Fornaciari Gino
Ancient Remnants:
Biomolecules in Paleopathology
Third ESBB annual conference
Verona, Italy
8th – 11th October 2013
The diagnoses of ancient tumors
and infectious diseases depend
on the specific detection of
cancer cells and pathogens in
buried individuals; these fields of
research are known as paleooncology and paleo-microbiology,
two emerging disciplines which
have benefited from
technological advances.
from Thi-Nguyen-Ny Tran et al, 2011
For almost more than 15 years the detection, identification, and characterization of
tumors and pathogens in ancient human specimens have emerged on the basis of
ancient DNA (aDNA) analyses. Over the last years, aDNA limitations due to potential
contamination by modern DNA and altered aDNA have led to the development of
alternative methods for the detection and characterization of non nucleotidic
biomolecules, including proteins.
Histology, histochemistry and immunohistochemistry techniques can again be useful,
in particular as preliminary approach.
Apse of S. Domenico
The nave
The Basilica of San Domenico Maggiore, which dates back to the beginning of
the 14th century, is one of the largest and important churches in Naples.
The Sacristy with the funerary gallery
The royal sarcophagi
The monumental Sacristy of the Basilica contains 37 wooden sarcophagi with the
mummies of ten Aragonese kings, princes and other Neapolitan nobles who died
between the end of the 15th and second half of the 16th century.
Artificial mummy of Giovanni d’Aragona (1566-1571)
Natural mummy of Antonello Petrucci (+1585)
Natural mummy of Ferdinando Orsini, 5th duke of Gravina in Apulia (+1549)
The sarcophagi of San Domenico Maggiore were carefully examined by a
team of the Institute of Pathology of Pisa University.
The tombs revealed many well preserved (natural and artificial) mummies.
Anthropological, radiological and
autoptic examinations of the
mummies were carried out on site.
The first X-ray of a mummy of S. Domenico
with a portable apparatus, in February 1983
Autopsy of the natural mummy of Pietro
d’Aragona, 3rd Duke of Montalto (1539-1552)
Guido Mazzoni (1491)
Naples, Capodimonte Museum
Ferrante I d’Aragona
King of Naples
(ca.1431-1494)
Ferrante I was King of Naples from 1458
to 1494 and died at the age of 63 years.
Low relief of Ferrante at Porta Capuana, Naples
Sarcophagus of Ferrante
(Basilica of S. Domenico Maggiore)
Autopsy of the mummy of Ferrante I
revealed a colon adenocarcinoma
extensively infiltrating the muscles of
the small pelvis.
The pelvic cavity of Ferrante at moment of
autopsy, with the anal canal and the site
of tumor (yellow arrow)
Section of sample of tumor after rehydration,
with the tumor massively infiltrating the
internal tissues of the abdomen (roundish
yellow-white formations)
Histology showed exceptionally well
preserved neoplastic cells, disposed in
cords and solid nests or forming
pseudo-glandular lumina, disseminated
in a fibrous tissue containing striated
muscular fibres.
The neoplastic cells still reacted with
cytokeratins.
Pan cytokeratin immunofluorescence, 200X
Hematoxilyn-Eosyn, 40X
Hematoxilyn-Eosyn, 100X
The cells were long and crowded, with
abundant cytoplasm and quite regular
hyperchromatic nuclei.
Hematoxilyn-Eosyn, 600X
Electron-Microscopy 10,000X
Artificial mummy of Antonio d’Aragona, IV duke of Montalto (1540-1584)
The excellent, almost exceptional preservation of the histological structure of
this unique tumor is probably due to the very dry microclimatic conditions of the
church and of the burial in a wooden sarcophagus 4 m high, and certainly to the
preserving substances used for embalming.
Ancient DNA amplification
of the neoplastic cells by
polymerase chain reaction
(PCR) in the mummy of
Ferrante I evidenced a
typical point mutation of
the K-ras gene codon 12.
The normal sequence GGT encoding
the amino acid glycin was turned
into GAT encoding the amino acid
aspartic acid.
The adenocarcinoma of King Ferrante I of Aragon
K-Ras: sequencing analysis
G A
T
C
C
C/T
A
Sequencing analysis of K-Ras exon 1:
The K-Ras codon 12 mutation (GGT>GAT)
was detected by direct sequencing in
tumor DNA of king Ferrante I of Aragon.
The other field of research that has benefited from
technological advances is paleomicrobiology, i.e. the
identification of microbes in ancient human remains.
The immunodetection of microbial antigens was used
as early as the 1980s in a case of smallpox.
Several studies have indicated that proteins may be
more resistant than aDNA to taphonomic decay.
Child mummy with smallpox
exanthema (Naples, XVI century)
Immunohistochemistry
Immunohistochemistry combines
morphology and immunochemical
detection of antigenic determinants
in tissues.
Detection of microbial antigens
can be done using
immunofluorescence analysis of
frozen tissues and
immunoperoxidase applied to
formalin-fixed, paraffin-embedded
tissues.
from Thi-Nguyen-Ny Tran et al, 2011
Immunohistochemistry is easy to perform and avoids any laboratory
contamination.
Direct visualization of ancient pathogens is also possible with this technique.
Among the mummies of S. Domenico, one of the most interesting was the artificial
mummy of a 2-year-old child, wearing the monastic clothes of the Dominican order.
The child died around the mid 16th century (14C: 1569±60)
The mummy showed a widespread vesiculopustular eruption: the scalp, the face,
the posterior trunk, the arms and the glutei were the most involved regions.
The impressive preservation of the mummy’s facial exanthema: the scalp, the face
with the palpebral regions and the lips are covered with vesicles and pustules.
The right forearm with large, confluent pustules:
the vesicles and pustules (10-20 mm in size),
completely dry, with crystalline content, show a
sort of central umbilication and a pale,
peripheral halo.
Fenner F, Henderson DA, Arita I, et al. Smallpox and Its Eradication. Geneva,
Switzerland: World Health Organization; 1988. Courtesy of DA Henderson.
At light microscopy the vesicles
showed an evident separation of the
superficial layers of the epidermis
from the basal layer.
Indirect immunofluorescence
with anti-variola virus antibody
showed focal positivity.
Negative staining with osmium tetroxide showed many tetrahedral
viral-like particles (so-called “brick-like”), peculiar to orthopox viruses.
Scale bar = 300 nm
Many egg shaped, dense structures (250 nm x 200 nm), composed by a
central dense region, or core, surrounded by a lower density area (inset).
Scale bars = 300 nm
Following incubation with anti-variola virus antiserum, after protein Agold complex, some of these particles were completely covered by the
A-gold protein (a), others were partially labelled (b, c).
Scale bars = 300 nm
The artificial mummy of Maria
d'Aragona (1503-1568), marquise of
Vasto (southern Italy), a noble woman
of the Italian Renaissance, was
exhumed in the Basilica of Saint
Domenico Maggiore.
Famed for her beauty, Maria
d'Aragona belonged to the intellectual
and religious circles of Ischia, whose
leading figures were Giulia Gonzaga
and the well-known poetess Vittoria
Colonna, friend of Michelangelo.
This very obese individual presented an asymmetrical swelling of the
lower limbs, right inguinal tumefaction and some white-yellowish
cicatricial areas of the skin.
The left arm showed a «dressing» , a linen
bandage intertwi-ned with ivy leaves (Hedera
helix L.)
The dressing covered an oval 15 X 10 mm ulcer with relatively regular
features, deep perpendicular borders and a brownish-black shaggy surface
at the base.
Inside the ulcer there was a free hard
spheroidal 5-mm diameter formation,
with irregular surface, which gave off an
intense aromatic smell when cut.
At light microscope the formation
appeared composed by vegetal
filaments immersed in an amorphous
substance. This substance,
microanalysed by EDS with a SEM
Stereoscan 250 coupled to a
microsound EDS link, revealed to be
prevalently sulphur, a substance largely
utilized at that time for skin diseases.
The immunohistochemical
technique was used on the
selected sections with antiTreponema pallidum antibody
(Sclavo-Italy), applying the
indirect immunofluorescence
method and secondary
fluorescin-label-led goat anti-IgG
antibody.
The results were excellent.
A large number of
filaments with intense
yellow-green
fluorescence and lying
between 10 and 20 µm.
were identified.
The dimensions and
the morpho-logical
characteristics of the
fluorescent treponemes
were well defined (a).
These sometimes formed a regular spire with a round formation at each extremity
(3c,e), at other times they were heaped (a) or isolated (d).
Ultrathin sections were observed at
SEM and TEM.
The ultrastructural study of the ulcerous
lesion showed, among a considerable
amount of necrotic material, several
spiral-like structures ranging in size
from 5 to 15 µm in Iength and 0.1-0.2
µm in width.
These structures were enclosed by an
outer-sheath composed of two
electrondense layers containing an
electron-transparent layer.
An internal electron-dense structure,
with a double spire irregularly rotating
around the cell, was identified as the
axial fibrils typical of the order of the
spirochetes.
Conclusions
The histological, immunological and
ultrastructural findings clearly show that we
are dealing with a treponemal infection.
The macroscopic characteristics of the
cutaneous ulcer are those typical of a third
stage luetic gumma containing numerous
treponemes.
Owing to the historical period (16th century)
and geographical position (Naples), a
diagnosis of tertiary venereal syphilis (the
Neapolitan disease of the Renaissance!),
seems appropriate.
This was the first example of
identification of treponemes in the
soft tissues of ancient human
remains, as until now the
paleopathological diagnosis of
syphilis has only been carried out on
skeletal remains.
The discovery of this mummy is
even more important since it dates
back to the 16th century when the
disease reached epidemic levels.
Further ultrastructural,
immunological, biochemical and
molecular investigations, so far
impossible, should help clarify the
biology of treponema in the
epidemic phase of the disease.
Physicians treating patients infected with
syphilis using mercury (woodcut , Wien, 1497)
Further examination of the mummy of Maria d’Aragona showed a large
pedunculated arborescent neoformation (2x7 mm) in the right inguinal region.
This neoformation was
rehydrated by Sandison's
solution and submitted to
histology by haematoxylineosin, Van Gieson and
Masson’s trichromic staining.
Light microscopy evidenced
an exophytic, papillary skin
lesion, with typical connective
axis and pronounced
parakeratosis.
(Masson’s Trichrome, 50X)
These macroscopic and histological aspects are peculiar of condyloma acuminatum,
a papillomavirus-induced squamous lesion also called “venereal wart”.
In order to detect the presence of HPV (Human papillomavirus) nucleic
acid, DNA extracted from a single 5 µm paraffin-embedded section was
amplified with L1 consensus primers GP5+/GP6+, promoting the
amplification of a 141-151 bp sequence from 25 distinct genital HPVs.
HPV 18
The automated sequencing of several clones revealed the presence of 100%
similarity sequences of both HPV 18 DNA, a virus with high oncogenic potential,
and JC9813 DNA, an HPV with low oncogenic potential.
HPV is a very ancient virus that has evolved
with mankind. Nowadays, HPV represents
one of the most common viruses sexually
transmitted, responsible for numerous
lesions at the level of the external and
internal genitals and, in particular, of cervix
cancer.
This study represents the first molecular
diagnosis of HPV in mummies and could
pave the way for further research on the
secular evolution of these viruses, very
important in human oncology.
A Peruvian mummy, a young
woman 20±3 years old from
Cuzco (Peru) from the National
Museum of Anthropology and
Ethnology of Florence, was
examined.
The bundle in twisted grass cords
is typical of the late intermediate
period (1000-1476 A.D.) of the
Andean highlands.
After preliminary X-ray,
showing good preservation of
the body, the mummy was
submitted to autopsy.
Macroscopically, a megavisceral
syndrome in the form of
cardiomegaly, megaesophagus,
gastric ectasia and megacolon,
with enormous amounts of feces,
was found.
Megacolon with abundant feces
Cardiomegaly
Giemsa staining evidenced in
the myocardium and
esophagus rare roundish
intratissutal nests, about
15-20 µm large, containing
several ovular formations (1-2
µm) with small nuclei.
The findings corresponded
morphologically to possible
intratissutal nests of
amastigotes of Trypanosoma
cruzi.
(Giemsa, 900X)
(Esophagus, Giemsa, 50X)
(Giemsa, 600X)
For the immunohistochemical study we
adopted the immunoperoxidase
method, and avidine-biotine system.
Monoclonal antibodies anti-T. cruzi aflagellar FCH-F8-1 and FCH-F8-4,
produced at the Institute Fatala Chaben
of Buenos Aires, were used.
(Esophagus, immunoperoxidase, 50X)
The nests of ovular formations and the
single formations, if separately
examined, showed intense, selective
immunoreactivity.
(Immunoperoxidase, 400X)
The ultrastructural study of the
esophageal wall showed some rare
ovular formations, with a diameter
maximum of 1 µm, on large deposits
of collagen fibers.
collagen
layer
These formations were characterized
by:
•  Double peripheral membrane;
•  Microtubules with diameter of
about 20 nm (T);
•  Lumps of electron dense material,
with a diameter of about 200 nm,
identifiable as nucleus (N);
•  A semilunar elongated body, of
about 300 X 80 nm, identifiable as
kinetoplast (K);
•  A cylinder-like structure, of about
300X30-40 nm, with more electron
dense thickening at one end,
identifiable as axoneme (A) with
its basal body (B).
These structures are peculiar to amastigotes of the Trypanosomatidae family.
The macroscopic, histological,
immunohistochemical and
ultrastructural findings clearly showed
that we were in presence of an old
case of chronic Chagas’ disease.
We thus obtained the first direct
demonstration of the existence of this
disease and its etiological agent in the
southern American continent, during
the pre-Inca civilization.
This specimen was used as positive
ancient control in a large study about
the diffusion of American
trypanosomiasis in pre-Columbian
south America.
The Egyptian mummy of a young
woman aged about 20, dating back to
the late Ptolemaic period (II-I century
B.C.) and currently exhibited in the
municipal museum of Narni (central
Italy), was studied.
The embalming technique of this
period, consisting of evisceration
followed by re-deposition of the
internal organs in the body
cavities, made it possible to study
these organs.
Autopsy revealed a bundle of
linen bandages, enveloping a
hollow muscular organ,
measuring 9x6x3 cm, with a 5mm thick wall, identified as the
stomach.
The stomach
Light microscopy showed a 6x4 mm
cystic structure, with a wall of
about 80 µm, humped by externally
projected protrusions.
A rostellum, with two rows of
hooklets and two roundish
structures, identifiable as suckers,
were visible under a layer of loose
fibrous tissue.
Morphology strongly suggested a
cysticercus of Taenia solium
(or “pig tapeworm”).
Immunohistochemistry with the indirect immunofluorescence method was strongly
positive.
Observation with a laser confocal microscope evidenced the wall of the cystic
structure coloured with an intense yellow-green fluorescence.
On the basis of these results, we can reliably assert that this young Egyptian
woman was affected by - and most probably died of - cysticercosis.
This discovery was of great scientific
importance because, as far as tapeworms
are concerned, only the presence of ova
has been documented in the intestinal tract
of an Egyptian male buried in the first half of
the 12th century BC.
This is the first paleopathological diagnosis
of cysticercosis, i.e. of human tissue
invasion by T. solium larvae, and confirms
the wide diffusion in Egypt of pig farming,
representing the most common intermediate
hosts of T. solium.
In this table we have summarized the different morphological methods
until now adopted in the Laboratory of Paleopathology of the University of
Pisa, to diagnose some soft tissue diseases without (or before) aDNA:
Histology Histo chemistry Colon carcinoma Immuno Electron aDNA histo Microscopy chemistry ++ + ++ + ++ + -­‐ ++ ++ -­‐ ++ + -­‐ -­‐ ++ Treponema -­‐ -­‐ ++ ++ -­‐ Trypanosoma -­‐ + ++ ++ ++ Cysticercus + + ++ -­‐ -­‐ Smallpox virus HPV +: auxiliary
++: diagnostic
In conclusion, immunohistochemistry and electron
microscopy, two morphological disciplines in the
last years rather neglected, compared to ancient
DNA studies, can be still useful and have to be
revaluated in Paleopathology.
The future potential of these materials should be
considered in managing the preservation of
bioarchaeological specimens stored in laboratories
and museums.
Histological study of the
tumor of King Ferrante I of
Aragon
University of Pisa
Division of Paleopathology
www.paleopatologia.it
Laboratories of Pisa
Laboratories at the Charterhouse of Calci