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wissenschaft & praxis
Paleoimmunology
What we can learn from the past and use for the future – introduction of a new subdiscipline.
Diese Zusammenfassung eines Vortrages, gehalten am
IFBLS-Welt-Kongress im Juni 2004 in Stockholm, gibt
einen Überblick über ein außergewöhnliches Forschungsfeld, zu dem MTAs wesentlich beigetragen
haben.
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Introduction
Our laboratory, the Institute of Pathophysiology of the Medical University Innsbruck
in Austria, has a long standing experience in
performing immunohistochemistry (IHC) and
immunofluorescence (IF) methods. In fact, we were the first
to introduce this technology to the analyses of extracellular
matrix (ECM) proteins in general. These proteins are the
major quantitative constituents of mammalian organisms.
Normally, we use these techniques in our research group
to elucidate the very beginning of atherosclerotic changes in
vessel walls or autoimmune diseases like scleroderma.
However, more than 20 years ago we were invited the
first time to apply our knowledge not only on recent patient
biopsies but also on historical specimens.
To give a short overview over the years: We investigated
biological material, which was 170 years to 50 million years
old. Human atherosclerotic vessels collected in Vienna, Austria, in the 19th century, 500-1,500 year-old human mummies
from Peru, skin and striated muscle from the 5,300 year-old
glacier mummy („Iceman“) from Tyrol, Austria, and a 50 million year-old bat with preserved soft body parts from the
fossil excavation site of Messel, Germany, were examined.
But why did we investigate historical specimens at all?
First, we could elucidate the causes of death and assess the occurrence of diseases in the past. This may be especially important for studies of rare diseases.
Second, it was the technical challenge, to introduce a new
tool for paleontological studies. Until now, only a limited
number of methods were available for the examination of
such sensitive and precious samples. In previous studies, IHC
and IF could apparently not be successfully implemented on
historical material by other groups.
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& praxis
Background
The historical biological material has been fixed either
„naturally“ by certain environmental conditions or in an
„artificial“ manner. Airdrying, freezing, or placement in an
oxygen poor surrounding are „natural“ ways of preservation. The embalming process performed e.g. on egyptian mummies is some kind of „artifical“ fixation.
In the 18th and 19th century pathologists began to place
samples in ethanol and other fixation solutions. Besides drawings and engravings this was the only way to preserve interesting anatomical and pathological specimens. Extensive
anatomical collections were founded all over Europe. The socalled „Collectio Rokitansky“, one of the largest historical
collections in the entire world, is situated in
Vienna (Austria). It comprises more than
50,000 objects, including formalin-fixed human and veterinarian specimens and is named
after the famous 19th century pathologist Carl
von Rokitansky.
A historical controversy
Does inflammation play a key role in atherogenesis? Our own work, summarized on the
„autoimmune hypothesis“ for atherogenesis,
clearly supports this notion. We have identified
heat shock protein 60 (HSP60) as the culprit
autoantigen triggering mononuclear cell infiltration of the arterial intima (Xu Q. et al.
1993). It was against this background that we
embarked on the present study on historical arterial specimens. The signs of inflammation in atherosclerotic plaques have been observed for centuries, but it`s role
was the subject of great controversy in the 19th century between the renowned pathologists Carl von Rokitansky (Fig. 1)
and Rudolf Virchow (Fig. 2), from Austria and Germany,
respectively.
Fig. 1: Carl von Rokitansky
(1804-1878)
Fig. 2: Rudolf Virchow
(1821-1902)
Both described cellular inflammatory changes in atherosclerotic vessel walls. However, while von Rokitansky considered these changes secondary in nature, Virchow supported their primary role in atherogenesis
We had the unique opportunity to illuminate this controversy by investigating atherosclerotic lesions from autopsies performed by Carl von Rokitansky in the middle of the
19th century.
Twelve atherosclerotic arteries, originally collected between the years 1827 and 1885, were selected from the
Collectio Rokitansky. Intriguingly, autopsy reports compiled approximately 170 years ago are often still available,
a remarkable sign of a well-organized storage system
throughout the centuries. In the beginning of the 19th century, specimens were stored in pure alcohol („Weingeist“),
later on formalin was used to preserve the tissue up to present. Historical and recent vascular and non-vascular tissues
were examined for control purposes. The historical and
contemporary tissues were dehydrated and paraffin embedded. After preparation of paraffin sections, modern sophisticated immunohistochemical and immunofluorescence methods were successfully applied. We showed that epi-
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Fig. 3: CD3+ T-lymphocytes (red) in arteriae lienalis of a 64 year old
female died in 1840
topes of certain atherosclerosis markers (CD3, collagen type I, collagen type III, collagen type IV, fibronectin and von
Willebrand factor) survived the extraordinary long fixation time of 170 years (Fig. 3). However, many other epitopes (e.g. macrophages) did not resist the long and harsh
storage conditions.
Together with earlier findings, these results support the hypothesis that CD3+ T cells are present in the earliest phases
of atherosclerosis and, therefore, agree with Virchow`s view
that inflammation plays a primary role in initiating the atherogenic process.
Not only „artificially“ preserved historical human tissue
served us as an origin for investigations on immunological
questions.
How to distinguish early from late scars in mummies?
On recent tissue we were able to show that scar formation
is characterized by increased production of collagen type III
followed by collagen type I. Using this approach, it is possible
to distinguish early from late stages of fibrosis and scar formation. We could so elucidate the causes of death and assess the occurrence of diseases in the past. But are the antigenic determinants of collagen still detectable in „naturally“ preserved human
tissue? To answer this question, we studied skin samples secured on the occasion of two expeditions to Peru, taking place between 1958 and 1960. The aim of these expeditions was to study the remnants of pre-Columbian culture. The extremely dry
soil in caves along the northern and southern coast of Peru afforded very favourable conditions for the preservation of organic
material. Thus, air-dried mummies aged from 500 to over 1,500
years were found with excellently preserved skeletal as well as
Fig. 4: Collagen type I positive fibers detected by immunofluorescence in peruvian mummy
soft parts without embalming. We analyzed skin biopsies from
three mummies to detect certain extracellular matrix proteins
such as collagens. Indirect immunofluorescence procedure was
applied on frozen sections of the mummies`s skin and on control skin from recent specimens. It should, however, be mentioned that producing frozen sections from this air-dried material turned out to be a considerable technical challenge.
We found collagen type I positive fibers (Fig. 4). Also the
fine reticular mesh of collagen type III was still detectable in
air-dried mummy skin. Pro-collagens and non-collagenous
ECM proteins like laminin and fibronectin gave negative results. Apparently, only epitopes localized in the so-called
„collagenous“ triple-helical trypsin resistant parts of the
molecules were preserved to a certain extent.
Based on this experience, we were invited to perform similar experiments on 50-million year old biological material.
Visible, but not real
The Eocene age, 50-million years ago, was the time when
mammalians began to take over a leading role in natural history. In Messel near Darmstadt, Germany, an old opencast mine composed of soft oil-shale exists. Here, a flora and fauna
of Eocene ecosystem is preserved with all soft parts in incredible detail due to apparently optimal environmental conditions. In addition to the information that can be obtained
with respect to the whole ecosystem itself, feeding habits of the
fossilized animals can also be deduced by analysing their stomach contents. In our case, we decided to analyse a bat. Taking advantage of the relatively easy accessibility of the flying
membranes of the wings (Fig. 5). To our surprise, it was relatively easy to freeze the membranes in liquid nitrogen and to
manufacture frozen, unfixed sections. But we used a trick:
We embedded the small fossil tissue in mouse liver before
freezing. This provided us an excellent quality of sections of
the fossil and the surrounding mouse liver served as an internal control for antibody specificity. With a classical methylene blue staining of the fossil sections, collagen-like fibrillar
structures were clearly evident (Fig. 6). However, we got only autofluorescence in the fossil sections whereas the recent
mouse liver, our internal positive control, was clearly positive for collagen type I, fribronectin and other ECM proteins.
We were quite disappointed when we could not demonstrate ECM components with immunofluorescence technique
in pre-historical material. Subsequently, we subjected a bat
specimen to amino acid analysis. Surprisingly, it turned out
that the characteristical amino acids for collagenous proteins did not appear.
Fig. 5: 50 million year-old pre-historical bat
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Fig. 7: 5300 year-old glacier mummy found in the Tyrolean alps
Fig. 6: Methylene blue staining of flying membrane of
pre-historical bat
To summarize it: Conventional histology showed us collagen like structures in the bat flying membrane whereas immunofluorescence and amino acid analysis failed to detect
collagenous fibers.
We did not know how to tie these strings together. However, several years later after completion of these experiments
we got an answer by means of scanning electron microscopy:
The „soft parts“ of the bat flying membrane had been replaced by lawns of bacteria. And the bacteria still provided
the microscopic appearance of preserved tissue!
Immunohistochemical studies in tissues of the Tyrolean
Iceman
In September 1991, a glacier mummy was found by tourists at a height of 3,200m at the Similaun glacier in the Tyrolean alps. It was an archeological sensation when this mummy turned out to be of Neolithic origin and was later dated
to about 5,300 years. The most surprising observation was,
of course, the fact that the mummy was perfectly preserved
with very little waxy degeneration that would preclude most
studies of pathological relevance. Not only skin, but also
eyeballs and internal organs were preserved.
For our analysis we secured skin and muscle from the
hip area (Fig. 7). Since we were experienced from our work
with the pre-historical bat tissue, we performed amino acid
analysis in advance. The amino acids 4-hydroxyproline, proline and glycine, typical for collagenous tissue, could be detected in appropriate amounts. When performing immunofluorescence tests on frozen sections of skin and muscle specimens from the „Iceman“ we got massive autofluorescence.
Therefore, in this case immunohistochemistry was exclusively
used to detect extracellular matrix proteins. We showed that
antigenic determinants, specific for collagen type I, type III,
pro-collagen type III and collagen type VI (Fig. 8) were still
detectable. Thus, in principle we are now e.g. able to distinguish early from late scars in glacier mummies.
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A few years ago scientists found a spearhead under the
scapula of the Neolithic „Iceman“. The common theory about the circumstances of his death is now, that he has been
chased around and died of exhaustion in the mountains.
Summary
Our laboratory has introduced immunohistological techniques successfully into paleontological research, thus creating a new subdiscipline, paleoimmunology.
Long time preserved historical and pre-historical tissue is
now accessible for pathological investigations in various
fields. This novel application of immunohistochemical and
immunofluorescence techniques gives us a strong tool for
further retrospective studies especially on anatomical collections from the last 200 years as well as archeological relicts
(Wick G. et al. 2001).
Acknowledgements
Over a periode of three decades medical laboratory technologists, physicians, biologists in our laboratory and archeologists
have put technical and intellectual input into this theme. The person who held the strings together and
incited all of us is my mentor Prof. Wick,
whom I want to thank especially.
■
Christina Mayerl, Dipl. MTA
Institut für Pathophysiologie
Medizinische Universität Innsbruck
[email protected]
Fig. 8: Collagen type VI positive fibers detected by immunohistochemistry in historical glacier mummy
Source of pictures:
Fig.1: http://clendening.kumc.edu/dc/pc/rokitansky.jpg
Fig. 2: www.uniwuerzburg.de/pathologie/
Virchow/bildtexte/text12.htm
Fig. 3: Reprinted from Immunol Allergy Clin N Am Vol 23, Knoflach M, Mayrl B, Mayerl C, Sedivy R and Wick G „Atherosclerosis
as a paradigmatic disease of the elderly: role of the immune system“. 117-132, copyright (2003), with permission from Elsevier.
Fig. 4: by courtesy of Prof. Dr. G. Wick
Fig. 5, 6, 8: Reprinted from Exp Gerontol Vol 36(9), Wick G, Kalischnig G, Maurer H, Mayerl C, Mueller P „Really old-palaeoimmunology: immunohistochemical analysis of extracellular matrix
proteins in historic and pre-historic material“. 1565-1579, copyright (2001), with permission from Elsevier
Fig. 7: by courtesy of Prof. Dr. J. Szilvássy
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