Nanobacteria Do Exist and Actively Participate in the Calcification of Arterial Plaque

By E. Olavi Kajander, MD

Department of Biochemistry, University of Kuopio , Kuopio , Finland

Email olavi.kajander@uku.fi

 

The cause of pathological calcification, including atherosclerosis, dental pulp stones and kidney stones, used to be an enigma, but our science is rapidly clarifying the relationship between nanobacterial infections and disease.  The life-long incidence of kidney stones appears to have increased throughout the whole 20th century, and now occurs in up to 15% of the population.1 Nanobacteria have been linked to human kidney stone and preliminary studies showed Koch’s postulates to be fulfilled.1, 2, 3, 4  Calcified hard plaques is now a common form of coronary heart disease but were surprisingly a clinical rarity 100 years ago5. Calcified plaques can lead to acute myocardial infarct, because apatite (calcium phosphate mineral) exposed to blood activates a thrombotic cascade. Nanobacteria were the first (may still be the only) calcium-phosphate mineral containing particles isolated from human blood. Radioactively labeled nanobacteria were shown to accumulate in rabbit aorta and aortic valve, although their main elimination route was excretion via kidneys into urine6. This study pointed to the potential role that nanobacteria could have in atherosclerosis, heart valve calcification and kidney stone formation. Nanobacteria were present and actively involved in the processes: 1. Nanobacteria were shown to be active nidi forming the right type of calcified mineral. Active nidus means a center of calcification that can mediate calcium-phosphate mineral formation under non-saturating calcium and phosphate concentrations. In fact, nanobacteria are so good in utilizing these minerals that they consume all free calcium and/or phosphate from their culture medium, whichever is first consumed to zero2. 2. Nanobacteria have and release endotoxin7 and thereby stimulate and mediate chronic local inflammatory reactions in atherosclerotic plaque. 3. Nanobacteria have been shown to infect humans and infections last possibly life-long. 4. Almost 100% of atherosclerotic patients in USA and in Finland have antinanobacteria antibodies in their serum, whereas in healthy blood donors antinanobacteria antibodies are present in about 15% (see web pages of Nanobacteria Minisymposium held at Kuopio last year). 5. Nanobacteria have been shown to be susceptible to several antibiotics and sequestering agents8. Since nanobacteria form calcific biofilm it is clear that their eradication needs combination chemotherapy directed at the biofilm, the calcified deposits and the agent. Such chemotherapy can be very demanding since nanobacteria grow very slowly. Thus lessons learned from the treatment of tuberculosis or leprosy should be remembered.

 

Dr. Cranton has not personally studied nanobacteria, but has pointed out that nanobacteria do not exist and cannot cause atherosclerosis. His motivation seems to be to stop ongoing combination drug trials that aim at verifying whether nanobacteria cause atherosclerosis and how to cure this infectious process. These studies use the same principles that vindicated Helicobacter pylori in peptic ulcer disease: curative therapy was the evidence for the causative role of the agent. That approach lead into a revolution in the therapy of Helicobacter pylori-mediated diseases. This was a good thing.

 

Nanobacteria form calcific biofilms and replicate under blood/serum conditions, as was first published by Kajander and Ciftctioglu9, a fact that has been reproduced and published by many research groups, e.g., NASA, Mayo Clinic, McGill University, Exeter University, University of Illinois, Alcala University and University of Ulm. Dr. Cranton refers only to one NIH researcher, Cisar10, who could also culture similar particles from serum and human saliva sources. Cisar had no positive or negative controlled controls and did not use valid published immunological control methods. Cisar verified our findings, and also confirmed the extreme difficulties in performing PCR, but finally suggested his opinion that the culturable particles cannot be bacteria, since they were too small, were not inhibited with a respiratory poison, nucleic acids could not be detected with standard procedures and their protein patterns revealed only few proteins, much less than one would expect from a common bacterium. Cisar did not sequence any proteins. He did not do any DNA work besides staining with Hoechst 33258, where he got the same weakly positive result than we did. To the contrary of Dr. Cranton’s claims, Cisar did not do a PCR phylogenetic analysis using 16S rDNA sequences simply because he did not get any: all his samples, including negative controls, were contaminated with Pseudomonas sp. This fact is clearly stated in his paper and means that he did not have any data on the bacterial status of nanobacteria.

 

We do totally agree with Cisar that nanobacteria are not common bacteria. Nanobacterial samples may contain pieces of DNA from common bacteria, which makes phylogenetic PCR analysis using universal primers practically impossible and worthless. PCR analysis assumes that the ribosomal gene has ‘universial’ sequences detectable by the primers, but this is not true for nanobacteria and other organisms. When we originally named nanobacteria in 1990, we wanted to separate them from common bacteria. Unfortunately, the “bacteria” part of the name still lures less-well informed scientists to compare nanobacteria with E. coli and other common bacteria, which are 100-fold bigger and produce biomass 10,000-fold faster than nanobacteria.

 

As pointed out by Dr. Cranton, apatite can be formed under super-saturating concentrations of calcium and phosphate via several mechanisms. To our knowledge, nanobacteria-mediated calcification is the only mechanism to make apatite at non-saturating levels of calcium and phosphate. Cisar did not follow saturation degree analysis in his studies although saliva is known to be highly super-saturated with calcium and phosphate. Yet Cisar suggested as an alternative explanation nanobacteria to be replicating apatite mineral particles.

 

Naming an agent as particles or nanobacteria, living or non-living but self-replicating, has relatively little meaning with respect to causing disease, e.g., the atherosclerotic process. The fundamental importance is that these self-replicating special particles that we call Nanobacterium sanguineum are found in blood and in atheroslerotic plaques. This fact was initially presented by Laszlo Puskas at Nanobacteria Minisymposium held at Kuopio last year, detected by us (unpublished data) and finally now has been verified by Mayo Clinic and University of Texas11 .  Macrophages in the aorta and other arteries internalise nanobacteria and stimulate a local chronic inflammatory cascade, that eventually proceeds to nanobacteria-mediated calcification. In fact, according to Dr. Cranton, Cisar10 also verified the medical importance of the nanobacteria phenomenon: he stated that submicroscopic crystals of calcium apatite, as occur in plasma, were shown to be nucleators of biomineralization. However, the presence of apatite crystals had earlier been shown only by us, but the role of apatite biofilms in blood clotting and blood vessels is well known. Thus the described presence of apatite particles could be potentially deadly, in fact a mechanism initiating myocardial infarction.

 

Dr. Cranton is putting forward ungrounded claims on nanobacteria and on therapy trials aiming at eradicating them. The claims need short comments:

  1. Nanobacteria have been shown to be unique calcifying agents. They can be cultured and passaged in cell culture media mimicking serum in composition. Atherosclerotic plaques contain nanobacteria as detected by researchers from Mayo Clinic and the University of Texas11 .
  2. The administration strategy of EDTA must maintain drug blood levels on a sustained therapeutically effective level.  I have personally conducted serum-EDTA levels on patients treated with NanobacTX, and this prescription combination is effective.
  3. Many drugs are successfully administered rectally. In most cases rectal administration is highly effective. The efficacy of NanobacTX to deliver sustained therapeutic levels of EDTA has been determined using analytical methods developed at Kuopio University .
  4. In antimicrobial therapy, administration route, dose and frequency has to be carefully considered in order to maintain high enough drug levels. In most infectious diseases, it would be unwise to administer a drug intravenously once or twice a week while knowing that the therapeutic concentrations are retained only for a short period after the administration.
  5. Dr. Cranton states: ‘It makes little sense to assume that calcification of artery walls are an important indicator of clinical significance of the disease state’. This statement is ungrounded and against the perspectives how myocardial infarcts develop.  Additionally, the clinical & scientific literature is heavily replete with evidence substantiating the pivotal importance of calcification processes in the development of atherosclerotic disease and contrary to Dr. Cranton’s statement.
  6. The purpose of NanobacTX treatment is to be effective. This means that it must decrease calcification in atherosclerotic plaques. It is obvious that EDTA alone is not sufficient to reach this goal, because under in vitro tests8 EDTA alone has no inhibitory action on nanobacteria at clinically achievable EDTA concentrations. Dr. Cranton has also stated that intravenous EDTA therapy does not decrease calcification scores. A combination therapy is required to reach this aim.  NanobacTX is uniquely effective in decreasing coronary artery calcification12.
  7. James Robert s, MD, FACC, has reported significant reduction in EBCT scores. This is an objective way to measure the effect of therapy. Benedict Maniscalco, MD, FACC, will publish a formal study on NanobacTX therapy in Circulation12.
  8. It is unknown at this point how nanobacteria infection affects homocysteine levels.

 

There is new published evidence that nanobacteria do exist8,11.12 and are biological entities reacting, e.g., to light13, cause kidney stones and are found in human atherosclerotic plaques. As discussed earlier, new evidence also indicates that several drugs are effective in-vitro against nanobacteria, but in-vivo eradication of nanobacterial biofilms and calcification requires combination therapy. Administration of EDTA alone is ineffective towards this goal, since it will not kill nanobacteria at the blood concentrations achievable and has been shown, by itself, to be ineffective in reducing coronary artery calcification scores.  NanobacTX, a unique prescription combinatory nanobiotic is specifically formulated to eradicate nanobacterial biofilm, calcification and the nanobacteria themselves and has been shown in validated IRB-monitored clinical cardiology studies to be uniquely effective in doing so as measured by significant decreases in coronary artery atherosclerotic plaque burden, and other measurement parameters soon to be announced12.

 

E. Olavi Kajander, MD

Email olavi.kajander@uku.fi

 

 

 


References

  1. Kajander, EO, Ciftcioglu, N, Miller-Hjelle, MA, Hjelle, JT. Nanobacteria: controversial pathogens in nephrolithiasis and polycystic kidney disease. Curr Opin Nephrol Hypertens 2001:445-451.
  2. Ciftcioglu N, Bjorklund M, Kuorikoski K, et al. Nanobacteria: an infectious cause for kidney stone formation. Kidney Int 1999; 56:1893-1898.
  3. Garcia Cuerpo E, Kajander EO, Ciftcioglu N, et al. Nanobacteria. Un modelo de neo-litogenesis experimental. Arch. Esp. Urol 2000; 53:291-303.
  4. Sommer, AP, Kajander, EO. Nanobacteria-induced kidney stone formation: novel paradigm based on the FERMIC model. Crystal Growth & Design 2002, in press.
  5. Meade, TW. Cardiovascular disease – linking pathology and epidemiology. Int J Epidemiol 2001, 30:1179-1183.
  6. Akerman KK, Kuikka JT, Ciftcioglu N, et al. Radiolabeling and in vivo distribution of nanobacteria in rabbit. Proc SPIE Int Soc Opt Eng 1997;3111:436-442.
  7. Hjelle JT, Miller-Hjelle MA, Poxton IR, et al.  Endotoxin and nanobacteria in polycystic kidney disease. Kidney Int 2000; 57:2360-2374.
  8. Ciftcioglu, N, Miller-Hjelle, MA, Hjelle, JT, Kajander, EO. Inhibition of nanobacteria by antimicrobial drugs as measured by a modified microdilution method. Antimicrob Agents Chemother 2002, 46:2077-2086.
  9. Kajander EO, Ciftcioglu N. Nanobacteria: An alternative mechanism for pathogenic intra- and extracellular calcification and stone formation. Proc Natl Acad Sci USA 1998; 95:8274-8279.
  10. Cisar JO, Xu D-Q, Thompson J, Swaim W, Hu L, Kopecko DJ. An alternative explanation of nanobacteria-induced biomineralization. Proc Natl Acad Sci USA 2000; 97:11511-11515.
  11. Rasmussen, TE, Kirkland , BL, Chalesworth, J et al. Electron microscopic and immunological evidence of nanobacterial-like structures in calcified carotid arteries, aortic aneurysms, and cardiac valves. JACC 2002, 39 (Suppl 1):206A.
  12. Maniscalco, BS. Letter to the editor. Circulation 2002, in press.
  13. Sommer, AP, Hassinen, HI, Kajander, EO. Light-induced replication of nanobacteria: a preliminary report. J Clin Laser Med Surg 2002, 20:241-244.