- Email: [email protected]
Interferon ß and suicide in multiple sclerosis
published, and it is therefore important to clarify that our RT-PCR amplifies an S fragment, which is identical for both Berkel (PUUNCPS) and Hallnas Bl1 (PUUSSEG)
increased pulmonary vascular permeability. Pulmonary involvement is a known complication of HFRS, especially if the fluid balance is not carefully monitored, but it is an infrequent component of the disease. None of the distinctive virological, clinical, pathogenetic, or pathological features that collectively form HPS have been satisfactorily described outside the Americas.
hantaviruses on aminoacid level. This may be the reason why Rollin et al misinterpret our RT-PCR results. Hantaviruses show a high degree of genetic variability. They have unique sequences in many patients and can be found in various rodents as well as in insectivores and birds. Dependent on the animal reservoir and the geographical area, the genetic repertoire of hantaviruses will cause different manifestations of the disease ranging from NE to HFRS and HPS. The spectrum of NE and HFRS includes pulmonary involvement as a known complication, which is not an infrequent component of the disease in Europe, and hantavirus infections without signs of renal involvement have been reported from various European countries.4,s Hantavirus infections in USA also produce combined pulmonary and renal complications, as can be seen in cases caused by Black Creek Canal and Bayou viruses, but most HPS cases are clearly linked to SN viruses. The hantavirus S sequences found in our patients are not identical to sequences of strains propagated in our laboratory (PUU and HAN) or to sequences amplified from other patients’ specimen nor to any other hanta S RNA sequences in the actual database.
*Pierre E Rollin, Thomas G Ksiazek, Sherif R Zaki, Stuart T Nichol, C J Peters Special Pathogens Branch and Molecular Pathology and Ultrastructure Activity, Division of Viral and Rickettsial Diseases, National Center for Infectious Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
Diseases,
1 Schreiber M, Laue T,Wolff C. Hantavirus pulmonary syndrome in Germany. Lancet 1996; 347: 336-37. 2 Duchin JS, Koster F, Peters CJ, et al. Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. N Engl J Med 1994; 330: 949-55. 3 Ksiazek TG, Peters CJ, Rollin PE, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995; 52: 1017-23. 4 Bowen MD, Kariwa H, Rollin PE, et al. Genetic characterization of a human isolate of Puumala hantavirus from France. Virus Res 1995; 38: 279-89. 5
Hjelle B, Spiropoulou CF, Torrez-Martinez N, et al. Detection of Muerto Canyon virus RNA in peripheral blood mononuclear cells from patients with hantavirus pulmonary syndrome. J Infect Dis 1994; 170: 1013-17.
*Michael Schreiber, Thomas Laue, Carsten Wolff Microbiology Section, Division of Virology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany; and Institut für Laboratoriums- und *Medical
Authors’
reply
Transfusiomsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfahlen, Bad
CDC screening criteria for HPS our first showed symptoms of febrile illness, bilateral patient interstitial pulmonary infiltrates, and required supplemental oxygen. None of the excluding factors described by the CDC workers were observed. For serology the antigens PUU and HAN, as recommended by WHO, were used. Since all tests were negative and only ELISA with recombinant HAN nucleocapsid showed antibody titres reaching cutoff level, we decided to screen for hanta S RNA by RT-PCR. The results obtained with PUU and HAN antigens, of course, do not rule out any other hantavirus strain-specific antibodies in our patients. Negative results with PUU or HAN antigens were also reported by McKenna et al,’ and Rollin and colleagues recorded an HPS case in Florida negative for Sin Nombre (SN)-specific antibody in primary screening.’ To avoid false-positive reactions the nested RT-PCR was done with the same precautions and safety conditions as described by Ksiazek and co-workers.3 In these reports nucleotide sequences of most of the PCR products were unique, a major argument against cross contamination. In accord with these reports we also found unique major sequences for each PCR product (including the rodent excreta). Each of our PCR products was cloned into pCRII, and twenty clones were sequenced, to detect variants of the hantavirus sequences. As a result of this procedure three sequences were detected in one patient. They differed in only one aminoacid exchange. None of these variant sequences, however, were identical to that of the second patient. These results may have been obtained because our sequencing procedure was different from Rollin and colleagues’ method. Their method of choice is direct sequencing of PCR products,3 a method unsuitable to detect intrapatient virus variability. Each sequence in the two patients and the rodent excreta differed by 2-4% of aminoacids from the PUUNCPS Known such as sequence. HPSSSEG, sequences HPSCC107SS, and HANSNC differed by 16%, 14%, and 12%, respectively. The figure we submitted with our report showing the aminoacid sequence alignment was not
SiR-According
Oeynhausen
to
1
2
3
4 5
McKenna P, Clement J, Matthys P, Coyle PV, McCaughey C. Serological evidence of hantavirus disease in Northern Ireland. J Med Virol 1994; 43: 33-38. Rollin PE, Ksiazek TG, Elliott LH, et al. Isolation of Black Creek Canal virus, a new hantavirus from Sigmodon hispidus in Florida. J Med Virol 1995; 46: 35-39. Ksiazek TG, Peters CJ, Rollin PE, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995; 52: 1017-23. Alexeyev OA, Baranov BA. Puumala virus infection without signs of renal involvement. Scand J Infect Dis 1993; 25: 525-27. Clement J, Coloson P, McKenna P. Hantavirus pulmonary syndrome in New England and Europe. N Engl J Med 1994; 331: 545-46.
Interferon ß
and suicide in
multiple sclerosis
SiR-Napier (April 6, p 968)’ suggests that your editorial (Feb 24, p 483)" may have given a negative impression of 0-interferon. Although just one trial has been undertaken with this drug.3 she cites Lublin and colleagues4 as believing that this therapy may "signal a new era in the management of MS (multiple sclerosis)", while neglecting to inform readers that Lublin was a member of the manuscript committee for the original report.3 Most impartial opinion considers the case for (3-interferon be unproven, and there are concerns about suicide and attempted suicide in patients given the drug. The IFNB Study Group seems to dismiss the possibility that these episodes represented a side-effect of therapy, and suggests that they were attributable to the disease. Whether these episodes were analysed as disease relapse, and, if so, how much weighting was given to the suicide component, are not clear. to
Nigel
TJ O’Connor
and
Therapeutics Committee, Royal Shrewsbury Hospital, Shropshire SY3 8PN, UK
Drugs
1
Napier JC. Reputation
of interferon
ß-1h.
Lancet 1996; 347: 968.
1417
2
Editorial. Euromedicines evaluation: the
striptease begins.
Lancet
l996; 347: 483. 3
4
IFNB Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group. Interferon beta-lb in the treatment of multiple sclerosis: final outcome of the randomised controlled trial. Neurology 1995; 45: 1277-85. Lublin FD, Whitaker JN, Eidelman BH, Miller AE, Arnason BGW, Burks JS. Management of patients receiving interferon beta-1b for multiple sclerosis: report of a consensus conference. Neurology 1996; 46: 12-18.
Mycoplasma fermentans in joints of patients with rheumatoid arthritis and other joint disorders and chronic arthritis in SIR-Mycoplasmas as candidates for animals and should be considered many causing human joint disease. Mycoplasma fermentans was suggested more than 20 years ago as a cause of rheumatoid arthritis (RA) on the basis of isolation from synovial fluids of a few patients.’ Although M fermentans has been isolated subsequently, many attempts have failed.2 With PCR M fermentans has been found in the throats of more than 20%, peripheral blood leucocytes of about 10%, and urine samples of about 5% of both HIV-positive and HIV-negative patients.3 Thus, we looked for PCR evidence of M fermentans in the joints of patients with RA and other types of arthritis. From May, 1992, to August, 1995, 154 samples of synovial fluids and ten synovial biopsies were collected in the Rheumatology Department of the Centre Hospitalier Universitaire de Bordeaux and stored at -80°C for 1-4 months. After thawing, a 500 )iL aliquot of each synovial fluid specimen was diluted with an equal volume of phosphate-buffered saline and centrifuged at 13 000 g for 15 min, the DNA being extracted from the pellet after resuspension in lysis buffer (10 mM Tris HCI, 1 mM EDTA, 1% sodium dodecyl sulphate, and 100 g/mL proteinase K) and incubation at 37°C for 2 h. Synovial biopsy tissue (approximately 1 g) was put directly into the lysis buffer with a double concentration of proteinase K and incubated at 37°C overnight. The final DNA pellet was resuspended in 100 L of distilled water and 5 L used in a semi-nested PCR procedurebut modified so that the thermal cycling profile for each round of amplification comprised 35 cycles at 95°C for 30 s (60 s in the first cycle), 55°C for 30 s, and 72°C for 60 s (increased to 10 min for the final cycle). The second-round product (10 L) was analysed on a 2% agarose gel and positive samples, indicated by a 104 bp product, were confirmed by Southern blot analysis with an internal oligonucleotide as a probe. M fermentans was detected in 15 specimens, each from a different patient, all of whom had an inflammatory arthritis. Synovial fluid from four (14%) of 28 patients with RA were positive, as were synovial biopsy specimens from 4 (40%) of ten patients with RA: the mycoplasma was detected in the joints of 8 (21 %) of 38 patients with RA. M fermentans was detected in synovial fluid from two (20%) of ten patients cause
1418
acute
spondyloarthropathy with peripheral arthritis, one (20%) of five patients with psoriatic arthritis and four (13%) of 31 patients with unclassified inflammatory arthritis. None of the specimens from seven patients with reactive arthritis, four with chronic juvenile arthritis, 16 with osteoarthritis, three with post-traumatic hydrarthrosis, and nine with gouty arthritis was positive. M fermentans seems to be associated with some of the inflammatory arthritic diseases, including rheumatoid arthritis. Whether this mycoplasma triggers or perpetuates disease or is behaving as a passenger remains conjectural. We believe that this study should be expanded to seek M fermentans, other mycoplasmas and other microorganisms, particularly chlamydiae,4 in patients from other
with
geographical areas and that emphasis should be placed on early-stage disease because of detection in early, but not latestage mycoplasmal arthritis in animals.5 Thierry Sohaeverbeke, Claire B Gilroy, Christiane Bébéar, Joël Dehais, *David Taylor-Robinson *MRC
Sexually Transmitted Diseases Research Group, Jefferiss Trust Laboratories, Imperial College School of Medicine at St Mary’s, Paddington, London W2 1NY, UK; and Service de Rhumatologie et Service de Bactériologie Hôpital Pellegrin, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France
Pathogenic mycoplasma in rheumatoid arthritis? In: Pathogenic mycoplasma-a Ciba Foundation symposium. Amsterdam: Associated Scientic Publishers, 1972: 251-62. Barile MF, Yoshida H, Roth H. Rheumatoid arthritis: new findings on the failure to isolate or detect mycoplasmas by multiple cultivation or serologic procedures and review of the literature. Rev Infect Dis 1991;
Williams MH.
1
2
3
4
5
13: 571-82. Katseni VL, Gilroy CB, Ryait BK, et al. Mycoplasma fermentans in individuals seropositive and seronegative for HIV-1. Lancet 1993; 341: 271-73. Pando JA, Yarboro C, Ellaban A, et al. Prevalence of Chlamydia trachomatis by PCR in the joint of patients with early rheumatoid arthritis. Arthritis Rheum 1995; 38: S287. Washburn LR, Cole BC, Gelman MI, Ward JR. Chronic arthritis of rabbits induced by mycoplasmas. I. Clinical, microbiologic and histologic features. Arthritis Rheum 1980; 23: 825-36.
The
diabetologist’s
SIR-Kriegstein
prayer
and
colleagues (April 13, p 1045)’ argue the "need for many types of insulin". I cannot agree with this, and I am reminded of a prayer for diabetologists that I came across many years ago (I cannot remember from where): "Teach me that it is not so much the number and types of insulin I use to treat my diabetic patients; but the skill with which I use them, which determines their control and well-being".
Geoffrey Gill Diabetes and Endocrine Unit, Diabetes Centre, Walton Hospital, UK
1
Liverpool L9 1AE,
E v, Wedemeyer H-J, Starm G-R. Need for many types of insulin. Lancet 1996; 347: 1045.
Kriegstein
increased pulmonary vascular permeability. Pulmonary involvement is a known complication of HFRS, especially if the fluid balance is not carefully monitored, but it is an infrequent component of the disease. None of the distinctive virological, clinical, pathogenetic, or pathological features that collectively form HPS have been satisfactorily described outside the Americas.
hantaviruses on aminoacid level. This may be the reason why Rollin et al misinterpret our RT-PCR results. Hantaviruses show a high degree of genetic variability. They have unique sequences in many patients and can be found in various rodents as well as in insectivores and birds. Dependent on the animal reservoir and the geographical area, the genetic repertoire of hantaviruses will cause different manifestations of the disease ranging from NE to HFRS and HPS. The spectrum of NE and HFRS includes pulmonary involvement as a known complication, which is not an infrequent component of the disease in Europe, and hantavirus infections without signs of renal involvement have been reported from various European countries.4,s Hantavirus infections in USA also produce combined pulmonary and renal complications, as can be seen in cases caused by Black Creek Canal and Bayou viruses, but most HPS cases are clearly linked to SN viruses. The hantavirus S sequences found in our patients are not identical to sequences of strains propagated in our laboratory (PUU and HAN) or to sequences amplified from other patients’ specimen nor to any other hanta S RNA sequences in the actual database.
*Pierre E Rollin, Thomas G Ksiazek, Sherif R Zaki, Stuart T Nichol, C J Peters Special Pathogens Branch and Molecular Pathology and Ultrastructure Activity, Division of Viral and Rickettsial Diseases, National Center for Infectious Centers for Disease Control and Prevention, Atlanta, GA 30333, USA
Diseases,
1 Schreiber M, Laue T,Wolff C. Hantavirus pulmonary syndrome in Germany. Lancet 1996; 347: 336-37. 2 Duchin JS, Koster F, Peters CJ, et al. Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. N Engl J Med 1994; 330: 949-55. 3 Ksiazek TG, Peters CJ, Rollin PE, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995; 52: 1017-23. 4 Bowen MD, Kariwa H, Rollin PE, et al. Genetic characterization of a human isolate of Puumala hantavirus from France. Virus Res 1995; 38: 279-89. 5
Hjelle B, Spiropoulou CF, Torrez-Martinez N, et al. Detection of Muerto Canyon virus RNA in peripheral blood mononuclear cells from patients with hantavirus pulmonary syndrome. J Infect Dis 1994; 170: 1013-17.
*Michael Schreiber, Thomas Laue, Carsten Wolff Microbiology Section, Division of Virology, Bernhard Nocht Institute for Tropical Medicine, 20359 Hamburg, Germany; and Institut für Laboratoriums- und *Medical
Authors’
reply
Transfusiomsmedizin, Herz- und Diabeteszentrum Nordrhein-Westfahlen, Bad
CDC screening criteria for HPS our first showed symptoms of febrile illness, bilateral patient interstitial pulmonary infiltrates, and required supplemental oxygen. None of the excluding factors described by the CDC workers were observed. For serology the antigens PUU and HAN, as recommended by WHO, were used. Since all tests were negative and only ELISA with recombinant HAN nucleocapsid showed antibody titres reaching cutoff level, we decided to screen for hanta S RNA by RT-PCR. The results obtained with PUU and HAN antigens, of course, do not rule out any other hantavirus strain-specific antibodies in our patients. Negative results with PUU or HAN antigens were also reported by McKenna et al,’ and Rollin and colleagues recorded an HPS case in Florida negative for Sin Nombre (SN)-specific antibody in primary screening.’ To avoid false-positive reactions the nested RT-PCR was done with the same precautions and safety conditions as described by Ksiazek and co-workers.3 In these reports nucleotide sequences of most of the PCR products were unique, a major argument against cross contamination. In accord with these reports we also found unique major sequences for each PCR product (including the rodent excreta). Each of our PCR products was cloned into pCRII, and twenty clones were sequenced, to detect variants of the hantavirus sequences. As a result of this procedure three sequences were detected in one patient. They differed in only one aminoacid exchange. None of these variant sequences, however, were identical to that of the second patient. These results may have been obtained because our sequencing procedure was different from Rollin and colleagues’ method. Their method of choice is direct sequencing of PCR products,3 a method unsuitable to detect intrapatient virus variability. Each sequence in the two patients and the rodent excreta differed by 2-4% of aminoacids from the PUUNCPS Known such as sequence. HPSSSEG, sequences HPSCC107SS, and HANSNC differed by 16%, 14%, and 12%, respectively. The figure we submitted with our report showing the aminoacid sequence alignment was not
SiR-According
Oeynhausen
to
1
2
3
4 5
McKenna P, Clement J, Matthys P, Coyle PV, McCaughey C. Serological evidence of hantavirus disease in Northern Ireland. J Med Virol 1994; 43: 33-38. Rollin PE, Ksiazek TG, Elliott LH, et al. Isolation of Black Creek Canal virus, a new hantavirus from Sigmodon hispidus in Florida. J Med Virol 1995; 46: 35-39. Ksiazek TG, Peters CJ, Rollin PE, et al. Identification of a new North American hantavirus that causes acute pulmonary insufficiency. Am J Trop Med Hyg 1995; 52: 1017-23. Alexeyev OA, Baranov BA. Puumala virus infection without signs of renal involvement. Scand J Infect Dis 1993; 25: 525-27. Clement J, Coloson P, McKenna P. Hantavirus pulmonary syndrome in New England and Europe. N Engl J Med 1994; 331: 545-46.
Interferon ß
and suicide in
multiple sclerosis
SiR-Napier (April 6, p 968)’ suggests that your editorial (Feb 24, p 483)" may have given a negative impression of 0-interferon. Although just one trial has been undertaken with this drug.3 she cites Lublin and colleagues4 as believing that this therapy may "signal a new era in the management of MS (multiple sclerosis)", while neglecting to inform readers that Lublin was a member of the manuscript committee for the original report.3 Most impartial opinion considers the case for (3-interferon be unproven, and there are concerns about suicide and attempted suicide in patients given the drug. The IFNB Study Group seems to dismiss the possibility that these episodes represented a side-effect of therapy, and suggests that they were attributable to the disease. Whether these episodes were analysed as disease relapse, and, if so, how much weighting was given to the suicide component, are not clear. to
Nigel
TJ O’Connor
and
Therapeutics Committee, Royal Shrewsbury Hospital, Shropshire SY3 8PN, UK
Drugs
1
Napier JC. Reputation
of interferon
ß-1h.
Lancet 1996; 347: 968.
1417
2
Editorial. Euromedicines evaluation: the
striptease begins.
Lancet
l996; 347: 483. 3
4
IFNB Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group. Interferon beta-lb in the treatment of multiple sclerosis: final outcome of the randomised controlled trial. Neurology 1995; 45: 1277-85. Lublin FD, Whitaker JN, Eidelman BH, Miller AE, Arnason BGW, Burks JS. Management of patients receiving interferon beta-1b for multiple sclerosis: report of a consensus conference. Neurology 1996; 46: 12-18.
Mycoplasma fermentans in joints of patients with rheumatoid arthritis and other joint disorders and chronic arthritis in SIR-Mycoplasmas as candidates for animals and should be considered many causing human joint disease. Mycoplasma fermentans was suggested more than 20 years ago as a cause of rheumatoid arthritis (RA) on the basis of isolation from synovial fluids of a few patients.’ Although M fermentans has been isolated subsequently, many attempts have failed.2 With PCR M fermentans has been found in the throats of more than 20%, peripheral blood leucocytes of about 10%, and urine samples of about 5% of both HIV-positive and HIV-negative patients.3 Thus, we looked for PCR evidence of M fermentans in the joints of patients with RA and other types of arthritis. From May, 1992, to August, 1995, 154 samples of synovial fluids and ten synovial biopsies were collected in the Rheumatology Department of the Centre Hospitalier Universitaire de Bordeaux and stored at -80°C for 1-4 months. After thawing, a 500 )iL aliquot of each synovial fluid specimen was diluted with an equal volume of phosphate-buffered saline and centrifuged at 13 000 g for 15 min, the DNA being extracted from the pellet after resuspension in lysis buffer (10 mM Tris HCI, 1 mM EDTA, 1% sodium dodecyl sulphate, and 100 g/mL proteinase K) and incubation at 37°C for 2 h. Synovial biopsy tissue (approximately 1 g) was put directly into the lysis buffer with a double concentration of proteinase K and incubated at 37°C overnight. The final DNA pellet was resuspended in 100 L of distilled water and 5 L used in a semi-nested PCR procedurebut modified so that the thermal cycling profile for each round of amplification comprised 35 cycles at 95°C for 30 s (60 s in the first cycle), 55°C for 30 s, and 72°C for 60 s (increased to 10 min for the final cycle). The second-round product (10 L) was analysed on a 2% agarose gel and positive samples, indicated by a 104 bp product, were confirmed by Southern blot analysis with an internal oligonucleotide as a probe. M fermentans was detected in 15 specimens, each from a different patient, all of whom had an inflammatory arthritis. Synovial fluid from four (14%) of 28 patients with RA were positive, as were synovial biopsy specimens from 4 (40%) of ten patients with RA: the mycoplasma was detected in the joints of 8 (21 %) of 38 patients with RA. M fermentans was detected in synovial fluid from two (20%) of ten patients cause
1418
acute
spondyloarthropathy with peripheral arthritis, one (20%) of five patients with psoriatic arthritis and four (13%) of 31 patients with unclassified inflammatory arthritis. None of the specimens from seven patients with reactive arthritis, four with chronic juvenile arthritis, 16 with osteoarthritis, three with post-traumatic hydrarthrosis, and nine with gouty arthritis was positive. M fermentans seems to be associated with some of the inflammatory arthritic diseases, including rheumatoid arthritis. Whether this mycoplasma triggers or perpetuates disease or is behaving as a passenger remains conjectural. We believe that this study should be expanded to seek M fermentans, other mycoplasmas and other microorganisms, particularly chlamydiae,4 in patients from other
with
geographical areas and that emphasis should be placed on early-stage disease because of detection in early, but not latestage mycoplasmal arthritis in animals.5 Thierry Sohaeverbeke, Claire B Gilroy, Christiane Bébéar, Joël Dehais, *David Taylor-Robinson *MRC
Sexually Transmitted Diseases Research Group, Jefferiss Trust Laboratories, Imperial College School of Medicine at St Mary’s, Paddington, London W2 1NY, UK; and Service de Rhumatologie et Service de Bactériologie Hôpital Pellegrin, Centre Hospitalier Universitaire de Bordeaux, Bordeaux, France
Pathogenic mycoplasma in rheumatoid arthritis? In: Pathogenic mycoplasma-a Ciba Foundation symposium. Amsterdam: Associated Scientic Publishers, 1972: 251-62. Barile MF, Yoshida H, Roth H. Rheumatoid arthritis: new findings on the failure to isolate or detect mycoplasmas by multiple cultivation or serologic procedures and review of the literature. Rev Infect Dis 1991;
Williams MH.
1
2
3
4
5
13: 571-82. Katseni VL, Gilroy CB, Ryait BK, et al. Mycoplasma fermentans in individuals seropositive and seronegative for HIV-1. Lancet 1993; 341: 271-73. Pando JA, Yarboro C, Ellaban A, et al. Prevalence of Chlamydia trachomatis by PCR in the joint of patients with early rheumatoid arthritis. Arthritis Rheum 1995; 38: S287. Washburn LR, Cole BC, Gelman MI, Ward JR. Chronic arthritis of rabbits induced by mycoplasmas. I. Clinical, microbiologic and histologic features. Arthritis Rheum 1980; 23: 825-36.
The
diabetologist’s
SIR-Kriegstein
prayer
and
colleagues (April 13, p 1045)’ argue the "need for many types of insulin". I cannot agree with this, and I am reminded of a prayer for diabetologists that I came across many years ago (I cannot remember from where): "Teach me that it is not so much the number and types of insulin I use to treat my diabetic patients; but the skill with which I use them, which determines their control and well-being".
Geoffrey Gill Diabetes and Endocrine Unit, Diabetes Centre, Walton Hospital, UK
1
Liverpool L9 1AE,
E v, Wedemeyer H-J, Starm G-R. Need for many types of insulin. Lancet 1996; 347: 1045.
Kriegstein