Lysis Information and Courses from MediaLab, Inc.

Tuomanen EI.: Pathogenesis of pneumococcal inflammation: otitis media Vaccine. 19 Suppl 1:S38-40, 2000 Pneumococci cause damage to the ear in otitis media with an association with bacterial meningitis. The pathogenesis of injury involves host response to cell wall constituents and the pore-forming toxin, pneumolysin. Release of cell wall constituents, particularly during antibiotic-induced bacterial lysis, leads to an influx of leukocytes and subsequent tissue injury. The signal transduction cascade for this response is becoming defined and includes CD14, Toll-like receptor 2, NFkB, and cytokine production. The second source of injury is the cytotoxicity of the pore forming toxin, pneumolysin. Decreasing the sequelae of otitis can be achieved by an increased understanding of the site-specific mechanisms of pneumococcal-induced inflammation.

Rouquette C. Berche P. The pathogenesis of infection by Listeria monocytogenes Microbiologia. 12:245-58, 1996 Listeria monocytogenes is a Gram-positive bacterium responsible for severe infections in human and a large variety of animal species. It is a facultative intracellular pathogen which invades macrophages and most tissue cells of infected hosts where it can proliferate. The molecular basis of this intracellular parasitism has been to a large extent elucidated. The virulence factors, including internalin, listeriolysin O, phospholipases and a bacterial surface protein, ActA, are encoded by chromosomal genes organized in operons. Following internalisation into host cells, the bacteria escape from the phagosomal compartment and enter the cytoplasm. They then spread from cell to cell by a process involving actin polymerisation. In infected hosts, the bacteria cross the intestinal wall at Peyer's patches to invade the mesenteric lymph nodes and the blood. The main target organ is the liver, where the bacteria multiply inside hepatocytes. Early recruitment of polymorphonuclear cells lead to hepatocyte lysis, and thereby bacterial release This causes prolonged septicaemia, particularly in immunocompromised hosts, thus exposing the placenta and brain to infection. The prognosis of listeriosis depends on the severity of meningoencephalitis, due to the elective location of foci of infection in the brain stem (rhombencephalitis). Despite bactericidal antibiotic therapy, the overall mortality is still high (25 to 30%).

Blood is normally not present in the urine of healthy individuals, apart from blood during menses that may be detected in urine samples from females, Hematuria is associated with renal or genital disorders in which the bleeding is the result of irritation to the involved organs or some type of trauma. Examples include:Renal calculiPyelonephritisGlomerulonephritisTumorsTraumaExposure to toxic chemicals or drugsStrenuous exerciseHemoglobinuria may be due to the lysis of red blood cells within the urinary tract. This can be caused by intravascular hemolysis, as the hemoglobin is filtered through the glomeruli. In a healthy, normal individual, the hemoglobin molecule attaches to haptoglobin and bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, hemoglobin passes into the urine. Hemoglobinuria may be associated with:Hemolytic anemiaSevere burnsTransfusion reactionInfection Strenuous exercise

No blood is found in the urine of healthy individuals although samples from menstruating females, frequently, but not always, test positive for blood. Hematuria is associated with renal or genital urinary disorders in which the bleeding is the result of irritation to the involved organs or trauma. Examples include renal calculi, pyelonephritis, glomerulonephritis, tumors, trauma or exposure to toxic chemicals or drugs and/or strenuous exercise. Hemoglobinuria may be due to the lysis of red cells within the urinary tract. If it is caused by intravascular hemolysis, the hemoglobin is then filtered through the glomeruli. In the normal individual, the hemoglobin molecule attaches to haptoglobin and in this way bypasses the kidney filtration system. When the hemoglobin/haptoglobin system is overwhelmed, as in cases of hemolytic anemia, severe burns, transfusion reaction, infection or strenuous exercise, hemoglobin passes into the urine.

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.

This assay targets the tcdB gene, and employs real time PCR and molecular beacon technology.After specimen preparation and lysis, the target (if present) is amplified through a real time PCR process. Amplified targets are detected with hybridization probes labeled with quenched fluorophores (molecular beacons). Amplification, detection, and interpretation of signals are performed automatically by the Cepheid SmartCycler® software.The amplified targets are detected with a molecular beacon, a hairpin-forming, single-stranded oligonucleotide labeled at one end with a quencher and at the other end with a fluorescent reporter dye. In the absence of target, the fluorescence is quenched. In the presence of target, the hairpin structure opens upon beacon/target hybridization, resulting in emission of fluorescence. The amount of fluorescence at any given cycle depends on the amount of amplicons present; the software continuously monitors the fluorescence emitted during amplification.

There are several methods for extracting genetic material from cells. Different methods use slightly different techniques or different combinations of reagents. However, the basic process is generally the same. The desired cells are put into a tube and mixed with a special cocktail. The cocktail usually includes detergents, enzymes, and salts. The sample is disrupted to cause cell lysis, or the breaking open of the cells. The detergent breaks down the membrane lipids, enzymes remove the proteins, and the salt pulls out the DNA. Centrifuging the sample will cause the DNA to form a pellet at the bottom of the tube. The remaining cocktail can be poured off and the DNA pellet can be resuspended for further use. Today, there are systems that will isolate and purify DNA automatically. Automated techniques can often work on more than one sample at a time, require little intervention, and produce more purified samples than manual techniques.

This slide shows residual yeast after red blood cells have been lysed with acetic acid.

Important events that occur in an immune-mediated hemolytic transfusion reaction (HTR) include: Antibody Binding to Red Blood Cells Antibodies may be either IgM or IgG class. IgM antibodies activate complement and lead to intravascular hemolysis where free hemoglobin is released into the plasma. IgG antibodies rarely activate complement but they are often involved in effecting phagocytosis. The concentration of the antibody is directly related to the severity of the HTR. Activation of Complement The end result of complement activation is red cell lysis. Activation of Mononuclear Phagocytes and Cytokines Sensitized red cells are removed from circulation by mononuclear phagocytes. Macrophages in the spleen and Kupffner cells in the liver are active in this process. Activation of Coagulation Antibody-antigen complexes may initiate coagulation and cause disseminated intravascular coagulation (DIC). Shock and Renal Failure Hemolysis can be intravascular or extravascular. In intravascular hemolysis, free hemoglobin, RBC stroma, and intracellular enzymes are released into the blood stream. This results in hemoglobulinemia and hemglobinuria which can lead to kidney damage. In extravascular hemolysis, there is no release of free hemoglobin. Sensitized red cells are removed from the circulation by the monocytes and macrophages in the reticuloendothelial system.