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This month in J Lab Clin Med
The Journal of LABORATORY and CLINICAL MEDICINE Copyright © 2000 by Mosby, Inc.
VOLUME 136
OCTOBER 2000
NUMBER 4
THIS MONTH IN J Lab Clin Med Issue Highlights for October 2000
Alcoholism and oxidative injury The metabolism of alcohol in the liver leads to the production of oxidants as by-products, a finding that has been held to be a credible explanation—at least in part—for the tendency of liver fibrosis to develop in patients with alcoholism. But of course, alcohol is metabolized in tissues other than the liver, and both alcohol and some of its metabolic by-products circulate in the bloodstream. Thus it’s reasonable to ask whether the oxidative damage done by alcohol might be a much more general phenomenon, with repercussions throughout the body. If so, the malnourished alcoholic patient might be a particularly susceptible subject. ˘ and colleagues from the University of Istanbul and Istanbul’s BalikDr Ümit Mutlu-Türkoglu li Rum Hospital asked just that question. They evaluated 28 patients with chronic alcoholism severe enough that they admitted to drinking an average 255 g of alcohol daily and had been admitted to a detoxification unit but who were all also free from viral hepatitis and cirrhosis. They compared these patients with 15 normal control subjects, all of whom used alcohol sparingly. As described beginning on page 287, the authors measured blood malondialdehyde (thiobarbituric acid–reactive substances), protein carbonyl compounds, and electrophoretic evidence of DNA damage. Each of these was modestly greater in the alcoholic patients than in the control subjects, suggesting an ongoing oxidative stress. The investigators also exposed lymphocytes to oxidative stress in vitro, finding that the alcoholic patients had both a greater amount of endogenous DNA damage in their lymphocytes and a greater increase in such damage on exposure to hydrogen peroxide. The authors conclude that ongoing oxidative stress is a credible contributor to many aspects of the disease we recognize as alcoholism. Of course, alcohol use is a complex issue, with some benefits seeming to accrue to those who use it in moderation. Alcoholism is also a complex phenomenon, with many social and nutritional features beyond the mere exposure to alcohol and its metabolites. On page 258, Dr Andrew Collins of the Rowett Research Institute (Aberdeen, Scotland) offers an editorial comment to put this study in perspective.
Platelet-neutrophil interactions Two articles in the current issue address the interaction between neutrophils and platelets; one explores a possible new context in which such interactions might be functionally important, and the other examines details of the interaction at a molecular level. doi:10.1067/mlc.2000.109598 253
254 In this issue
J Lab Clin Med October 2000
Beginning on page 304, Drs Christopher Kirton and Gerard Nash, from the University of Birmingham (England), describe a series of experiments suggesting that activated platelets may promote the transfer of neutrophils from the bloodstream to the endothelial surface. Endothelial cells were grown to confluence in a glass capillary device and were exposed to perfusions of isolated neutrophils. A variety of manipulations were tested, including the presedimentation of small numbers of (activated or unactivated) platelets on the monolayer and including the stimulation of the neutrophils with N-formyl-methionyl-leucyl-phenylalanine. The behavior of the passing neutrophils was observed microscopically. There were no adhesive interactions between unstimulated neutrophils and platelet-free monolayers. When platelets had been allowed to attach to the monolayer (covering less than 1% of the available surface), there were many short-lived interactions, but very few of the neutrophils became adherent in a stationary manner. The use of stimulated platelets, or stimulation of the neutrophils, led to a higher incidence of stationary adhesion as well as migration of some neutrophils—initially adherent to platelets—onto endothelial cells. Blocking antibodies were then used to show that the adhesion was dependent on P-selectin exposure on the surface of the platelets as well as on β2 integrin expression on the neutrophils. A related article appears on page 292. Dr A. Majluf-Cruz and several coworkers from Temple University (Philadelphia) pursued the finding that thrombospondin appears to be important in neutrophil-platelet interactions, asking what portions of the thrombospondin molecule mediated the effect. A number of thrombospondin fragments were prepared, either by enzymatic cleavage or by synthetic techniques. Several such peptides containing a heparin-binding motif would compete successfully for the binding of thrombospondin to the heparin-agarose beads. Only those containing the residues F16-G33, however, were competitors for binding of thrombospondin to neutrophils. There is also binding to neutrophils by thrombospondin that has had its heparin-binding domain removed; competitive binding studies again were used to localize a second binding site to residues A784-N823. These two articles contribute to our understanding of the interactions between platelets and neutrophils and may help us develop tools for modulating those interactions to clinical benefit.
From the Chicago Meetings: Hereditary pancreatitis Pancreatitis is a complex clinical syndrome with many possible pathogeneses. In some cases, the pathophysiology may be obvious—or at least intuitively satisfying. Gallstones may obstruct the pancreatic duct, or toxins (such as organophosphates or the venom of the Trinidadian scorpion) may promote excessive pancreatic secretion against an obstructing sphincter of Oddi. In many other circumstances, however, the pathophysiology is a bit more puzzling, both in the triggering of events and in the continuation of the process. The enzymes released by the pancreas are largely secreted as proenzymes, so they have to get turned on to do their mischief; moreover, the body is richly endowed with proteinase inhibitors and inactivators, so the activated enzymes must escape such regulation to keep on doing damage. And something has to make these enzymes get into the interstices in the first place. Mechanistically, one may imagine that there are situations in which the amount of released proenzyme is simply huge, situations in which proenzymes are too easily activated to their catalytically active forms, situations in which the active enzymes resist the host’s control mechanisms, and situations in which the host’s control mechanisms are simply not up to the task. Of course, it is also possible to construct scenarios in which the primary phlogistic event is oxidative or otherwise divorced from the enzymatic machinery of the pancreas. A rare syndrome—hereditary pancreatitis—provides an “experiment of nature” for the study of the genesis of pancreatitis. In some kindreds, mutations in the gene for trypsinogen render the resulting trypsin resistant to proteolytic cleavage; once a small amount of trypsinogen is activated, the system is hard to brake.
J Lab Clin Med Volume 136, Number 4
In this issue
An invited review this month, prepared by Dr Charles Ulrich and associates from the University of Cincinnati, discusses the pathophysiology of pancreatitis, with a special emphasis on the lessons we’ve been taught by the study of hereditary pancreatitis (see page 260).
Apoptosis, Fas, and uremia: Still a confusing story For many clinical reasons, it is easy to believe that a patient with uremia may have accelerated programmed cell death in a number of tissues; it’s been shown that apoptosis is greater than normal in circulating blood cells from uremic patients, and it has been reported that serum from uremic patients promotes apoptosis in a variety of cultured cells. One of the recognized triggers for apoptosis is a cell surface receptor known as Fas; its naturally occurring ligand (FasL) or even antibodies directed against Fas may trigger programmed death of cells displaying Fas on their surfaces. Might an imbalance between Fas and FasL explain the pro-apoptotic effect of uremic serum and in turn explain the observed excessive apoptosis in circulating cells in uremic patients? Dr Mary C. Perianayagam and colleagues from the New England Medical center (Boston) explored this issue, and they present their findings beginning on page 320. In addition to Fas on the surface of cells, there exists a soluble form that may be shed and may be detected in the plasma or serum (soluble Fas, or sFas). These investigators measured sFas and (s)FasL in the blood of normal subjects, uremic patients not yet undergoing dialysis, and uremic patients who were dialysis dependent (hemo- or peritoneal). They found that the level of sFasL was pretty uniform among the several groups of individuals. However, the level of sFas was higher in the patients with kidney failure, strongly (inversely) correlating with the creatinine clearance. That’s certainly not what one would have predicted if an imbalance between Fas and FasL were the explanation for the pro-apoptotic effect of uremic serum. On the other hand, one can speculate that sFas may be released as a protection against excessive production of sFasL, or in circumstances where the effect of sFasL is somehow potentiated. The story’s complexity is enhanced by two observations: a low-molecular-weight band on electrophoresis that has sFas immunoreactivity (likely a degradation product) and a high-molecular-weight band that likely represents Fas/FasL aggregates. In fact, the contribution of the Fas system to apoptosis in uremia is probably the net result of a complex choreography. For the editors Dale E. Hammerschmidt, MD Editor-in-Chief
255
VOLUME 136
OCTOBER 2000
NUMBER 4
THIS MONTH IN J Lab Clin Med Issue Highlights for October 2000
Alcoholism and oxidative injury The metabolism of alcohol in the liver leads to the production of oxidants as by-products, a finding that has been held to be a credible explanation—at least in part—for the tendency of liver fibrosis to develop in patients with alcoholism. But of course, alcohol is metabolized in tissues other than the liver, and both alcohol and some of its metabolic by-products circulate in the bloodstream. Thus it’s reasonable to ask whether the oxidative damage done by alcohol might be a much more general phenomenon, with repercussions throughout the body. If so, the malnourished alcoholic patient might be a particularly susceptible subject. ˘ and colleagues from the University of Istanbul and Istanbul’s BalikDr Ümit Mutlu-Türkoglu li Rum Hospital asked just that question. They evaluated 28 patients with chronic alcoholism severe enough that they admitted to drinking an average 255 g of alcohol daily and had been admitted to a detoxification unit but who were all also free from viral hepatitis and cirrhosis. They compared these patients with 15 normal control subjects, all of whom used alcohol sparingly. As described beginning on page 287, the authors measured blood malondialdehyde (thiobarbituric acid–reactive substances), protein carbonyl compounds, and electrophoretic evidence of DNA damage. Each of these was modestly greater in the alcoholic patients than in the control subjects, suggesting an ongoing oxidative stress. The investigators also exposed lymphocytes to oxidative stress in vitro, finding that the alcoholic patients had both a greater amount of endogenous DNA damage in their lymphocytes and a greater increase in such damage on exposure to hydrogen peroxide. The authors conclude that ongoing oxidative stress is a credible contributor to many aspects of the disease we recognize as alcoholism. Of course, alcohol use is a complex issue, with some benefits seeming to accrue to those who use it in moderation. Alcoholism is also a complex phenomenon, with many social and nutritional features beyond the mere exposure to alcohol and its metabolites. On page 258, Dr Andrew Collins of the Rowett Research Institute (Aberdeen, Scotland) offers an editorial comment to put this study in perspective.
Platelet-neutrophil interactions Two articles in the current issue address the interaction between neutrophils and platelets; one explores a possible new context in which such interactions might be functionally important, and the other examines details of the interaction at a molecular level. doi:10.1067/mlc.2000.109598 253
254 In this issue
J Lab Clin Med October 2000
Beginning on page 304, Drs Christopher Kirton and Gerard Nash, from the University of Birmingham (England), describe a series of experiments suggesting that activated platelets may promote the transfer of neutrophils from the bloodstream to the endothelial surface. Endothelial cells were grown to confluence in a glass capillary device and were exposed to perfusions of isolated neutrophils. A variety of manipulations were tested, including the presedimentation of small numbers of (activated or unactivated) platelets on the monolayer and including the stimulation of the neutrophils with N-formyl-methionyl-leucyl-phenylalanine. The behavior of the passing neutrophils was observed microscopically. There were no adhesive interactions between unstimulated neutrophils and platelet-free monolayers. When platelets had been allowed to attach to the monolayer (covering less than 1% of the available surface), there were many short-lived interactions, but very few of the neutrophils became adherent in a stationary manner. The use of stimulated platelets, or stimulation of the neutrophils, led to a higher incidence of stationary adhesion as well as migration of some neutrophils—initially adherent to platelets—onto endothelial cells. Blocking antibodies were then used to show that the adhesion was dependent on P-selectin exposure on the surface of the platelets as well as on β2 integrin expression on the neutrophils. A related article appears on page 292. Dr A. Majluf-Cruz and several coworkers from Temple University (Philadelphia) pursued the finding that thrombospondin appears to be important in neutrophil-platelet interactions, asking what portions of the thrombospondin molecule mediated the effect. A number of thrombospondin fragments were prepared, either by enzymatic cleavage or by synthetic techniques. Several such peptides containing a heparin-binding motif would compete successfully for the binding of thrombospondin to the heparin-agarose beads. Only those containing the residues F16-G33, however, were competitors for binding of thrombospondin to neutrophils. There is also binding to neutrophils by thrombospondin that has had its heparin-binding domain removed; competitive binding studies again were used to localize a second binding site to residues A784-N823. These two articles contribute to our understanding of the interactions between platelets and neutrophils and may help us develop tools for modulating those interactions to clinical benefit.
From the Chicago Meetings: Hereditary pancreatitis Pancreatitis is a complex clinical syndrome with many possible pathogeneses. In some cases, the pathophysiology may be obvious—or at least intuitively satisfying. Gallstones may obstruct the pancreatic duct, or toxins (such as organophosphates or the venom of the Trinidadian scorpion) may promote excessive pancreatic secretion against an obstructing sphincter of Oddi. In many other circumstances, however, the pathophysiology is a bit more puzzling, both in the triggering of events and in the continuation of the process. The enzymes released by the pancreas are largely secreted as proenzymes, so they have to get turned on to do their mischief; moreover, the body is richly endowed with proteinase inhibitors and inactivators, so the activated enzymes must escape such regulation to keep on doing damage. And something has to make these enzymes get into the interstices in the first place. Mechanistically, one may imagine that there are situations in which the amount of released proenzyme is simply huge, situations in which proenzymes are too easily activated to their catalytically active forms, situations in which the active enzymes resist the host’s control mechanisms, and situations in which the host’s control mechanisms are simply not up to the task. Of course, it is also possible to construct scenarios in which the primary phlogistic event is oxidative or otherwise divorced from the enzymatic machinery of the pancreas. A rare syndrome—hereditary pancreatitis—provides an “experiment of nature” for the study of the genesis of pancreatitis. In some kindreds, mutations in the gene for trypsinogen render the resulting trypsin resistant to proteolytic cleavage; once a small amount of trypsinogen is activated, the system is hard to brake.
J Lab Clin Med Volume 136, Number 4
In this issue
An invited review this month, prepared by Dr Charles Ulrich and associates from the University of Cincinnati, discusses the pathophysiology of pancreatitis, with a special emphasis on the lessons we’ve been taught by the study of hereditary pancreatitis (see page 260).
Apoptosis, Fas, and uremia: Still a confusing story For many clinical reasons, it is easy to believe that a patient with uremia may have accelerated programmed cell death in a number of tissues; it’s been shown that apoptosis is greater than normal in circulating blood cells from uremic patients, and it has been reported that serum from uremic patients promotes apoptosis in a variety of cultured cells. One of the recognized triggers for apoptosis is a cell surface receptor known as Fas; its naturally occurring ligand (FasL) or even antibodies directed against Fas may trigger programmed death of cells displaying Fas on their surfaces. Might an imbalance between Fas and FasL explain the pro-apoptotic effect of uremic serum and in turn explain the observed excessive apoptosis in circulating cells in uremic patients? Dr Mary C. Perianayagam and colleagues from the New England Medical center (Boston) explored this issue, and they present their findings beginning on page 320. In addition to Fas on the surface of cells, there exists a soluble form that may be shed and may be detected in the plasma or serum (soluble Fas, or sFas). These investigators measured sFas and (s)FasL in the blood of normal subjects, uremic patients not yet undergoing dialysis, and uremic patients who were dialysis dependent (hemo- or peritoneal). They found that the level of sFasL was pretty uniform among the several groups of individuals. However, the level of sFas was higher in the patients with kidney failure, strongly (inversely) correlating with the creatinine clearance. That’s certainly not what one would have predicted if an imbalance between Fas and FasL were the explanation for the pro-apoptotic effect of uremic serum. On the other hand, one can speculate that sFas may be released as a protection against excessive production of sFasL, or in circumstances where the effect of sFasL is somehow potentiated. The story’s complexity is enhanced by two observations: a low-molecular-weight band on electrophoresis that has sFas immunoreactivity (likely a degradation product) and a high-molecular-weight band that likely represents Fas/FasL aggregates. In fact, the contribution of the Fas system to apoptosis in uremia is probably the net result of a complex choreography. For the editors Dale E. Hammerschmidt, MD Editor-in-Chief
255