Inflammatory Information and Courses from MediaLab, Inc.

The punch or trephine biopsy is one of the most popular methods of sampling for diagnosis of inflammatory skin conditions. A special instrument is used to cut out a cylindrical shaped piece of skin. The skin core contains the full skin thickness with the main skin layers (epidermis, dermis, and subcutaneous) usually being present. The punch biopsy must be embedded so that sections are taken perpendicular to the epidermis. This typically means that the specimen will be laid on its side (it is rounded and has no clear edges), with the epidermis disc being clearly visible on one side. If the punch biopsy is larger than 0.4 cm in diameter, it may be bisected. In bisected punch biopsies, each half will be laid on its side (cut surface) into the block face.

Reactive mesothelial cells can be found when there is an infection or an inflammatory response present in a body cavity. This condition can be due to the presence of a bacterial, viral or fungal infection. It can also be the result of trauma or the presence of metastatic tumor.Reactive mesothelial cells tend to come in clusters and clumps and have a more washed out cytoplasm in body fluids. Notice in the image on the right, how indistinct the cytoplasmic borders are in this clump compared to normal mesothelial cells. The wide separation of the nuclei and the well defined nucleoli help to identify these as reactive mesothelial cells. However if there is any doubt, the smear should be sent for hematology or pathology review.Note: It is not uncommon for macrophages to be mixed into a reactive mesothelial clump.

Atherosclerosis is one of the leading causes of heart disease and its presence is an important risk factor for events leading to acute myocardial infarction (AMI). In the past, atherosclerosis was described as a cholesterol and lipid storage event. Now we know it is a chronic inflammatory disorder of the arterial vessels with lipid components. Atherosclerosis begins with damage to the cells that line the blood vessels. Some possible causes of this cell injury are bacterial infection, hyperlipidemia, hypertension, glycosylated products of diabetes, cytokines from adipose tissue, or exposure to toxins such as pollution and second-hand smoke. Monocytes and lymphocytes adhere to the injured site; macrophages enter and ingest proteins and, along with modified lipoproteins, create foam cells. An inflammatory milieu results as cytokines and other inflammatory molecules become involved; foam cells and white blood cells begin secreting cytokines and metalloproteinases. Myeloperoxidase is also released by degranulated white blood cells and macrophages. As inflammation and accumulation of these products continues, fatty dots and streaks are formed on the vessel lining and the formation of plaque begins. As the atherosclerotic process continues, involved cells proliferate forming a complex extracellular matrix and a fibrous cap. If development continues, possibly over decades, the plaque formations are distributed throughout various vessels, become calcified or collagenized and make the vessel walls rigid. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

Previously, screening for cardiovascular disease (CVD) focused on hyperlipidemia, obesity, and hypertension. However, approximately one half of AMIs occur in healthy men and women with normal or only slightly elevated plasma lipids. With new insights into cardiac disease and the ACS, novel biomarkers such as inflammatory markers, hormones, and other biomolecules indicating myocardial stress are required. Some new screening markers are in use today and many more are in study and evaluation for future use. New screening markers for CVD and ACS are: Highly Sensitive C-Reactive Protein (hs-CRP) Homocysteine Ischemial Modified Albumin (IMA) Myeloperoxidase (MPO)

In 2002, the AHA and CDC recommended measurement of hs-CRP as an aid in the diagnosis and treatment of CVD. At low levels, it can detect those at risk for cardiac heart disease. At high levels in those with no history of heart disease, it indicates a high risk for AMI, stroke, or peripheral vascular disease. For patients with ACS or stable coronary disease, hs-CRP is used to predict future coronary events.Nephelometry and immunoturbidimetric measurement methods provide lower limits needed for hs-CRP assays. Due to variation in results among clinical laboratories, work is underway for standardization of measurements. Ranges of hs-CRP in prediction of risk for CVD are: <1.0 mg/L Low CVD risk 1.0-3.0 mg/L Average risk for CVD >3.0 mg/L High risk for future CVDIf results are >10.0 mg/L, the patient should be evaluated for an acute inflammatory condition.

The H & E section of the brain biopsy (left frame of image)revealed edema of the parencymya with the accumulation of inflammatory cells in the perivascular spaces. The close in view of the exudate (right frame of image) reveals that the inflammatory exudate is comprised primarily of polymorphonuclear luekocytes. The histologic diagnosis therefore is suppurative meningitis, with culture results necessary to establish the etiologic agent.

Atherosclerosis is a clogging, narrowing and hardening of the body's large and medium-sized blood vessels. Atherosclerosis can lead to hypertension, stroke, myocardial infarction (heart attack), renal problems, etc. Not surprisingly, cardiovascular risk markers tend to reflect a person's degree of atherosclerosis.Atherosclerosis is actually a chronic inflammatory response within the walls of arteries. Small lipoproteins like LDL are able to diffuse through the endothelial wall of blood vessels and accumulate. The inflammatory component of atherosclerosis results from the migration of leukocytes (mainly macrophages) that enter the blood vessel walls. These macrophages seek to remove the deposited LDL as well as intermediate-density lipoproteins (IDL). As macrophages phagocytose these lipoproteins, they become foam cells that get trapped in the endothelial space. This eventually leads to "hardening" or "furring" of the arteries and plaque formation. Arteriosclerosis is a general term describing any hardening (loss of elasticity) of medium or large arteries whereas atherosclerosis is a hardening of an artery specifically due to plaque. The risk to patients with significant atherosclerosis is that eventually a narrowing of the artery (stenosis) can cause a reduction in oxygen delivery to tissues and plaque rupture can lead to an acute coronary event.

Recall that the inflammatory events leading to atherosclerosis are due to the presence of LDL particles which diffuse through the endothelium and into the vessel wall. It makes sense that the more LDL particles there are, the more risk there would be for LDL depositing in the vessel wall. It would seem therefore that measuring the number of LDL particles could be more useful than measuring the cholesterol content of the particles. Traditional measurements of LDL-C quantify the amount of cholesterol associated with all the LDL in a patient sample; they don't tell us how many LDL particles there are. An analogy can be made with battleships. If you wanted to measure the size of a navy that was sailing for your shores, it makes more sense to count the number of ships than to count the amount of cargo the ships carry in order to estimate the number of ships. Of course, it is intuitive that the more LDL-C there is, the greater the number of LDL particles. In that sense, LDL particle number should correlate to LDL cholesterol, and this is indeed true. However, studies now show that measurement of the number of LDL particles is a more powerful predictor of cardiovascular risk. The exact relationship between LDL particle number and cholesterol content actually varies due to the fact that the lipoproteins vary in size and in the ratio of triglycerides to cholesterol. So, although cholesterol is related to LDL particle number, it is not in perfect proportion.How can we then measure LDL particle number? The most obvious way would be to measure apolipoprotein B100 (often abbreviated ApoB). Each LDL particle has one molecule of ApoB attached to it. Therefore, if we measured ApoB, we would be measuring the number of LDL particles, not the contents of those particles, and number appears to be more important with regard to adverse outcomes.

The traditional CRP test uses immunoassay methods that are sensitive to concentrations of 5-20 mg/L. The hs-CRP test, with its increased sensitivity, is able to detect C-reactive protein in lower levels, 0.5-10.0 mg/L. As with most risk markers, the results of hs-CRP testing are generally interpreted on a relative scale; the higher the value, the higher the risk of a future cardiovascular event.The American Heart Association and Centers for Disease Control and Prevention has defined risk groups with hs-CRP as follows: Low risk: < 1.0 mg/L Average risk: 1.0 to 3.0 mg/L High risk: > 3.0 mg/L It is important to note that hs-CRP assays are measuring the same protein as traditional CRP assays. Thus, in patients with active inflammation (such as chronic, active arthritis; lupus; infection; etc.) hs-CRP values would be expected to be high and would not necessarily implicate cardiovascular risk. If values greater than 10 mg/L are seen in repeated measurements, a non-cardiovascular cause should be considered. Taking anti-inflammatory drugs (NSAIDs, aspirin, etc.) or the statin-class of cholesterol-lowering drugs may reduce CRP levels in patients. This is not an artifact, but is thought to be an effect of treating the underlying inflammatory process.

Atherosclerosis. U.S. Department of Health & Human Services National Institutes of Health. Available at http://www.nhlbi.nih.gov/health/dci/Diseases/Atherosclerosis/Atherosclerosis_WhatIs.html Accessed March 25, 2013.Daniels LB, Barrett-Connor E, Sarno M, Laughlin GA,Bettencourt R, Wolfert RL. Lipoprotein-associated phospholipase A2 (Lp-PLA2) independently predicts incident coronary heart disease (CHD) in an apparently healthy older population: The Rancho Bernardo study. J Am Coll Cardiol. 2008;51:913-919.Executive Summary of the third report of the National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III). JAMA. 2001; 285:2486-2497. Frostegard, J, Wu R, Lemne C, Thulin T, Witztum JL and de Faire U. Circulating oxidized low-density lipoprotein is increased in hypertension, Clin Sci 2003; 105, 615.Garza CA, Montoir VM, McConnell JP, et al. Association between lipoprotein-associated phospholipase A2 and cardiovascular disease: a systematic review. Mayo Clin Proc. 2007;82(2):159-165.Interpretive Handbook, (MC0440rev0407) Mayo Clinic, RochesterMN;2007. Maksimowicz-McKinnon K, Bhatt DL, Calabrese LH: Recent advances in vascular inflammation: C-reactive protein and other inflammatory biomarkers. Curr Opin Rheumatol. 2004;16:18-24.Mora S, Szklo M, Otvos JD, et al. LDL particle subclasses, LDL particle size, and carotid atherosclerosis in the multi-ethnic study of atherosclerosis. Atherosclerosis. 2007;192:211-217.NACB Laboratory Medicine Practice Guidelines. Emerging biomarkers of cardiovascular disease and stroke. NationalAcademy of Clinical Biochemistry Laboratory Medicine Practice Guidelines. 2006.PLACtest animation, diaDexus. http://www.plactest.com/laboratorians/action.php Accessed March 25, 2013.Rifai N, Warnick GR. Lipids, lipoproteins, apolipoproteins, and other cardiovascular risk factors. In: BurtisCA, Ashwood ER. BrunsDE. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 4th ed. St. Louis, MO: Elsevier Saunders: 2006; chap. 26.Ridker PM, Rifai N, Rose L, et al. Comparison of C-reactive protein and low-density lipoprotein cholesterol levels in the prediction of first cardiovascular events. N Engl J Med. 2002;347:1557-1565.Sniderman AD. Differential response of cholesterol and particle measures of atherogenic lipoproteins to LDL-lowering therapy: Implications for clinical practice. J Clin Lipidol 2008;2:36-42.Tsimikas, S, Brilakis ES, Miller ER, et al. Oxidized phospholipids, Lp(a) lipoprotein, and coronary artery disease, N Engl J Med: 2005;353:46.Tsimikas S, Bergmark C, Beyer RW, et al. Temporal increases in plasma markers of oxidized low-density lipoprotein strongly reflect the presence of acute coronary syndromes. J Am Coll Cardiol. 2003; 41: 360.Tsimikas, S, Lau HK, Han KR, et al. Percutaneous coronary intervention results in acute increases in oxidized phospholipids and lipoprotein(a): Short-term and long-term immunologic responses to oxidized low-density lipoprotein. Circulation. 2004;109, 3164.Tsimikas S, Witztum JL, Miller ER, Sasiela WJ, et al. High-dose atorvastatin reduces total plasma levels of oxidized phospholipids and immune complexes present on apolipoprotein B-100 in patients with acute coronary syndromes in the MIRACL trial, Circulation: 2004;110, 1406. Walldius G, Jungner I, Holme I, et al. High apolipoprotein B, low apolipoprotein A-I, and improvement in the prediction of fatal myocardial infarction (AMORIS study): a prospective study. Lancet. 2001;358:2026-2033.Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable risk factors associated with myocardial infarction in 52 countries (the INTERHEART study): case-control study. Lancet. 2004;364:937-952.

HbSS blood may contain reticulocytes with an abnormal presence of CD36 on their membranes, allowing platelets to form a bridge between these young sickle cells and endothelial cells in post-capillary venules. This initial slow down of blood flow creates an environment in which cells containing HbSS can easily form sickled cells.The rigid cells, which are formed as a result of the sickling process, can collect in and plug small blood vessels. This can then cause tissue damage and organ infarction. In addition, the polymerized hemoglobin is thought to disrupt the RBC membrane, exposing phosphatidylserine that can trigger hemostasis. Subsequently, white blood cells (WBCs) may adhere to endothelium in response to recruitment in the inflammatory process. This leads to further occlusion as neutrophils capture additional RBCs in the post-capillary venules.Contributing further to vaso-occlusion is the decreased level of L-arginine in patients with sickle cell disease. L-arginine is a substrate needed to produce nitric oxide (NO). NO has vasodilatory properties as well as anti-inflammatory and anti-platelet properties. Thus a decrease in NO may lead to increased cellular adherence to endothelium.

Transferrin saturation (TS) is usually reported along with the serum iron (SI) and total iron binding capacity (TIBC). TS indicates the percent of iron binding sites on transferrin that are carrying iron. TS is derived from a calculation using the formula:TS =(SI/TIBC) x 100TS results are reported as percentages. Typical reference intervals for TS are 20% to 55% for males and 15% to 50% for females. TS is currently considered to be a good test for screening persons for hereditary hemochromatosis (HH) due to its sensitivity and specificity for iron overload. It may be elevated prior to significant deposition of tissue iron. TS levels increase as additional iron is accumulated.A drawback to using the TS is that it is dependent on performing both the SI and TIBC. The unsaturated iron-binding capacity UIBC may be a lower cost alternative.The optimal TS criterion for detecting HH is controversial. Using a TS of >60% for males and >50% for females has been found highly accurate in detecting abnormal iron metabolism in persons with HH. Others studies suggest using lower TS levels, e.g. 45%, as a criterion indicating further testing is warranted. Current guidelines from the American College of Physicians include a TS cutoff level of >55% for identifying iron overload. (11)Patients with initially increased TS should be followed by performing a second TS from a fasting morning specimen. The patient should also be advised not to take vitamins supplemented with iron or oral contraceptives for several days prior to the repeated test. TS levels may be affected by diurnal variation, dietary factors, and co-existing disease states such as inflammation and hepatitis. Patients with HH may have falsely normal TS if chronic blood loss or inflammatory disease is present.

IL-6 responds to tissue injury. IL-6 is synthesized and secreted by many different cells in addition to adipocytes including immune cells, fibroblasts, endothelial cells and skeletal muscle. IL-6 is increased in obesity and insulin resistance and those with elevated levels are at higher risk for type 2 diabetes and myocardial infarction. Similar to TNF-a, IL-6 increases NEFA release and reduces adiponectin secretion. IL-6 increases insulin resistance by inhibiting insulin receptor signal transduction in liver cells. It also increases other inflammatory cytokines, interleukin-1 (IL-1) and TNF-a, and stimulates the liver to produce C-reactive protein (CRP), an important protein marker of inflammation.

Adiponectin is very different from TNF-a, IL-6, and PAI-1. It is synthesized and secreted almost exclusively by the adipocytes and is an anti-inflammatory cytokine. Levels of adiponectin are decreased in weight gain, obesity and in those who are insulin resistant. Secretions of TNF-a and IL-6 reduce adipocyte secretion of adiponectin. Adiponectin is a protective adipokine. It inhibits several steps in the inflammatory process and increases insulin sensitivity by enhancing glucose transport into muscle cells. Adiponectin also decreases liver glucose production. Adiponectin slows and inhibits steps in plaque formation in blood vessels and is thus antiatherogenic.

Adipokines play several roles in the atherosclerotic inflammatory process: TNF-a activity produces inflammatory changes in vascular tissue and adhesion molecules. This increases the ability of monocytes to adhere to vessel walls. Resistin also promotes cell adhesion. Angiotensin II from angiotensinogen enhances the adhesion process of monocytes and platelets to vessel walls. When glucose levels are increased, leptin assists in the incorporation of lipids by enhancing uptake of cholesterol by macrophages. IL-6 enhances the inflammatory process and increases CRP. If there are ruptured atherosclerotic plaques, PAI-1 increases probability of thrombus formation and inhibits fibrin clot lysis.

Clostridium difficile is the cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC). PMC is an inflammatory disease of the colon caused by toxins of C. difficile.C. difficile produces two potent toxins: Toxin A (TcdA), an enterotoxinToxin B (TcdB), a cytotoxin It is the production of these toxins in the gastrointestinal tract that ultimately leads to disease. There is a relationship between toxin levels, the development of pseudomembranous colitis (PMC), and the duration of diarrhea. For many years, toxin A was regarded as more important than toxin B in the disease process. Later on, disease producing strains producing only toxin B were identified. These strains produced serious disease, and toxin B was found to be responsible for more serious damage to intestinal cells.

Most Clostridium infections arise from endogenous sources. That is, many of the Clostridium species that are associated with disease in humans are part of the normal intestinal microflora, which is true of Clostridium difficile.The organism was originally isolated in 1935 as a component of the normal intestinal flora of healthy newborns. It was dubbed difficile because the organism grows slowly and is difficult to culture. Early investigators also noted that the organism produced a potent toxin, but the relationship between C. difficile antibiotic-associated diarrhea (AAD) and pseudomembranous colitis (PMC) was not elucidated until the 1970's. PMC is an inflammatory disease of the colon caused by toxins of Clostridium difficile. Normal intestinal flora is an important factor in host response to an infectious microorganism. Resistance to intestinal infection is significantly reduced when there is a reduction in the normal flora as a result of antibiotic treatment. The most common manifestation of this decreased host resistance is the development of PMC.

Eosinophils have a circulating half-life of approximately 18 hours and a tissue life span of at least 6 days. They are capable of locomotion and phagocytosis and can enter inflammatory sites, but do so less readily than neutrophils. In tissues the primary location for eosinophils is in the epithelial barriers to the outside world such as, lungs, skin and GI tract. They are capable of returning to the circulating blood and bone marrow after they enter the tissues. Eosinophils are active in parasitic infections and in allergic reactions such as asthma and hay fever, and may be present in great numbers in the peripheral blood during these conditions. Stress, shock, or burns may also cause an increase in this type of cell. Eosinophils modulate an allergic response by liberating substances which can neutralize mast cell and basophil products. The image on the right shows malarial ring forms, which are parasites. This patient showed an increased eosinophil count due to his parasitic infection.

Eosinophils have a circulating half-life of approximately 18 hours and a tissue life span of at least 6 days.They are capable of locomotion and phagocytosis and can enter inflammatory sites, but do so less readily than neutrophils.In tissues the primary location for eosinophils is in the epithelial barriers to the outside world such as, lungs, skin and GI tract.They are capable of returning to the circulating blood and bone marrow after they enter the tissues.

The cytoplasm of eosinophils is evenly filled by numerous orange-red granules of uniform size. They do not overlie the nucleus. The eosinophil granules contain numerous enzymes including peroxidase, phospholipase D, catalase, acid phosphatase, and vitamin B12-binding proteins. The eosinophil's ability to kill bacteria is less than that of neutrophils. Their main purpose is to counteract parasitic infections and to participate in immune allergic reactions. They may also be increased in a variety of nonimmunologic inflammatory responses from bacteria and fungi causing chronic infections. Malignancies, collagen vascular diseases, and myeloproliferative disorders may also may be settings for prominent eosinophils.