855
comparison of equimolar amounts of porcine and semisynthetic human insulin. J Intern Med 1991; 229: 293-96. 25. Clausen Sjobom N, Lins P-E, Adamson U, Theodorsson E. A comparative study on the hormonal responses to insulin-induced hypoglycaemia using semisynthetic human insulin and pork insulin in patients with type 1 diabetes mellitus. Diabetic Med 1990; 7: 775-79. 26. Patrick AW, Bodmer CW, Tieszen KL, White MC, Williams G. Human insulin and awareness of acute hypoglycaemia in insulin-dependent diabetes. Lancet 1991; 338: 528-32. 27. Mühlhauser I, Heinemann L, Fritsche E, von Lenhep K, Berger M. Hypoglycemic symptoms and frequency of severe hypoglycemia in patients treated with human and animal insulin preparations. Diabetes Care 1991; 14: 745-49. 28. Orchard TJ, Maser RE, Becker DJ, Dorman JS, Drash AL. Human insulin use and hypoglycaemia: insights from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetic Med 1991; 8: 469-74. 29. Heine RJ, van der Veen EA. Human insulin and hypoglycaemia. Lancet 1990; 335: 62. 30. Anderson JH, Holcomber JH, Grimes JA, Galloway JA. Hypoglycemia unawareness and human insulin. Diabetologia 1990; 33 (suppl): 122A. 31. Stocks AE. Human insulin. Med J Aust 1991. 154: 295-96.
Calprotectin, zinc, and abscesses Bacteria can survive, without proliferation, in an established abscess for many weeks unless the abscess is drained.1 Although neutrophils are plentiful, most are dead or disintegrating and abscess fluid can itself prevent neutrophil killing.2Other factors must inhibit but not kill bacteria and possibly render them less susceptible to bactericidal antibiotics. One candidate is the calcium-binding protein known as calprotectin or Ll protein that is found mainly in neutrophil cytoplasm and is stable in neutrophil lysates.3 This protein seems to act by binding zinc, an element that affects microbial growth and the host immune
response.4 Calprotectin
has a molecular weight of 36-5 kDa and is present in large amounts in human granulocytes and in normal mucosal squamous epithelium.5°6 There are three polypeptide chains in the protein-two heavy chains and a light chain--each of which can bind two calcium ions.’ Plasma concentrations are high in patients with bacterial infection, rising to 40-130 times normal in those with meningitis, septicaemia, and pneumonia.8 Calprotectin is also present in the epidermis of patients with psoriasis and allergic contact dermatitis.6The light chain seems to be identical to cystic fibrosis antigen (calgranulin), which is found in the serum of patients with the disease and in carriers of the cystic fibrosis gene.9 The light and heavy chains are the same as the MRP-8 and MRP-14 calcium-binding proteins derived from macrophages in chronic polyarthritis. 10 Portions of the aminoacid sequence of calprotectin are those of neutrophil-immobilising proteins (NIF-1 and NIF-2), so calprotectin might be important in the adherence of myeloid cells to the endothelium during the inflammatory response Release of an inhibitory factor from disrupted neutrophils but not from intact ones was shown by McNamara et al12, who studied growth of Candida albicans pseudohyphae. The same group 13 later found that the factor had a similar aminoacid sequence to calprotectin and incubation with a specific
antibody to calprotectin eliminated its inhibitory activity. A concentration of 21 mg/1 prevented the growth of 90% of strains of Candida albicans; at higher concentrations (288 and 77 mg/1), Staphylococcus aureus and Escherichia coli were inhibited. Steinbakk et al’ found minimum inhibitory concentrations of calprotectin of 4-32 mg/1 for C albicans, 256 mg/l for E coli and Klebsiella spp, 64 mg/1 for S aureus, and 64-256 mg/l for S epidermidis; killing was observed at concentrations 2-4 times the minimum inhibitory concentration. monoclonal
Sohnle et aP have now shown that the inhibition of C albicans growth by human neutrophil lysates or abscess fluid supernatant is reversed by a low concentration of zinc (1 -4-14 pmol/1); other trace elements, including iron, were ineffective. Addition of neutrophil lysate protein prevented zinc passing through a dialysis membrane. The presence of calprotectin was confirmed by application of monoclonal antibody to western blots of fluid. human abscess electrophoresis gels containing However, human abscess fluid protein only inhibited and did not kill C albicans. Calprotectin released from dead neutrophils seemed to exert its antifungal effect by depriving the yeast of zinc, but some supporting data were not presented and effects on bacteria were not examined. Most zinc ingested in the diet is lost in the faeces but a serum concentration of 1 mg/1 is maintained, largely in a bound form; concentrations fall by 10-60% in infections and other acute illnesses.4 The metal probably stabilises cellular membranes; neutrophil phagocytosis is inhibited in the physiological range. In bacteria, zinc is needed for functioning of certain enzymes (eg, nucleic acid polymerases), and for production of virulence factors.4Apart from a few yeasts, microorganisms have no mechanism for sequestering zinc and might be susceptible to zinc deprivation. However, bacteria require only very low concentrations (10-4 to 10-2 mmol/1) for growth, so in-vitro study of zinc deficiency is difficult. Bacteria are inhibited by zinc concentrations of 0.5-32 mmol/1 and zinc-impregnated dressings are widely used to reduce bacterial counts in wounds and promote healing.14 Thus host defences might inhibit bacterial growth in pus by reducing available zinc whereas medicine achieves the same end by raising it. We do not know whether calprotectin is a sufficiently powerful chelator of zinc to inhibit bacteria in this way, but it is remarkable that only now is the function of one of the most abundant proteins in pus becoming understood. Joiner KA, Onderdonk AB, Gelfand JA, Bartlett JG, Gorbach SL. A quantitative model for subcutaneous abscess formation in mice. Br J Exp Pathol 1980; 61: 97-107. 2. Bamberger DM, Herndon BL. Bactericidal capacity of neutrophils in rabbits with experimental acute and chronic abscesses. J Infect Dis 1.
1990; 162: 186-92. 3. Sohnle PG, Collins-Lech C, Wiessner JH. The zinc-reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J Infect Dis 1991; 164: 137-42. 4. Sugarman B. Zinc and infection. Rev Infect Dis 1983; 5: 137-47.
856
5. Dale I, Fagerhol MK, Naesgaard I. Purification and partial characterization of a highly immunogenic human leucocyte protein, the L1 antigen. Eur J Biochem 1983; 134: 1-6. 6. Kelly SE, Jones DB, Fleming S. Calgranulin expression in inflammatory dermatoses. J Pathol 1989; 159: 17-21. 7. Steinbakk M, Naess-Andresen CF, Lingaas E, Dale I, Brandtzaeg P, Fagerhol MK. Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet 1990; 336: 763-65. 8. Sander J, Fagerhol MK, Bakken JS, Dale I. Plasma levels of the leucocyte L1 protein in febrile conditions: relation to aetiology, number of leucocytes in blood, blood sedimentation reaction and C-reactive protein. Scand J Clin Lab Invest 1984; 44: 357-62. 9. Andersson KB, Sletten K, Berntzen HB, et al. The leucocyte L1 protein: identity with the cystic fibrosis antigen and the calcium-binding MRP-8 and MRP-14 macrophage components. Scand J Immunol 1988; 28: 241-45. 10. Odink K, Cerletti N, Bruggen J, et al. Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis. Nature 1987; 330: 80-82. 11. Freemont P, Hogg N, Edgeworth J. Sequence identity. Nature 1989; 339: 516. 12. McNamara MP, Wiessner JH, Collins-Lech C, Hahn BL, Sohnle PG. Neutrophil death as a defence mechanism against Candida albicans infections. Lancet 1988; ii: 1163-65. 13. Sohnle PG, Collins-Lech C, Weissner JH. Antimicrobial activity of an abundant calcium-binding protein in the cytoplasm of human neutrophils. J Infect Dis 1991; 163: 187-92. 14. Soderberg T, Agren M, Tengrup I, Hallmans G, Banck G. The effects of an occlusive zinc medicated dressing on the bacterial flora in excised wounds in the rat. Infection 1989; 17: 81-85.
DMPA and breast cancer: the has had its day
dog
By the mid-1980s injectable contraceptives, especially depot-medroxyprogesterone acetate had been used as a contraceptive by more (DMPA), than 11 million women world wide. So why does one still encounter newspaper headlines such as "birth control jabs all-clear opposed by MPs" or "pressing the needle not the facts"? DMPA is available for contraception in Europe, Asia, Africa, and the Far East but not in the USA; this discrepancy has fuelled some of the adverse media publicity. The US Food and Drug Administration’s Gynecology Advisory Committee twice recommended approval of DMPA for contraceptive use in that country for certain groups of women. The FDA controls the details of labelling but the product is in every pharmacy for "legitimate" for certain conditions prescription (eg, A small of but bold endometriosis). group physicians prescribe the drug as a contraceptive and, over the years, many thousands of women in the USA have used injectable contraceptives.’ Much of the original controversy surrounding DMPA was related to the use of beagle dogs as test subjects and the relevance of animal tests to human beings. National and international bodies such as the World Health Organisation and the UK Committee on Safety of Medicines have now recognised that the beagle is a very poor predictor of the consequences of steroid administration to women,2 and no longer require testing to be done in these dogs. In this issue (p 833) WHO researchers report the results of a hospital-based case-control study of DMPA use. The study was conducted over 9 years (1979-1988) in five hospitals in developing countries. The risk of breast cancer did not increase with increased duration of use
of DMPA, nor did it increase in women who began to use DMPA more than 5 years previously. The risk was slightly above average in the first 4 years after initial exposure, mainly in women under 35. The marginal increase in risk in young women is remarkably similar to that in oral contraceptive users.3 Confirmation of these findings will undoubtedly direct attention to the possible effect of progestagens breast cancer development. Are they bringing forward the appearance of cancers that would have occurred later? A meta-analysis of oral contraceptive data suggests that this may be the case.’ A publication by the Washington-based Institute of Medicines draws attention to the need for more research into contraceptives and breast cancer,
on
including improved post-marketing surveillance. Data are needed to establish the effect of artificial steroids on subgroups of users, and the possible effect of different progestagens. This report recommends that drug regulatory agencies should "consider premarketing and post-marketing requirements as an integrated whole and devise ways of spreading the investment already required by manufacturers, and ultimately paid for by users, in such a way as to maximize the information on safety that will result". Unfortunately, day-to-day practice of medicine cannot wait until that information is to hand. For DMPA, the WHO study confirms that the advantages outweigh possible adverse effects. Injectable contraceptives lower the risk of ovarian and endometrial cancer and also reduce the incidence of pelvic inflammatory disease. The advantages of DMPA are especially pronounced in developing countries. Haemoglobin concentrations tend to increase in women who use DMPA and this will help to prevent anaemia. In such areas the compliance of pill users is often poor and pregnancy rates are high ;6 DMPA is acceptable and has a lower failure rate than any other widely used reliable method. Over 500 000 women die each year from causes related to pregnancy and childbirth99% of them in developing countries. More than half the deaths are in women who had unplanned pregnancies. In some countries as many as two-thirds of women interviewed say they do not want more children or that their last pregnancy was unplannedbut these women are not using any form of reliable contraception and most do not have access to safe, effective methods.7 Contraceptives save lives--of both mothers and their children. Health care workers therefore have a moral responsibility to provide reliable contraceptives to all who request them. DMPA should find a place among the "cafeteria" of methods available-in developed as well as developing nations. Countries such as the USA, Australia, and Japan would do well to review their existing position on injectable preparations, otherwise they may deprive their female citizens of a reliable, effective, and safe method of
contraception.
comparison of equimolar amounts of porcine and semisynthetic human insulin. J Intern Med 1991; 229: 293-96. 25. Clausen Sjobom N, Lins P-E, Adamson U, Theodorsson E. A comparative study on the hormonal responses to insulin-induced hypoglycaemia using semisynthetic human insulin and pork insulin in patients with type 1 diabetes mellitus. Diabetic Med 1990; 7: 775-79. 26. Patrick AW, Bodmer CW, Tieszen KL, White MC, Williams G. Human insulin and awareness of acute hypoglycaemia in insulin-dependent diabetes. Lancet 1991; 338: 528-32. 27. Mühlhauser I, Heinemann L, Fritsche E, von Lenhep K, Berger M. Hypoglycemic symptoms and frequency of severe hypoglycemia in patients treated with human and animal insulin preparations. Diabetes Care 1991; 14: 745-49. 28. Orchard TJ, Maser RE, Becker DJ, Dorman JS, Drash AL. Human insulin use and hypoglycaemia: insights from the Pittsburgh Epidemiology of Diabetes Complications Study. Diabetic Med 1991; 8: 469-74. 29. Heine RJ, van der Veen EA. Human insulin and hypoglycaemia. Lancet 1990; 335: 62. 30. Anderson JH, Holcomber JH, Grimes JA, Galloway JA. Hypoglycemia unawareness and human insulin. Diabetologia 1990; 33 (suppl): 122A. 31. Stocks AE. Human insulin. Med J Aust 1991. 154: 295-96.
Calprotectin, zinc, and abscesses Bacteria can survive, without proliferation, in an established abscess for many weeks unless the abscess is drained.1 Although neutrophils are plentiful, most are dead or disintegrating and abscess fluid can itself prevent neutrophil killing.2Other factors must inhibit but not kill bacteria and possibly render them less susceptible to bactericidal antibiotics. One candidate is the calcium-binding protein known as calprotectin or Ll protein that is found mainly in neutrophil cytoplasm and is stable in neutrophil lysates.3 This protein seems to act by binding zinc, an element that affects microbial growth and the host immune
response.4 Calprotectin
has a molecular weight of 36-5 kDa and is present in large amounts in human granulocytes and in normal mucosal squamous epithelium.5°6 There are three polypeptide chains in the protein-two heavy chains and a light chain--each of which can bind two calcium ions.’ Plasma concentrations are high in patients with bacterial infection, rising to 40-130 times normal in those with meningitis, septicaemia, and pneumonia.8 Calprotectin is also present in the epidermis of patients with psoriasis and allergic contact dermatitis.6The light chain seems to be identical to cystic fibrosis antigen (calgranulin), which is found in the serum of patients with the disease and in carriers of the cystic fibrosis gene.9 The light and heavy chains are the same as the MRP-8 and MRP-14 calcium-binding proteins derived from macrophages in chronic polyarthritis. 10 Portions of the aminoacid sequence of calprotectin are those of neutrophil-immobilising proteins (NIF-1 and NIF-2), so calprotectin might be important in the adherence of myeloid cells to the endothelium during the inflammatory response Release of an inhibitory factor from disrupted neutrophils but not from intact ones was shown by McNamara et al12, who studied growth of Candida albicans pseudohyphae. The same group 13 later found that the factor had a similar aminoacid sequence to calprotectin and incubation with a specific
antibody to calprotectin eliminated its inhibitory activity. A concentration of 21 mg/1 prevented the growth of 90% of strains of Candida albicans; at higher concentrations (288 and 77 mg/1), Staphylococcus aureus and Escherichia coli were inhibited. Steinbakk et al’ found minimum inhibitory concentrations of calprotectin of 4-32 mg/1 for C albicans, 256 mg/l for E coli and Klebsiella spp, 64 mg/1 for S aureus, and 64-256 mg/l for S epidermidis; killing was observed at concentrations 2-4 times the minimum inhibitory concentration. monoclonal
Sohnle et aP have now shown that the inhibition of C albicans growth by human neutrophil lysates or abscess fluid supernatant is reversed by a low concentration of zinc (1 -4-14 pmol/1); other trace elements, including iron, were ineffective. Addition of neutrophil lysate protein prevented zinc passing through a dialysis membrane. The presence of calprotectin was confirmed by application of monoclonal antibody to western blots of fluid. human abscess electrophoresis gels containing However, human abscess fluid protein only inhibited and did not kill C albicans. Calprotectin released from dead neutrophils seemed to exert its antifungal effect by depriving the yeast of zinc, but some supporting data were not presented and effects on bacteria were not examined. Most zinc ingested in the diet is lost in the faeces but a serum concentration of 1 mg/1 is maintained, largely in a bound form; concentrations fall by 10-60% in infections and other acute illnesses.4 The metal probably stabilises cellular membranes; neutrophil phagocytosis is inhibited in the physiological range. In bacteria, zinc is needed for functioning of certain enzymes (eg, nucleic acid polymerases), and for production of virulence factors.4Apart from a few yeasts, microorganisms have no mechanism for sequestering zinc and might be susceptible to zinc deprivation. However, bacteria require only very low concentrations (10-4 to 10-2 mmol/1) for growth, so in-vitro study of zinc deficiency is difficult. Bacteria are inhibited by zinc concentrations of 0.5-32 mmol/1 and zinc-impregnated dressings are widely used to reduce bacterial counts in wounds and promote healing.14 Thus host defences might inhibit bacterial growth in pus by reducing available zinc whereas medicine achieves the same end by raising it. We do not know whether calprotectin is a sufficiently powerful chelator of zinc to inhibit bacteria in this way, but it is remarkable that only now is the function of one of the most abundant proteins in pus becoming understood. Joiner KA, Onderdonk AB, Gelfand JA, Bartlett JG, Gorbach SL. A quantitative model for subcutaneous abscess formation in mice. Br J Exp Pathol 1980; 61: 97-107. 2. Bamberger DM, Herndon BL. Bactericidal capacity of neutrophils in rabbits with experimental acute and chronic abscesses. J Infect Dis 1.
1990; 162: 186-92. 3. Sohnle PG, Collins-Lech C, Wiessner JH. The zinc-reversible antimicrobial activity of neutrophil lysates and abscess fluid supernatants. J Infect Dis 1991; 164: 137-42. 4. Sugarman B. Zinc and infection. Rev Infect Dis 1983; 5: 137-47.
856
5. Dale I, Fagerhol MK, Naesgaard I. Purification and partial characterization of a highly immunogenic human leucocyte protein, the L1 antigen. Eur J Biochem 1983; 134: 1-6. 6. Kelly SE, Jones DB, Fleming S. Calgranulin expression in inflammatory dermatoses. J Pathol 1989; 159: 17-21. 7. Steinbakk M, Naess-Andresen CF, Lingaas E, Dale I, Brandtzaeg P, Fagerhol MK. Antimicrobial actions of calcium binding leucocyte L1 protein, calprotectin. Lancet 1990; 336: 763-65. 8. Sander J, Fagerhol MK, Bakken JS, Dale I. Plasma levels of the leucocyte L1 protein in febrile conditions: relation to aetiology, number of leucocytes in blood, blood sedimentation reaction and C-reactive protein. Scand J Clin Lab Invest 1984; 44: 357-62. 9. Andersson KB, Sletten K, Berntzen HB, et al. The leucocyte L1 protein: identity with the cystic fibrosis antigen and the calcium-binding MRP-8 and MRP-14 macrophage components. Scand J Immunol 1988; 28: 241-45. 10. Odink K, Cerletti N, Bruggen J, et al. Two calcium-binding proteins in infiltrate macrophages of rheumatoid arthritis. Nature 1987; 330: 80-82. 11. Freemont P, Hogg N, Edgeworth J. Sequence identity. Nature 1989; 339: 516. 12. McNamara MP, Wiessner JH, Collins-Lech C, Hahn BL, Sohnle PG. Neutrophil death as a defence mechanism against Candida albicans infections. Lancet 1988; ii: 1163-65. 13. Sohnle PG, Collins-Lech C, Weissner JH. Antimicrobial activity of an abundant calcium-binding protein in the cytoplasm of human neutrophils. J Infect Dis 1991; 163: 187-92. 14. Soderberg T, Agren M, Tengrup I, Hallmans G, Banck G. The effects of an occlusive zinc medicated dressing on the bacterial flora in excised wounds in the rat. Infection 1989; 17: 81-85.
DMPA and breast cancer: the has had its day
dog
By the mid-1980s injectable contraceptives, especially depot-medroxyprogesterone acetate had been used as a contraceptive by more (DMPA), than 11 million women world wide. So why does one still encounter newspaper headlines such as "birth control jabs all-clear opposed by MPs" or "pressing the needle not the facts"? DMPA is available for contraception in Europe, Asia, Africa, and the Far East but not in the USA; this discrepancy has fuelled some of the adverse media publicity. The US Food and Drug Administration’s Gynecology Advisory Committee twice recommended approval of DMPA for contraceptive use in that country for certain groups of women. The FDA controls the details of labelling but the product is in every pharmacy for "legitimate" for certain conditions prescription (eg, A small of but bold endometriosis). group physicians prescribe the drug as a contraceptive and, over the years, many thousands of women in the USA have used injectable contraceptives.’ Much of the original controversy surrounding DMPA was related to the use of beagle dogs as test subjects and the relevance of animal tests to human beings. National and international bodies such as the World Health Organisation and the UK Committee on Safety of Medicines have now recognised that the beagle is a very poor predictor of the consequences of steroid administration to women,2 and no longer require testing to be done in these dogs. In this issue (p 833) WHO researchers report the results of a hospital-based case-control study of DMPA use. The study was conducted over 9 years (1979-1988) in five hospitals in developing countries. The risk of breast cancer did not increase with increased duration of use
of DMPA, nor did it increase in women who began to use DMPA more than 5 years previously. The risk was slightly above average in the first 4 years after initial exposure, mainly in women under 35. The marginal increase in risk in young women is remarkably similar to that in oral contraceptive users.3 Confirmation of these findings will undoubtedly direct attention to the possible effect of progestagens breast cancer development. Are they bringing forward the appearance of cancers that would have occurred later? A meta-analysis of oral contraceptive data suggests that this may be the case.’ A publication by the Washington-based Institute of Medicines draws attention to the need for more research into contraceptives and breast cancer,
on
including improved post-marketing surveillance. Data are needed to establish the effect of artificial steroids on subgroups of users, and the possible effect of different progestagens. This report recommends that drug regulatory agencies should "consider premarketing and post-marketing requirements as an integrated whole and devise ways of spreading the investment already required by manufacturers, and ultimately paid for by users, in such a way as to maximize the information on safety that will result". Unfortunately, day-to-day practice of medicine cannot wait until that information is to hand. For DMPA, the WHO study confirms that the advantages outweigh possible adverse effects. Injectable contraceptives lower the risk of ovarian and endometrial cancer and also reduce the incidence of pelvic inflammatory disease. The advantages of DMPA are especially pronounced in developing countries. Haemoglobin concentrations tend to increase in women who use DMPA and this will help to prevent anaemia. In such areas the compliance of pill users is often poor and pregnancy rates are high ;6 DMPA is acceptable and has a lower failure rate than any other widely used reliable method. Over 500 000 women die each year from causes related to pregnancy and childbirth99% of them in developing countries. More than half the deaths are in women who had unplanned pregnancies. In some countries as many as two-thirds of women interviewed say they do not want more children or that their last pregnancy was unplannedbut these women are not using any form of reliable contraception and most do not have access to safe, effective methods.7 Contraceptives save lives--of both mothers and their children. Health care workers therefore have a moral responsibility to provide reliable contraceptives to all who request them. DMPA should find a place among the "cafeteria" of methods available-in developed as well as developing nations. Countries such as the USA, Australia, and Japan would do well to review their existing position on injectable preparations, otherwise they may deprive their female citizens of a reliable, effective, and safe method of
contraception.
