histidine to glutamate. Folate coenzymes also play a cruical role in the synthesis of purine needed for DNA. Folate deficiency can be diagnosed when megaloblastic, macrocytic red blood cells are present and serum folate and red cell folate are decreased, while serum B12 remains within normal limits. If folate levels are inadequate for conversion of histidine to glutamate, an intermediate product, formiminoglutamate (FIGlu), is formed. Urinary levels of FIGlu are thus elevated in folate deficiency and serve as a diagnostic tool for the condition.

Serum B12 Anemia associated with B12 (cobalamin) deficiency can be diagnosed in several ways. Clinically, it will be similar to folate deficiency but can be distinguished by measuring serum B12 levels, including serum total cobalamin and serum holo-transcobalamin II (the transport protein for B12). Biomarkers of B12 include homocysteine and methylmalonic acid levels, which change early on in the development of B12 deficiency. Historically, the Schilling test allowed for determination of defective absorption (gastric vs. intestinal). In this test, B12 is given as an injection and the amount excreted in urine is measured. This allows problems with different steps of B12 absorption to be distinguished. The Schilling test is no longer used in clinical practice, though to date no other test has replaced it specific function.

Specific Immunosuppressive Therapy

July 20, 2016 Hematology, Immunology, Infectious Diseases, Oncology, Pharmacology, Transplantation No comments , , , , , , , , , , , , , , , ,

The ideal immunosuppressant would be antigen-specific, inhibiting the immune response to the alloantigens present in the graft (or vice versa alloantigens present in recipient in GVHD) while preserving the recipient's ability to respond to other foreign antigens. Although this goal has not yet been achieved, several more targeted immunosuppressive agents have been developed. Most involve the use of monoclonal antibodies (mAbs) or soluble ligands that bind specific cell-surface molecules. On limitation of most first-generation of mAbs came from their origin in animals. Recipients of these frequently developed an immune response to the nonhuman epitopes, rapidly clearing the mAbs from the body. This limitation has been overcome by the construction of humanized mAbs and mouse-human chimeric antibodies.

Many different mAbs have been tested in transplantation settings, and the majority work by either depleting the recipient of a particular cell population or by blocking a key step in immune signaling. Antithymocyte globulin (ATG), prepared from animals exposed to human lymphocytes, can be used to deplete lymphocytes in patients prior to transplantation, but has significant side effects. A more subset-specific strategy uses a mAb to the CD3 molecule of the TCR, called OKT3, and rapidly depletes mature T cells from the circulation. This depletion appears to be caused by binding of antibody-coated T cells to Fc receptors on phagocytic cells, which then phagocytose and clear the T cells from the circulation. In a further refinement of this strategy, a cytotoxic agent such as diphtheria toxin is coupled with the mAb. Antibody-bound cells then internalize the toxin and die. Another technique uses mAbs specific for the high-affinity IL-2 receptor CD25. Since this receptor is expressed only on activated T cells, this treatment specifically blocks proliferation of T cells activated in response to the alloantigens of the graft. However, since TREG cells also express CD25 and may aid in alloantigen tolerance, this strategy may have drawbacks. More recently, a mAb against CD20 has been used to deplete mature B cells and is aimed at suppressing AMR (antibody-mediated rejection) responses. Finally, in cases of bone marrow transplantation, mAbs against T-cell-specific markers have been used to pretreat the donor's bone marrow to destory immunocompetent T cells that may react with the recipient tissues, causing GVHD.

Because cytokines appear to play an important role in allograft rejection, these compounds can also be specifically targeted. Animal studies have explored the use of mAbs specific for the cytokines implicated in transplant rejection, particularly TNF-alpha, IFN-gamma, and IL-2. In mice, anti-TNF-alpha mAbs prolong bone marrow transplants and reduce the incidence of GVHD. Antibodies to IFN-gamma and to IL-2 have each been reported in some cases to prolong cardiac transplants in rats.

TH-cell activation requires a costimulatory signal in addition to the signal mediated by the TCR. The interaction between CD80/86 on the membrane of APCs and the CD28 or CTLA-4 molecule on T cells provides one such signal. Without this costimulatory signal, antigen-activated T cells become anergic. CD28 is expressed on both resting and activated T cells, while CTLA-4 is expressed only on activated T cells and binds CD80/86 with a 20-fold-higher affinity. In mice, D. J. Lenschow, J. A. Bluestone, and colleagues demonstrated prolonged graft survival by blocking CD80/86 signaling with a soluble fusion protein consisting of the extracellular domain of CTLA-4 fused to human IgG1 heavy chain. This new drug, belatacept, was shown to induce anergy in T cells directed against the graft tissue and has been approved by the FDA for prevention of organ rejection in adult kidney transplant pateints.

The Barriers, The Innate Immune System, and Correlations to Inflammation

October 27, 2015 Immunology, Infectious Diseases No comments , , , , , ,


11th_Annual_Randy_Oler_Memorial_Operation_Toy_Drop_at_Fort_Bragg_N.C.,_Dec._6,_2008Defenses of the skin and Mucosa

Epidermis

The epidermis consists of stratified squamous cells, most of which are keratinocytes. Keratinocytes produce the protein keratin, which is not readily degraded by most microorganisms. As cells from the dermis are pushed outward into the epidermal region, they produce copious amounts of keratin and then die. This layer of dead keratinized cells forms the surface of the skin. The dead cells of the epidermis are continuously shed (desquamation). Thus, bacteria that manage to bind to epidermal cells are constantly being removed from the body.

Skin is dry and has an acidic pH (pH 5), two features that inhibit the growth of many pathogenic bacteria, which prefer a wet environment with a neutral pH (pH 7). The temperature of the skin (34 C to 35 C) is lower than that of body interior. Accordingly, bacteria that succeed in colonizing the skin must be able to adapt to the very different internal environment of the body if they manage to reach underlying tissue.

Hair follicles, sebaceous (fat) glands, and sweat glands are composed of simple epithelial cells and offer sites for potential breaches in the skin that could be used by some bacteria to move past the skin surface. These sites are protected by the peptidoglycan-degrading lysozyme and by lipids that are toxic to many bacteria.

The defenses of the skin do not completely prevent bacterial growth, as is evident from the fact that there are bacteria capable of colonizing the surface of the skin. The consist primarily of gram-positive bacteria, a mixture of cocci and rods. The commensal microbiota of the skin helps to protect against pathogenic bacteria by occupying sites that might be colonized by pathogenic bacteria. It also competes with incoming pathogens for essential nutrients. Some resident bacteria also produce bactericidal compounds which target other bacteria. The commensal microbiota does not completely prevent colonization of the skin by potential pathogens but hampers it enough so that the colonization by pathogenic bacteria is usually transient.

Mucosal Surfaces

The respiratory tract, gastrointestinal tract, and urogenital tract are topologically “inside” the body, but they are exposed constantly to the outer environment and foreign materials.

Internal surface areas/mucosal epithelia are comprised of only one epithelial layer. Mucosal epithelia have a temperature of around 37 C and a pH of 7.0 to 7.4. Mucosal epithelia are continuously bathed in fluids.

Mucosal cells are regularly replaced and old cells are ejected into the lumen. Thus, bacteria that manage to reach and colonize a mucosal surface are constantly being eliminated from the mucosal surface and can remain in the area only if they can grow rapidly enough to colonize newly produced cells.

Chemical and other innate defenses help to reduce the growth rates of bacteria sufficiently to allow ejection of mucus blobs and sloughing of mucosal cells to clear the bacteria from the area.

Mucus is an important defense that protect mucosal from bacteria. Mucus is a mixture of glycoproteins produced by goblet cells, a specialized cell type incorporated into the epithelial layer. Mucus has a viscous, slimy consistency, which allows it to act as a lubricant. Mucus plays a protective role because it traps bacteria and prevents them from reaching the surfaces of the mucosal. Mucus is constantly being produced, and excess mucus is shed in blobs that are expelled. Bacteria trapped in mucus are thus eliminated from the site.

In the gastrointestinal and urinary tracts, peristalsis and the rapid flow of liquids through the area remove the mucus blobs, along with the lumen contents.

In the respiratory tract and in the fallopian tubes, there are specialized cells, ciliated columnar cells, whose elongated protrusions (cilia) are continuously waving in the same direction. The waving action of the cilia propels mucus blob out of the area. Mucus has proteins that have antibacterial activity and these proteins include lysozyme, lactoperoxidase, toxic antimicrobial peptides (defensins, cathelicidins, histatins). Lactoferrin sequesters iron and deprives bacteria of this essential nutrient.

Most mucosal surfaces are protected by a normal resident microbiota, except uterus and upper female genital tract and the urinary tract. Resident microbiota on mucosal surface predominately consists of gram-positive bacteria.

Special defenses of the gastrointestinal tract

The lumen of the stomach is an extremely acidic environment (pH ~2), which acts as a protective barrier to prevent bacteria from reaching more vulnerable areas, such as the small intestine and colon, where conditions are more favorable for bacterial growth. Bacteria ingested in foods are probably protected somewhat from the full impact of stomach acid by the buffering capacity of the food. Food increase the chance that some of the bacteria might survive long enough in the stomach to reach the small intestine.

Bile salts are steroids with detergent-like properties that are produced in the liver, stored in the gall bladder, and then released through the bile duct into the intestine.  The detergent-like properties of bile salts help to disrupt bacterial membranes, especially those of gram-negative bacteria.


Defenses of The Innate Immune System

Skin and mucosal surfaces (barriers) are highly effective in preventing pathogenic bacteria from entering tissue and blood, but from time to time, bacteria succeed in breaching these surfaces. Bacteria that get this far encounter a formidable defense force, the phagocytic cells (neutrophils, monocytes, macrophages, and dendritic cells), natural killer cells, and the proteins that help organize their activity. These cells, together with a set of blood proteins called complement and another set of proteins called cytokines are called the innate immune system. Innate immune system plays a key role in the defence reactions against foreign invaders and correlates with inflammation.

The Firepower of Innate Immune System

The firepower of the innate immune system is very effective in killing bacteria. The phagocyte first forms pseudopods that engulf the bacterium. After engulfment, the bacterium is encased in an endocytic vesicle called phagosome. Various lysosomal enzymes, antimicrobial peptides, membrane-permeabilizing proteins, and degrading proteins mediate nonoxidative killing. Oxidative killing occurs through the formation of toxic reactive oxygen species.

Unlike phagocytic cells, NK cells do not ingest their targets, although their mode of killing resembles that of phagocytes in many respects. NK cells store their toxic substances in granules. Binding to an infected human target cell stimulates the release of these granules. To distinguish a infected cell from a healthy cell, the NK cell use the MHC-I molecule. Healthy cells express MHC-I protein on their surfaces, and MHC-I binds to a second inhibitory receptor on the NK cell surface and halts the activation of the cytotoxic response. In contrast, infected cells express much less MHC-I on their surfaces than normal cells, and the activation response of the NK cell proceeds, leading to an attack on the infected cell. Thus, instead of ingesting a bacterium or infected cell, the innate cytotoxic NK cells bombard infected cells. Cytotoxic-cell granules contain a protein called perforin that insets into the membrane of a target cell and causes channels to form. These channels allow other granule proteins, a set of proteases called granzymes, to enter the target cell. One effect of this assult appears to be forcing the target cell to initiate apoptosis.

C3a and C5a are proinflammatory molecules that stimulate mast cells to release their granules, which contain vasoactive substance that increase the permeability of blood vessels and thus facilitate the movement of phagocytes from blood vessels into tissue. C5a also acts together with cytokines to signal phagocytes to leave the bloodstream and to guide them to the infection site. Once PMNs or monocytes have left the bloodstream, they move along a gradient of C5a to find the locus of infection. At the site of infection, C3b binds to the surface of the invading bacterium and makes it easier for phagocytes to ingest the bacterium. This activity is called opsonization.

Another role of activated complement components is direct killing of the bacterium. Activated components C5b recruits C6, C7, C8, and C9 to form a membrane-damaging complex in the membranes of some types of microorganisms. This complex is called the membrane attack complex (MAC). Formation of the MAC inactivates enveloped viruses and kills bacteria by punching holes in their membranes.


Correlations Between Innate Immune System and Inflammation

Inflammation is one imporant response of vascular tissues to harmful stimuli, such as damaged tissues and the release of irritants, caused by infection. The inflammatory response recruits innate-immune cells from the blood vessels to the site of infection. Proinflammatory cytokines are induced by the complement cascade (C3a and C5a, phagocytes have receptors for C3b on their surface) and by mast cells (mast cells secrete vasoactive amines including histamine and serotonin) and activated phagocytes.