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This month in J Lab Clin Med
The Journal of LABORATORY and CLINICAL MEDICINE Copyright © 2002 by Mosby, Inc.
VOLUME 140
JULY 2002
NUMBER 1
THIS MONTH IN J Lab Clin Med Issue Highlights for July 2002
Growth factors and neutrophil priming Since it has become possible to produce lineage-specific marrow-stimulating hormones in pharmacologically (and commercially) useful quantities, their use has become routine in several contexts. Erythropoietin has limited the need for transfusion in patients with renal failure (even as it has tempted endurance athletes); granulocyte colony stimulating factor (G-CSF) has shortened the period of severe neutropenia in a variety of chemotherapeutic contexts. In some cases, the ability to use growth factors pharmacologically has led to new insights into their activities. For example, G-CSF seems to improve granulocyte function as well as increase the rate at which the cells are produced; this clinical observation has its functional correlate in the ability of G-CSF to prime neutrophil oxidative responses. That is, G-CSF is not an agonist for the activation of neutrophil oxidase by itself, but pre-treating neutrophils with G-CSF leads to an enhanced oxidase response when they are subsequently exposed to a different agonist, such as a formylated bacterial peptide. In this month’s issue (pages 9-16), Dr Pamela Mansfield and several colleagues from the University of Michigan explored the mechanism of neutrophil priming by G-CSF. It was already known that several neutrophil functions—including oxidase activation and degranulation— depended upon phospholipase D and could be inhibited by certain exogenous ceramides. The researchers first demonstrated that G-CSF would cause (not merely prime) the release of gelatinase from the cells’ tertiary granules but would not cause release from secondary granules. Similar doses (within the range known to prime oxidase activation) also led to an increase in phospholipase D activity. C2 ceramide inhibited the gelatinase release, in parallel with its known effect as an inhibitor of phospholipase D. These findings allowed one to postulate a mechanism for priming: G-CSF might release something from the tertiary granules that contributed to the activity of the cytochrome oxidase. In support of this notion, neutrophils treated with 50 ng/mL G-CSF were found to contain 20% more cytochrome b558 in the plasma membrane fraction than did unstimulated cells, and this change was blocked by C2 ceramide. When another component of the oxidase, p47 phox, was similarly examined, it was not found to be increased after G-CSF treatment. This difference provides a plausible explanation for the ability of G-CSF to prime oxidase activity but not to activate the oxidase in the absence of additional agonists.
J Lab Clin Med 2002;140:1-3. Copyright © 2002 by Mosby, Inc. All rights reserved. 0022-2143/2002 $35.00 ⫹ 0 5/8/126362 doi:10.1067/mlc.2002.126362
1
2
In this issue
J Lab Clin Med July 2002
Adrenergic selectivity in cardiac arrest It is common to use adrenergic agonists such as epinephrine during resuscitation of patients who have suffered cardiac arrest. But their use is not always successful, and repeated administrations after an initial failure are not often fruitful. Part of the problem may be that these drugs are a bit of a two-edged sword, having problematic effects—such as increasing myocardial irritability and increasing post-ischemic oxygen demand—in addition to their sought-after positive effects. Noting that the deleterious effects derive disproportionately from -adrenergic actions of epinephrine, Dr Kada Klouche and associates from the Institute of Critical Care Medicine (Palm Springs, Calif) and from the University of Southern California suggested that post-resuscitative myocardial dysfunction might be less severe if a selective ␣2 agent were used instead of epinephrine. They tested this idea in a porcine model. Ventricular fibrillation was provoked in anesthetized pigs; after seven minutes, artificial ventilation and chest compressions were begun. After two minutes of such mechanical resuscitation, the animals received either epinephrine or the ␣2-selective agent, ␣-methyl norepinephrine (␣-MNE). After an additional four minutes, electrical defibrillation was carried out. The two drugs produced comparable increases in coronary artery perfusion pressure, and all animals in both groups were successfully defibrillated. The post-resuscitation ejection was reduced by 35% in the animals that had received epinephrine, but only by 14% in those that had received ␣-MNE. Post-resuscitative tachycardia was also less pronounced in the animals that had received the more selective agent. These findings suggest that the (peripheral) effects of ␣2 adrenergic stimulation may provide resuscitative benefits comparable to those of nonselective ␣/ stimulation, while avoiding some of the problematic central effects of the traditional approach.
Angling for cancer-related genes: An example of an emerging technology Many cancers are known to occur at increased frequency in relatives of diagnosed patients; for most of them, however, the familial associations are relatively weak and only a few specific risk genes have been characterized. Many (perhaps most) cancers result from genetic events at the cellular level, but only a relatively small number of specific mutations/activations/translocations have been identified. Unless a cancer has a commonly recurrent cytogenetic or biochemical or functional abnormality, researchers will have trouble knowing what to seek and where to look. Broader knowledge of the human genome and the development of improved micro-scale assay techniques are changing this picture; one may now screen for a large number of candidate genes quite efficiently. An example of this process is provided this month by Drs Keith and Amy Skubitz of the University of Minnesota. These researchers extracted RNA from renal cell carcinomas, from normal kidneys and from kidneys harboring non-malignant diseases. The extracts were subjected to microarray analysis using an affinity matrix capable of detecting (expression of) 12,000 known genes and an even larger number of transcripts. A computer analysis then allowed them to determine which were overexpressed or underexpressed to a degree unlikely to be explained by chance alone. This was done in a couple different ways. A “genetic signature” could be prepared for each group of samples, identifying the genes that were expressed in ⬎75% (or other arbitrarily chosen proportion) of the samples, as well as genes that were infrequently expressed in the samples. This “signature” could be compared among the groups to identify patterns that might suggest further, more focused study. Alternatively, one could examine the intensity of gene expression in individual samples, rather than its frequency. Both types of analysis were applied to the samples, yielding a set of fifty genes that could discriminate among the types of tissue studied. It is likely that this relatively small set of genes includes some that are of importance to the biology of renal cell cancer. A more detailed study of this subset may yield clinically useful insights. The paper may be found on page 52.
J Lab Clin Med Volume 140, Number 1
In this issue
Monocytes in renal injury Although one thinks of glomerulonephritis as a disorder of the kidney’s filtration apparatus, there is often tubular dysfunction as well. There may be tubulointerstitial changes histologically, and these may include evidence of inflammatory injury. Monocyte chemoattractant protein-1 (MCP-1) has been found to be upregulated in the tubulointerstitium and/or glomeruli of many types of human and experimental renal disorders, and seems a credible contributor to tubular dysfunction. Dr Takayuka Ota et alii, from the University of Occupational and Environmental Health (School of Medicine), Kitakyushu, Japan, and the Nagasaki University School of Medicine, investigated the localization of MCP-1 expression in a rat model of progressive renal failure. Rats underwent either subtotal nephrectomy or sham operation, then had immunohistochemical, ultrastructural, and in situ hybridization studies performed to examine the expression of MCP-1 protein and mRNA in the kidney. MCP-1 protein and mRNA were barely detectable in the kidneys of of sham-operated control rats. However, MCP-1 expression was elevated in tubular cells of the remnant kidneys of the experimental group. Accompanying this increased expression was significant macrophage infiltration. Most of the MCP-1 was observed in the proximal tubular cells; relatively little was seen in distal tubular cells. MCP-1 protein and mRNA were not expressed in the glomeruli. Immunoelectron microscopy showed the presence of MCP-1 in the rough endoplasmic reticulum of proximal tubular cells, confirming the findings of light microscopy and indicating that proximal tubular cells were the site of production. MCP-1 was also observed in endocytic vesicles adjacent to the brush border of proximal tubular cells, suggesting incorporation of MCP-1 from the tubular lumen. These findings suggest that MCP-1 in proximal tubular cells might be involved in tubulointerstitial damage in chronic renal failure associated with glomerular hypertension.
Peroxisomes and esophageal cancer Some human cancer cells have been found to express receptors that can be activated by the peroxisome proliferator. The growth of cells expressing the peroxisome-proliferator-activated receptor ␥ (PPAR␥) has been reported to be inhibited by ligands to that receptor. Esophageal squamous-cell carcinoma is one of the tumors in which this mechanism seems to be operative. Dr M. A. K. Rumi and fellow researchers from the Shimane Unuiversity (Japan) studied several esophageal cancer cell lines. They detected expression of PPAR␥ in several such lines (T.T, T.Tn and EC-GI-10), and they studied the effects of PPAR␥ ligands on their growth in vitro. Troglitazone, pioglitazone, and a reagent known as 15d-PGJ2 were selected as the test ligands, and each of them inhibited the growth of each of the cell lines.The growth inhibitory effect was characterized by a dose-dependent inhibition of DNA synthesis and an arrest of the cell cycle in the G1 phase. Cell cycle arrest in T.Tn cells induced by troglitazone could be correlated with an increased level of cyclin-dependent kinase inhibitors (p27Kip1, p21Cip1/Waf1, and p18Ink4c). Moreover, troglitazone treatment increased the expression of interleukin 1␣, a multifunctional cytokine involved in anti-tumor immunity. It is of particular interest that some of the PPAR␥ ligands tested were agents already known to be safe enough to give to humans; these findings therefore may eventually point the way to manipulating this phenomenon to therapeutic advantage. The paper from the Shimane researchers may be found on page 17. Drs Tadahito Shimada and Akira Terano, of theDokkyo University School of Medicine (Mibu, Tochigi, Japan) provide an editorial putting this finding in context (see page 4). For the editors Dale E. Hammerschmidt, MD Editor-in-Chief
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VOLUME 140
JULY 2002
NUMBER 1
THIS MONTH IN J Lab Clin Med Issue Highlights for July 2002
Growth factors and neutrophil priming Since it has become possible to produce lineage-specific marrow-stimulating hormones in pharmacologically (and commercially) useful quantities, their use has become routine in several contexts. Erythropoietin has limited the need for transfusion in patients with renal failure (even as it has tempted endurance athletes); granulocyte colony stimulating factor (G-CSF) has shortened the period of severe neutropenia in a variety of chemotherapeutic contexts. In some cases, the ability to use growth factors pharmacologically has led to new insights into their activities. For example, G-CSF seems to improve granulocyte function as well as increase the rate at which the cells are produced; this clinical observation has its functional correlate in the ability of G-CSF to prime neutrophil oxidative responses. That is, G-CSF is not an agonist for the activation of neutrophil oxidase by itself, but pre-treating neutrophils with G-CSF leads to an enhanced oxidase response when they are subsequently exposed to a different agonist, such as a formylated bacterial peptide. In this month’s issue (pages 9-16), Dr Pamela Mansfield and several colleagues from the University of Michigan explored the mechanism of neutrophil priming by G-CSF. It was already known that several neutrophil functions—including oxidase activation and degranulation— depended upon phospholipase D and could be inhibited by certain exogenous ceramides. The researchers first demonstrated that G-CSF would cause (not merely prime) the release of gelatinase from the cells’ tertiary granules but would not cause release from secondary granules. Similar doses (within the range known to prime oxidase activation) also led to an increase in phospholipase D activity. C2 ceramide inhibited the gelatinase release, in parallel with its known effect as an inhibitor of phospholipase D. These findings allowed one to postulate a mechanism for priming: G-CSF might release something from the tertiary granules that contributed to the activity of the cytochrome oxidase. In support of this notion, neutrophils treated with 50 ng/mL G-CSF were found to contain 20% more cytochrome b558 in the plasma membrane fraction than did unstimulated cells, and this change was blocked by C2 ceramide. When another component of the oxidase, p47 phox, was similarly examined, it was not found to be increased after G-CSF treatment. This difference provides a plausible explanation for the ability of G-CSF to prime oxidase activity but not to activate the oxidase in the absence of additional agonists.
J Lab Clin Med 2002;140:1-3. Copyright © 2002 by Mosby, Inc. All rights reserved. 0022-2143/2002 $35.00 ⫹ 0 5/8/126362 doi:10.1067/mlc.2002.126362
1
2
In this issue
J Lab Clin Med July 2002
Adrenergic selectivity in cardiac arrest It is common to use adrenergic agonists such as epinephrine during resuscitation of patients who have suffered cardiac arrest. But their use is not always successful, and repeated administrations after an initial failure are not often fruitful. Part of the problem may be that these drugs are a bit of a two-edged sword, having problematic effects—such as increasing myocardial irritability and increasing post-ischemic oxygen demand—in addition to their sought-after positive effects. Noting that the deleterious effects derive disproportionately from -adrenergic actions of epinephrine, Dr Kada Klouche and associates from the Institute of Critical Care Medicine (Palm Springs, Calif) and from the University of Southern California suggested that post-resuscitative myocardial dysfunction might be less severe if a selective ␣2 agent were used instead of epinephrine. They tested this idea in a porcine model. Ventricular fibrillation was provoked in anesthetized pigs; after seven minutes, artificial ventilation and chest compressions were begun. After two minutes of such mechanical resuscitation, the animals received either epinephrine or the ␣2-selective agent, ␣-methyl norepinephrine (␣-MNE). After an additional four minutes, electrical defibrillation was carried out. The two drugs produced comparable increases in coronary artery perfusion pressure, and all animals in both groups were successfully defibrillated. The post-resuscitation ejection was reduced by 35% in the animals that had received epinephrine, but only by 14% in those that had received ␣-MNE. Post-resuscitative tachycardia was also less pronounced in the animals that had received the more selective agent. These findings suggest that the (peripheral) effects of ␣2 adrenergic stimulation may provide resuscitative benefits comparable to those of nonselective ␣/ stimulation, while avoiding some of the problematic central effects of the traditional approach.
Angling for cancer-related genes: An example of an emerging technology Many cancers are known to occur at increased frequency in relatives of diagnosed patients; for most of them, however, the familial associations are relatively weak and only a few specific risk genes have been characterized. Many (perhaps most) cancers result from genetic events at the cellular level, but only a relatively small number of specific mutations/activations/translocations have been identified. Unless a cancer has a commonly recurrent cytogenetic or biochemical or functional abnormality, researchers will have trouble knowing what to seek and where to look. Broader knowledge of the human genome and the development of improved micro-scale assay techniques are changing this picture; one may now screen for a large number of candidate genes quite efficiently. An example of this process is provided this month by Drs Keith and Amy Skubitz of the University of Minnesota. These researchers extracted RNA from renal cell carcinomas, from normal kidneys and from kidneys harboring non-malignant diseases. The extracts were subjected to microarray analysis using an affinity matrix capable of detecting (expression of) 12,000 known genes and an even larger number of transcripts. A computer analysis then allowed them to determine which were overexpressed or underexpressed to a degree unlikely to be explained by chance alone. This was done in a couple different ways. A “genetic signature” could be prepared for each group of samples, identifying the genes that were expressed in ⬎75% (or other arbitrarily chosen proportion) of the samples, as well as genes that were infrequently expressed in the samples. This “signature” could be compared among the groups to identify patterns that might suggest further, more focused study. Alternatively, one could examine the intensity of gene expression in individual samples, rather than its frequency. Both types of analysis were applied to the samples, yielding a set of fifty genes that could discriminate among the types of tissue studied. It is likely that this relatively small set of genes includes some that are of importance to the biology of renal cell cancer. A more detailed study of this subset may yield clinically useful insights. The paper may be found on page 52.
J Lab Clin Med Volume 140, Number 1
In this issue
Monocytes in renal injury Although one thinks of glomerulonephritis as a disorder of the kidney’s filtration apparatus, there is often tubular dysfunction as well. There may be tubulointerstitial changes histologically, and these may include evidence of inflammatory injury. Monocyte chemoattractant protein-1 (MCP-1) has been found to be upregulated in the tubulointerstitium and/or glomeruli of many types of human and experimental renal disorders, and seems a credible contributor to tubular dysfunction. Dr Takayuka Ota et alii, from the University of Occupational and Environmental Health (School of Medicine), Kitakyushu, Japan, and the Nagasaki University School of Medicine, investigated the localization of MCP-1 expression in a rat model of progressive renal failure. Rats underwent either subtotal nephrectomy or sham operation, then had immunohistochemical, ultrastructural, and in situ hybridization studies performed to examine the expression of MCP-1 protein and mRNA in the kidney. MCP-1 protein and mRNA were barely detectable in the kidneys of of sham-operated control rats. However, MCP-1 expression was elevated in tubular cells of the remnant kidneys of the experimental group. Accompanying this increased expression was significant macrophage infiltration. Most of the MCP-1 was observed in the proximal tubular cells; relatively little was seen in distal tubular cells. MCP-1 protein and mRNA were not expressed in the glomeruli. Immunoelectron microscopy showed the presence of MCP-1 in the rough endoplasmic reticulum of proximal tubular cells, confirming the findings of light microscopy and indicating that proximal tubular cells were the site of production. MCP-1 was also observed in endocytic vesicles adjacent to the brush border of proximal tubular cells, suggesting incorporation of MCP-1 from the tubular lumen. These findings suggest that MCP-1 in proximal tubular cells might be involved in tubulointerstitial damage in chronic renal failure associated with glomerular hypertension.
Peroxisomes and esophageal cancer Some human cancer cells have been found to express receptors that can be activated by the peroxisome proliferator. The growth of cells expressing the peroxisome-proliferator-activated receptor ␥ (PPAR␥) has been reported to be inhibited by ligands to that receptor. Esophageal squamous-cell carcinoma is one of the tumors in which this mechanism seems to be operative. Dr M. A. K. Rumi and fellow researchers from the Shimane Unuiversity (Japan) studied several esophageal cancer cell lines. They detected expression of PPAR␥ in several such lines (T.T, T.Tn and EC-GI-10), and they studied the effects of PPAR␥ ligands on their growth in vitro. Troglitazone, pioglitazone, and a reagent known as 15d-PGJ2 were selected as the test ligands, and each of them inhibited the growth of each of the cell lines.The growth inhibitory effect was characterized by a dose-dependent inhibition of DNA synthesis and an arrest of the cell cycle in the G1 phase. Cell cycle arrest in T.Tn cells induced by troglitazone could be correlated with an increased level of cyclin-dependent kinase inhibitors (p27Kip1, p21Cip1/Waf1, and p18Ink4c). Moreover, troglitazone treatment increased the expression of interleukin 1␣, a multifunctional cytokine involved in anti-tumor immunity. It is of particular interest that some of the PPAR␥ ligands tested were agents already known to be safe enough to give to humans; these findings therefore may eventually point the way to manipulating this phenomenon to therapeutic advantage. The paper from the Shimane researchers may be found on page 17. Drs Tadahito Shimada and Akira Terano, of theDokkyo University School of Medicine (Mibu, Tochigi, Japan) provide an editorial putting this finding in context (see page 4). For the editors Dale E. Hammerschmidt, MD Editor-in-Chief
3