- Email: [email protected]
Selective toxicity of vincristine against chronic lymphocytic leukaemia in vitro
of 5, further weakened the association between respiratory symptoms and gas cooking, especially in women. Was the reported association in females in the British study still found when correction was made for such personal and other environmental factors?
when those of possibly equal exposure (single people) considered separately. This finding is of interest, but information on marital status is not available from most centres and this form of analysis cannot be included in the international comparison. Leynaert and colleagues2 are wrong to attribute to us the view that differences between men and women are necessarily more likely to be due to differences in exposure. Wieringa and colleagues show inconsistent associations between gas appliances and respiratory disease in Belgium. They also suggest that adjustment for asthma in a sibling and respiratory infection before the age of 5 years may reduce our observed associations. We can think of no a priori reason why these factors should be confounding our observed association, and adjustment for both factors marginally but not significantly increases the odds in women and decreases the odds in men. As soon as the full international analysis is completed it will be submitted for publication. At present there is little evidence that additional variables to those used in the original East Anglian data set should be included.
*M Wieringa, J Weyler, P Vermeire University of Antwerp, Department of Epidemiology Universiteitsplein 1, B-2610, Antwerp, Belgium
Department of Public Health Medicine, United Medical and Dental Schools, St Thomas’s Hospital, London SE1 7EH, UK
even are
*Adjusted for area of residence, ever having smoked >1 year, and age-group. tall variables: in past 12 months. tp
gas cooking
*D Jarvis, S Chinn, and
Respiratory Medicine,
1 Jarvis D, Chinn S, Luczynska C, Burney P. Association of respiratory symptoms and lung function in young adults with use of domestic gas appliances. Lancet 1996; 347: 426-31.
1
2
Authors’ reply SIR-The publication of our study,’ which was based on data collected in England as part of the multicentre ECRHS, has prompted research groups from other countries that also took part in ECRHS to test whether our findings-a strong and consisted association between respiratory symptoms and the use of gas cooking in women-can be replicated in their own local data sets.2 The ECRHS has collected information on respiratory disease and exposure to known and suspected risk factors for asthma from over 17 000 people living in more than 30 centres in 14 different countries (some of which are not in Europe). The project management group agreed the following two-stage plan for analysis. First, data from individual centres could be aggregated to country level and analysed to identify potential risk factors for disease. Second, assuming that the analysis was accepted by the wider research community as being valid (for example by being accepted for publication), researchers would be provided with the appropriate variables from the combined data set for all centres to test whether their hypothesis held in all the populations studied. This approach was designed to prevent false associations arising from trawling through the immense combined ECRHS data set. However, analyses on the international data sets are complex and an inevitable delay occurs between the two stages. Data anomalies need clarification and supplementary information may be required for results to be appropriately interpreted. In the meantime, individual centres may choose to analyse their local data with methods that may or may not be adopted for the final
international analysis. We are examining the relation of domestic gas appliances and respiratory disease in the ECRHS international data set. There is wide variation in exposure to gas appliances, the nature of these appliances, and the type of gas they use. It would be premature to comment on the conclusions of colleagues from France and Belgium, but we can make the
following observations. Combined data from two of the four centres in France show that the difference between men and women persists
Luczynska,
P
Burney
Jarvis D, Chinn S, Luczynska C, Burney P. Association of respiratory symptoms and lung function in young adults with the use of domestic gas appliances. Lancet 1996; 347: 426-31. Leynaert B, Liard R, Bousquet J, Mesbah H, Neukirch F. Gas cooking and respiratory health in women. Lancet 1996; 347: 1052-53.
Selective toxicity of vincristine against chronic lymphocytic leukaemia in vitro SiR-It is generally assumed that vincristine, as are other Vinca alkaloids, is a cell-cycle-specific agent that blocks mitosis through metaphase arrest’ by specifically binding to tubulin. Other metabolic effects have been noticed, but only tubulin inhibition has been related to the cytotoxic effects of vincristine. Vincristine is used in several regimens for chronic lymphocytic leukaemia (CLL). However, a note on the use of vincristine in CLL has been cautionary sounded: the value of vincristine has not been proved in this disease, but because combination regimens previously established for other B-cell cancers happened to include vincristine, this drug has been given to a large number of patients with CLL. Since the majority of CLL cells are not mitotically active, the use of vincristine does not seem rational. While investigating the cytotoxicity of a number of chemotherapeutic agents against CLL cells in vitro, we noted a striking action of vincristine against lymphocytes from patients with CLL but not from normal healthy blood donors. We isolated peripheral blood mononuclear cells from 20 patients with CLL (>90% leukaemic B cells) and from 11 volunteers healthy by density gradient centrifugation. The cells were cultured in duplicate in 200 ilL aliquots (2X10/mL) on microtitre plates in RPMI 1640 medium containing 10% fetal calf serum. Vincristine concentrations were 0, 2, 10, 50, 250, and 1250 nmol/L. Toxicity was assessed by the reduction of macromolecular
protein synthesis or [3H]-leucine incorporation as an endpoint.3 The cells were incubated for 4 days. The tracer was added for the final 24 h, whereafter the proteins were precipitated with perchloric acid and the radioactivity in the macromolecular protein fraction was counted.3 Dose-response curves (figure) showed a remarkable difference in the response of CLL lymphocytes and normal lymphocytes. A statistically significant difference (t test) 1491
vessels, and the number of blood vessels showing PN-1
immunoreactivity, were greatly reduced in the diseased brain tissue samples.’ PN-1, like other serpins, is highly sensitive to oxidation.’’ In retrospect, the reduced PN-1 activity observed in the Alzheimer samples could reflect P-amyloidpeptide-mediated oxidation of extracellular matrix proteins, including PN-1. These observations seem to favour the important notion on vascular oxidative damage in Alzheimer’s disease. David Gurwitz National Laboratory for the Genetics of Israeli Tel Aviv 69978, Israel
Populations, Tel-Aviv University,
Bradbury J. ß-amyloid vasoactivity in Alzheimer’s
1
disease. Lancet
1996;
347: 750. 2
of vincristine
Figure: Cytotoxicity lymphocytes
against normal lymphocytes
3
and CLL
Results presented
as mean
(SD).
4
between the groups was already noted at clinically achievable vincristine concentrations. Our results showed selective action of vincristine against CLL lymphocytes. The mechanisms involved require careful investigation. Re-evaluation of the part played by vincristine in the treatment of CLL is justified. *Juhani Vilpo, Leena Vilpo, for the Tampere CLL Group *Laboratory of Molecular Haematology, Department of Clinical Chemistry, Tampere University Hospital, and Department of Medicine, University of Tampere Medical School, PO Box 2000, FIN-33521 Tampere, Finland
1
2
3
Pouillart P. Vincristine. In: Cvitkovic E, Droz JP, Armand JP, Khoury S, eds. Handbook of chemotherapy in clinical oncology. Channel Islands: Scientific Communication International Ltd, 1993: 309-11. Han T, Rai K. Chronic lymphocytic leukemia. In: Henderson ES, Lister TA, eds. Leukemia. Philadelphia: WB Saunders Company, 1990: 565-611. Vilpo JA, Vilpo LM. Metabolism, incorporation into DNA, and interactions with 1-b-D-arabinofuranosylcytosine of 5-hydroxymethyl2’-deoxyuridine in human promyelocytic leukemia cells (HL-60). Cancer Res 1988; 48: 33117-22.
(3-amyloid vasoactivity
in Alzheimer’s disease
SiR-Studies of Alzheimer’s disease brain tissue
might lend described support theory, by Bradbury (March 16, p 750),’ of a role for vascular p-amyloid peptidemediated oxidative damage in the aetiology of this disease. As she states, there is no doubt that this theory may open new avenues for treatment of Alzheimer’s patients-eg, by the use of superoxide scavengers or antioxidants. Until such studies are published, it is intriguing to note that human histological data that favour this theory have already been to
the
new
5
Wagner SL, Geddes JW, Cotman CW, et al. Protease nexin-1, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer’s disease. Proc Natl Acad Sci USA 1989; 86: 8284-88. Choi BH, Suzuki M, Kim T, Wagner SL, Cunningham DD. Protease nexin-1: localization in the human brain suggests a protective role against extravasated serine proteases. Am J Pathol 1990; 137: 741-47. Vaughan PJ, Su J, Cotman CW, Cunningham DD. Protease nexin-1, a potent thrombin inhibitor, is reduced around cerebral blood vessels in Alzheimer’s disease. Brain Res 1994; 668: 160-70. Scott RW, Bergman BL, Bajpai A, et al. Protease nexin: properties and a modified purification procedure. J Biol Chem 1985; 260: 7029-34. 1
SiR-Bradbury’ comments on Thomas and colleagues’= proposal that imbalances (in rats) in free-radical generating mechanisms lead to degeneration of the cerebral vascular endothelium in Alzheimer’s disease (AD). Although this would be difficult to demonstrate physiologically in man, our recent morphological observations attest that such degeneration is linked to A(3 deposition in cortical regions of people with AD.3 We used immunocytochemical methods to define microvascular abnormalities in post-mortem brain tissue obtained from people with AD and normally ageing controls. Double immunostaining of neocortical tissue sections with antibodies to endothelial cell markers such as CD34 or CD31 and to basement lamina-namely, collagen IVrevealed differential staining of numerous capillary profiles.’ The differential labelling was characterised by attenuation or degeneration of the endothelium, indicated by CD34negative capillary profiles that still retained their basement lamina shown by collagen IV reactivity alone. This occurrence was seen in virtually all A(3-laden cortical lobes of more than 90% of AD as well as Down’s syndrome patients, but was not readily evident in non-AD disorders, brain regions of people with AD, or age-matched healthy
published. 7 years ago,
Wagner
brain serine
et
of the nexin-1 protease
aF showed that the
activity
ubiquitous proteinase-inhibitor (PN-1) was strikingly reduced (about six-fold) compared with age-matched controls in postmortem hippocampus and cerebral cortex tissue from individuals with Alzheimer’s disease. Yet, the levels of mRNA for PN-1 in the same samples were unchanged, indicating that an unidentified process-possibly raised serine proteinase activity-was consuming PN-1. Subsequent attempts to identify such activities in these samples failed (D Gurwitz, unpublished observations). Immunostaining has shown that human brain PN-1 was localised mainly around small brain capillaries.3 Additional studies of postmortem Alzheimer tissue samples showed that both PN-1 immunoreactivity around blood
1492
Figure: Relation between microvascular abnormalities and neocortical A(3 deposits in frontal cortex Direct correlation between number of degenerated microvessels per unit area and density of Ap deposits (r=0.80, p<0001, df=148). Data points represent average density counts of five fields from ten sections of 25 um thickness double stained for microvasculature (basement lamina marker) and Ap, neurofibrillary tangles, or neurons.
when those of possibly equal exposure (single people) considered separately. This finding is of interest, but information on marital status is not available from most centres and this form of analysis cannot be included in the international comparison. Leynaert and colleagues2 are wrong to attribute to us the view that differences between men and women are necessarily more likely to be due to differences in exposure. Wieringa and colleagues show inconsistent associations between gas appliances and respiratory disease in Belgium. They also suggest that adjustment for asthma in a sibling and respiratory infection before the age of 5 years may reduce our observed associations. We can think of no a priori reason why these factors should be confounding our observed association, and adjustment for both factors marginally but not significantly increases the odds in women and decreases the odds in men. As soon as the full international analysis is completed it will be submitted for publication. At present there is little evidence that additional variables to those used in the original East Anglian data set should be included.
*M Wieringa, J Weyler, P Vermeire University of Antwerp, Department of Epidemiology Universiteitsplein 1, B-2610, Antwerp, Belgium
Department of Public Health Medicine, United Medical and Dental Schools, St Thomas’s Hospital, London SE1 7EH, UK
even are
*Adjusted for area of residence, ever having smoked >1 year, and age-group. tall variables: in past 12 months. tp
gas cooking
*D Jarvis, S Chinn, and
Respiratory Medicine,
1 Jarvis D, Chinn S, Luczynska C, Burney P. Association of respiratory symptoms and lung function in young adults with use of domestic gas appliances. Lancet 1996; 347: 426-31.
1
2
Authors’ reply SIR-The publication of our study,’ which was based on data collected in England as part of the multicentre ECRHS, has prompted research groups from other countries that also took part in ECRHS to test whether our findings-a strong and consisted association between respiratory symptoms and the use of gas cooking in women-can be replicated in their own local data sets.2 The ECRHS has collected information on respiratory disease and exposure to known and suspected risk factors for asthma from over 17 000 people living in more than 30 centres in 14 different countries (some of which are not in Europe). The project management group agreed the following two-stage plan for analysis. First, data from individual centres could be aggregated to country level and analysed to identify potential risk factors for disease. Second, assuming that the analysis was accepted by the wider research community as being valid (for example by being accepted for publication), researchers would be provided with the appropriate variables from the combined data set for all centres to test whether their hypothesis held in all the populations studied. This approach was designed to prevent false associations arising from trawling through the immense combined ECRHS data set. However, analyses on the international data sets are complex and an inevitable delay occurs between the two stages. Data anomalies need clarification and supplementary information may be required for results to be appropriately interpreted. In the meantime, individual centres may choose to analyse their local data with methods that may or may not be adopted for the final
international analysis. We are examining the relation of domestic gas appliances and respiratory disease in the ECRHS international data set. There is wide variation in exposure to gas appliances, the nature of these appliances, and the type of gas they use. It would be premature to comment on the conclusions of colleagues from France and Belgium, but we can make the
following observations. Combined data from two of the four centres in France show that the difference between men and women persists
Luczynska,
P
Burney
Jarvis D, Chinn S, Luczynska C, Burney P. Association of respiratory symptoms and lung function in young adults with the use of domestic gas appliances. Lancet 1996; 347: 426-31. Leynaert B, Liard R, Bousquet J, Mesbah H, Neukirch F. Gas cooking and respiratory health in women. Lancet 1996; 347: 1052-53.
Selective toxicity of vincristine against chronic lymphocytic leukaemia in vitro SiR-It is generally assumed that vincristine, as are other Vinca alkaloids, is a cell-cycle-specific agent that blocks mitosis through metaphase arrest’ by specifically binding to tubulin. Other metabolic effects have been noticed, but only tubulin inhibition has been related to the cytotoxic effects of vincristine. Vincristine is used in several regimens for chronic lymphocytic leukaemia (CLL). However, a note on the use of vincristine in CLL has been cautionary sounded: the value of vincristine has not been proved in this disease, but because combination regimens previously established for other B-cell cancers happened to include vincristine, this drug has been given to a large number of patients with CLL. Since the majority of CLL cells are not mitotically active, the use of vincristine does not seem rational. While investigating the cytotoxicity of a number of chemotherapeutic agents against CLL cells in vitro, we noted a striking action of vincristine against lymphocytes from patients with CLL but not from normal healthy blood donors. We isolated peripheral blood mononuclear cells from 20 patients with CLL (>90% leukaemic B cells) and from 11 volunteers healthy by density gradient centrifugation. The cells were cultured in duplicate in 200 ilL aliquots (2X10/mL) on microtitre plates in RPMI 1640 medium containing 10% fetal calf serum. Vincristine concentrations were 0, 2, 10, 50, 250, and 1250 nmol/L. Toxicity was assessed by the reduction of macromolecular
protein synthesis or [3H]-leucine incorporation as an endpoint.3 The cells were incubated for 4 days. The tracer was added for the final 24 h, whereafter the proteins were precipitated with perchloric acid and the radioactivity in the macromolecular protein fraction was counted.3 Dose-response curves (figure) showed a remarkable difference in the response of CLL lymphocytes and normal lymphocytes. A statistically significant difference (t test) 1491
vessels, and the number of blood vessels showing PN-1
immunoreactivity, were greatly reduced in the diseased brain tissue samples.’ PN-1, like other serpins, is highly sensitive to oxidation.’’ In retrospect, the reduced PN-1 activity observed in the Alzheimer samples could reflect P-amyloidpeptide-mediated oxidation of extracellular matrix proteins, including PN-1. These observations seem to favour the important notion on vascular oxidative damage in Alzheimer’s disease. David Gurwitz National Laboratory for the Genetics of Israeli Tel Aviv 69978, Israel
Populations, Tel-Aviv University,
Bradbury J. ß-amyloid vasoactivity in Alzheimer’s
1
disease. Lancet
1996;
347: 750. 2
of vincristine
Figure: Cytotoxicity lymphocytes
against normal lymphocytes
3
and CLL
Results presented
as mean
(SD).
4
between the groups was already noted at clinically achievable vincristine concentrations. Our results showed selective action of vincristine against CLL lymphocytes. The mechanisms involved require careful investigation. Re-evaluation of the part played by vincristine in the treatment of CLL is justified. *Juhani Vilpo, Leena Vilpo, for the Tampere CLL Group *Laboratory of Molecular Haematology, Department of Clinical Chemistry, Tampere University Hospital, and Department of Medicine, University of Tampere Medical School, PO Box 2000, FIN-33521 Tampere, Finland
1
2
3
Pouillart P. Vincristine. In: Cvitkovic E, Droz JP, Armand JP, Khoury S, eds. Handbook of chemotherapy in clinical oncology. Channel Islands: Scientific Communication International Ltd, 1993: 309-11. Han T, Rai K. Chronic lymphocytic leukemia. In: Henderson ES, Lister TA, eds. Leukemia. Philadelphia: WB Saunders Company, 1990: 565-611. Vilpo JA, Vilpo LM. Metabolism, incorporation into DNA, and interactions with 1-b-D-arabinofuranosylcytosine of 5-hydroxymethyl2’-deoxyuridine in human promyelocytic leukemia cells (HL-60). Cancer Res 1988; 48: 33117-22.
(3-amyloid vasoactivity
in Alzheimer’s disease
SiR-Studies of Alzheimer’s disease brain tissue
might lend described support theory, by Bradbury (March 16, p 750),’ of a role for vascular p-amyloid peptidemediated oxidative damage in the aetiology of this disease. As she states, there is no doubt that this theory may open new avenues for treatment of Alzheimer’s patients-eg, by the use of superoxide scavengers or antioxidants. Until such studies are published, it is intriguing to note that human histological data that favour this theory have already been to
the
new
5
Wagner SL, Geddes JW, Cotman CW, et al. Protease nexin-1, an antithrombin with neurite outgrowth activity, is reduced in Alzheimer’s disease. Proc Natl Acad Sci USA 1989; 86: 8284-88. Choi BH, Suzuki M, Kim T, Wagner SL, Cunningham DD. Protease nexin-1: localization in the human brain suggests a protective role against extravasated serine proteases. Am J Pathol 1990; 137: 741-47. Vaughan PJ, Su J, Cotman CW, Cunningham DD. Protease nexin-1, a potent thrombin inhibitor, is reduced around cerebral blood vessels in Alzheimer’s disease. Brain Res 1994; 668: 160-70. Scott RW, Bergman BL, Bajpai A, et al. Protease nexin: properties and a modified purification procedure. J Biol Chem 1985; 260: 7029-34. 1
SiR-Bradbury’ comments on Thomas and colleagues’= proposal that imbalances (in rats) in free-radical generating mechanisms lead to degeneration of the cerebral vascular endothelium in Alzheimer’s disease (AD). Although this would be difficult to demonstrate physiologically in man, our recent morphological observations attest that such degeneration is linked to A(3 deposition in cortical regions of people with AD.3 We used immunocytochemical methods to define microvascular abnormalities in post-mortem brain tissue obtained from people with AD and normally ageing controls. Double immunostaining of neocortical tissue sections with antibodies to endothelial cell markers such as CD34 or CD31 and to basement lamina-namely, collagen IVrevealed differential staining of numerous capillary profiles.’ The differential labelling was characterised by attenuation or degeneration of the endothelium, indicated by CD34negative capillary profiles that still retained their basement lamina shown by collagen IV reactivity alone. This occurrence was seen in virtually all A(3-laden cortical lobes of more than 90% of AD as well as Down’s syndrome patients, but was not readily evident in non-AD disorders, brain regions of people with AD, or age-matched healthy
published. 7 years ago,
Wagner
brain serine
et
of the nexin-1 protease
aF showed that the
activity
ubiquitous proteinase-inhibitor (PN-1) was strikingly reduced (about six-fold) compared with age-matched controls in postmortem hippocampus and cerebral cortex tissue from individuals with Alzheimer’s disease. Yet, the levels of mRNA for PN-1 in the same samples were unchanged, indicating that an unidentified process-possibly raised serine proteinase activity-was consuming PN-1. Subsequent attempts to identify such activities in these samples failed (D Gurwitz, unpublished observations). Immunostaining has shown that human brain PN-1 was localised mainly around small brain capillaries.3 Additional studies of postmortem Alzheimer tissue samples showed that both PN-1 immunoreactivity around blood
1492
Figure: Relation between microvascular abnormalities and neocortical A(3 deposits in frontal cortex Direct correlation between number of degenerated microvessels per unit area and density of Ap deposits (r=0.80, p<0001, df=148). Data points represent average density counts of five fields from ten sections of 25 um thickness double stained for microvasculature (basement lamina marker) and Ap, neurofibrillary tangles, or neurons.