- Email: [email protected]
Trypsin In poliovaccine manufacture
3
4
Cooksley WGE, Scott JSD, Pace R, Ferman A, Sheridan J. Pathobiology of hepatitis B in aboriginals. Hepatology 1990; 12: 398 (abstr). Deivanayagam N, Vasudevan S, Ashok TP, et al. Potency of oral polio vaccine stored at distribution centres in Madras. Indian J Pediatr 1990; 57: 757-61.
*Dement!a cases; p value (Fisher’s exact test) =0 461 for genotypes across all cells, and p= 0 499 for allele frequencies across all cells.
Trypsin In poliovaccine manufacture SiR-Stricker and Elswood (Jan 1, p 52) refer to our work1 in an effort to support their contention that "cell cultures in the past were exposed to trypsin for only 25 min". Our work was done on monolayer cultures of normal human cells and, although we may have used a 25 min trypsin exposure when subcultivating, this time varied widely among laboratories. More to the point, however, is that the monkey kidney used in "poliovaccines manufactured between 1975 and 1984" was intact tissue that was exposed to trypsin for hours and usually overnight.2-5 Only primary cultures of monkey kidney cells were permitted by the control authorities to be used for poliovaccine manufacture so no subsequent exposure to trypsin occurred. Contrary to the conclusion reached by Stricker and Elswood, during poliovirus vaccine manufacture there was a long exposure of primary monkey kidney tissue to trypsin, thus decreasing the likelihood of putative HIV infection. Leonard Hayflick University of California, San Francisco, PO Box 89, The Sea Ranch, CA 95497, USA
1 Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961; 25: 585-621. 2 Youngner JS. Monolayer tissue cultures I: preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells. Proc Soc Exp Biol Med 1954; 85: 202-05. 3 Bodian D. Simplified method of dispersion of monkey kidney cells with trypsin. Virology 1956; 4: 575-77. 4 Rappaport C. Trypsinization of monkey kidney tissue: an automatic method for the preparation of cell suspensions. Bull WHO 1956; 14: 147-66. 5 Montes de Oca H, Probst P, Grubbs R. High yield method for dispersing simian kidney for cell cultures. Appl Microbiol 1971; 21: 90-94.
Apolipoprotein E allele in Chamorros with amyotrophic lateral sclerosis/parkinsonismdementia complex SiR-The association of apolipoprotein E type 4 allele (E4) with Alzheimer’s disease (AD) has been reported by Strittmatter et al,l and confirmed by others,2,3 leading to speculation that the e4 allele may play a functional role in the pathogenic cascade associated with development of AD. Roses and Strittmatter have hypothesised that AD may not result from a direct detrimental effect of E4 per se but rather from the absence of other alleles, E3 and e2, suggesting a protective effect of these two alleles by stabilising tau protein and thereby preventing subsequent formation of neurofibrillary tangles (NFT). We report findings from ongoing and laboratory studies of the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of Guam, a spectrum of neurodegenerative diseases known to occur with high frequency among the indigenous Chamorro population. The hallmark neuropathological feature of ALS/PDC is widespread NFT formation in the brain and spinal cord. Ultrastructural studies indicate that the NFT in ALS/PDC of Guam are morphologically similar to NFT in ADand are immunoreactive to anti-amyloid antibodies, indicating the presence of p/A4-protein as is found in senile plaques, cerebrovascular amyloid deposits, and NFT in AD.5 Additionally, and perhaps most relevant, is the fact that NFT formation occurs with high frequency in neurologically normal
Table:
Genotype and allele frequency for cases and controls
Chamorros, particularly over 50 years of age, and may be more extensive than NFT formation in AD patients in other
populations.6 To
measure
allele
frequencies,
we
examined 12
patients
(6 males) with manifestations of the neurodegenerative diseases of Guam. The predominant features were PDC (in 6), dementia (in 3), parkinsonism (in 2), and ALS (in 1). A
comparison was made of the allele frequencies in the cases with that of 12 controls (2 males) from Guam. All patients and controls were Chamorro, with the median age of the cases being higher than controls (62 vs 50 years, p < 0-01). The e4 allele frequencies for Chamorro cases and controls (42%, see table), are considerably lower than allele frequencies of 19-50% in AD cases and 10-16% in controls reported from other studies.1-3 The frequency of the e3 allele in the Chamorro patient group (91 -7%) is notably higher than the frequencies of 44-58% reported among AD cases, whereas the frequency for Chamorro controls (792%) is similar to the 73-78% reported in other control populations. The e2 allele frequencies in the Chamorro patient group (4-2%) are similar to the 3-6% reported for AD cases, but higher in the Chamorro controls (16-7%) than the 8-11 % reported for normals in other studies. The genotype 84/e3 was present in only 1 patient (with dementia) and 1 control, whereas the majority of patients (83%) and controls (58%) had the 83/s3 genotype. None of the cases or controls was genotype e4/E4. To our knowledge, this is the first report of apoE allele studies in ALS/PDC of Guam. Our findings, although limited by age and sex differences and small sample size, indicate that the presence of the e3 allele may not be protective against neurofibrillary tangle formation (and subsequent disease manifestation) in this population and that the E4 allele is not associated with the prevalent neurodegenerative diseases of Guam. These results may represent a significant departure from the pathogenic mechanisms associated with development of clinically similar neurodegenerative diseases that occur as a consequence of widespread neurofibrillary degeneration. S C Waring, P C O’Brien, L T Kurland, S N Thibodeau, M-S Tsai, R C Petersen, C E Esteban-Santillan Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA; Department of Laboratory Medicine and Pathology, Mayo Clinic; Department of Neurology and Department of Health Sciences Research, Mayo Clinic; and School of Allied Health and Nursing, University of Guam, Mangilao, Guam ,
1
2
3 4
5
6
Strittmatter WJ, Saunders AM, Schmechel D, et al. Apolipoprotein E: High-avidity binding to &bgr;-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci 1993; 90: 1977-81. Poirier J, Davignon J, Bouthillier D, et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993; 342: 697-99. Mayeux R, Stern Y, Ottman R, et al. The apolipoprotein ∈4 allele in patients with Alzheimer’s disease. Ann Neurol 1993; 34: 752-54. Hirano A. Progress in the pathology of motor neuron disease. In: Zimmerman HM, ed. Progress in neuropathology, Vol 2, Grune and Stratton, Inc. New York 1973; 181-215. Guiroy DC, Mellini M, Miyazaki M, et al. Neurofibrillary tangles of Guamanian amyotrophic lateral sclerosis, parkinsonism-dementia and neurologically normal Guamanians contain a 4- to 4·5-kilodalton protein which is immunoreactive to anti-amyloid &bgr;/A4-protein antibodies. Acta Neuropathol 1993; 86: 265-74. Anderson FH, Richardson EP, Okazaki H, Brody JA. Neurofibrillary degeneration on Guam: frequency in Chamorros and non-Chamorros with no known neurological disease. Brain 1979; 102: 65-77.
611
4
Cooksley WGE, Scott JSD, Pace R, Ferman A, Sheridan J. Pathobiology of hepatitis B in aboriginals. Hepatology 1990; 12: 398 (abstr). Deivanayagam N, Vasudevan S, Ashok TP, et al. Potency of oral polio vaccine stored at distribution centres in Madras. Indian J Pediatr 1990; 57: 757-61.
*Dement!a cases; p value (Fisher’s exact test) =0 461 for genotypes across all cells, and p= 0 499 for allele frequencies across all cells.
Trypsin In poliovaccine manufacture SiR-Stricker and Elswood (Jan 1, p 52) refer to our work1 in an effort to support their contention that "cell cultures in the past were exposed to trypsin for only 25 min". Our work was done on monolayer cultures of normal human cells and, although we may have used a 25 min trypsin exposure when subcultivating, this time varied widely among laboratories. More to the point, however, is that the monkey kidney used in "poliovaccines manufactured between 1975 and 1984" was intact tissue that was exposed to trypsin for hours and usually overnight.2-5 Only primary cultures of monkey kidney cells were permitted by the control authorities to be used for poliovaccine manufacture so no subsequent exposure to trypsin occurred. Contrary to the conclusion reached by Stricker and Elswood, during poliovirus vaccine manufacture there was a long exposure of primary monkey kidney tissue to trypsin, thus decreasing the likelihood of putative HIV infection. Leonard Hayflick University of California, San Francisco, PO Box 89, The Sea Ranch, CA 95497, USA
1 Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res 1961; 25: 585-621. 2 Youngner JS. Monolayer tissue cultures I: preparation and standardization of suspensions of trypsin-dispersed monkey kidney cells. Proc Soc Exp Biol Med 1954; 85: 202-05. 3 Bodian D. Simplified method of dispersion of monkey kidney cells with trypsin. Virology 1956; 4: 575-77. 4 Rappaport C. Trypsinization of monkey kidney tissue: an automatic method for the preparation of cell suspensions. Bull WHO 1956; 14: 147-66. 5 Montes de Oca H, Probst P, Grubbs R. High yield method for dispersing simian kidney for cell cultures. Appl Microbiol 1971; 21: 90-94.
Apolipoprotein E allele in Chamorros with amyotrophic lateral sclerosis/parkinsonismdementia complex SiR-The association of apolipoprotein E type 4 allele (E4) with Alzheimer’s disease (AD) has been reported by Strittmatter et al,l and confirmed by others,2,3 leading to speculation that the e4 allele may play a functional role in the pathogenic cascade associated with development of AD. Roses and Strittmatter have hypothesised that AD may not result from a direct detrimental effect of E4 per se but rather from the absence of other alleles, E3 and e2, suggesting a protective effect of these two alleles by stabilising tau protein and thereby preventing subsequent formation of neurofibrillary tangles (NFT). We report findings from ongoing and laboratory studies of the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS/PDC) of Guam, a spectrum of neurodegenerative diseases known to occur with high frequency among the indigenous Chamorro population. The hallmark neuropathological feature of ALS/PDC is widespread NFT formation in the brain and spinal cord. Ultrastructural studies indicate that the NFT in ALS/PDC of Guam are morphologically similar to NFT in ADand are immunoreactive to anti-amyloid antibodies, indicating the presence of p/A4-protein as is found in senile plaques, cerebrovascular amyloid deposits, and NFT in AD.5 Additionally, and perhaps most relevant, is the fact that NFT formation occurs with high frequency in neurologically normal
Table:
Genotype and allele frequency for cases and controls
Chamorros, particularly over 50 years of age, and may be more extensive than NFT formation in AD patients in other
populations.6 To
measure
allele
frequencies,
we
examined 12
patients
(6 males) with manifestations of the neurodegenerative diseases of Guam. The predominant features were PDC (in 6), dementia (in 3), parkinsonism (in 2), and ALS (in 1). A
comparison was made of the allele frequencies in the cases with that of 12 controls (2 males) from Guam. All patients and controls were Chamorro, with the median age of the cases being higher than controls (62 vs 50 years, p < 0-01). The e4 allele frequencies for Chamorro cases and controls (42%, see table), are considerably lower than allele frequencies of 19-50% in AD cases and 10-16% in controls reported from other studies.1-3 The frequency of the e3 allele in the Chamorro patient group (91 -7%) is notably higher than the frequencies of 44-58% reported among AD cases, whereas the frequency for Chamorro controls (792%) is similar to the 73-78% reported in other control populations. The e2 allele frequencies in the Chamorro patient group (4-2%) are similar to the 3-6% reported for AD cases, but higher in the Chamorro controls (16-7%) than the 8-11 % reported for normals in other studies. The genotype 84/e3 was present in only 1 patient (with dementia) and 1 control, whereas the majority of patients (83%) and controls (58%) had the 83/s3 genotype. None of the cases or controls was genotype e4/E4. To our knowledge, this is the first report of apoE allele studies in ALS/PDC of Guam. Our findings, although limited by age and sex differences and small sample size, indicate that the presence of the e3 allele may not be protective against neurofibrillary tangle formation (and subsequent disease manifestation) in this population and that the E4 allele is not associated with the prevalent neurodegenerative diseases of Guam. These results may represent a significant departure from the pathogenic mechanisms associated with development of clinically similar neurodegenerative diseases that occur as a consequence of widespread neurofibrillary degeneration. S C Waring, P C O’Brien, L T Kurland, S N Thibodeau, M-S Tsai, R C Petersen, C E Esteban-Santillan Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA; Department of Laboratory Medicine and Pathology, Mayo Clinic; Department of Neurology and Department of Health Sciences Research, Mayo Clinic; and School of Allied Health and Nursing, University of Guam, Mangilao, Guam ,
1
2
3 4
5
6
Strittmatter WJ, Saunders AM, Schmechel D, et al. Apolipoprotein E: High-avidity binding to &bgr;-amyloid and increased frequency of type 4 allele in late-onset familial Alzheimer disease. Proc Natl Acad Sci 1993; 90: 1977-81. Poirier J, Davignon J, Bouthillier D, et al. Apolipoprotein E polymorphism and Alzheimer’s disease. Lancet 1993; 342: 697-99. Mayeux R, Stern Y, Ottman R, et al. The apolipoprotein ∈4 allele in patients with Alzheimer’s disease. Ann Neurol 1993; 34: 752-54. Hirano A. Progress in the pathology of motor neuron disease. In: Zimmerman HM, ed. Progress in neuropathology, Vol 2, Grune and Stratton, Inc. New York 1973; 181-215. Guiroy DC, Mellini M, Miyazaki M, et al. Neurofibrillary tangles of Guamanian amyotrophic lateral sclerosis, parkinsonism-dementia and neurologically normal Guamanians contain a 4- to 4·5-kilodalton protein which is immunoreactive to anti-amyloid &bgr;/A4-protein antibodies. Acta Neuropathol 1993; 86: 265-74. Anderson FH, Richardson EP, Okazaki H, Brody JA. Neurofibrillary degeneration on Guam: frequency in Chamorros and non-Chamorros with no known neurological disease. Brain 1979; 102: 65-77.
611