Wednesday, November 30, 2011
Egypt Ellection
Iam really very happy for the ellection in Egypt. Iam near fivty years old and I did not see free ellection in my country before. Sorry Ican not now write in microbiology as my mind is really occupied. Iwill writ again, but now it is the time of my country.
Tuesday, November 29, 2011
Monday, November 28, 2011
Pneumonia
Pneumonia is an important cause of morbidity and mortality in adults and children. It results from the host inflammatory response to infection of the distal airways and the lung alveoli (Schutter et.al, 2011). The most useful classification is based on the site of acquisition: community-acquired (CAP) or hospital-acquired pneumonia (HAP) (Esperatti and Marti, 2008).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Pneumonia
Pneumonia is an important cause of morbidity and mortality in adults and children. It results from the host inflammatory response to infection of the distal airways and the lung alveoli (Schutter et.al, 2011). The most useful classification is based on the site of acquisition: community-acquired (CAP) or hospital-acquired pneumonia (HAP) (Esperatti and Marti, 2008).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Hospital-associated pneumonia (HAP) is the second most common nosocomial infection (after urinary tract infection) and the most common nosocomial infection acquired in the intensive care unit (ICU). HAP associated with mechanical ventilation is called ventilator-associated pneumonia (VAP) (Franzetti et.al., 2010). VAP has an estimated incidence of 8–28% and is associated with an excess of ICU stay, increased costs, and attributable mortality (Safdar et.al., 2005).
The term atypical pneumonia was originally used to describe an unusual presentation of pneumonia. It is now more widely used in reference to either pneumonia caused by a relatively common group of pathogens, or to a distinct clinical syndrome the existence of which is difficult to demonstrate (Murdoch and Chambers, 2009).
The “atypical pathogen” group includes Mycoplasma pneumoniae; Legionella; Chlamydia pneumoniae; Coxiella burnettii; and the respiratory viruses, especially
Introduction and Aim of the Work
influenza A and B, parainfluenza 1, 2, and 3, respiratory syncytial virus (RSV) and Epstein-Barr virus (EBV) (Lieberman, 2005).
The diagnosis of pneumonia in the hospitalized patient is even more challenging than the diagnosis of CAP (Richards et.al., 2000). Clinical findings alone are not sufficient for a definitive diagnosis. Therfore, a variety of noninvasive and invasive tests have been proposed as guides for diagnosis and treatment of hospital-acquired pneumonia. Methods include sputum Gram stain and culture, serologic studies, antigen detection tests, and nucleic acid amplification methods (Carroll, 2002).
Many patient- and disease-specific factors contribute to the pathophysiology of HAP, particularly in the surgical population. Risk-factor modification and inpatient prevention strategies can have a significant impact on the incidence of HAP (Kieninger and Lipsett, 2009).
Saturday, November 26, 2011
Innate Immune Response to Pathogens and Recent Advances in Microbiology Researches
An immune system is a system of biological structures and processes within an organism that protects against disease by identifying and killing pathogens and tumor cells. It detects a wide variety of agents, from viruses to parasitic worms, and needs to distinguish them from the organism's own healthy cells and tissues in order to function properly. Detection is complicated as pathogens can evolve rapidly, and adapt to avoid the immune system and allow the pathogens to successfully infect their hosts.
To survive this challenge, multiple mechanisms evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include antimicrobial peptides called defensins, phagocytosis, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms. The typical vertebrate immune system consists of many types of proteins, cells, organs, and tissues that interact in an elaborate and dynamic network. As part of this more complex immune response, the human immune system adapts over time to recognize specific pathogens more efficiently. This adaptation process is referred to as "adaptive immunity" or "acquired immunity" and creates immunological memory. The following chapters will discuss various mechanisms of immune response to pathogens based on laboratory evidence studies.
To survive this challenge, multiple mechanisms evolved that recognize and neutralize pathogens. Even simple unicellular organisms such as bacteria possess enzyme systems that protect against viral infections. Other basic immune mechanisms evolved in ancient eukaryotes and remain in their modern descendants, such as plants and insects. These mechanisms include antimicrobial peptides called defensins, phagocytosis, and the complement system. Jawed vertebrates, including humans, have even more sophisticated defense mechanisms. The typical vertebrate immune system consists of many types of proteins, cells, organs, and tissues that interact in an elaborate and dynamic network. As part of this more complex immune response, the human immune system adapts over time to recognize specific pathogens more efficiently. This adaptation process is referred to as "adaptive immunity" or "acquired immunity" and creates immunological memory. The following chapters will discuss various mechanisms of immune response to pathogens based on laboratory evidence studies.
Friday, November 25, 2011
Qualitative and quantitative detection of endotoxin in serum
Endotoxins are large (molecular weight, 200,000 to 1,000,000), heat-stable lipopolysaccharides (LPS) which are the major components of the cell wall of the gram-negative bacterium.There are more than 20 assays for the detection of endotoxin (McCabe, W. R., 1980), of which three have been used for the detection of endotoxin in clinical specimens: the rabbit pyrogen assay, the LAL
bioassay, and immunoassays. The method of choice would appear to be the LAL assay. The advantages of this assay are increased sensitivity, potential for quantitation, reactivity with the biologically active component lipid A, and relative convenience of operation.
The LAL Assay
In 1956, Bang (Bang, 1956) discovered that the endotoxin of a Vibrio species from seawater, pathogenic for the horseshoe crab (Limulus polyphemus), caused fatal intravascular coagulation and that endotoxin induced activation of this process in vitro. Levin, Bang, and coworkers subsequently showed that this coagulation was the result of an endotoxin-initiated reaction causing the enzymatic conversion of a clottable protein derived from the circulating blood cell (amebocyte) of the crab (Levin, et al.,1968; Young, et al., 1972). They recognized the potential for this biological reagent as a diagnostic tool and characterized its properties. A lysate from the amebocyte is extremely sensitive to the presence of endotoxin.
Coagulation system of L. polyphemus.
The coagulation system of L. polyphemus consists of several enzymes which are arranged in three pathways in a fashion which resembles the classic, alternate, and common mammalian coagulation cascade pathways, the components of which activate each other in a ‘‘cascade’’ sequence. The coagulation system of the Japanese horseshoe crab, T. tridentatus, which is considered homologous to the L. polyphemus American horseshoe crab, has been studied extensively (Fig.3) (Iwanaga, S., 1993; Iwanaga, et al., 1985). This
cascade sequence results in an amplification of the original stimulus which accounts for the sensitivity of the Limulus coagulation system to endotoxin at picogram-per-milliliter (10-12 g/ml) concentrations. An additional component of Limulus amebocytes is an anti-LPS factor which has anti-endotoxin properties (Warren, et al., 1992).
Gel clot LAL assay.
In the original version of the gel clot test, the endotoxin-activated clotting enzyme cleaves the coagulogen to form a clot. To perform this test, a small amount of LAL solution is added to an equal volume of a sample or a standard dilution in a small test tube. If, after an appropriate incubation time, a firm gel clot is formed, the test is scored positive. A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to
enhance the visualization of clot formation in microtiter volumes have been described (Gardi, et al, 1980; Hussaini, et al., 1987; Prior, et al., 1979). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.
Coagulogen-based LAL assay.
The limitations of the gel clot LAL test are the subjective endpoint and the relative lack of sensitivity. To overcome these limitations, various methods to quantitate the progress of the reaction leading to coagulogen conversion have been employed, for example, through monitoring the increase in turbidity (Dubczak, et al., 1979; Urbaschek, et al., 1985), the loss of coagulogen as the clot forms (Baek, 1983; Zhang, et al., 1988), the increase in precipitated protein (Nandan, et al., 1977; Nandan, et al, 1977), or the appearance of a peptide cleavage fragment of coagulogen (Zhang, et al., 1994).
Chromogenic LAL assay.
In the chromogenic LAL assay method (Iwanga, et al ., 1978), the coagulogen is completely or partially removed to be replaced by a chromogenic substrate (Scully, et al., 1980), a small synthetic peptide linked to a chromophore (para-nitroaniline) containing an amino acid sequence similar to that present at the site in the clotting protein cleaved by the clotting enzyme (X-Y-Gly-Arg-pNA). The chromogenic LAL assay usually has two stages: a LAL activation stage and, following the addition of the chromogenic substrate to the reaction mixture, a chromophore release stage. Release of the chromophore imparts a yellow color to the solution. The strength of the yellow color (as measured by optical density [OD] at 405 nm in a spectrophotometer) is a function of the amount of active clotting enzyme (and indirectly to the amount of endotoxin)
present in the solution. Both phases of the chromogenic reaction are critically time and temperature dependent, but within these limitations the chromogenic assay is sensitive to 10 pg/ml (Thomas, et al., 1981). A single-step chromogenic assay has been described (Duner, K. I., 1993; Lindsay, et al., 1989).
Specificity of the LAL Assay
The two pathways leading to the coagulation of LAL, one activated by endotoxin triggered by factor C and the other activated by b-glucans triggered by a glucan-reactive factor G, can be specifically blocked by polymyxin and laminarin, respectively (Zhang, et al., 1994). Hence, reactivity with the LAL assay that is inhibited by polymyxin B can be used as specific evidence for endotoxin. LAL derived from the Japanese horseshoe crab and from which this factor G has been removed has been promoted as an endotoxin-specific reagent (Obayashi, et al., 1985, Obayashi, et al., 1986).
LAL Endotoxin Assay for Blood Samples
When the LAL assay is used to detect endotoxin in blood, two obstacles are encountered: (i) the complex and poorly understood inhibitory factors and (ii) the levels of endotoxemia generally being at the limit of test detection. Schematic overview 1. illustrates the complex interaction among components of blood, endotoxin, and LAL. Endotoxin interacts with several components of plasma, including bile salts, proteins, and lipoproteins, leading to disaggregation, some inactivation, and the formation of complexes. These multiple effects of plasma on the activity of endotoxin are not always apparent as inactivation. (Beller, et al., 1963).
Inhibition by plasma and serum.
The loss of reactivity to LAL on addition of endotoxin to plasma or serum is partly reversible, in that reactivity can be restored by dilution with distilled water or saline (Levin, et al., 1970), and partly irreversible (Johnson, et al., 1977). The ability of plasma or serum to inhibit endotoxin activity is time dependent and temperature sensitive, being maximal at 37 to 45°C and abolished after plasma or serum is heated at 60°C for 5 min, and varies in proportion to the endotoxin potency. These characteristics imply an enzymatic inactivation of endotoxin by native plasma (Johnson, et al., 1977; Novitsky, et al., 1985, Novitsky, et al., 1985, Obayashi, T., 1984, Olofsson, et al., 1986, Webster, et al., 1980), although this has yet to be definitively demonstrated.
Endotoxemia without Sepsis
Liver disease. Endotoxemia has been suspected of having pathogenic properties in patients with liver disease even in the absence of overt gram-negative sepsis (Nolan, J. P., 1975). The origin of endotoxin in this setting is also believed to be from the gastrointestinal tract because several studies have found a portal-to-systemic gradient of endotoxin level, with higher level in portal venous blood than in peripheral blood ( Bigatello, et al, 1987; Jacob, et al, 1977; Lumsden, et al, 1988; Prytz, et al, 1976).
Hemodialysis. pyrogenic reactions are an important problemwith hemodialysis, and there is concern that this is due to contamination of the dialysis water with bacteria or endotoxin (Pegues, et al, 1992; Raij, et al, 1973) or contamination resulting from the use of reprocessed dialyzers (Flaherty, et al, 1993; Gordon, et al, 1988). There is uncertainty as to whether endotoxin is able to cross the different types of dialyzer membranes and also whether the LAL-
reactive material (LAL-RM) found in the plasma of patients undergoing hemodialysis is something other than endotoxin. It is suggested that the LAL-RM is a cellulose-based material, possibly (1-3)-β-D-glucans, which has properties distinct from endotoxin (Roslansky, et al, 1991) and reacts with the factor G-drive pathway of LAL (Zhang, et al,1994). An endotoxin specific assay which does not react with (1-3)- β-D-glucans has been developed and applied (Taniguchi, et al, 1990). In any event, LAL testing of plasma of hemodialysis patients has limited ability to detect pyrogenic reactions, having positive and negative predictive values of less than 70% (Gordon, et al, 1992).
Intestinal endotoxemia. An origin from the gastrointestinal tract has often been presumed for endotoxemia in patients with gastrointestinal diseases (Cooperstock, et al, 1985; Wellmann, et al, 1984) and also in patients receiving radiotherapy to the abdomen in association with symptoms of nausea (Maxwell, et al, 1986).
Other conditions. Transient endotoxemia occurs in patients undergoing minimally invasive procedures of the urinary (Garibaldi, et al, 1973; Robinson, et al, 1975; Tanaka, et al, 1988), biliary (Lumsden, et al, 1989), or gastrointestinal (Kelley, et al, 1985) tract. In general, the severity of symptoms and the degree or frequency of detection of endotoxemia in these patients are higher when gram-negative bacteria are found at the sites of these procedures. In premature neonates, there is an association between endotoxin in cord blood and growth of gram-negative bacteria from placental samples (Scheifele, et al, 1984).
bioassay, and immunoassays. The method of choice would appear to be the LAL assay. The advantages of this assay are increased sensitivity, potential for quantitation, reactivity with the biologically active component lipid A, and relative convenience of operation.
The LAL Assay
In 1956, Bang (Bang, 1956) discovered that the endotoxin of a Vibrio species from seawater, pathogenic for the horseshoe crab (Limulus polyphemus), caused fatal intravascular coagulation and that endotoxin induced activation of this process in vitro. Levin, Bang, and coworkers subsequently showed that this coagulation was the result of an endotoxin-initiated reaction causing the enzymatic conversion of a clottable protein derived from the circulating blood cell (amebocyte) of the crab (Levin, et al.,1968; Young, et al., 1972). They recognized the potential for this biological reagent as a diagnostic tool and characterized its properties. A lysate from the amebocyte is extremely sensitive to the presence of endotoxin.
Coagulation system of L. polyphemus.
The coagulation system of L. polyphemus consists of several enzymes which are arranged in three pathways in a fashion which resembles the classic, alternate, and common mammalian coagulation cascade pathways, the components of which activate each other in a ‘‘cascade’’ sequence. The coagulation system of the Japanese horseshoe crab, T. tridentatus, which is considered homologous to the L. polyphemus American horseshoe crab, has been studied extensively (Fig.3) (Iwanaga, S., 1993; Iwanaga, et al., 1985). This
cascade sequence results in an amplification of the original stimulus which accounts for the sensitivity of the Limulus coagulation system to endotoxin at picogram-per-milliliter (10-12 g/ml) concentrations. An additional component of Limulus amebocytes is an anti-LPS factor which has anti-endotoxin properties (Warren, et al., 1992).
Gel clot LAL assay.
In the original version of the gel clot test, the endotoxin-activated clotting enzyme cleaves the coagulogen to form a clot. To perform this test, a small amount of LAL solution is added to an equal volume of a sample or a standard dilution in a small test tube. If, after an appropriate incubation time, a firm gel clot is formed, the test is scored positive. A firm gel clot is one that remains solid in the bottom of the reaction tube when the tube is inverted. Methods to
enhance the visualization of clot formation in microtiter volumes have been described (Gardi, et al, 1980; Hussaini, et al., 1987; Prior, et al., 1979). With all gel clotbased techniques, a semiquantitative result can be obtained through serial dilution of samples and standards.
Coagulogen-based LAL assay.
The limitations of the gel clot LAL test are the subjective endpoint and the relative lack of sensitivity. To overcome these limitations, various methods to quantitate the progress of the reaction leading to coagulogen conversion have been employed, for example, through monitoring the increase in turbidity (Dubczak, et al., 1979; Urbaschek, et al., 1985), the loss of coagulogen as the clot forms (Baek, 1983; Zhang, et al., 1988), the increase in precipitated protein (Nandan, et al., 1977; Nandan, et al, 1977), or the appearance of a peptide cleavage fragment of coagulogen (Zhang, et al., 1994).
Chromogenic LAL assay.
In the chromogenic LAL assay method (Iwanga, et al ., 1978), the coagulogen is completely or partially removed to be replaced by a chromogenic substrate (Scully, et al., 1980), a small synthetic peptide linked to a chromophore (para-nitroaniline) containing an amino acid sequence similar to that present at the site in the clotting protein cleaved by the clotting enzyme (X-Y-Gly-Arg-pNA). The chromogenic LAL assay usually has two stages: a LAL activation stage and, following the addition of the chromogenic substrate to the reaction mixture, a chromophore release stage. Release of the chromophore imparts a yellow color to the solution. The strength of the yellow color (as measured by optical density [OD] at 405 nm in a spectrophotometer) is a function of the amount of active clotting enzyme (and indirectly to the amount of endotoxin)
present in the solution. Both phases of the chromogenic reaction are critically time and temperature dependent, but within these limitations the chromogenic assay is sensitive to 10 pg/ml (Thomas, et al., 1981). A single-step chromogenic assay has been described (Duner, K. I., 1993; Lindsay, et al., 1989).
Specificity of the LAL Assay
The two pathways leading to the coagulation of LAL, one activated by endotoxin triggered by factor C and the other activated by b-glucans triggered by a glucan-reactive factor G, can be specifically blocked by polymyxin and laminarin, respectively (Zhang, et al., 1994). Hence, reactivity with the LAL assay that is inhibited by polymyxin B can be used as specific evidence for endotoxin. LAL derived from the Japanese horseshoe crab and from which this factor G has been removed has been promoted as an endotoxin-specific reagent (Obayashi, et al., 1985, Obayashi, et al., 1986).
LAL Endotoxin Assay for Blood Samples
When the LAL assay is used to detect endotoxin in blood, two obstacles are encountered: (i) the complex and poorly understood inhibitory factors and (ii) the levels of endotoxemia generally being at the limit of test detection. Schematic overview 1. illustrates the complex interaction among components of blood, endotoxin, and LAL. Endotoxin interacts with several components of plasma, including bile salts, proteins, and lipoproteins, leading to disaggregation, some inactivation, and the formation of complexes. These multiple effects of plasma on the activity of endotoxin are not always apparent as inactivation. (Beller, et al., 1963).
Inhibition by plasma and serum.
The loss of reactivity to LAL on addition of endotoxin to plasma or serum is partly reversible, in that reactivity can be restored by dilution with distilled water or saline (Levin, et al., 1970), and partly irreversible (Johnson, et al., 1977). The ability of plasma or serum to inhibit endotoxin activity is time dependent and temperature sensitive, being maximal at 37 to 45°C and abolished after plasma or serum is heated at 60°C for 5 min, and varies in proportion to the endotoxin potency. These characteristics imply an enzymatic inactivation of endotoxin by native plasma (Johnson, et al., 1977; Novitsky, et al., 1985, Novitsky, et al., 1985, Obayashi, T., 1984, Olofsson, et al., 1986, Webster, et al., 1980), although this has yet to be definitively demonstrated.
Endotoxemia without Sepsis
Liver disease. Endotoxemia has been suspected of having pathogenic properties in patients with liver disease even in the absence of overt gram-negative sepsis (Nolan, J. P., 1975). The origin of endotoxin in this setting is also believed to be from the gastrointestinal tract because several studies have found a portal-to-systemic gradient of endotoxin level, with higher level in portal venous blood than in peripheral blood ( Bigatello, et al, 1987; Jacob, et al, 1977; Lumsden, et al, 1988; Prytz, et al, 1976).
Hemodialysis. pyrogenic reactions are an important problemwith hemodialysis, and there is concern that this is due to contamination of the dialysis water with bacteria or endotoxin (Pegues, et al, 1992; Raij, et al, 1973) or contamination resulting from the use of reprocessed dialyzers (Flaherty, et al, 1993; Gordon, et al, 1988). There is uncertainty as to whether endotoxin is able to cross the different types of dialyzer membranes and also whether the LAL-
reactive material (LAL-RM) found in the plasma of patients undergoing hemodialysis is something other than endotoxin. It is suggested that the LAL-RM is a cellulose-based material, possibly (1-3)-β-D-glucans, which has properties distinct from endotoxin (Roslansky, et al, 1991) and reacts with the factor G-drive pathway of LAL (Zhang, et al,1994). An endotoxin specific assay which does not react with (1-3)- β-D-glucans has been developed and applied (Taniguchi, et al, 1990). In any event, LAL testing of plasma of hemodialysis patients has limited ability to detect pyrogenic reactions, having positive and negative predictive values of less than 70% (Gordon, et al, 1992).
Intestinal endotoxemia. An origin from the gastrointestinal tract has often been presumed for endotoxemia in patients with gastrointestinal diseases (Cooperstock, et al, 1985; Wellmann, et al, 1984) and also in patients receiving radiotherapy to the abdomen in association with symptoms of nausea (Maxwell, et al, 1986).
Other conditions. Transient endotoxemia occurs in patients undergoing minimally invasive procedures of the urinary (Garibaldi, et al, 1973; Robinson, et al, 1975; Tanaka, et al, 1988), biliary (Lumsden, et al, 1989), or gastrointestinal (Kelley, et al, 1985) tract. In general, the severity of symptoms and the degree or frequency of detection of endotoxemia in these patients are higher when gram-negative bacteria are found at the sites of these procedures. In premature neonates, there is an association between endotoxin in cord blood and growth of gram-negative bacteria from placental samples (Scheifele, et al, 1984).
Thursday, November 24, 2011
Cellular and Humoral Response Involved in Gram-Negative Bacterial Sepsis and Septic Shock
As soon as a bacterium enters the body, it is confronted with two lines of defense: a humoral line and a cellular line. The humoral factors comprise complement, antibodies, and acute phase proteins. In the cellular line of defense, in particular the mononuclear cells (monocytes and macrophages) and the neutrophils are of great significance since these cells may recognize bacterial cell wall constituents directly or indirectly after complement and antibody bind to the bacterium and its constituents. It is now thought that continuous challenges with small amounts of bacterial constituents may be necessary to keep the immune system alert to infections. Indeed, low levels of LPS are present in healthy individuals without causing disease (Takakuwa, et al., 1994; Vogel, et al., 1990).
Lipopolysaccharide
While the terms endotoxin and LPS are used interchangeably, the former term to emphasize the biological activity and the latter term to refer particularly to the chemical structure and composition of the molecule (Hitchcock, et al., 1986; Qureshi, et al., 1991). LPS is a major constituent of the outer membrane of gram-negative bacteria and is the only lipid constituent of the outer leaflet (Rietschel, et al., 1994). LPS is an essential compound of the cell wall and is a
prerequisite for bacterial viability. It is not a toxic molecule when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse (Hellman, et al., 2000, Rietschel, et al., 1994). Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS (De Bleser, et al., 1994). In addition, some antibiotics are known to cause the release of LPS from bacteria (Crosby, et al., 1994).
The LPS molecule consists of four different parts (Fig1,2). (Lugtenberg, et al., 1983; Raetz, et al., 1990; Rietschel, et al., 1994).The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. All bacterial species carry unique LPS. Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (Kotani, et al 1985). The second part of the LPS molecule is the inner core, which consists of two or more 2-keto-3-deoxyoctonic acid (KDO) sugars linked to the lipid A glucosamine and two or three heptose (L-glycero-D-manno-heptose) sugars linked to the KDO. Both sugars are unique to bacteria. The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core. The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the
terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length (Edwin S, et al., 2003).
Cellular defense:
LPS and other bacterial (surface) components are recognized by complement and antibodies, leading to opsonisation and lysis of the bacterium. Phagocytes (monocytes, macrophages, and polymorphonuclear leukocytes [PMN]) are able to recognize opsonized bacterial components by complement receptors and Fc receptors (which bind immunoglobulin G [IgG] antibodies) (Frank, et al., 1991).
In the host response to bacteria, the mononuclear phagocytes (monocytes and macrophages) are of major importance. Recognition of LPS or other bacterial components by these cells initiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators (Hiemstra, et al., 1993; Mayer, et al., 1991; Roitt, I. M., 1994). One of the key mediators is TNF-α which is one of the first cytokines released by macrophages (Beutler, et al., 1985). The release of TNF-α,
IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (Hack, et al., 1997; Katori, et al., 2000; Lukacs, et al., 1999).
The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (Jaeschke, H., and C. W. Smith., 1997; Kawamura, et al., 1995; Van Oosten, et al., 1995). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-α, leukotriene B4, and PAF (Mulligan, et al., 1993; Van Epps, et al., 1993). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability increasing protein (BPI), enzymes, and oxygen free radicals (Chatham, et al., 1993; Roitt, I. M., 1994). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (Bone, et al., 1991). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor, endothelin-1, chemokines, and colony stimulating factors (CSF) (Mahalingam, et al., 1999).
The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as
IL-2, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)-CSF. IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN-γ enhances the effects of LPS on mononuclear cells (Bone, R. C., 1991; Heinzel, et al., 1994; Jaeschke, H., 1996; Ying, et al.,1993). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (Bone, et al., 1991).
Humeral response
Bacteria activate both complement pathways: i) alternative pathway which is triggered by binding polysaccharide surface components (O antigen, capsule, and LPS) to complement factor 3 (C3) (Joiner, et al., 1984; Quezado, et al., 1994; Tesh, et al., 1988) ii) classical pathway which is activated by binding Lipid A to C1q (Ying, et al., 1993). The classical complement pathway is also activated in the presence of specific antibodies (IgG and IgM) against gram-negative bacterial constituents. In all three cases, C3b is deposited on the molecule or cell surface, which promotes phagocytosis by macrophages and neutrophils and leads to insertion of C5–C9 (membrane attack complex) into the cell surface, leading to lysis of the bacterium (De Boer, et al., 1993; Frank, et al., 1991). However, long O-antigen chains in gram-negative bacteria may protect the bacteria from complement-mediated lysis (Haeney, M. R., 1998). With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate
these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such ashistamine), facilitating the invasion of phagocytes. (Espevik, et al., 1993; Hsueh, et al., 1990; Kuipers, et al., 1994; Mulligan, et al., 1993; Pu¨schel, et al., 1993; Roitt, I. M., 1994; Tesh, et al., 1988; Van Epps, et al., 1993)
During infection, liver parenchymal cells are stimulated by TNF-α, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide- binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, α1-acid glycoprotein, α2-macroglobulin, and some proteinase inhibitors (476, 498). The expression is differentially upregulated from several fold (C3 and C9) to even 1,000-fold (C-reactive protein). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (Fey, et al., 1994; Kuipers, et al., 1994; Ramadori, et al., 1990). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (Fey, et al., 1994).
Lipopolysaccharide
While the terms endotoxin and LPS are used interchangeably, the former term to emphasize the biological activity and the latter term to refer particularly to the chemical structure and composition of the molecule (Hitchcock, et al., 1986; Qureshi, et al., 1991). LPS is a major constituent of the outer membrane of gram-negative bacteria and is the only lipid constituent of the outer leaflet (Rietschel, et al., 1994). LPS is an essential compound of the cell wall and is a
prerequisite for bacterial viability. It is not a toxic molecule when it is incorporated into the bacterial outer membrane, but after release from the bacterial wall, its toxic moiety, lipid A, is exposed to immune cells, thus evoking an inflammatory response. LPS and other cell wall constituents are released from the bacterial cells when they multiply but also when bacteria die or lyse (Hellman, et al., 2000, Rietschel, et al., 1994). Various endogenous factors like complement and bactericidal proteins can cause disintegration of bacteria, resulting in the release of LPS (De Bleser, et al., 1994). In addition, some antibiotics are known to cause the release of LPS from bacteria (Crosby, et al., 1994).
The LPS molecule consists of four different parts (Fig1,2). (Lugtenberg, et al., 1983; Raetz, et al., 1990; Rietschel, et al., 1994).The first and most essential part is lipid A, the covalently linked lipid component of LPS. Six or more fatty acid residues are linked to two phosphorylated glucosamine sugars. All bacterial species carry unique LPS. Experiments with synthetic lipid A have shown that this part of the LPS molecule represents the toxic moiety (Kotani, et al 1985). The second part of the LPS molecule is the inner core, which consists of two or more 2-keto-3-deoxyoctonic acid (KDO) sugars linked to the lipid A glucosamine and two or three heptose (L-glycero-D-manno-heptose) sugars linked to the KDO. Both sugars are unique to bacteria. The outer core, the third part of the LPS molecule, consists of common sugars and is more variable than the inner core. It is normally three sugars long with one or more covalently bound sugars as side chains. LPS serotypes consisting of lipid A and the complete inner and outer core are denoted Ra-LPS, whereas the Rb- and Rc-LPS serotypes only contain a part of the outer core. The fourth moiety of the LPS molecule is the O antigen. This part of the LPS molecule is attached to the
terminal sugar of the outer core, extends from the bacterial surface, and is highly immunogenic. It is composed of units of common sugars, but there is a huge interspecies and interstrain variation in the composition and length (Edwin S, et al., 2003).
Cellular defense:
LPS and other bacterial (surface) components are recognized by complement and antibodies, leading to opsonisation and lysis of the bacterium. Phagocytes (monocytes, macrophages, and polymorphonuclear leukocytes [PMN]) are able to recognize opsonized bacterial components by complement receptors and Fc receptors (which bind immunoglobulin G [IgG] antibodies) (Frank, et al., 1991).
In the host response to bacteria, the mononuclear phagocytes (monocytes and macrophages) are of major importance. Recognition of LPS or other bacterial components by these cells initiates a cascade of release of inflammatory mediators, vascular and physiological changes, and recruitment of immune cells. An LPS-activated macrophage becomes metabolically active and produces intracellular stores of oxygen free radicals and other microbicidal agents (lysozyme, cationic proteins, acid hydrolases, and lactoferrin) and secretes inflammatory mediators (Hiemstra, et al., 1993; Mayer, et al., 1991; Roitt, I. M., 1994). One of the key mediators is TNF-α which is one of the first cytokines released by macrophages (Beutler, et al., 1985). The release of TNF-α,
IL-1, IL-6, IL-8, IL-12, platelet-activating factor (PAF), chemokines, and eicosanoids has profound effects on the surrounding tissue (Hack, et al., 1997; Katori, et al., 2000; Lukacs, et al., 1999).
The extravasation of PMN is enabled by vasodilatation and upregulation of adhesion molecules on endothelial cells, PMN, and macrophages (Jaeschke, H., and C. W. Smith., 1997; Kawamura, et al., 1995; Van Oosten, et al., 1995). The PMN react to these stimuli by intravascular aggregation, adherence to the endothelium, diapedesis, and the production of inflammatory mediators like TNF-α, leukotriene B4, and PAF (Mulligan, et al., 1993; Van Epps, et al., 1993). The (activated) PMN express CD14, CD11/CD18, and several complement and Fc receptors and are thus able to recognize and phagocytose LPS, bacterial fragments, and whole bacteria. As specialized phagocytes, PMN produce an impressive series of microbicidal agents, such as lysozyme, bactericidal/permeability increasing protein (BPI), enzymes, and oxygen free radicals (Chatham, et al., 1993; Roitt, I. M., 1994). These agents are used mainly for lysosomal killing of microorganisms. However, adherence of the PMN to endothelial cells and the presence of high concentrations of stimuli may also result in the release of microbicidal agents; much of the endothelial damage observed in sepsis is caused by these agents (Bone, et al., 1991). Endothelial cells respond to LPS (via soluble CD14) and to the circulating cytokines by the release of IL-1, IL-6, eicosanoids, the vasoactive agents endothelium derived relaxation factor, endothelin-1, chemokines, and colony stimulating factors (CSF) (Mahalingam, et al., 1999).
The inflammatory mediators secreted by the different cell populations attract and activate B and T lymphocytes. In turn, the latter release mediators such as
IL-2, gamma interferon (IFN-γ), and granulocyte-macrophage (GM)-CSF. IL-2 and GM-CSF are involved in proliferation and activation of PMN and mononuclear cells, whereas IFN-γ enhances the effects of LPS on mononuclear cells (Bone, R. C., 1991; Heinzel, et al., 1994; Jaeschke, H., 1996; Ying, et al.,1993). The actions of the activated immune cells combined with the effects of the inflammatory mediators cause symptoms such as fever, endothelial damage, capillary leakage, peripheral vascular dilatation, coagulation disorders, microthrombi, and myocardial depression. These phenomena may finally result in multiple organ dysfunction, shock, and death (Bone, et al., 1991).
Humeral response
Bacteria activate both complement pathways: i) alternative pathway which is triggered by binding polysaccharide surface components (O antigen, capsule, and LPS) to complement factor 3 (C3) (Joiner, et al., 1984; Quezado, et al., 1994; Tesh, et al., 1988) ii) classical pathway which is activated by binding Lipid A to C1q (Ying, et al., 1993). The classical complement pathway is also activated in the presence of specific antibodies (IgG and IgM) against gram-negative bacterial constituents. In all three cases, C3b is deposited on the molecule or cell surface, which promotes phagocytosis by macrophages and neutrophils and leads to insertion of C5–C9 (membrane attack complex) into the cell surface, leading to lysis of the bacterium (De Boer, et al., 1993; Frank, et al., 1991). However, long O-antigen chains in gram-negative bacteria may protect the bacteria from complement-mediated lysis (Haeney, M. R., 1998). With the cleavage of C3 and C5, the chemoattractive and vasoactive agents C3a and C5a are released. They cause increased vascular permeability, upregulate adhesion molecule expression on endothelial cells and neutrophils, and attract and activate
these phagocytes. Furthermore, they activate basophilic granulocytes and mast cells: these cells release a variety of vasoactive compounds (such ashistamine), facilitating the invasion of phagocytes. (Espevik, et al., 1993; Hsueh, et al., 1990; Kuipers, et al., 1994; Mulligan, et al., 1993; Pu¨schel, et al., 1993; Roitt, I. M., 1994; Tesh, et al., 1988; Van Epps, et al., 1993)
During infection, liver parenchymal cells are stimulated by TNF-α, IL-1, and IL-6 to produce acute-phase proteins. These proteins comprise C-reactive protein, serum amyloid A, lipopolysaccharide- binding protein (LBP), serum amyloid P, hemopexin, haptoglobin, complement C3 and C9, α1-acid glycoprotein, α2-macroglobulin, and some proteinase inhibitors (476, 498). The expression is differentially upregulated from several fold (C3 and C9) to even 1,000-fold (C-reactive protein). Some of the acute-phase proteins, like LBP modulate the immune response reactions by activation of phagocytes and antigen-presenting cells, but basically the acute-phase response is considered to alleviate the damage caused during infection (Fey, et al., 1994; Kuipers, et al., 1994; Ramadori, et al., 1990). Albumin is a so-called negative acute-phase protein since its production is down regulated during inflammation (Fey, et al., 1994).
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