To Townsend Letter
Response to November 2002 issue and Elmer M. Cranton's letter
'Alleged Nanobacteria Do Not Cause Calcification of Arterial
Plaque'

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

Dear Editor;  Jonathan Collin, MD:
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 labelled 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 Roberts, 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.