Paleopathologic diagnosis based on experimental mummification

The difficulties of diagnosis of pathologic conditions are immensely magnified when the subject of a postmortem examination has been postmortem for several hundreds to thousands of years. Artefacts of decomposition and bacterial and fungal invasion are compounded upon those of rehydration when mummified tissue is examined. As an approach to these problems, a study of the changes seen in experimentally mummified and rehydrated tissues was undertaken. Normal and pathologic tissues were studied in comparison to sections prepared from the fresh tissue. The experimentally mummified tissues were generally similar to, but somewhat better preserved than, actual human mummies. There was organ and tissue specific variability in preservation, and different classes of pathology likewise showed differential preservation. Inflammatory reactions were not very well-preserved although infecting microorganisms were easily identified. Degenerative processes such as atherosclerosis and others characterized by the accumulation of abnormal products, were well preserved, while necrosis, as in acute myocardial infarction, was not. Malignancies were particularly well preserved. The implications of these findings for previous and future mummy studies is discussed in terms of our understanding of the evolution of disease processes.     

Histological examination of experimentally mummified tissues

Tissue mummified by desiccation can be examined histologically after rehydration with Ruffer's solution, and a number of natural mummies have been studied by this technique. In order to study the efficacy of the rehydration process, and to evaluate the degree of artefact introduced, normal human tissues were desiccated, rehydrated, and examined histologically. There was generally good preservation of architecture and moderate preservation of cellular detail, with some organ specific variability. The prospect of an atlas of mummified pathological lesions is discussed, as an aid to the determination of disease patterns in prehistoric populations.     

Hybridization screening of very short PCR products for paleoepidemiological studies of Chagas' disease

Single strands of very short PCR products can be covalently immobilized to a slide and then easily detected by probe hybridization. In this work, the PCR product was a 70-nucleotide segment of ancient DNA, representing a portion of repeat mini-circle DNA from the kinetoplast of Trypanosoma cruzi, the infectious agent of Chagas' disease (American Trypanosomiasis). The target segment was initially established to be present in soft tissue samples taken from four "naturally" mummified Andean bodies using PCR followed by cloning and sequencing. Hybridization screening of the covalently immobilized PCR products positively identified products from 25 of 27 specimens of different tissues from these four mummies. The method appears to be ideal for the purpose of screening a large number of specimens when the target PCR product is very short.     

Preservation of proteins in mummified tissues

Protein material was extracted from the dessicated tissues of several Egyptian mummies and a frozen Eskimo. The distribution and degree of preservation of high molecular weight protein was analyzed by gel filtration, protein assays, amino acid analysis, and polyacrylamide gel electrophoresis. The protein has undergone considerable degradation although some high molecular weight protein (C. 130,000 daltons) remains intact. Amino acid analysis of the extracted protein indicates the basic amino acids have undergone a chemical modification and may represent a point of preferential breakdown in the polypeptide chain. Atomic absorption spectrophotometry of tissue cations suggests a correlation between degree of preservation of mummified tissue and levels of sodium salts (natron) in the tissue.     

Deficiency of uridine monophosphate synthase (DUMPS) and X-chromosome deletion in fetal mummification in cattle

Ten mummified fetuses were tested for the deficiency of uridine monophosphate synthase (DUMPS), which is known to contribute to the embryonic and fetal mortality in cattle. Genomic DNAs of the mummified fetuses were extracted from tissue samples collected from the mummies and were amplified by GenomiPhi DNA amplification kit. UMPS gene of the mummies was amplified by polymerase chain reaction (PCR) with DUMPS primers. Out of ten mummies examined, two fetuses were heterozygous (carriers) for DUMPS as indicated by the presences of three bands of 89, 53 and 36 bp. Estimated stage of gestation when the death occurred in the two mummies was 3.5 and 2.5 months, respectively. The other fetuses exhibited only two bands of 53 and 36 bp on the polyacrylamide gel indicated that they were normal. On the other hand, all the mummies were sexed using AMX/Y primers. Specific regions of Y and X chromosomes were amplified by PCR using AMX/Y. The expected 280 bp fragment in the female sample and the 280 and 217 bp in the male sample were observed. Nine mummies had a normal X and Y chromosome bands; however, the other mummified fetus exhibited only Y chromosome band, while the constitutive X chromosome fragment was missing. The estimated stage of gestation when the death occurred in this mummified fetus was 100 days. This might be the first report of DUMPS and X-chromosome deletion at the amelogenin gene in bovine-mummified fetuses in Japan.     

Technical note: Terahertz imaging of ancient mummies and bone

Ancient mummified soft‐tissues are a unique source to study the evolution of disease. Diagnostic imaging of such historic tissues is of foremost interest in paleoanthropology or paleopathology, with conventional x‐ray and computed tomography (CT) being the gold‐standard. Longer wavelength radiation in the far‐infrared or Terahertz region allows diagnostic close‐to‐surface tissue differentiation of bone morphology while being harmless to human cells. The aim of this study is to show the feasibility and the morpho‐diagnostic impact of THz imaging of historic remains. Images of an artificially embalmed ancient Egyptian human mummy hand, an artificially embalmed ancient Egyptian mummified fish and a macerated human lumbar vertebra were obtained by THz‐pulse imaging and compared with conventional X‐ray and CT images. Although conventional x‐ray imaging provides higher spatial resolution, we found that THz‐imaging is well‐suited for the investigation of ancient mummified soft tissue and embalming‐related substances / wrappings. In particular, bone and cartilaginous structures can be well differentiated from surrounding soft‐tissues and bandage‐wrappings by THz imaging. Furthermore, THz‐pulse imaging also measures the time‐delay of the pulsed signal when passing through the sample, which provides supplementary information on the optical density of the sample that is not obtained by X‐ray and CT. Terahertz radiation provides a completely non‐invasive diagnostic imaging modality for historic dry specimens. We anticipate this modality also to be used for detection of hidden objects in historic samples such as funerary amulets still in situ in wrapped mummies, as well as potentially for the identification of spectral signatures from chemical substances, e.g., in embalming essences.. Am J Phys Anthropol, 2010. © 2010 Wiley‐Liss, Inc.     

Evidence based palaeopathology: Meta-analysis of Pubmed-listed scientific studies on pre-Columbian,South American mummies

The aim of this study was to review all Pubmed^®-listed palaeopathological studies performed on pre-Columbian South American mummies. A total of 61 studies were found (1977–2005). Review criteria included e.g. method of examination, method of mummification, palaeopathological diagnoses and individual age of mummies as well as dating, which ranged from 7500 to 500 years BP, mainly (if reported) originating from the Chiribaya and Chinchorro cultures. The average age of the 99 individually reported mummies was about 25 years. Only six studies included computed tomography, thirteen studies used classical radiography as an examination method. Three studies analysed parasite related diseases, especially caused by Trypanosoma cruzi. Among all of the reported infectious diseases (n = 9), there were seven studies presenting cases of tuberculosis. The results were also compared inter-culturally. In 61 studies (37 epidemiological and 24 case reports) more than 6400 mummified individuals were analysed. By contrast, meta-analytic data for ancient Egyptian mummies ( Zweifel et al., 2009) included about 3000 analysed individuals in 131 studies (85 case reports and 46 epidemiological studies). In general, ancient Egyptian mummies were shown to be intentionally mummified, whereas the Pre-Columbian American mummies showed a great diversity of spontaneous mummification. However, ritualistic mummification methods were also practised (n = 2). This study's results shall assist to improve evidence-based research in palaeopathology.     

Histological analysis and staining techniques modified and verified on ancient mummified tissues to study microorganism infestations.

The purpose of this work is to give a brief account of the possibility to estimate the preservation of human mummified tissues using histological analysis. This method can be useful to identify injuries and to plan qualified conservative actions on ancient human remains. Some preliminary results are presented here regarding the study on 16 ancient mummies from the Egyptian Museum of Turin. Samples of mummified tissues were taken without damaging the remains; they were hydrated again and dyed with histological techniques which were specifically modified and verified in same cases. Our research identifies some agents (biological and chemical) that appear to be responsible, among others, of mummified tissue destruction. The microscopic examination reveals features that might refer to fungal and bacterial infestation. Using special staining methods on seried sections we were able to suspect, at least in one case, the presence of biologically active forms. Microbiological assays confirm the vitality of fungi. Histological tissue analysis can then be useful to guide any conservative intervention for preservation and protection of the integrity of biological remains from museum collections.     

Chagas disease and human migration

Human Chagas disease is a purely accidental occurrence. As humans came into contact with the natural foci of infection might then have become infected as a single addition to the already extensive host range of Trypanosoma cruzi that includes other primates. Thus began a process of adaptation and domiciliation to human habitations through which the vectors had direct access to abundant food as well as protection from climatic changes and predators. Our work deals with the extraction and specific amplification by polymerase chain reaction of T. cruzi DNA obtained from mummified human tissues and the positive diagnosis of Chagas disease in a series of 4, 000-year-old Pre-Hispanic human mummies from the northern coast of Chile. The area has been inhabited at least for 7,000 years, first by hunters, fishers and gatherers, and then gradually by more permanent settlements. The studied specimens belonged to the Chinchorro culture, a people inhabiting the area now occupied by the modern city of Arica. These were essentially fishers with a complex religious ideology, which accounts for the preservation of their dead in the way of mummified bodies, further enhanced by the extremely dry conditions of the desert. Chinchorro mummies are, perhaps, the oldest preserved bodies known to date.     

Fatty acid composition and preservation of the Tyrolean Iceman and other mummies

In anthropology, objective parameters to adequately describe storage conditions and the preservation of mummies have yet to be identified. Considering that fatty acids degrade to stable products, we analysed their profile in human mummies and in control samples by gas chromatography coupled to mass spectrometry (GC/MS). Originating from different epochs and civilizations, samples of the Tyrolean Iceman, other glacier corpses, a freeze dried mummy, corpses from a permafrost region, a corpse mummified immersed in water, and a desert mummy were evaluated. Chemometric analysis based on the concentrations of 16 fatty acids revealed the degree of similarity between anthropologic and fresh corpse samples, which was mainly influenced by the content of palmitic acid, oleic acid, and 10-hydroxystearic acid. The presence of 10-hydroxystearic acid was associated with immersion in water, whereas dry mummification was accompanied by high contents of oleic acid. Samples of the Tyrolean Iceman clustered between fresh tissue and those of other glacier corpses indicating the good preservation of this mummy. Thus, environmental post-mortem conditions were associated with characteristic fatty acid patterns suggesting that chemometric analysis of fatty acid contents may add to our knowledge about post-mortem storage conditions and the preservation of human corpses.     

Techniques of DNA-studies on prehispanic ectoparasites (Pulex sp., Pulicidae,Siphonaptera) from Animal mummies of the Chiribaya culture,southern Peru

During a paleoparasitological survey of several animal mummies (Cavia aperea f. porcellus and Canis familiaris) from Chiribaya Baja, an archaeological site in Southern Peru, an unexpected find was made. In the well preserved fur, large numbers of mummified fleas (Pulex simulans/irritans) that parasitized the animals during life were encountered. Due to the relative recent event of the host mummification and the outstanding preservation of the fleas, an attempt for the retrieval of DNA was made. A DNA extraction and sequencing protocol for archaeological ectoparasitic remains has been established, taking additional studies for tissue and protein preservation into account. Tissue preservation was assessed with transmission electron microscopy and the protein preservation was tested through the racemisation ratios of aspartic acid. Regions of the 28S rDNA gene were successfully amplified and sequenced. Further research perspectives are outlined.     

Nuclear magnetic resonance imaging of mummified corpses

Clinical applications of nuclear magnetic resonance (NMR) imaging have demonstrated its great potential for noninvasive examinations of soft tissues. Here preliminary experiments are reported on mummified human tissue by means of NMR cross-sectional and transmission imaging. For this purpose the natural viscosity of the tissues has been lowered by rehydration with an aqueous solution of 20% acetone.     

Hounsfield Units ranges in CT-scans of bog bodies and mummies

Mummification processes, either artificial or natural, preserve the tissues from postmortem decay, but change them from their original state. In this study we provided the first comprehensive set of Hounsfield Unit (HU) ranges specific for tissues mummified under different environmental conditions (peat bog, cold-dry and hot-dry environment). We also analyzed the impact of different museal preservation techniques on the HU ranges, as e.g. in the Tollund Man and Grauballe Man, two bog bodies from Denmark. The HU results for mummies were compared with HU results from forensic cases, cremated and inhumated ancient human skeletal remains, and fossil animal bones. Knowledge of the typical HU range for the different tissues in mummies may help to avoid misinterpretation of increased or reduced radiodensity as evidence of paleopathological conditions. Finally, we demonstrate the practical benefit of using our re-defined HU ranges by showing the improved results of 3D visualization from automatic segmentation in an Inca mummy from Mount Llullaillaco.     

Tissue identification and histologic study of six lung specimens from Egyptian mummies

Twenty-eight specimens obtained either from organ bundles in the body cavities of intact mummies, from damaged mummies, or from isolated canopic jars were examined for tissue identification and histopathologic study. The methods of rehydration and fixation were optimized by application to 40 dehydrated modern samples before studies of mummified tissue were undertaken. The tissue of origin could be definitely identified in 24 of the 28 specimens. Even small fragments obtained from isolated canopic jars proved suitable for histologic study. Six lung specimens were selected for more detailed study. All six showed focal deposition of anthracotic pigment. Electron diffraction and electron microprobe analysis of one of the small, polarizable crystals associated with the anthracosis indicated a mineral content of silica, aluminum, and iron. Two specimens showed focal areas of calcification consistent with old mycobacterial disease. Other histopathologic findings included evidence of pulmonary edema, emphysema, and pneumonia.     

mtDNA from hair and nail clarifies the genetic relationship of the 15th century Qilakitsoq Inuit mummies

The 15th century Inuit mummies excavated at Qilakitsoq in Greenland in 1978 were exceptionally well preserved and represent the largest find of naturally mummified specimens from the Arctic. The estimated ages of the individuals, their distribution between two adjacent graves, the results of tissue typing, and incomplete STR results led researchers to conclude that the eight mummies formed two distinct family groups: A grandmother (I/5), two daughters (I/3, I/4), and their two children (I/1, I/2) in one grave, and two sisters (II/6, II/8) and a daughter (II/7) of one of them in the other. Using mtDNA from hair and nail, we have reanalyzed the mummies. The results allowed the unambiguous assignment of each of the mummies to one of three mtDNA haplogroups: A2b (I/5); A2a (I/2, I/3, II/6, II/8); A2a-311 (I/1, I/4, II/7), excluded some of the previous relations, and pointed to new ones. I/5 is not the grandmother/mother of the individuals in Grave I, and she is not maternally related to any of the seven other mummies; I/3 and I/4 are not sisters and II/7 is neither the daughter of II/6 nor of II/8. However, I/1 may be the child of either I/4 or II/7 and these two may be sisters. I/2 may be the son of I/3, who may be the daughter of either II/6 or II/8, and these two may be sisters. The observation of haplogroups A2a and A2b amongst the 550-year-old Inuit puts a lower limit on the age of the two lineages in Greenland.     

Effects of intraguild predation on aphid parasitoid survival

To assess the potential selection pressure caused by intraguild predation between predators and parasitoids of aphids an estimate was made of the predation risk to Aphis fabaeScop. mummified by Lysiphlebus fabarum(Marshall, 1896) on sugar beet. Mummified aphids were exposed to a natural community of predators. Their survival time was estimated during a 10-day field survey. Additionallythe role of alternative prey on parasitoid survival was investigated by adding unparasitised aphids to half of the mummy aggregations.The field data were evaluated by survival analysis. Two covariates were tested within a Cox proportional hazard model: (i) the presence of the alternative prey and (ii) the patch structure (number of proximal mummies attacked). Within 4–5 days after exposure predators destroyed approx. 50% of the mummies. The model with both covariates revealed a significant difference concerning survival of the mummies in the two treatments (Likelihood ratio test, ²=78.03, P=0.0001). Alternative prey reduced the predation risk on mummies by 29%, while a high level of predation on proximal mummies increased the individual predation risk by 4%. The results are discussed in the context of prey location by predators and the evolution of anti-predator mechanisms.     

Determination of optimal rehydration,fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.     

Determination of optimal rehydration, fixation and staining methods for histological and immunohistochemical analysis of mummified soft tissues.

During an excavation headed by the German Institute for Archaeology, Cairo, at the tombs of the nobles in Thebes-West, Upper Egypt, three types of tissues from different mummies were sampled to compare 13 well known rehydration methods for mummified tissue with three newly developed methods. Furthermore, three fixatives were tested with each of the rehydration fluids. Meniscus (fibrocartilage), skin, and a placenta were used for this study. The rehydration and fixation procedures were uniform for all methods. The stains used were standard hematoxylin and eosin, elastica van Gieson, periodic acid-Schiff, and Grocott, and five commercially obtained immunohistochemical stains including pancytokeratin, vimentin, alpha-smooth-muscle-actin, basement membrane collagen type IV, and S-100 protein. The sections were examined by transmitted light microscopy. Our study showed that preservation of the tissue is dependent on the quality and effectiveness of the combination of the rehydration and fixation solutions, and that the quality of the histological and histochemical stains is dependent on the tissue quality. In addition, preservation of the antigens in the tissues is dependent on tissue quality, and fungal permeation had no influence on the tissue. Finally, the results are tissue specific. For placenta the best solution combination was Sandison and solution III (both fixed with formaldehyde) while results for skin were best with Ruffer I (using formaldehyde and Schaffer as fixatives), Grupe et al. (using formaldehyde as a fixative) and solution III (in combination with formaldehyde and Bouin fixatives). Ruffer II (using formaldehyde as a fixative) and solution III (in combination with Schaffer fixative) gave the best results for fibrocartilage.     

More Realistic Face Model Surface Improves Relevance of Pediatric In-Vitro Aerosol Studies

Background

Various hard face models are commonly used to evaluate the efficiency of aerosol face masks. Softer more realistic “face” surface materials, like skin, deform upon mask application and should provide more relevant in-vitro tests. Studies that simultaneously take into consideration many of the factors characteristic of the in vivo face are lacking. These include airways, various application forces, comparison of various devices, comparison with a hard-surface model and use of a more representative model face based on large numbers of actual faces.

Aim

To compare mask to “face” seal and aerosol delivery of two pediatric masks using a soft vs. a hard, appropriately representative, pediatric face model under various applied forces.

Methods

Two identical face models and upper airways replicas were constructed, the only difference being the suppleness and compressibility of the surface layer of the “face.” Integrity of the seal and aerosol delivery of two different masks [AeroChamber (AC) and SootherMask (SM)] were compared using a breath simulator, filter collection and realistic applied forces.

Results

The soft “face” significantly increased the delivery efficiency and the sealing characteristics of both masks. Aerosol delivery with the soft “face” was significantly greater for the SM compared to the AC (p< 0.01). No statistically significant difference between the two masks was observed with the hard “face.”

Conclusions

The material and pliability of the model “face” surface has a significant influence on both the seal and delivery efficiency of face masks. This finding should be taken into account during in-vitro aerosol studies.     

Assessment of the Reporting Quality of Randomized Controlled Trials on the Treatment of Diabetes Mellitus with Traditional Chinese Medicine: A Systematic Review

Background

After the publication of the CONSORT 2010 statement, few studies have been conducted to assess the reporting quality of randomized clinical trials (RCTs) on treatment of diabetes mellitus with Traditional Chinese Medicine (TCM) published in Chinese journals.

Objective

To investigate the current situation of the reporting quality of RCTs in leading medical journals in China with the CONSORT 2010 statement as criteria.

Methods

The China National Knowledge Infrastructure (CNKI) electronic database was searched for RCTs on the treatment of diabetes mellitus with TCM published in the Journal of Traditional Chinese Medicine, Chinese Journal of Integrated Traditional & Western Medicine, and the China Journal of Chinese Materia Medica from January to December 2011. We excluded trials reported as “animal studies”, “in vitro studies”, “case studies”, or “systematic reviews”. The CONSORT checklist was applied by two independent raters to evaluate the reporting quality of all eligible trials after discussing and comprehending the items thoroughly. Each item in the checklist was graded as either “yes” or “no” depending on whether it had been reported by the authors.

Results

We identified 27 RCTs. According to the 37 items in the CONSORT checklist, the average reporting percentage was 45.0%, in which the average reporting percentage for the “title and abstract”, the “introduction”, the “methods”, the “results”, the “discussion” and the “other information” was 33.3%, 88.9%, 36.4%, 54.4%, 71.6% and 14.8%, respectively. In the Journal of Traditional Chinese Medicine, Chinese Journal of Integrated Traditional & Western Medicine, and the China Journal of Chinese Materia Medica the average reporting percentage was 42.2%, 56.8%, and 46.0%, respectively.

Conclusions

The reporting quality of RCTs in these three journals was insufficient to allow readers to assess the validity of the trials. We recommend that editors require authors to use the CONSORT statement when reporting their trial results as a condition of publication.