- Email: [email protected]
EFFECT OF EXTRACELLULAR SLIME SUBSTANCE FROM STAPHYLOCOCCUS EPIDERMIDIS ON THE HUMAN CELLULAR IMMUNE RESPONSE
365 suppress gonadotropin secretion6,7 and gonadal maturation32 in rodents, may be such an inhibitor. We thank the staff of Krankenhaus der Barmherzigen Brueder, Eisenstadt for their cooperation, and Dr Harry Lynch for advice in developing our melatonin assay. F. W. was supported by the Postdoctoral Research Exchange Program of the Max Kade Foundation Inc, New York.
Correspondence should be addressed to F. W. at UniversitatsKinderklinik, Waehringer Guertel 74-76, A-1090, Vienna, Austria, or to R. J. W. at MIT, E25-604, Cambridge, Mass, 02139, USA.
REFERENCES
F, Waldhauser M, Wurtman RJ. A possible role for melatonin in human sexual maturation. 3rd International Symposium on Psycho-Neuro Endocrinology in Reproduction, July 9-12, 1982, Spoleto, Italy (in press). 2. Heubner O. Tumor der glandula pinealis. Dtsch Med Wochenschr 1898; 24: 214-15. 3. Marburg O. Zur Kenntnis der normalen und pathologischen Histologie der Zirebeldruese. Arb Neur Inst Wien 1909; 12: 217-79. 4 Kitay JI, Altschule MD. The pineal gland. A review of the physiologic literature. Cambridge, Massachusetts: Harvard University Press, 1954. 5. Waldhauser F, Wurtman RJ. The secretion and actions of melatonin. In: Litwack G, ed. Biochemical actions of hormones. New York Academic Press, 1983: 187-225. 6. Wurtman RJ, Axelrod J, Chu EW. Melatonin, a pineal substance: effect on the rat ovary. Science 1963; 141: 277-78. 7. Reiter RJ. Reproductive effects of the pineal gland and pineal indoles in the Syrian hamster and the albino rat. In: Reiter RJ, ed. The pineal gland. Boca Raton, Florida: CRC Press, 1981: 45-81. 8. Arendt J. Current status of assay methods of melatonin. Adv Biosci 1981; 29: 3-7. 9. Lynch HJ. Assay methodology. In: Relkin R, ed. The pineal gland. New York: Elsevier Biomedical, 1983: 129-50. 10. Lewy AJ. Biochemistry and regulation of mammalian melatonin production. In: Relkin R, ed. The pineal gland. New York: Elsevier Biomedical, 1983: 77-128. 11. Pelham RW, Vaughan GM, Sandock KL, Vaughn MK. Twenty-four-hour cycle of a melatonin-like substance in the plasma of human males. J Clin Endocrinol Metab 1973; 37: 341-44. 12. Lynch HJ, Wurtman RJ, Moskowitz MA, Archer MC, Ho MH. Daily rhythm in human melatonin. Science 1975; 17: 169-71. 13. Lynch HJ, Wurtman RJ. Melatonin levels as they relate to reproductive physiology. In: Reiter RJ, ed. The pineal gland, vol. II. Boca Raton, Florida: CRC Press, 1982: 103-23. 14. Ozaki Y, Lynch HJ, Wurtman RJ. Melatonin in rat pineal, plasma and urine: 24-hour rhythmicity and effect of chlorpromazine. Endocrinology 1976; 98: 1418-24. 15. Pang SF, Brown GM, Grota LJ, Chambers JW, Rodman RL. Determination of N-acetylserotonin and melatonin activities in the pineal gland, retina, Harderian gland, brain and serum’of rats and chickens. Neuroendocrinology 1977; 23: 1-13. 16. Tanner JM. Growth and endocrinology of the adolescent. In: Gardner LI, ed. Endocrine and genetic diseases of childhood. Philadelphia: W. B. Saunders, 1969: 19-69. 17. Arendt J, Wilkmson M. Melatonin. In: Jaffe BM, Behrman MR, eds. Methods of hormone radioimmunoassay. Second ed. New York: Academic Press, 1978 18. Arendt J, Hampton S, English J, Kwasowkis P, Marks V. 24-Hour profiles of melatonin, cortisol, insulin, c-peptide, and GIP following a meal and subsequent fasting. Clin Endocrinol 1982; 16: 89-95. 19 Lewy AJ, Markey SP. Analysis of melatonin in human plasma by gas chromatography: negative chemical ionization mass spectormetry. Science 1978; 201: 741-43. 20. Iguchi H, Ken-Ichi Kato, Ibayashi H. Melatonin serum levels and metabolic clearance rate in patients with liver cirrhosis. J Clin Endocrinol 1982; 54: 1025-27. 21. Levine L, Riceberg LR. Radioimmunoassay for melatonin. Res Commun Chem Pathol Pharmacol 1975; 10: 693-701. 22. Statistical Analytical System, SAS-Institute, Cary, N. Caroline, 1979. 23. Lenko HL, Lang U, Aubert ML, Paunier L, Sizonenko PC. Homonal changes in puberty. VII. Lack of variation of daytime plasma melatonin. J Clin Endocrinol Metab 1981; 54: 1056-58. 24. Ehrenkranz JRL, Tamarkin L, Comite F, et al. Daily rhythm of plasma melatonin in normal and precocious puberty. J Clin Endocrinol Metab 1982; 55: 307-10. 25. Tamarkin L, Abastillas P, Chen H, McNemar A, Sidbury JB. The daily profile of plasma melatonin in obese and Prader-Willi syndrome children. J Clin Endocrinol Metab 1982; 55: 491-95 26. Waldhauser F, Frisch M, Weissenbacher G, Zeitlhuber U, Toifl K. Day-and-nighttime serum melatonin in children and adults. Pediatr Res 1981; 15: 1566 (abstr). 27. Silman RE, Leone RM, Hooper RJL, Preece MA. Melatonin, the pineal gland and human puberty. Nature 1979; 282: 301-03. 28 Hartmann L, Roger M, Lemaitre BJ, Massias JF, Chaussain JL. Plasma and urinary melatonin in male infants during the first 12 months of life. Clin Chim Acta 1982; 121: 37-42. 29. Come FA, Grumbach MM, Kaplan SL, Reiter EO. Correlation of luteinizing hormone-releasing factor-induced luteinizing hormone and follicle-stimulating hormone release from infancy to 19 years with the changing pattern of gonadotropin secretion in agonadal patients: relation to the restraint of puberty. J Clin Endocrinol Metab 1980; 50: 163-68. 30. Dierschke DJ, Karsch FJ, Weick RF, Weiss G, Hotckiss J, Knobil E Hypothalamicpituitary regulation of puberty: feedback control of gonadotropin secretion in the rhesus monkey. In: Grumbach MM, Grave GD, Mayer FG, eds. Control of the onset of puberty. New York: John Wiley and Sons, 1974: 104. 31. Winter JSD Comparative aspects of animal and human puberty. 2nd International Conference on the Control of the Onset of Puberty, September 15-18, Stresa, Italy. 32. Wurtman RJ, Axelrod J. The pineal gland. Sci Am 1965; 213: 50-60. 1. Waldhauser
EFFECT OF EXTRACELLULAR SLIME SUBSTANCE FROM STAPHYLOCOCCUS EPIDERMIDIS ON THE HUMAN CELLULAR IMMUNE RESPONSE ERNEST D. GRAY GEORG PETERS* WARREN E. REGELMANN MARJORIE VERSTEGEN
Departments of Pediatrics and Biochemistry, University of Minnesota, Minneapolis, Minnesota
Summary
Staphylococcus epidermidis infection of plastic catheters is often associated with
heavy deposits of slime. To test whether this slime affects the human cellular immune response, its effect on the lymphoproliferative response of mononuclear cells to polyclonal stimulators was measured. Slime drastically reduces this response. Its inhibitory action was not immediate but took place over a few days and resulted in destruction of affected cells. The effect is dose related. This inhibition of cellular response may contribute to S epidermidis infection of
implanted prostheses. Introduction
Staphylococcus epidermidis
is a ubiquitous commensal on membranes. It can be an in the opportunistic pathogen presence of permanently or transiently implanted prosthetic devices. The earliest observations of this behaviour concerned infected ventriculoatrial shunts in children.I-3 The infections usually started long after the operation and persisted. Septic episodes improved with antimicrobial therapy but bacterial foci could seldom be eradicated. S epidermidis infections with similar characteristics have been observed with a variety of implanted plastic devices4-7 as well as intravascular
human skin and
mucous
catheters.8,9 Catheters isolated from patients with S epidermidis infection contain heavy deposits ofaslimy substance in which staphylococcal cells are embedded.1O In-vitro studies have shown that the organisms adhere to plastic surfaces and eventually become embedded in a matrix of slime.11, 12, 14 The presence of slime seems to be important in the adherence of the organisms to plastic. 12, 14-16 Slime may also be involved in the persistence of foreign body S epidermidis infections through some action on the host defences. The present report describes the effects of slime on the human cellular immune response. Methods
Staphylococcus epidermidis slime.-Slime was produced during the growth of S epidermidis K11(an isolate from a catheter tip obtained from a patient with catheter-related septicaemia). This strain is known for its ability to adhere to plastic surfaces and to produce slime.11-14 The staphylococcal strain was grown in brainheart infusion overnight at 37 °C. After being washed thrice with physiological saline the cells were resuspended in saline (approximately 108 cells/ml). Agar plates were inoculated with 5 ml of the bacteria-saline suspension and incubated for 48 h at 37 °C. Nutrient agar containing 3% casamino acids and 1% glucose was used as growth medium to promote abundant production of slime. Thereafter the liquid bacteria-slime mixture was collected in sterile flasks and treated with an ’Ultraturax’ (Janke und Kunkel GmbH, Staufen, West Germany) for 5 min to disrupt the ropy clots of slime. After centrifugation (12000 g for 30 min) the supernatant was extensively dialysed against distilled water for 48 h at 4°C, concentrated by filtration (Amicon filter PM 10, Amicon GmbH, Heidelberg, West Germany), and lyophilised. (The slime used for this study was bacteria-free but was not fractionated-. We have *Present address:
Germany.
Hygiene Institute, University of Cologne, Cologne,
West
-
366
subjected it to phenol extraction and ion-exchange chromatography. One of the fractions from a DEAE column seems
since
contain the substance that affects lymphoproliferation.) Cell culture.-Mononuclear cells (MNC) were isolated from peripheral blood by ’Ficoll-HyPaque’ density gradient centrifugation. For assay of blastogenic response, 105 MNC were incubated in wells of flat-bottomed microtitre plates in Eagle’s minimal essential medium (GIBCO, Grand Island, NY) containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/ml L-glutamine with 5% fetal calf serum. To triplicate wells of the cells were added 100 ng phytohaemagglutinin (PHA) (HA16, Wellcome) or 4 ng streptococcal blastogen A 17 and various amounts of staphylococcal slime. The cultures were incubated at 37°C in an atmosphere containing 5% CO2 for 5 days; then 0.5 IACI 3H-thymidine specific activity 6 - 7 Ci/mmol (New England Nuclear Corp) was added and the cells were harvested after a further 18 h of incubation. The amount of radioactivity incorporated was measured by scintillation counting. 18 To assess whether the effects of staphylococcal slime on MNC in culture could be due to a direct and rapid toxic action of some slime components, experiments were also performed in which slime (100 µg/well) was added at various times (0, 2, and 4 days) after stimulation with PHA. The number of blastic cells were counted on day 6 and expressed as a percentage of those in control cultures with PHA but no slime. Enumeration of blast cells.-To determine whether the effects of staphylococcal slime were directed at 3H-thymidine incorporation rather than blastogenesis, cell cultures were set up as described and after 6 days of incubation, 6 replicate wells were combined and washed with MEM, and the proportion of blasts determined by to
2-Effect of staphylococcal slime on blast formation by mononuclear cells in response to PHA (0 - 0) and streptococcal
Fig .
blastogen A (• -0).
.
phase-contrast light microscopy.
Cell viability.-To test the effect of varying periods of exposure to staphylococcal slime on cell viability, the proportion ofviable cells in ’Cultures with PHA plus slime were compared with that in control cultures without slime. Cell viability was assessed by trypan blue dye exclusion.
Fig 3-Effect of varying periods of exposure to staphylococcal slime on
the number of blastic cells formed in response to PHA.
Results slime levels (10 and 100 µg/well) drastically reduced proliferative response to both phytohaemagglutinin and streptococcal blastogen A (fig 1). The effects of staphylococcal slime on blast formation reflected those on thymidine incorporation-it produced a profound decrease in the numbers of blastic cells (fig 2). The effects of slime thus seem to be related to interference with induction of the normal T-cell proliferation by PHA and blastogen A rather than to inhibition of thymidine uptake or incorporation into DNA. The action of slime was not immediate; its effect was expressed only when slime was present for 4-6 days of the
High
the
«
Fig
4-Effect of varying periods of exposure to staphylococcal slime the number of viable cells in cultures with PHA.
on
cell culture period (fig 3). Also, the total numbers of both viable cells and blasts decreased as incubation time with slime increased (fig 4). Some lytic activity seems to be associated with the action of slime. The proportion of viable cells remaining in culture was indistinguishable from the proportion in control cultures, which suggests that affected cells are totally destroyed. Discussion
The extracellular slime produced by Staphylococcus is associated with the ability of the organism to establish foci of infection on implanted plastic foreign bodies, 10,14 perhaps because of the adhesivel2-16 and protective properties of slime. Often such infections are controlled only by removal of the plastic device. The findings reported here suggest that slime also interferes with the function of the cellular immune system. The lymphoproliferative response to two distinct18 polyclonal T-cell stimulators is strongly inhibited by slime, which eventually
epidermidis
of staphylococcal slime on the blastogenic response of mononuclear cells to PHA (0-0) and streptococcal blastogen A
Fig 1—Effect (•-•).
367
destroys affected cells. The inhibitory effect is not due to a general toxic action on cells since surviving cells are as viable as those in control cultures. Slime produces its effect only after it has been incubated with cells for some time, so its action is unlikely to be mediated by rapid cytolysis. Slime may act by activating a subpopulation of cytotoxic cells which are responsible for the lysis of affected cells.19, 20 Preliminary analysis of the surface antigens of surviving cells has revealed that, as in control cultures, most cells are ofT lineage-that is, they react with a monoclonal antibody (Clone 9 - 6, New England Nuclear Lyt-3)21directed against the sheep redblood-cell receptor. In slime-treated cultures, however, much fewer T cells had the surface structures associated with either
helper/inducer (OKT4+) cells.22 It is
or
cytotoxic/suppressor (OKT8)
clear whether slime exposure represses the expression of these antigens borne by differentiated T cells or whether the surviving cells are partially differentiated T cells. The dramatic effects of staphylococcal slime in-vitro suggest that this substance may contribute to the extended course ofS epidermidis foreign-body infections. Although the in-vitro effects of slime are observable only with high concentrations of the substance, slime is presented to the immune system in-vivo as a solid phase-that is, effectively in high concentration. Suppression of normal host responses may not only prolong the course of S epidermidis infection but also increase the risk of other opportunistic infections. not
A preliminary account of this research was presented at the Twenty-Third Interscience Conference on Antimicrobial Agents and Chemotherapy, October, 1983. The research was supported by US Public Health Service research grant HL-30058. We are grateful to the late Dr L. W. Wannamaker and Dr G. Pulverer for valuable discussions and encouragement.
Correspondence should be addressed to E. D. G., Department of Pediatrics and Biochemistry, Division of Infectious Diseases, University of Minnesota Medical School, Box 296, Mayo Memorial Building, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA. REFERENCES 1.
Callaghan RP, Cohen SJ, Stewart JT. Septicemia due to colonization of Spitz-Holter valves by staphylococci. Five cases treated with methicillin. Br Med J 1961; i:
PARATHYROID HORMONE DOES NOT INHIBIT PLATELET AGGREGATION C. LEITHNER H. SINZINGER
2nd Department of Internal Medicine, University of Vienna, and *Ludwig Boltzmann-Institute of Clinical Endocrinology, Vienna, Austria
The suggestion that parathyroid hormone (PTH) is a major uraemic toxin was examined by testing the effects of synthetic human PTH fragments and synthetic bovine PTH on ADP- induced and collagen-induced platelet aggregation. Whereas the bovine parathyroid-gland extracts inhibited platelet aggregation in a dose-dependent manner, none of the synthetic compounds was effective even at high concentrations. It is suggested that the inhibition of platelet aggregation by extracts of bovine parathyroid glands is not caused by PTH fragments and is probably an effect of other constituents contained in the extract. These findings argue against a role of PTH in the pathogenesis of platelet dysfunction and bleeding tendency in uraemia and were supported by platelet-aggregation studies in 6 patients with primary hyperparathyroidism. Platelet aggregation was normal before and unchanged after surgery of the parathyroid glands.
Summary
Introduction
tendency in uraemia has a multifactorial particular, platelet aggregation is inhibited by medium-sized molecules that accumulate in plasma during uraemia,2 and the interaction between platelets and the vascular wall might be disturbed by an increased production of prostacyclin (PGI2).3,4 Remuzzi and co-workers reported that natural bovine parathyroid hormone (PTH) strongly inhibited platelet aggregation and secretion induced in vitro by different stimuli,s and on the basis of these findings the suggestion that PTH is a major uraemic toxin6 was put forward. We have now investigated platelet aggregation with several pure synthetic PTH preparations. BLEEDING
origin.
In
860-63.
RJ Bacteriological studies on colonized ventriculoatrial shunts. Dev Med Child 1970; 12 (suppl 22): 83-87. Schimke RT, Black PH, Mark VH, Schwartz MN. Indolent Staphylococcus albus or aureus bacteremia after ventriculoatriostomy. N Engl J Med 1961; 264: 264-70 Masur H, Johnson WD. Prosthetic valve endocarditis. J Thorac Cardiovasc Surg 1980;
Methods
2 Holt
Neurol
3 4.
80: 31-37. 5. Stinchfield FE,
Bigliani LU, Neu HC, Gross TP, Foster CR. Late hematogenous infection of total joint replacement. J Bone Joint Surg 1980; 62A: 1345-50. 6. Choo MH, Holmes DR, Gersh BJ, et al. Permanent pacemaker infections: Characterization and management. Am J Cardiol 1981; 48: 559-64. 7. Rubin J, Rogers WA, Taylor HM, et al. Peritonitis during continuous ambulatory peritoneal dialysis. Ann Intern Med 1980; 92: 7-13. 8. Duma RJ, Warner JF, Dalton HP. Septicema from intravenous infusions. N Engl J Med 1977; 284: 257-60. 9. Bender JW, Hughes WT. Fatal Streptococcus epidermidis sepsis following bone marrow transplantation. Johns Hopkins Med J 1980; 146: 13-15. 10. Peters G, Locci R, Pulverer G. Microbial colonization of prosthetic devices. II. Scanning electron microscopy of naturally infected intravenous catheters. Zbl Bakt Hyg, I Abt Orig B 1981; 173: 293-99. 11. Locci R, Peters G, Pulverer G Microbial colonization of prosthetic devices. III. Adhesion of staphylococci to lumma of intravenous catheters perfused with bacterial suspensions. Zbl Bakt Hyg, I Abt Orig B 1981; 173: 300-07. 12. Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters.J Infect Dis 1982; 146: 479-82. 13. Ludwicka A, Locci R, Jansen B, Peters G, Pulverer G. Microbial colonization of prosthetic devices. V. Attachment of coagulase-negative staphylococci and "slime"-production on chemically pure synthetic polymers. Zbl Bakt Hyg, I Abt Orig B 1983; 177: 527-32. 14. Peters G, Saborowski F, Locci R, Pulverer G Investigations on staphylococcal infection of intravenous endocardial pacemaker-electrodes. Am Heart J (in press). 15 Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime-producing strains of Staphyloccocus epidermidis to smooth surfaces. Infect Immun 1982; 37: 318-26. 16. Bayston R, Penny SR. Excessive production of mucoid substance in Staphylococcus SIIA a possible factor in colonization of Holter shunts. Dev-Med Child Neurol 1972, 27 (suppl) 25-28.
J. KOVARIK W. WOLOSZCZUK*
The following synthetic peptides were used: bovine PTH 1-84, human PTH 1-44, 28-48, and 53-84 (Bachem Inc, Torrance, CA, USA), and human PTH 1-34 (Peninsula Laboratories Inc, Belmont, CA, USA). The lyophilised peptides were dissolved in 10 mM acetic acid and diluted in 0.1 M phosphate-buffered saline (PBS, pH 7-4) containing 107o human serum albumin. Bovine parathyroid gland extracts were obtained from Sigma and (’Parathorm’) from Hormon Chemie, Munich, West Germany. These preparations were dialysed exhaustively against isotonic saline (cut-off 2000 daltons) and then diluted in PBS-albumin solution. 20 healthy male volunteers, aged 20-35, who had been free of medication for 4 weeks, served as blood donors. After preparation of platelet-rich plasma (PRP), aggregation tests were carried out with a
17.
18.
Gray ED. Purification and properties of an extracellular blastogen produced by group A streptococci. J Exp Med 1979; 149: 1438-49. Regelmann WE, Gray ED, Wannamaker LW. Characterization of the human cellular immune response to purified group A streptococcal blastogen A. J Immunol 1982; 128: 1631-36.
LV, Rowley DA. Homeostasis of the antibody response. Immunoregulation by NK cells. Science 1983, 222: 581-85. Rubens RP, Henney CS. Studies on the mechanism oflymphocyte mediated cytolysis. VIII. The use of co nA to delineate a distinctive killer T cell subpopulation. J
19 Abruzzo 20
Immunol 1977; 118: 180-86. 21. Kamoun M, Martin P-J, Hansen JA, Brown MA, Siadak AW, Nowinski RC Identification of a human T lymphocyte surface protein associated with the E-rosette receptor. J Exp Med 1981; 153: 207-12. 22. Kung PC, Goldstein G Functional and developmental compartments of human T lymphocytes. Vox Sang 1980; 39: 121-27.
Correspondence should be addressed to F. W. at UniversitatsKinderklinik, Waehringer Guertel 74-76, A-1090, Vienna, Austria, or to R. J. W. at MIT, E25-604, Cambridge, Mass, 02139, USA.
REFERENCES
F, Waldhauser M, Wurtman RJ. A possible role for melatonin in human sexual maturation. 3rd International Symposium on Psycho-Neuro Endocrinology in Reproduction, July 9-12, 1982, Spoleto, Italy (in press). 2. Heubner O. Tumor der glandula pinealis. Dtsch Med Wochenschr 1898; 24: 214-15. 3. Marburg O. Zur Kenntnis der normalen und pathologischen Histologie der Zirebeldruese. Arb Neur Inst Wien 1909; 12: 217-79. 4 Kitay JI, Altschule MD. The pineal gland. A review of the physiologic literature. Cambridge, Massachusetts: Harvard University Press, 1954. 5. Waldhauser F, Wurtman RJ. The secretion and actions of melatonin. In: Litwack G, ed. Biochemical actions of hormones. New York Academic Press, 1983: 187-225. 6. Wurtman RJ, Axelrod J, Chu EW. Melatonin, a pineal substance: effect on the rat ovary. Science 1963; 141: 277-78. 7. Reiter RJ. Reproductive effects of the pineal gland and pineal indoles in the Syrian hamster and the albino rat. In: Reiter RJ, ed. The pineal gland. Boca Raton, Florida: CRC Press, 1981: 45-81. 8. Arendt J. Current status of assay methods of melatonin. Adv Biosci 1981; 29: 3-7. 9. Lynch HJ. Assay methodology. In: Relkin R, ed. The pineal gland. New York: Elsevier Biomedical, 1983: 129-50. 10. Lewy AJ. Biochemistry and regulation of mammalian melatonin production. In: Relkin R, ed. The pineal gland. New York: Elsevier Biomedical, 1983: 77-128. 11. Pelham RW, Vaughan GM, Sandock KL, Vaughn MK. Twenty-four-hour cycle of a melatonin-like substance in the plasma of human males. J Clin Endocrinol Metab 1973; 37: 341-44. 12. Lynch HJ, Wurtman RJ, Moskowitz MA, Archer MC, Ho MH. Daily rhythm in human melatonin. Science 1975; 17: 169-71. 13. Lynch HJ, Wurtman RJ. Melatonin levels as they relate to reproductive physiology. In: Reiter RJ, ed. The pineal gland, vol. II. Boca Raton, Florida: CRC Press, 1982: 103-23. 14. Ozaki Y, Lynch HJ, Wurtman RJ. Melatonin in rat pineal, plasma and urine: 24-hour rhythmicity and effect of chlorpromazine. Endocrinology 1976; 98: 1418-24. 15. Pang SF, Brown GM, Grota LJ, Chambers JW, Rodman RL. Determination of N-acetylserotonin and melatonin activities in the pineal gland, retina, Harderian gland, brain and serum’of rats and chickens. Neuroendocrinology 1977; 23: 1-13. 16. Tanner JM. Growth and endocrinology of the adolescent. In: Gardner LI, ed. Endocrine and genetic diseases of childhood. Philadelphia: W. B. Saunders, 1969: 19-69. 17. Arendt J, Wilkmson M. Melatonin. In: Jaffe BM, Behrman MR, eds. Methods of hormone radioimmunoassay. Second ed. New York: Academic Press, 1978 18. Arendt J, Hampton S, English J, Kwasowkis P, Marks V. 24-Hour profiles of melatonin, cortisol, insulin, c-peptide, and GIP following a meal and subsequent fasting. Clin Endocrinol 1982; 16: 89-95. 19 Lewy AJ, Markey SP. Analysis of melatonin in human plasma by gas chromatography: negative chemical ionization mass spectormetry. Science 1978; 201: 741-43. 20. Iguchi H, Ken-Ichi Kato, Ibayashi H. Melatonin serum levels and metabolic clearance rate in patients with liver cirrhosis. J Clin Endocrinol 1982; 54: 1025-27. 21. Levine L, Riceberg LR. Radioimmunoassay for melatonin. Res Commun Chem Pathol Pharmacol 1975; 10: 693-701. 22. Statistical Analytical System, SAS-Institute, Cary, N. Caroline, 1979. 23. Lenko HL, Lang U, Aubert ML, Paunier L, Sizonenko PC. Homonal changes in puberty. VII. Lack of variation of daytime plasma melatonin. J Clin Endocrinol Metab 1981; 54: 1056-58. 24. Ehrenkranz JRL, Tamarkin L, Comite F, et al. Daily rhythm of plasma melatonin in normal and precocious puberty. J Clin Endocrinol Metab 1982; 55: 307-10. 25. Tamarkin L, Abastillas P, Chen H, McNemar A, Sidbury JB. The daily profile of plasma melatonin in obese and Prader-Willi syndrome children. J Clin Endocrinol Metab 1982; 55: 491-95 26. Waldhauser F, Frisch M, Weissenbacher G, Zeitlhuber U, Toifl K. Day-and-nighttime serum melatonin in children and adults. Pediatr Res 1981; 15: 1566 (abstr). 27. Silman RE, Leone RM, Hooper RJL, Preece MA. Melatonin, the pineal gland and human puberty. Nature 1979; 282: 301-03. 28 Hartmann L, Roger M, Lemaitre BJ, Massias JF, Chaussain JL. Plasma and urinary melatonin in male infants during the first 12 months of life. Clin Chim Acta 1982; 121: 37-42. 29. Come FA, Grumbach MM, Kaplan SL, Reiter EO. Correlation of luteinizing hormone-releasing factor-induced luteinizing hormone and follicle-stimulating hormone release from infancy to 19 years with the changing pattern of gonadotropin secretion in agonadal patients: relation to the restraint of puberty. J Clin Endocrinol Metab 1980; 50: 163-68. 30. Dierschke DJ, Karsch FJ, Weick RF, Weiss G, Hotckiss J, Knobil E Hypothalamicpituitary regulation of puberty: feedback control of gonadotropin secretion in the rhesus monkey. In: Grumbach MM, Grave GD, Mayer FG, eds. Control of the onset of puberty. New York: John Wiley and Sons, 1974: 104. 31. Winter JSD Comparative aspects of animal and human puberty. 2nd International Conference on the Control of the Onset of Puberty, September 15-18, Stresa, Italy. 32. Wurtman RJ, Axelrod J. The pineal gland. Sci Am 1965; 213: 50-60. 1. Waldhauser
EFFECT OF EXTRACELLULAR SLIME SUBSTANCE FROM STAPHYLOCOCCUS EPIDERMIDIS ON THE HUMAN CELLULAR IMMUNE RESPONSE ERNEST D. GRAY GEORG PETERS* WARREN E. REGELMANN MARJORIE VERSTEGEN
Departments of Pediatrics and Biochemistry, University of Minnesota, Minneapolis, Minnesota
Summary
Staphylococcus epidermidis infection of plastic catheters is often associated with
heavy deposits of slime. To test whether this slime affects the human cellular immune response, its effect on the lymphoproliferative response of mononuclear cells to polyclonal stimulators was measured. Slime drastically reduces this response. Its inhibitory action was not immediate but took place over a few days and resulted in destruction of affected cells. The effect is dose related. This inhibition of cellular response may contribute to S epidermidis infection of
implanted prostheses. Introduction
Staphylococcus epidermidis
is a ubiquitous commensal on membranes. It can be an in the opportunistic pathogen presence of permanently or transiently implanted prosthetic devices. The earliest observations of this behaviour concerned infected ventriculoatrial shunts in children.I-3 The infections usually started long after the operation and persisted. Septic episodes improved with antimicrobial therapy but bacterial foci could seldom be eradicated. S epidermidis infections with similar characteristics have been observed with a variety of implanted plastic devices4-7 as well as intravascular
human skin and
mucous
catheters.8,9 Catheters isolated from patients with S epidermidis infection contain heavy deposits ofaslimy substance in which staphylococcal cells are embedded.1O In-vitro studies have shown that the organisms adhere to plastic surfaces and eventually become embedded in a matrix of slime.11, 12, 14 The presence of slime seems to be important in the adherence of the organisms to plastic. 12, 14-16 Slime may also be involved in the persistence of foreign body S epidermidis infections through some action on the host defences. The present report describes the effects of slime on the human cellular immune response. Methods
Staphylococcus epidermidis slime.-Slime was produced during the growth of S epidermidis K11(an isolate from a catheter tip obtained from a patient with catheter-related septicaemia). This strain is known for its ability to adhere to plastic surfaces and to produce slime.11-14 The staphylococcal strain was grown in brainheart infusion overnight at 37 °C. After being washed thrice with physiological saline the cells were resuspended in saline (approximately 108 cells/ml). Agar plates were inoculated with 5 ml of the bacteria-saline suspension and incubated for 48 h at 37 °C. Nutrient agar containing 3% casamino acids and 1% glucose was used as growth medium to promote abundant production of slime. Thereafter the liquid bacteria-slime mixture was collected in sterile flasks and treated with an ’Ultraturax’ (Janke und Kunkel GmbH, Staufen, West Germany) for 5 min to disrupt the ropy clots of slime. After centrifugation (12000 g for 30 min) the supernatant was extensively dialysed against distilled water for 48 h at 4°C, concentrated by filtration (Amicon filter PM 10, Amicon GmbH, Heidelberg, West Germany), and lyophilised. (The slime used for this study was bacteria-free but was not fractionated-. We have *Present address:
Germany.
Hygiene Institute, University of Cologne, Cologne,
West
-
366
subjected it to phenol extraction and ion-exchange chromatography. One of the fractions from a DEAE column seems
since
contain the substance that affects lymphoproliferation.) Cell culture.-Mononuclear cells (MNC) were isolated from peripheral blood by ’Ficoll-HyPaque’ density gradient centrifugation. For assay of blastogenic response, 105 MNC were incubated in wells of flat-bottomed microtitre plates in Eagle’s minimal essential medium (GIBCO, Grand Island, NY) containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mmol/ml L-glutamine with 5% fetal calf serum. To triplicate wells of the cells were added 100 ng phytohaemagglutinin (PHA) (HA16, Wellcome) or 4 ng streptococcal blastogen A 17 and various amounts of staphylococcal slime. The cultures were incubated at 37°C in an atmosphere containing 5% CO2 for 5 days; then 0.5 IACI 3H-thymidine specific activity 6 - 7 Ci/mmol (New England Nuclear Corp) was added and the cells were harvested after a further 18 h of incubation. The amount of radioactivity incorporated was measured by scintillation counting. 18 To assess whether the effects of staphylococcal slime on MNC in culture could be due to a direct and rapid toxic action of some slime components, experiments were also performed in which slime (100 µg/well) was added at various times (0, 2, and 4 days) after stimulation with PHA. The number of blastic cells were counted on day 6 and expressed as a percentage of those in control cultures with PHA but no slime. Enumeration of blast cells.-To determine whether the effects of staphylococcal slime were directed at 3H-thymidine incorporation rather than blastogenesis, cell cultures were set up as described and after 6 days of incubation, 6 replicate wells were combined and washed with MEM, and the proportion of blasts determined by to
2-Effect of staphylococcal slime on blast formation by mononuclear cells in response to PHA (0 - 0) and streptococcal
Fig .
blastogen A (• -0).
.
phase-contrast light microscopy.
Cell viability.-To test the effect of varying periods of exposure to staphylococcal slime on cell viability, the proportion ofviable cells in ’Cultures with PHA plus slime were compared with that in control cultures without slime. Cell viability was assessed by trypan blue dye exclusion.
Fig 3-Effect of varying periods of exposure to staphylococcal slime on
the number of blastic cells formed in response to PHA.
Results slime levels (10 and 100 µg/well) drastically reduced proliferative response to both phytohaemagglutinin and streptococcal blastogen A (fig 1). The effects of staphylococcal slime on blast formation reflected those on thymidine incorporation-it produced a profound decrease in the numbers of blastic cells (fig 2). The effects of slime thus seem to be related to interference with induction of the normal T-cell proliferation by PHA and blastogen A rather than to inhibition of thymidine uptake or incorporation into DNA. The action of slime was not immediate; its effect was expressed only when slime was present for 4-6 days of the
High
the
«
Fig
4-Effect of varying periods of exposure to staphylococcal slime the number of viable cells in cultures with PHA.
on
cell culture period (fig 3). Also, the total numbers of both viable cells and blasts decreased as incubation time with slime increased (fig 4). Some lytic activity seems to be associated with the action of slime. The proportion of viable cells remaining in culture was indistinguishable from the proportion in control cultures, which suggests that affected cells are totally destroyed. Discussion
The extracellular slime produced by Staphylococcus is associated with the ability of the organism to establish foci of infection on implanted plastic foreign bodies, 10,14 perhaps because of the adhesivel2-16 and protective properties of slime. Often such infections are controlled only by removal of the plastic device. The findings reported here suggest that slime also interferes with the function of the cellular immune system. The lymphoproliferative response to two distinct18 polyclonal T-cell stimulators is strongly inhibited by slime, which eventually
epidermidis
of staphylococcal slime on the blastogenic response of mononuclear cells to PHA (0-0) and streptococcal blastogen A
Fig 1—Effect (•-•).
367
destroys affected cells. The inhibitory effect is not due to a general toxic action on cells since surviving cells are as viable as those in control cultures. Slime produces its effect only after it has been incubated with cells for some time, so its action is unlikely to be mediated by rapid cytolysis. Slime may act by activating a subpopulation of cytotoxic cells which are responsible for the lysis of affected cells.19, 20 Preliminary analysis of the surface antigens of surviving cells has revealed that, as in control cultures, most cells are ofT lineage-that is, they react with a monoclonal antibody (Clone 9 - 6, New England Nuclear Lyt-3)21directed against the sheep redblood-cell receptor. In slime-treated cultures, however, much fewer T cells had the surface structures associated with either
helper/inducer (OKT4+) cells.22 It is
or
cytotoxic/suppressor (OKT8)
clear whether slime exposure represses the expression of these antigens borne by differentiated T cells or whether the surviving cells are partially differentiated T cells. The dramatic effects of staphylococcal slime in-vitro suggest that this substance may contribute to the extended course ofS epidermidis foreign-body infections. Although the in-vitro effects of slime are observable only with high concentrations of the substance, slime is presented to the immune system in-vivo as a solid phase-that is, effectively in high concentration. Suppression of normal host responses may not only prolong the course of S epidermidis infection but also increase the risk of other opportunistic infections. not
A preliminary account of this research was presented at the Twenty-Third Interscience Conference on Antimicrobial Agents and Chemotherapy, October, 1983. The research was supported by US Public Health Service research grant HL-30058. We are grateful to the late Dr L. W. Wannamaker and Dr G. Pulverer for valuable discussions and encouragement.
Correspondence should be addressed to E. D. G., Department of Pediatrics and Biochemistry, Division of Infectious Diseases, University of Minnesota Medical School, Box 296, Mayo Memorial Building, 420 Delaware Street SE, Minneapolis, Minnesota 55455, USA. REFERENCES 1.
Callaghan RP, Cohen SJ, Stewart JT. Septicemia due to colonization of Spitz-Holter valves by staphylococci. Five cases treated with methicillin. Br Med J 1961; i:
PARATHYROID HORMONE DOES NOT INHIBIT PLATELET AGGREGATION C. LEITHNER H. SINZINGER
2nd Department of Internal Medicine, University of Vienna, and *Ludwig Boltzmann-Institute of Clinical Endocrinology, Vienna, Austria
The suggestion that parathyroid hormone (PTH) is a major uraemic toxin was examined by testing the effects of synthetic human PTH fragments and synthetic bovine PTH on ADP- induced and collagen-induced platelet aggregation. Whereas the bovine parathyroid-gland extracts inhibited platelet aggregation in a dose-dependent manner, none of the synthetic compounds was effective even at high concentrations. It is suggested that the inhibition of platelet aggregation by extracts of bovine parathyroid glands is not caused by PTH fragments and is probably an effect of other constituents contained in the extract. These findings argue against a role of PTH in the pathogenesis of platelet dysfunction and bleeding tendency in uraemia and were supported by platelet-aggregation studies in 6 patients with primary hyperparathyroidism. Platelet aggregation was normal before and unchanged after surgery of the parathyroid glands.
Summary
Introduction
tendency in uraemia has a multifactorial particular, platelet aggregation is inhibited by medium-sized molecules that accumulate in plasma during uraemia,2 and the interaction between platelets and the vascular wall might be disturbed by an increased production of prostacyclin (PGI2).3,4 Remuzzi and co-workers reported that natural bovine parathyroid hormone (PTH) strongly inhibited platelet aggregation and secretion induced in vitro by different stimuli,s and on the basis of these findings the suggestion that PTH is a major uraemic toxin6 was put forward. We have now investigated platelet aggregation with several pure synthetic PTH preparations. BLEEDING
origin.
In
860-63.
RJ Bacteriological studies on colonized ventriculoatrial shunts. Dev Med Child 1970; 12 (suppl 22): 83-87. Schimke RT, Black PH, Mark VH, Schwartz MN. Indolent Staphylococcus albus or aureus bacteremia after ventriculoatriostomy. N Engl J Med 1961; 264: 264-70 Masur H, Johnson WD. Prosthetic valve endocarditis. J Thorac Cardiovasc Surg 1980;
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80: 31-37. 5. Stinchfield FE,
Bigliani LU, Neu HC, Gross TP, Foster CR. Late hematogenous infection of total joint replacement. J Bone Joint Surg 1980; 62A: 1345-50. 6. Choo MH, Holmes DR, Gersh BJ, et al. Permanent pacemaker infections: Characterization and management. Am J Cardiol 1981; 48: 559-64. 7. Rubin J, Rogers WA, Taylor HM, et al. Peritonitis during continuous ambulatory peritoneal dialysis. Ann Intern Med 1980; 92: 7-13. 8. Duma RJ, Warner JF, Dalton HP. Septicema from intravenous infusions. N Engl J Med 1977; 284: 257-60. 9. Bender JW, Hughes WT. Fatal Streptococcus epidermidis sepsis following bone marrow transplantation. Johns Hopkins Med J 1980; 146: 13-15. 10. Peters G, Locci R, Pulverer G. Microbial colonization of prosthetic devices. II. Scanning electron microscopy of naturally infected intravenous catheters. Zbl Bakt Hyg, I Abt Orig B 1981; 173: 293-99. 11. Locci R, Peters G, Pulverer G Microbial colonization of prosthetic devices. III. Adhesion of staphylococci to lumma of intravenous catheters perfused with bacterial suspensions. Zbl Bakt Hyg, I Abt Orig B 1981; 173: 300-07. 12. Peters G, Locci R, Pulverer G. Adherence and growth of coagulase-negative staphylococci on surfaces of intravenous catheters.J Infect Dis 1982; 146: 479-82. 13. Ludwicka A, Locci R, Jansen B, Peters G, Pulverer G. Microbial colonization of prosthetic devices. V. Attachment of coagulase-negative staphylococci and "slime"-production on chemically pure synthetic polymers. Zbl Bakt Hyg, I Abt Orig B 1983; 177: 527-32. 14. Peters G, Saborowski F, Locci R, Pulverer G Investigations on staphylococcal infection of intravenous endocardial pacemaker-electrodes. Am Heart J (in press). 15 Christensen GD, Simpson WA, Bisno AL, Beachey EH. Adherence of slime-producing strains of Staphyloccocus epidermidis to smooth surfaces. Infect Immun 1982; 37: 318-26. 16. Bayston R, Penny SR. Excessive production of mucoid substance in Staphylococcus SIIA a possible factor in colonization of Holter shunts. Dev-Med Child Neurol 1972, 27 (suppl) 25-28.
J. KOVARIK W. WOLOSZCZUK*
The following synthetic peptides were used: bovine PTH 1-84, human PTH 1-44, 28-48, and 53-84 (Bachem Inc, Torrance, CA, USA), and human PTH 1-34 (Peninsula Laboratories Inc, Belmont, CA, USA). The lyophilised peptides were dissolved in 10 mM acetic acid and diluted in 0.1 M phosphate-buffered saline (PBS, pH 7-4) containing 107o human serum albumin. Bovine parathyroid gland extracts were obtained from Sigma and (’Parathorm’) from Hormon Chemie, Munich, West Germany. These preparations were dialysed exhaustively against isotonic saline (cut-off 2000 daltons) and then diluted in PBS-albumin solution. 20 healthy male volunteers, aged 20-35, who had been free of medication for 4 weeks, served as blood donors. After preparation of platelet-rich plasma (PRP), aggregation tests were carried out with a
17.
18.
Gray ED. Purification and properties of an extracellular blastogen produced by group A streptococci. J Exp Med 1979; 149: 1438-49. Regelmann WE, Gray ED, Wannamaker LW. Characterization of the human cellular immune response to purified group A streptococcal blastogen A. J Immunol 1982; 128: 1631-36.
LV, Rowley DA. Homeostasis of the antibody response. Immunoregulation by NK cells. Science 1983, 222: 581-85. Rubens RP, Henney CS. Studies on the mechanism oflymphocyte mediated cytolysis. VIII. The use of co nA to delineate a distinctive killer T cell subpopulation. J
19 Abruzzo 20
Immunol 1977; 118: 180-86. 21. Kamoun M, Martin P-J, Hansen JA, Brown MA, Siadak AW, Nowinski RC Identification of a human T lymphocyte surface protein associated with the E-rosette receptor. J Exp Med 1981; 153: 207-12. 22. Kung PC, Goldstein G Functional and developmental compartments of human T lymphocytes. Vox Sang 1980; 39: 121-27.