E. Olavi Kajander

Interaction of nanobacteria with cultured mammalian cells

Abstract Nanobacteria were recently isolated from human blood and commercial fetal bovine serum (FBS) and were located in the α -2 subgroup of proteobacteria based upon their 16S rRNA gene sequence. They can be cultured even in the absence of mammalian cells, and have extraordinary properties, like very slow growth rate and an impermeable cell wall, making their detection difficult by standard microbiological techniques. Since they are present in FBS, and thus in cell cultures, it is essential to clarify their effects on cultured mammalian cells. In this study, we show that four out of six nanobacterial isolates from different sera exerted a cytotoxic effect on 3T6 fibroblasts verified by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] viability assay, lactate dehygrogenase (LDH) release and by direct microscopy. The cytotoxic effect of nanobacteria was attenuated after they had been subcultured several times. The cytotoxic effect was similar with all tested murine and human fibroblastoid cell lines. Differential interference contrast and electron microscopy, and FITC staining with specific monoclonal antibodies indicated selective, possibly receptor-mediated adherence, followed by internalization and cytotoxicity in the 3T6 fibroblasts used as a model in these interaction studies. Thus, nanobacteria have a special way of invading mammalian cells: they trigger cells that are not normally phagocytic to engulf them. These organisms seem to be an important cause for cell vacuolization, poor thriving and unexpected cell lysis, problems frequently encountered in mammalian cell culture.

Characteristics of nanobacteria and their possible role in stone formation

Kidney stone formation is a multifactorial disease in which the defence mechanisms and risk factors are imbalanced in favour of stone formation. We have proposed a novel infectious agent, mineral forming nanobacteria (NB), to be active nidi that attach to, invade and damage the urinary epithelium of collecting ducts and papilla forming the calcium phosphate center(s) found in most kidney stones. Stone formation may proceed in urine supersaturated with calcium phosphate, calcium oxalate and uric acid/urate under the influence of crystallization promoters and inhibitors. Our hypothesis underlines the role of active nidi: even supersaturated urine requires nidi for crystallization to appear.

Nanoforms: A new type of protein-associated mineralization

Abstract Controversy surrounds the interpretation of various nano-phenomena as being living organisms. Incubation of fetal bovine serum under standard cell culture conditions results in the formation of free entities in solution, here referred to as nanoforms. These nanoforms, when examined by transmission electron microscopy, have a distinct ovoid morphology ranging in size from tens to hundreds of nanometers. They are composed of hydroxyapatite and proteins and constitute a novel form of protein-associated mineralization. No detectable cell structure resembling bacteria is apparent. However, immunodetection of the proteins associated with the nanoforms, by two specific monoclonal antibodies, suggests a possible biogenic origin. The significance of nanoforms for the recognition of biological activity in ancient geological systems is discussed. The mode of mineralization in nanoforms is also compared to matrix-mediated calcification in vertebrates.

Metabolism, cellular actions, and cytotoxicity of selenomethionine in cultured cells

Selenomethionine metabolism and the biochemical basis for its cytotoxicity were analyzed in cultured human and murine lymphoid cells. The metabolic pathways were also addressed, using purified mammalian enzymes and crude tissue extracts. Selenomethionine was found to be effectively metabolized toS-adenosylmethionine analog, and that analog was further metabolized in transmethylation reactions and in polyamine synthesis, similarly to the corresponding sulphur metabolites of methionine. Selenomethionine did not block these pathways, nor was there a specific block on the synthesis of DNA, RNA, or proteins when added to the culture medium. Selenomethionine showed cytotoxicity at above 40 μM levels. Yet, low selenomethionine levels (10 μM) could replace methionine and support cell growth in the absence of methionine. Selenomethionine toxicity took place concomitantly with changes inS-adenosylmethionine pools. D-form was less cytotoxic than L-form. Methionine concentration modified the cytotoxicity. Together, this indicates that selenomethionine uptake and enzymic metabolism are involved in the cytotoxicity in a yet unknown way.

Association between Randall's Plaque and Calcifying Nanoparticles

Objectives Randall initially described calcified subepithelial papillary plaques, which he hypothesized as nidi for urinary calculi. The discovery of calcifying nanoparticles (CNP), also referred to as nanobacteria, in calcified soft tissues has raised another hypothesis about their possible involvement in urinary stone formation. This research is the first attempt to investigate the potential association of these two hypotheses. Methods We collected renal papilla and blood samples from 17 human patients who had undergone laparoscopic nephrectomy. Immunohistochemical staining (IHS) was applied using monoclonal antibody (mAb) against CNP. Homogenized papillary tissues and serum samples were cultured for CNP. Scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDS) were performed on papillary samples. Serum samples were tested for CNP antigen and antibody with enzyme-linked immunosorbent assay (ELISA). Results Randall’s plaques (RP) were visible on gross inspection in 11 out of 17 samples. IHS was positive for CNP antigen in 8 of the visually positive samples, but in only 1 of the remaining samples. SEM revealed spherical apatite-formations in 14 samples confirmed by EDS analysis. In cultures, all serum samples and 13 tissue homogenates grew CNP. In ELISA, 14 samples were positive for CNP-antigen and 11 samples were positive for CNP-antibody. Conclusion There was evidence of a link between detection of CNP and presence of RP. Although causality was not demonstrated, these results suggest that further studies with negative control samples should be made to explore the etiology of RP formation, thus leading to a better understanding of the pathogenesis of stone formation.

Radiolabeling and in-vivo distribution of nanobacteria in rabbits

Nanobacteria are minute bacteria recently isolated from mammalian blood. They encapsulate themselves with apatite mineral. Cultured nanobacteria were radiolabeled with 99mTc, using a method which has been previously used for labeling red blood cells with 99mTc, and in vivo distribution of nanobacteria was followed with Single Photon Emission Computed Tomography (SPECT) imaging. The labeling yield was over 30%. Two rabbits were studied using dynamic planar imaging performed in the AP-position immediately after injection. Serial SPECT scans were acquired up to 24 h and one planar image was taken at 45 h. A control study was performed administering a similar dose of [99mTc] labeled albumin nanocolloids. Regional nanobacteria-to- nanocolloid ratios were calculated along with time and tissues (45 h) were analyzed for radioactivity and for nanobacteria. The main finding was that radiolabeled nanobacteria remained intact and showed a tissue specific distribution with a high accumulation in the kidneys and also in urine. Spleen, stomach, heart and intestine also showed increased uptake. Excretion into urine started 10 - 15 min after injection. These were live nanobacteria in the urine, which had better capabilities to penetrate into cells in vitro. The nanobacteria accessed the urine via tubular cells since nanobacteria were found in their cytoplasm and tubular surfaces. The results suggest that nanobacteria utilize endocytic transport of tubular cells and may be involved in the pathogenesis of mineral formation in mammalian kidney stones.

Association Between Nanobacteria and Periodontal Disease

To the Editor: Hung et al1 recently reported that the association between periodontal disease and incident tooth loss and peripheral arterial disease (PAD) is probably mediated via the oral infection-inflammation pathway. Discussions on the same hypothesis have been continuing for many decades. But so far, no clear cause-and-effect relationship has been found. Part of the link between these two diseases may be discovered through novel investigations of the opportunistic, infectious bacteria that colonize the mouth, form biofilm, cause periodontal disease, and activate white blood cells to release proinflammatory mediators that may contribute to heart disease and stroke. There are >300 species of known bacteria forming populations of several hundred billion in the human oral cavity. The number of bacteria reaches a thousand billion when the mouth is not sufficiently cleaned. Coronary atherosclerosis (CA) has been suggested to be an inflammatory disease in which chronic dental infections may trigger pathogenic mechanisms in the walls of arteries. Vascular …

Extraordinary survival of nanobacteria under extreme conditions

Nanobacteria show high resistance to gamma irradiation. To further examine their survival in extreme conditions several disinfecting and sterilizing chemicals as well as autoclaving, UV light, microwaves, heating and drying treatments were carried out. The effect of antibiotics used in cell culture were also evaluated. Two forms of nanobacteria were used in the tests: nanobacteria cultured in serum containing medium, and nanobacteria cultured in serum-free medium, the latter being more mineralized. Nanobacteria, having various amounts of apatite on their surfaces, were used to analyze the degree of protection given by the mineral. The chemicals tested included ethanol, glutaraldehyde, formalin, hypochlorite, hydrogen peroxide, hydrochloric acid, sodium hydroxide, detergents, and commercial disinfectants at concentrations generally used for disinfection. After chemical and physical treatments for various times, the nanobacteria were subcultered to detect their survival. The results show unique and wide resistance of nanobacteria to common agents used in disinfection. It can also be seen that the mineralization of the nanobacterial surface furthermore increases the resistance. Survival of nanobacteria is unique among living bacteria, but it can be compared with that observed in spores. Interestingly, nanobacteria have metabolic rate as slow as bacterial spores. A slow metabolic rate and protective structures, like mineral, biofilm and impermeable cell wall, can thus explain the observations made.

Sedimentary rocks in our mouth: dental pulp stones made by nanobacteria

The mechanisms of dental pulp stone formation are still largely unknown. Pulp stones are mainly composed of carbonate apatite. Only few experimental reports have elucidated the potential of some selected bacteria to produce apatite under in vitro conditions using special calcification media. The tested stone forming bacteria were, in fact, often better known for their cariogenic potential. Our preliminary work with 18 dental pulp stones from Turkey, selected only by severity of the stone formation, indicated the presence of nanobacterial antigens in the demineralized stones. Furthermore, high incidence of kidney stones and gall stones in the patient group and in their parents was found. This raises the implication that nanobacteria may enter the body also via oral route, in addition to the parenteral and transplacental routes. The role of nanobacteria in dental pulp stone formation was further studied by following nanobacterial colonization and mineral formation on human tooth in vitro. Two molar teeth, one having pulp stone and one without, were vertically cut into two pieces, sterilized by autoclaving and incubated with or without nanobacteria in DMEM. Electron microscopic observations indicate that nanobacteria can cause apatite stone formation on tooth surface. The sever from of dental pulp stone formation might be associated with nanobacteria. This form of dental disease results in loss of teeth due to osteolytic processes. This addresses the necessity for a study on unconventional mineral-forming bacteria as a cause for human diseases.

Stone formation and calcification by nanobacteria in the human body

The formation of discrete and organized inorganic crystalline structures within macromolecular extracellular matrices is a widespread biological phenomenon generally referred to as biomineralization. Recently, bacteria have been implicated as factors in biogeochemical cycles for formation of many minerals in aqueous sediments. We have found nanobacterial culture systems that allow for reproducible production of apatite calcification in vitro. Depending on the culture conditions, tiny nanocolloid-sized particles covered with apatite, forming various size of aggregates and stones were observed. In this study, we detected the presence of nanobacteria in demineralized trilobit fossil, geode, apatite, and calcite stones by immunofluorescence staining. Amethyst and other quartz stones, and chalk gave negative results. Microorganisms are capable of depositing apatite outside the thermodynamic equilibrium in sea water. We bring now evidence that this occurs in the human body as well. Previously, only struvite kidney stones composed of magnesium ammonium phosphate and small amounts of apatite have been regarded as bacteria related. 90 percent of demineralized human kidney stones now screened, contained nanobacteria. At least three different distribution patterns of nanobacteria were conditions, and human kidney stones that are formed from small apatite units. Prerequisites for the formation of kidney stones are the supersaturation of urine and presence of nidi for crystallization. Nanobacteria are important nidi and their presence might be of special interest in space flights where supersaturation of urine is present due to the loss of bone. Furthermore, we bring evidence that nanobacteria may act as crystallization nidi for the formation of biogenic apatite structures in tissue calcification found in e.g., atherosclerotic plaques, extensive metastatic and tumoral calcification, acute periarthritis, malacoplakia, and malignant diseases. In nanaobacteria-infected fibroblasts, electron microscopy revealed intra- and extra-cellular needle-like crystal deposits, which were stainable with von Kossa stain and resemble calcospherules found in pathological calcification. Thus bacteria-mediated apatite formation takes place in aqueous environments, in humans and in geological sediments.