Nanopathology :

a new vision of the interaction environment-human life

by
Antonietta M. Gatti
Lab. of  Biomaterials, Dept. of Neurosciences, University of Modena & Reggio Emilia, Modena, Italy.
gatti@unimo.it

Summary

This article takes into consideration a new field of  research called Nanopathology and describes the fundamentals of this new discipline. The paper is divided into three main sections: the in-vitro and in-vivo experiments with 5 types of nanoparticles, and the clinical pieces of evidence of the presence of particulate matter in human pathological tissues affected by diseases of unknown  origin. The in-vitro experiments verified the interaction of 5 different types of nanoparticles with endothelial, gut and liver epithelial cells and macrophages from a toxicological and immunological point of view. The in-vivo experiments carried out on rats investigated the response to the implantation of 5 materials as nanoscaled particles and bulk disks in rats’ dorsal muscles. The clinical investigations verified the presence of inorganic micro and nano-particles in pathological tissues affected by cancer in different organs, leukaemia and lymphoma.
The main in-vitro results indicated that:
a- in the presence of metallic nanoparticles (Cobalt), macrophages become unable to mount appropriate defence to bacterial challenge and danger exists of increased susceptibility to infections;
b- the in-vivo tests indicated that metallic nanoparticles (and not the bulk samples) induced rhabdomyosarcoma in rats in 6 months;
c- the clinical samples showed the constant presence of micro and nanoparticles in the tissues with variable chemistry, sometimes directly related to the working place exposure.
Indications of new directions of the research are discussed.

1. Preface
Objectives of the Project Nanopathology were:

This technology was employed to check the interaction with 5 different types of engineered nanoparticles on cells and animals. At the same time, observations on pathological human tissues were carried out in order to verify the presence of micro- and nano-inorganic contaminants.
In a few circumstances, the study of the chemistry of this internal pollution allowed to identify the same pollutants in the environment and to understand the actual exposure the patient underwent.
This piece of evidence allowed to put forward possible mechanisms of dissemination of this pollution inside the body,  either through inhalation and  ingestion. That led to understand the possible sources of micro- and nano-environmental pollution, and its role on human and animal life.
Possible mechanisms of toxicity of this nanopollution versus cells were identified and a new research project will verify the conditions for nanoparticles to trigger specific diseases like, for instance, some forms of cancer.

2. Introduction
In 2001, we coined a new word: nanopathology, meaning by that the collection of pathologies due to micro- and nanoparticles. At that time, this was a void concept, since a comparatively small number of pathologies were recognized worldwide as triggered by particulate matter. Silica particles and asbestos fibres can induce lung diseases as silicosis and asbestosis. The inhalation of dust containing particles of quartz or fibres of a silicate can also induce lethal  pathologies of the lungs.  It is well-known that the inhalation of mine dust or of cigarette smoke are risk factors for the onset of  lung cancer. But a possible correlation with nanoscaled particulate matter was completely unknown, and that for 2 reasons: 1- in 2001, submicronic matter was recognized to be dispersed in the environment, though very few studies existed, and none of them was exhaustive, on its behaviour, but nothing had been done for nanosized particles; 2- no evidence identified nanodust as responsible of pathologies. Just micronic and (in very few instances) submicronic particles were considered as causing pathologies(e.g. pneumoconioses).
Now that nanotechnologies are a rapidly growing discipline, society is scared by possible side effects of the production, manipulation and use of nanoparticles, and nanosafety has become a priority. Many researchers are investigating the toxicity of nanoparticles towards cells, while our project has already gone further by investigating the in-vivo interaction and thir impact they have to humans.
Among the most abundant air pollutants in urban areas is particulate matter with a mean dia­meter < 10 µm (also called PM10 by environmental toxicologists, defined as particulate matter with an aerodynamic size of 10 µm or smaller). Over the years, it has become clear that particles with very low sizes (especially < 100 nm) are more significant health-wise than larger particles, since they have been shown to induce far more severe effects [1]. The sur­face/size-ratio increases exponentially with the decreasing of particle sizes, leading to enhanced surface reac­tivity. This enhanced surface reactivity might lead to greater biological activity per given mass, com­pared to larger particles, which in turn might have effects on, e.g., the inter­nalization of particles into tissues, cells and organelles, tox­icity, or the induction of oxi­dative stress [2]. By definition, particles with sizes below 100 nm are called nano-scaled particles (short: nanoparticles) or ultrafine particles by toxicologists.
Due to the minute size of nanoparticles, the internalization into the body’s tissues appears to be extremely easy. This was shown by experiments in human volun­teers with radioactive-labelled carbon nanoparticles (i.e. `Technegas`) that were shown to pass rapidly into the sys­temic circulation after inhalation. Radioactivity could already be detected in the blood 1 minute after inhalation [3]. Furthermore, ani­mal studies revealed that inhaled nanoparticles were relocated into the liver [4] and the brain [5]. Thus, nanoparticles seem to be able to circum­vent the tight blood-brain-barrier and possibly cross the blood-placenta barrier [6, 7]. Moreover, it has been sug­gested that nanoparticles are involved in thrombus formation in the blood [8, 9]. Today we know that particulate air pollution is associated with enhanced mortality from respiratory and cardiovascular diseases [10].
As the sources of internalized nanopar­ticles (food, air, etc.) and the location of parti­cle detec­tion are generally far apart, a distribution via the blood stream must have occurred. Thus, en­dothelial cells, which line the inner surface of blood ves­sels, will have direct contact with the particles. Endothelial cells are important in inflammation and wound healing. Upon pro-inflam­matory stimulation of the endothelium, adhesion molecules are expressed on the cell surface, thus mediating leukocyte attachment (e.g. E-selectin and intercellular adhesion mole­cule-1/ICAM-1). Besides, endo­thelial cells can release cytokines, such as inter­leukin-8 (IL-8, a key factor in neutrophil chemotaxis). Thus, these features con­tribute to the pro-inflammatory endothelial phe­notype that permits the transmigration of leu­kocytes from the blood into the perivascular space [11]. The activation of IL-8, E-selectin and ICAM-1 is regu­lated by the same transcription factors NF-kB (nu­clear fac­tor-kB) and AP-1 (acti­vator protein-1) [12-14].

3.Materials and Methods
The project was divided into 3 main sections: 1. in-vitro tests, 2. in-vivo tests  and 3. clinical evaluations.

3.1  The in-vitro tests considered the interaction of 5 different engineered nanoparticles (TiO2, SiO2, Co, Ni  and PVC) with cells (human endothelial cells ) and organs (in-vivo tests with rats and sheep). Nanoparticles of TiO2, SiO2, PVC, Co and Ni were examined with respect to the cellular internalisation and their influence on the cell viability, proliferative activity, and the pro-inflammatory endothelial pheno­type.
SiO2- and TiO2-particles were produced by flame spray pyrolysis (TAL Materials Inc.). The size spectrum of SiO2-particles was between 4 and 40 nm with 14 nm mean parti­cle size. The size of TiO2-particles was between 20 and 160 nm with 70 nm mean particle size. The size of Co-particles (Sigma) was between 50 and 200 nm (mean particle size 120 nm). The mean particle size of Ni-particles was about 62 nm (Nanoamor). Nanoparticles of PVC (polyvinylcloride) without phtalate were also used, but  their floating in the medium due to no weight did not allow an interaction with cells. Also the aggregation of Nickel particles and their deepening in the medium did not allow a real interaction with cells, so the results, being  function of  a non interaction, must be fully understood.
The particles were added to the cell culture medium and tested at three different concentrations (0.5, 5, and 50 µg/ml cul­ture medium) and the Cytotoxicity assays verified the  following parameters:
Macrophage and cell survival, citokine production, modulation of TLR expression,
Also a model system that leads to the formation of in-vitro capillaries within a three-dimen­sional extracellular matrix (fibrin and type I collagen) after stimulation with pro-angio­genic factors was developed. This model system allowed the evaluation and quantifica­tion of pro- and anti-angiogenic characteristics of different compounds. The addition of the different particles (TiO2, SiO2, Ni, Co, PVC) into the three-dimensional matrix revealed that only the presence of Co particles led to changes in the in-vitro capillary pheno­type. A number of abortive sprouts were formed and the developing sprouts were not so pronounced as in the positive control. However, cell viability ap­peared unaffected. Software-supported quantification of these Co-particle-induced changes revealed a significant reduction in the degree of angiogenesis in vitro.
3.2- In-vivo tests
The 5 mentioned, different materials were implanted in rats under two forms (bulk samples and nano-particulate materials). Each rat was implanted in the dorsal muscle with two similar implants. For each group, 2 rats were sacrificed after 6 months (short term), and 3 rats were sacrificed after 12 months (long term). In addition, 5 control rats were implanted with a reference material. Two of them were sacrificed after 6 months and 3 of them were sacrificed after 12 months. Five rats were fed by gavage with a mixture of nano-particles (50% Ni and 50% Zr with two different ranges) (about 0.030 – 60 mg by day): 2 were sampled after 6 months and 3 were sampled after 12 months, in order to see if the small-size particles can negotiate the bowel barrier and contribute to the pathogenesis of some diseases. Animals were sacrificed after 6 and 12 months.
After the short- and long-term implantation, samples were explanted and the gross necropsy of the animals performed. All samples were formalin-fixed and paraffin-embedded, microtome-sliced and Hematoxylin-Eosin-stained. The sections were analyzed histopathologically and under ESEM in order to verify the interface of the bulk and nanosized materials after the interaction.
3.3- The clinical evaluations.
More than 300 cases of tissues affected by a-cryptogenic granulomatosis of the liver; b-sarcoidosis; c- Crohn’s disease; d-cancer of the liver, the lungs, the colon and the brain; e-lymphoma.
The new diagnostic tool allowed to identify micro and nanoparticulate matter inside the tissues and the Energy Dispersive Spectroscopy allowed to identify its chemical composition.
New tests were developed with body fluids like the blood, the sperm and the follicular fluid in order to verify similar foreign presences.

The project was granted a 6-month extension as some findings needed to be investigated better. The following 4 tasks were investigated:
1. Analysis of pro-angiogenic M2 biasing effect of Co nanoparticles, by means of the evaluation of the angiogenic factor VEGF.
2.  3D co-cultivation system of endothelial cells and macrophages. Through the  quantification of E-selectin cell surface protein expression, the detection of Ki67 expression, the quantification of IL-8 release in cell culture supernatant and the quantification of E-selectin cell surface protein expression.3.
3.  Interaction of Co nanoparticles with endothelial cells.
Since it is known that monocytes and macro­phages play a role in angiogenesis, the effects of the co-cultivation of endothelial cells together with the monocytic cell line U937 in the angiogenesis model system was tested. It was important to verify the hypothesis according to which the addition of PMA-stimulated U937 cells to the culture could lead to a distinct decrease of the length of in-vitro-capillaries and also to a reduction of endothelial cell number.
4.  Correlation of clinical evidence with in-vitro mechanisms  The task wanted to  investigate further clinical cases of different diseases and, from a certain point of view,  try to identify the exposure starting from the analysis of the particles detected in the patient’s pathological tissues, and from another try to correlate this possible trigger of the pathology, putting forward possible mechanisms of dissemination of the particulate matter inside the body and the pathomechanism versus cell.

4. Results
4.1   The  in-vitro results verified that:
Whereas the ceramic particles of TiO2 and SiO2 did not show significant cytotoxic effects, the nanoparticles of Co and Ni induced a significant, concentration-depend­ent impairment of cel­lular viability. This impairment was observed at different levels (i.e. decrease of cell number, protein expression of the proliferation marker Ki67, and the metabolic activity).
A pro-inflammatory effect in HDMEC occurred after exposure to SiO2-, Co-, and Ni-par­ticles and was apparent by an enhanced release of IL-8. Only higher particle con­centrations (25 and 50 µg/ml) induced this increase in IL-8 release. The E-selectin protein expression was en­hanced by high amounts of Co-particles whereas Ni-parti­cles induced no protein expression of E-selectin. In contrast to the particles, divalent Co- and Ni-ions induced the expression of all pro-inflammatory markers tested (i.e. IL-8, E-selectin, ICAM-1).
In detail:
a- The investigated nano-particles do not affect cell survival or proliferation. Only Co nano-particles are toxic at  >100 mg/106 cells. They inhibit the expression of LPS receptors TLR4 and CD14 and induce a down-regulation of mRNA expression for TLR4 and CD14 ( The two receptor chains which recognise bacterial lipopolysaccharide and activate macrophage defence functions).
An interesting result is that Co nano-particles impair macrophages activation by bacterial LPS, in fact a negligible cytokine production was detected. These results mean that in presence of these metallic nanoparticles the macrophages become unable to mount  appropriate defences to bacterial challenge. That means that danger exists of increased susceptibility to infections. These very interesting results were not obtained with Nickel nanoparticles since, due to their forming micrometric aggregates and sinking in the medium, they did not interact with the cells.b- The Cobalt nanoparticles with endothelial cells showed an angiogenic behaviour,

Fig. 1: 3D-angiogenesis a) untreated control (arrowheads mark a contiguous in vitro capil­lary-network consisting of a number of endothelial cells), b) Co-particles (arrows: abortive sprouts), c) SiO2-particles, d) ZrO2-particles (fluorescence im­ages of a vital staining)

The analysis of images by an optical phenotype evaluation revealed that only Co-particles induced an impairment of angiogenesis in vitro(Fig. 2b). This im­pairment was characterized by the occurrence of a number of abortive sprouts (arrows). Fur­thermore, the developing sprouts were not as pronounced as in the positive control (i.e. shorter sprouts with a larger diameter). However, the cell viability appeared un­affected, as the cell number was not re­duced. All other particles (TiO2, SiO2, ZrO2, Ni, PVC) did not induce apparent devia­tions (2c - SiO2-particles, 2d - ZrO2-particles) in comparison to the un­treated control (Fig. 1)
Our study has shown that human endothelial cells possess a large capacity for the internaliza­tion of nanoparticles. All nanoparticles tested were taken up by the endo­thelial cells and to a major extent into vacuoles
The Cobalt nanoparticles showed an angiogenic behaviour, that can trigger a carcinogenic reaction.
The results during the extension of the project induced to put forward the hypothesis that the observed pro-inflammatory activation after Co-particle ex­posure may be attrib­uted to a release of divalent Co-ions by the particles, since the expo­sure of en­dothe­lial cells to these ions leads to the impaired endothelial viability and pro-in­flamma­tory stimula­tion. This contrasts with the effects of the Ni-particles. Here, the suggestion of a Ni-ion release by the particles resulting in an induced pro-inflammatory stimulation is not congruent with the pro-inflammatory effects induced by the respective ions, since Ni-ions induced both an increase in the release of IL-8 and the protein expression of endothelial cells adhesion mole­cules (i.e. E-selectin and ICAM-1), whereas Ni-parti­cles induced only an increased IL-8 release and the expression of adhesion mole­cules was not initiated. This indicates an activa­tion mechanism for the Ni-particles that deviates from the Ni-ion-induced activation that is shown to occur via a coopera­tion of the above-mentioned transcription factors NF-kB and AP-1. Since oxidative stress is also a relevant aspect in the mechanisms of (Ni-) particulate matter-induced effects [32], this mecha­nism of differential activation of pro-inflammatory gene pro­moters might play a role. Thus, it can be suggested that the Ni-ion release by the nanoparticles remains under the criti­cal limit for pro-inflam­matory activation but further Ni-nanoparticle induced effects (possibly oxidative stress) are responsible for the enhanced IL-8 release. Also the tests with the co-cultures (macrophage-endothelial cells) indicate a deep interaction of Cobalt nanoparticles with the cells. The addition of PMA-stimulated U937 cells to the culture led to a distinct decrease in the length of in-vitro capillaries and also to a reduction of endothelial cell number. In addition to that, the number of U937 colonies within the 3-dimensional extracellular matrix rose.
These in-vitro results indicate some mechanisms of actions of nanoparticles with cells and  explain the in-vivo results and the clinical evidence.

4.2 In-vivo tests
Our animal studies verified that  the metallic nanoparticles were cancerogenic, while the bulk material induced just a fibrotic capsule with a granulomatosic reaction

Fig.2 Cobalt and Nickel nanoparticles induced rhabdomyiosarcoma in the rats’ back . The metallic disks induced only fibrotic capsules.

The other materials (PVC, Silica, and Titania) developed  only a fibrotic capsule or a granulomatous tissue. The reason could be the agglomeration of the nanoparticles that transform them to micrometric debris. In this case, the body reacts to the size of particulate matter, as the materials are chemically inert.
The feeding of rats with Zirconia and Nickel showed that there is a passage to the internal organs for Zirconia, but not for Nickel. The ESEM analyses carried out in the internal organs verified only the presence of few particles of Zirconia. The Nickel particles were not found . A possible explanation is that their aggregation formed microparticles eliminated through the faeces.
The observation of the section of the site of implantation of nanoparticles in the sheep’s knee joint
was negative, since the nanoparticles were not found in the implantation site. That was the demonstration of their dissemination in other parts of the body due to the extracellular fluids.

4.3  Clinical observations.
The study of our series of pathological samples showed the following interesting findings:

 
The new diagnostic tool developed allows to detect inorganic particulate matter present in pathological tissues. Its identification is a fundamental step to identify the possible source of pollution and the kind of exposure the patient suffered [15-24].

5. Conclusions

The Project allowed to ascertain that environmental pollution exerts a clear effect on human health.
Through an innovative diagnostic tool it was possible to verify that micro and nanoparticles, involuntary  released in the environment  by industrial processes, are present in pathological tissues.  The evidence of such presence inside the internal tissues of the body proves that the physiological barriers available to the organism are not efficient enough to prevent the entrance. The observations carried out verified that a size threshold exists below which particulate matter can negotiate the physiological barriers. The presence of debris in tissues affected by pathologies of unknown origin, sharing an inflammatory nature, represents a cue to be verified in a systematic way by checking homogenous cohorts of patients.
Especially in mild inflammatory pathologies, the possible presence of foreign bodies should always be suspected and, in case such presence is proved, the origin of those pollutants should be investigated in order to remove the primary cause of disease from the patient’s lifestyle.
Pathologies like cryptogenic single- or multi-organ granulomatoses, sarcoidosis, Crohn’s disease, fibroses and scleroses should be checked for the presence of nanometric and micrometric debris, and its composition, size and shape assessed, as these are essential pieces of information to make a correct anamnesis.
Many forms of  cancer should be checked as well for the presence of micro- and nano-debris, since, when debris is actually present, it is generally to be found at the interface between healthy and pathologic tissue. The in-vivo tests carried out, with their focused implantation of bulk and nano-sized particles belonging to three different classes of materials, have already shown that metallic nanoparticles implanted in a muscle can induce rhabdomyosarcoma. Ceramic and polymeric particles, on the other hand, induced only fibrosis and granulomatosis, probably due to their coalescence.
Other diseases like Gulf and Balkan syndrome should be analysed to check if a specific nanopollution due to the blasts of weapons is present in the tissues.
Other pathologies of unknown origin (like diabetes or autoimmune syndromes) or some other less common cryptogenic diseases should be studied in view of a triggering mechanism involving nano-sized foreign bodies. Also some neurological diseases like, for instance, Alzheimer’s (already related to the presence of aluminium) and  ALS (amyotrophic lateral sclerosis) should be investigated for the presence of metals, possibly under the form of proteinates, arranged around the neuraxis.

The pathological mechanism through which micro- and nanoparticles become pathogenetic was in part investigated with the in-vitro tests carried out . It was clearly seen that nanoparticles can enter the cell, cross the nuclear membrane and reach the nucleus. The tests proved also that some nanoparticles can induce angiogenetic mechanisms, that can trigger or support a carcinogenic reaction, but they can also bypass the immune response.
The results obtained, had us put forward some mechanism of interaction between materials and cell/tissue/organ. The knowledge acquired could be enough to set up simulation models (either in vivo and in vitro) to check if those mechanisms are reproducible and repeatable.

6.  Discussion
The results obtained through our research should induce us to revise our way of understanding the impact of environmental pollution on our health. We should start by considering a simple natural law, stating that nothing can be created and nothing destroyed, but everything can just be transformed. It is a matter of fact that all forms of combustion change matter and produce micro- and nanoparticulate whose size depends mainly on the temperature reached in the crucible, and that most of that particulate, which, in many instances, is neither biodegradable nor biocompatible, gets dispersed in the environment. So, the air, the soil, water, vegetables are polluted and both humans and animals are the victims of that condition through the inhalation of the air and the ingestion of water and vegetables. The situation grows worse to humans who eat animals whose flesh is polluted, and nowadays that pollution may be imported from places which are very far from where those humans live. In our century, it is particularly hard to set boundaries to everything and pollution is no exception.
What the majority of the systems aimed at getting rid of wastes do is reduce them to a very small size, sometimes incomparably smaller than the original, and disperse them. The problem is that the smaller the size of that dust, the more penetrating and aggressive to the organism it is, and making it smaller and smaller does not seem particularly wise.
One of the most promising results of our research is the possibility it offers to trace back the source of pollution and the kind of exposure the subject underwent, by comparing what we detect in his pathological specimens with the environment where that subject lives or work, or the food he eats. So, it will not be too difficult to understand whether the strategy of having wastes “disappear” by making them small enough to become hard to detect or we had better resort to different solutions.

7.  The next future
In conclusion, what is necessary to do is:

Further directions of research can be the study of the so-called industrial sterility, malformed foeti in specific industrial areas, rare diseases and new pathologies investigated, but not yet understood. Epidemiological studies with homogenous cohorts of patients should be carried out, where, starting from the analysis of the embedded particulate matter, exposure is verified and the source of pollution is identified.

The results of our research have been readily applied by the Italian senatorial commission dealing with the problem of pathologies contracted by peacekeeping troops. A not negligible number of them were in the vicinity of the explosion of high-temperature rounds (e.g. depleted-uranium or tungsten weaponry) or were involved in the destruction of weapons which was carried out by heap them up in large holes dug in the ground and have them burn. Some of those soldiers were also close to burning oil rigs. In all those cases, they inhaled particulates produced be the combustion of large quantities of matter and, inevitably, inhaled and, sometimes, ingested them. Part of those soldiers developed diseases that can be classified as nanopathologies.

8.  References

[1]        Oberdorster, G., Pulmonary effects of inhaled ultrafine particles. Int Arch Occup Environ Health 2001, 74, 1-8.
[2]        Oberdorster, G., Oberdorster, E., and Oberdorster, J., Nanotoxicology: An emerging discipline evolving from studies of ultrafine particles. Environ Health Perspect 2005.
[3]        Nemmar, A., Hoet, P. H., Vanquickenborne, B., Dinsdale, D., Thomeer, M., Hoylaerts, M. F., Vanbilloen, H., Mortelmans, L., and Nemery, B., Passage of inhaled particles into the blood circulation in humans. Circulation 2002, 105, 411-414.
[4]        Oberdorster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Lunts, A., Kreyling, W., and Cox, C., Extrapulmonary translocation of ultrafine carbon particles following whole-body inhalation exposure of rats. J Toxicol Environ Health A 2002, 65, 1531-1543.
[5]        Oberdorster, G., Sharp, Z., Atudorei, V., Elder, A., Gelein, R., Kreyling, W., and Cox, C., Translocation of inhaled ultrafine particles to the brain. Inhal Toxicol 2004, 16, 437-445.
[6]        Reichrtova, E., Dorociak, F., and Palkovicova, L., Sites of lead and nickel accumulation in the placental tissue. Hum Exp Toxicol 1998, 17, 176-181.
[7]        Kaiglova, A., Reichrtova, E., Adamcakova, A., and Wsolova, L., Lactate dehydrogenase activity in human placenta following exposure to environmental pollutants. Physiol Res 2001, 50, 525-528.
[8]        Nemmar, A., Hoylaerts, M. F., Hoet, P. H., Dinsdale, D., Smith, T., Xu, H., Vermylen, J., and Nemery, B., Ultrafine particles affect experimental thrombosis in an in vivo hamster model. Am J Respir Crit Care Med 2002, 166, 998-1004.
[9]        Gatti, A. M., Montanari, S., Monari, E., Gambarelli, A., Capitani, F., and Parisini, B., Detection of micro- and nano-sized biocompatible particles in the blood. J Mater Sci Mater Med 2004, 15, 469-472.
[10]      Pope, C. A., 3rd, Epidemiology of fine particulate air pollution and human health: biologic mechanisms and who's at risk?, Environ Health Perspect 2000, 108 Suppl 4, 713-723.
[11]      Cook-Mills, J. M., and Deem, T. L., Active participation of endothelial cells in inflammation. J Leukoc Biol 2005, 77, 487-495.
[12]      Montgomery, K. F., Osborn, L., Hession, C., Tizard, R., Goff, D., Vassallo, C., Tarr, P. I., Bomsztyk, K., Lobb, R., Harlan, J. M., and et al., Activation of endothelial-leukocyte adhesion molecule 1 (ELAM-1) gene transcription. Proc Natl Acad Sci U S A 1991, 88, 6523-6527.
[13]      Roebuck, K. A., Rahman, A., Lakshminarayanan, V., Janakidevi, K., and Malik, A. B., H2O2 and tumor necrosis factor-alpha activate intercellular adhesion molecule 1 (ICAM-1) gene transcription through distinct cis-regulatory elements within the ICAM-1 promoter. J Biol Chem 1995, 270, 18966-18974.
[14]      Mukaida, N., Okamoto, S., Ishikawa, Y., and Matsushima, K., Molecular mechanism of interleukin-8 gene expression. J Leukoc Biol 1994, 56, 554-558.
[15]      Peters, K., Schmidt, H., Unger, R. E., Otto, M., Kamp, G., and Kirkpatrick, C. J., Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility. Biomaterials 2002, 23, 3413-3419.
[16]      A.M. Gatti , F. Rivasi “ Biocompatibility of micro- and nanoparticles Part I in liver and kidney.” Biomaterials  june 2002, vol 23 , issue 11 , 2381-2387 [22]                                                                      
[17]      AM Gatti  Biocompatibility of micro- and nano-particles in the colon (part II)  Biomaterials vol.25, 3, Feb 2004 385-392   
[18]      Kirkpatrick, C. J., Barth, S., Gerdes, T., Krump-Konvalinkova, V., and Peters, K., [Pathomechanisms of impaired wound healing by metallic corrosion products]. Mund Kiefer Gesichtschir 2002, 6, 183-190.
[19]      Peters, Unger, Gatti, Monari, Kirkpatrick Effects of nano-scaled particles on endothelial cell function in vitro:Studies on viability, proliferation and inflammation, J. of Material Science: Mat. in Medicine 15 (4),  321-325, 2004.
[20]      AM. Gatti, Montanari, Monari, Gambarelli, Capitani, Parisini Detection of micro and nanosized biocompatible particles in blood. J. of Mat. Sci. Mat in Med. 15 (4): 469-472, April 2004
[21]      AM Gatti Risk assessment of micro and nanoaprticles and the human health, Chapter  of Handbook  of  Nanostructured biomaterials and their applications ed American Scientific Publisher USA 2005, cap. 12, 347-369.                                                               
 [22]     Gatti, AM, Symposium Keynote Presentation “Risk Assessment of Nano-Particles and Nano-Technologies for Human Health. 7th World Biomaterials Congress- Sindney (Australia)  2004 (pag. 748-749).
[23]      M. Lucarelli, A.M. Gatti, G. Savarino, P. Quattroni, L. Martinelli, E. Monari, D. Boraschi “Innate defence function of macrophages can be biased by nano-sized ceramic and metallic particles”  Cytokin Network, Decembre 2004, Vol 15 No. 4,pag 339-346
[24]      A. Gatti, S. Montanari, A. Gambarelli, F. Capitani, R. Salvatori “ In-vivo short- and long-term evaluation of the interaction material-blood” Journal of Materials Science Materials in Medicine, 2005, 16, 1213-19.