Acta Biologica Hungarica

, Volume 56, Issue 3–4, pp 233–245 | Cite as

Detection of Nanobacteria-Like Particles in Human Atherosclerotic Plaques

  • L. G. PuskásEmail author
  • L. Tiszlavicz
  • Zs. Rázga
  • L. L. Torday
  • T. Krenács
  • J. Gy. Papp


Recent and historical evidence is consistent with the view that atherosclerosis is an infectious disease or microbial toxicosis impacted by genetics and behavior. Because small bacterial-like particles, also known as nanobacteria have been detected in kidney stones, kidney and liver cyst fluids, and can form a calcium apatite coat we posited that this agent is present in calcified human atherosclerotic plaques. Carotid and aortic atherosclerotic plaques and blood samples collected at autopsy were examined for nanobacteria- like structures by light microscopy (hematoxylin-eosin and a calcium-specific von Kossa staining), immuno-gold labeling for transmission electron microscopy (TEM) for specific nanobacterial antigens, and propagation from homogenized, filtered specimens in culture medium. Nanobacterial antigens were identified in situ by immuno-TEM in 9 of 14 plaque specimens, but none of the normal carotid or aortic tissue (5 specimens). Nanobacteria-like particles were propagated from 26 of 42 sclerotic aorta and carotid samples and were confirmed by dot immunoblot, light microscopy and TEM. [3H]L-aspartic acid was incorporated into high molecular weight compounds of demineralized particles. PCR amplification of 16S rDNA sequences from the particles was unsuccessful by traditional protocols. Identification of nanobacteria-like particles at the lesion supports, but does not by itself prove the hypothesis that these agents contribute to the pathogenesis of atherosclerosis, especially vascular calcifications.


Nanobacteria-like particles atherosclerosis carotid artery aorta vascular plaque 



Dulbecco’s Eagle’s medium


transmission electron microscopy






fetal bovine serum


low density lipoprotein




bovine serum albumin


phosphate-buffered saline




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  1. 1.
    Sifil, A., Cavdar, C., Uretmen, P., Yavuzsen, T., Nazli, C., Camsari, T. (2000) Atherosclerotic heart disease in dialysis patients. Nephron 86, 387–388.CrossRefGoogle Scholar
  2. 2.
    Kiechl, S., Egger, G., Mayr, M., Wiedermann, C. J., Bonora, E., Oberhollenzer, F., Muggeo, M., Xu, Q., Wick, G., Poewe, W., Willeit, J. (2001) Chronic infections and the risk of carotid atherosclerosis: Prospective results from a large population study. Circulation 103, 1064–1070.CrossRefGoogle Scholar
  3. 3.
    Kessler, M. Atherosclerosis and hemodialysis. Nephrologie 21, 349–350.Google Scholar
  4. 4.
    Zoccali, C., Benedetto, F. A., Mallamaci, F., Tripepi, G., Fermo, I., Foca, A., Paroni, R., Malatoni, L. S. (2000) Inflammation is associated with carotid atherosclerosis in dialysis patients. Creed Investigators. Cardiovascular Risk Extended Evaluation in Dialysis Patients. J. Hypertens. 18, 1207–1213.CrossRefGoogle Scholar
  5. 5.
    Lonnenmann, G. (2000) Chronic inflammation in hemodialysis: the role of contaminated dialysate. Blood Purif. 18, 214–223.CrossRefGoogle Scholar
  6. 6.
    Zimmermann, J., Herrlinger, S., Pruy, A., Metzger, T., Wanner, C. (1999) Inflammation enhances cardiovascular risk and mortality in hemodialysis patients. Kidney Int. 55, 648–658.CrossRefGoogle Scholar
  7. 7.
    Lopes-Virella, M. F., Virella, G. (1985) Immunological and microbiological factors in the pathogenesis of atherosclerosis. Clin. Immunol. Immunopathol. 37, 377–386.CrossRefGoogle Scholar
  8. 8.
    Bachmaier, K., Le, J., Penninger, J. M. (2000) Catching heart disease: antigenic mimicry and bacterial infections. Nat. Med. 6, 841–842.CrossRefGoogle Scholar
  9. 9.
    Burian, K., Kis, Z., Virok, D., Endresz, V., Prohaszka, Z., Duba, J., Berencsi, K., Boda, K., Horvath, L., Romics, L., Fust, G., Gonczol, E. (2001) Independent and joint effects of antibodies to human heat-shock protein 60 and Chlamydia pneumoniae infection in the development of coronary atherosclerosis. Circulation 103, 1503–1508.CrossRefGoogle Scholar
  10. 10.
    Chiu, B. (1999) Multiple infections in carotid atherosclerotic plaques. Am. Heart. J. 138, S534–536.CrossRefGoogle Scholar
  11. 11.
    Leionen, M., Saikku, P. (2000) Infections and atherosclerosis. Scand. Cardiovasc. J. 34, 12–20.CrossRefGoogle Scholar
  12. 12.
    Ellis, R. W. (1997) Infection and coronary heart disease. J. Med. Microbiol. 46, 535–539.CrossRefGoogle Scholar
  13. 13.
    Markus, H. S., Sitzer, M., Carrington, D., Mendall, M. A., Steinmetz, H. (1999) Chlamydia pneumoniae infection and early asymptomatic carotid atherosclerosis. Circulation 100, 832–837.CrossRefGoogle Scholar
  14. 14.
    Stenvinkel, P., Heimburger, O., Jogestrand, T., Karnell, A., Samuelsson, A. (1999) Does persistent infection with Chlamydia pneumoniae increase the risk of atherosclerosis in chronic renal failure? Kidney Int. 55, 2531–2532.CrossRefGoogle Scholar
  15. 15.
    Coles, K. A., Plant, A. J., Riley, T. V., Smith, D. W., McQuillan, B. M., Thompson, P. L. (1999) Lack of association between seropositivity to Chlamydia pneumoniae and carotid atherosclerosis. Am. J. Cardiol. 84, 825–828.CrossRefGoogle Scholar
  16. 16.
    Gasbarrini, A., Cremonini, F., Armuzzi, A., Ojetti, V., Candelli, M., Di Campli, C., Sanz-Torre, E., Pola, R., Gasbarrini, G., Pola, P. (1999) The role of Helicobacter pylori in cardiovascular and cerebrovascular diseases. J. Physiol. Pharmacol. 50, 735–742.PubMedGoogle Scholar
  17. 17.
    Adam, E. (1987) High levels of cytomegalovirus antibody in patients requiring vascular surgery for atherosclerosis. Lancet 2, 291–293.CrossRefGoogle Scholar
  18. 18.
    Horvath, R., Cerny, J., Benedik, J. Jr, Hokl, J., Jelinkova, I., Benedik, J. (2000) The possible role of human cytomegalovirus (HCMV) in the origin of atherosclerosis. J. Clin. Virol. 16, 17–24.CrossRefGoogle Scholar
  19. 19.
    Chiu, B., Viira, E., Tucker, W., Fong, I. W. (1997) Chlamydia pneumoniae, cytomegalovirus, and herpes simplex virus in atherosclerosis of the carotid artery. Circulation 96, 2144–2148.CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Mininck, C. R., Fabricant, C. G., Fabricant, J., Litrenta, M. M. (1979) Atheroarteriosclerosis induced by infection with a herpesvirus. Am. J. Pathol. 96, 673–706.Google Scholar
  21. 21.
    Wainwright, M. (1999) Nanobacteria and associated ‘elementary bodies’ in human disease and cancer. Microbiology 145, 2623–2624.CrossRefGoogle Scholar
  22. 22.
    Wilson, P. W. F., Kauppila, L. I., O’Donnell, C. J., Kiel, D. P., Hannan, M., Polak, J. M., Cupples, A. (2001) Abdominal aortic calcific deposits are an important predictor of vascular morbidity and mortality. Circulation 103, 1529–1534.CrossRefGoogle Scholar
  23. 23.
    Huang, H., Virmani, R., Younis, H., Burke, A. P., Kamm, R. D., Lee, R. T. (2001) The impact of calcification on the biomechanical stability of atherosclerotic plaques. Circulation 103, 1051–1056.CrossRefGoogle Scholar
  24. 24.
    Shioi, A., Mori, K., Jono, S., Wakikawa, T., Hiura, Y., Koyama, H., Okuno, Y., Nishizawa, Y., Morii, H. (2000) Mechanism of atherosclerotic calcification. Z. Kardiol. 89 Suppl. 2, 75–79.CrossRefGoogle Scholar
  25. 25.
    Shanahan, C. M., Proudfoot, D., Tyson, K. L., Cary, N. R., Edmonds, M., Weissberg, P. L. (2000) Expression of mineralisation-regulating proteins in association with human vascular calcification. Z. Kardiol. 89 Suppl. 2, 63–68.CrossRefGoogle Scholar
  26. 26.
    Jeziorska, M., McCollum, C., Woolley, D. E. (1998) Calcification in atherosclerotic plaque of human carotid arteries: associations with mast cells and macrophages. J. Pathol. 185, 10–17.CrossRefGoogle Scholar
  27. 27.
    Hsu, H. H., Tawfik, O., Sun, F. (2000) Effects of lectins on calcification by vesicles isolated from aortas of cholesterol-fed rabbits. Biochim. Biophys. Acta 1464, 262–272.CrossRefGoogle Scholar
  28. 28.
    Kajander, E. O., Ciftcioglu, N. (1998) Nanobacteria: an alternative mechanism for pathogenic intraand extracellular calcification and stone formation. Proc. Natl. Acad. Sci. USA 95, 8274–8279.CrossRefGoogle Scholar
  29. 29.
    Kajander, E. O., Kuronen, I., Åkerman, K., Pelttari, A., Ciftcioglu, N. (1997) Nanobacteria from blood, the smallest culturable autonomously replicating agent on Earth. Proc. SPIE Int. Soc. Opt. Eng. 3111, 420–428.Google Scholar
  30. 30.
    Ciftcioglu, N., Bjorklund, M., Kuorikoski, K., Bergstrom, K., Kajander, E. O. (1999) Nanobacteria: an infectious cause for kidney stone formation. Kidney Int. 56, 1893–1898.CrossRefGoogle Scholar
  31. 31.
    Hjelle, J. T., Miller-Hjelle, M. A., Poxton, I. R., Kajander, E. O., Ciftcioglu, N., Jones, M. L., Caughey, R. C., Brown, R., Millikin, P. D., Darras, F. S. (2000) Endotoxin and nanobacteria in polycystic kidney. Kidney Int. 57, 2360–2374.CrossRefGoogle Scholar
  32. 32.
    Kajander, E. O., Ciftcioglu, N., Miller-Hjelle, M. A., Hjelle, J. T. (2001) Nanobacteria: controversial pathogens in nephrolithiasis and polycystic kidney disease. Curr. Opin. Nephrol. Hypertens. 10, 445–452.CrossRefGoogle Scholar
  33. 33.
    Franklin, R. M., Martin, M. T. (1980) Staining and histochemistry of undecalcified bone embedded in a water-miscible plastic. Stain Technol. 55, 313–321.CrossRefGoogle Scholar
  34. 34.
    Cisar, J. O., Xu, D. Q., Thompson, J., Swaim, W., Hu, L., Kopecko, D. J. (2000) An alternative interpretation of nanobacteria-induced biomineralization. Proc. Natl. Acad. Sci. USA 97, 11511–11515.CrossRefGoogle Scholar
  35. 35.
    Ciftcioglu, N., Kajander, E. O. (1998) Interaction of nanobacteria with cultured mammalian cells. Pathophysiology 4, 259–270.CrossRefGoogle Scholar
  36. 36.
    Ciftcioglu, N., Miller-Hjelle, M. A., Hjelle, J. T., Kajander, E. O. (2002) Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method. Antimicrob. Agents Chemother. 46, 2077–2086.CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Barr, S. C., Linke, R. A., Janssen, D., Guard, C. L., Smith, M. C., Daugherty, C. S., Scarlett, J. M. (2003) Detection of biofilm formation and nanobacteria under long-term cell culture conditions in serum samples of cattle, goats, cats, and dogs. Am. J. Vet. Res. 64, 176–182.CrossRefGoogle Scholar
  38. 38.
    Kajander, E. O., Ciftcioglu, N., Aho, K., Garcia-Cuerpo, E. (2003) Characteristics of nanobacteria and their possible role in stone formation. Urol. Res. 31, 47–54.PubMedGoogle Scholar
  39. 39.
    Conte, V. A., Grases, F. F., Costa-Bauza, A., Piza, R. P. (2001) Microinfections and kidney lithiasis. Arch. Esp. Urol. 54, 855–860.Google Scholar
  40. 40.
    Li, Y., Wen, Y., Yang, Z., Wei, H., Liu, W., Tan, A., Wu, X., Wang, Q., Huang, S., Kajander, E. O., Ciftcioglu, N. (2002) Culture and identification of nanobacteria in bile. Zhonghua Yi Xue Za Zhi. 82, 1557–1560.PubMedGoogle Scholar
  41. 41.
    Morgan, M. B. (2002) Nanobacteria and calcinosis cutis. J. Cutan Pathol. 29, 173–175.CrossRefGoogle Scholar
  42. 42.
    Drancourt, M., Jacomo, V., Lepidi, H., Lechevallier, E., Grisoni, V., Coulange, C., Ragni, E., Alasia, C., Dussol, B., Berland, Y., Raoult, D. (2003) Attempted isolation of Nanobacterium sp. microorganisms from upper urinary tract stones. J. Clin. Microbiol. 41, 368–372.CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Price, P. A., Thomas, G. R., Pardini, A. W., Figueira, W. F., Caputo, J. M., Williamson, M. K. (2002) Discovery of a high molecular weight complex of calcium, phosphate, fetuin, and matrix gamma-carboxyglutamic acid protein in the serum of etidronate-treated rats. J. Biol. Chem. 277, 3926–3934.CrossRefGoogle Scholar
  44. 44.
    Price, P. A., Lim, J. E. (2003) The inhibition of calcium phosphate precipitation by fetuin is accompanied by the formation of a fetuin-mineral complex. J. Biol. Chem. 278, 22144–22152.CrossRefGoogle Scholar
  45. 45.
    Khullar, M., Sharma, S. K., Singh, S. K., Bajwa, P., Sheikh, F. A., Relan, V., Sharma, M. (2004) Morphological and immunological characteristics of nanobacteria from human renal stones of a north Indian population. Urol. Res. 32, 190–195.CrossRefGoogle Scholar
  46. 46.
    Ciftcioglu, N., McKay, D. S., Kajander, E. O. (2003) Association between nanobacteria and periodontal disease. Circulation 108, 58–59.Google Scholar
  47. 47.
    Sedivy, R., Battistutti, W. B. (2003) Nanobacteria promote crystallization of psammoma bodies in ovarian cancer. APMIS 111, 951–4.CrossRefGoogle Scholar

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© Akadémiai Kiadó, Budapest 2005

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Authors and Affiliations

  • L. G. Puskás
    • 1
    Email author
  • L. Tiszlavicz
    • 3
  • Zs. Rázga
    • 3
  • L. L. Torday
    • 4
  • T. Krenács
    • 3
  • J. Gy. Papp
    • 2
    • 4
  1. 1.Laboratory of Functional GenomicsBiological Research CenterSzegedHungary
  2. 2.Research Unit for Cardiovascular Pharmacology of the Hungarian Academy of SciencesSzegedHungary
  3. 3.Department of PathologyUniversity of SzegedSzegedHungary
  4. 4.Department of Pharmacology and PharmacotherapyUniversity of SzegedSzegedHungary

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