Imaging intact human organs with local resolution of cellular structures using hierarchical phase-contrast tomography.

Imaging intact human organs from the organ to the cellular scale in three dimensions is a goal of biomedical imaging. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique using the European Synchrotron Radiation Facility (ESRF)'s Extremely Brilliant Source (EBS). The spatial coherence of the ESRF-EBS combined with our beamline equipment, sample preparation and scanning developments enabled us to perform non-destructive, three-dimensional (3D) scans with hierarchically increasing resolution at any location in whole human organs. We applied HiP-CT to image five intact human organ types: brain, lung, heart, kidney and spleen. HiP-CT provided a structural overview of each whole organ followed by multiple higher-resolution volumes of interest, capturing organotypic functional units and certain individual specialized cells within intact human organs. We demonstrate the potential applications of HiP-CT through quantification and morphometry of glomeruli in an intact human kidney and identification of regional changes in the tissue architecture in a lung from a deceased donor with coronavirus disease 2019 (COVID-19).

[Mesothelial proliferation of the tunica vaginalis testis]. / Mesotheliale Proliferate der Tunica vaginalis testis.

Proliferative changes seen in reactive mesothelial hyperplasia of a hydrocele sac may mimic malignant mesothelioma. There is no immunohistochemical staining that reliably separates benign from malignant mesothelial proliferations. However, the combined analysis of BAP1 by immunohistochemistry and CDKN2A by FISH has been reported to yield both a high specificity and sensitivity in this differential diagnosis. In addition, the evaluation of risk factors such as asbestos exposure or prior traumata may be helpful for the correct diagnosis. Exclusion of stromal invasion, which is diagnostic for malign mesothelioma, is of utmost importance. Therefore, extended histological workup is essential.

[German Guideline for Idiopathic Pulmonary Fibrosis]. / S2K-Leitlinie zur Diagnostik der idiopathischen Lungenfibrose.

Idiopathic pulmonary fibrosis (IPF) is a severe and often fatal disease. Diagnosis of IPF requires considerable expertise and experience. Since publication of the international IPF guideline in the year 2011 and Update 2018 several studies and technical advances occurred, which made a new assessment of the diagnostic process mandatory. In view of the antifibrotic drugs which have been approved for the treatment of IPF patients, the goal of this guideline is to foster early, confident and effective diagnosis of IPF. The guideline focusses on the typical clinical setting of an IPF patient and provides tools to exclude known causes of interstitial lung disease including standardised questionnaires, serologic testing and cellular analysis of bronchoalveolar lavage. High resolution computed tomography remains crucial in the diagnostic work-up. If it is necessary to obtain specimen for histology transbronchial lung cryobiopsy is the primary approach, while surgical lung biopsy is reserved for patients who are fit for it and in whom bronchoscopic diagnosis did not provide the information needed. Despite considerable progress, IPF remains a diagnosis of exclusion and multidisciplinary discussion remains the golden standard of diagnosis.

[Lung transplantation : Histomorphological diagnosis and clinical aspects]. / Lungentransplantation : Histomorphologische Diagnostik und klinische Aspekte.

The function of pulmonary allografts is regularly impaired by alloimmune reactions with quite variable clinical outcomes, different involved effector cells and molecules, as well as affected anatomical compartments. Acute rejection of grafts after lung transplantation (LuTx) is not only associated with the subsequent development of acute graft dysfunction, but can also contribute - among other immunological and nonimmunological factors - to the development of chronic lung allograft dysfunction (CLAD), which is the main reason for the limited long-term survival after LuTx. In addition to ACR and analogous to other solid organ transplants, the importance of antibody-mediated (humoral) rejection (AMR) in LuTx has also been recognized. There are currently no specific laboratory, radiological, or clinical tests available for either ACR or AMR. Only by the synoptic examination of histopathological changes and interpretation against the background of microbiological, virological, serological, and functional findings, can adequate sensitivity and specificity be achieved in the diagnostics of rejection. In this article, the current criteria for histopathological diagnostics of rejection following LuTx are summarized and the most important differential diagnoses are discussed.

[Injury patterns in interstitial lung diseases]. / Schädigungsmuster interstitieller Lungenerkrankungen.

Interstitial lung diseases (ILD) consist of a complex group of hundreds of non-neoplastic pulmonary diseases with divergent clinical presentation, morphology and progression tendency. This great number of clinical entities contrasts with a limited number of injury patterns. By definition, an adequate classification requires a synopsis of the clinical, radiological and morphological findings. The ATS/ERS (American Thoracic Society/ European Respiratory Society) guidelines recommend an open lung biopsy if high-resolution computed tomography does not provide conclusive results. Due to the focal nature and overlapping features of injury patterns, microscopic categorization is not always possible. In order to broaden the diagnostic criteria by using molecular patterns the Lung Research Working Group of the Institute of Pathology of Hannover Medical School, Europe's leading transplant center, is working up fresh explanted human lungs in a standardized manner. These fresh specimens are used for translational research by means of functional, morphological and molecular techniques in order to identify disease-specific regulatory processes and to make them usable diagnostically and therapeutically.

Endobronchial Ultrasound in Suspected Non-Malignant Mediastinal Lymphadenopathy.

BACKGROUND: Endobronchial ultrasound (EBUS) bronchoscopy with transbronchial needle aspiration (TBNA) is a well-established tool in mediastinal staging in lung cancer and gains importance in exploration of non-malignant lymphadenopathy. The aim of this study was to evaluate the role of EBUS-TBNA in suspected non-malignant diseases. METHODS: A retrospective, single-center, observation analysis of endobronchial ultrasound bronchoscopy procedures was performed in a university medical center between March 2013 and July 2015. All patients with suspected non-malignant mediastinal lymphadenopathy were included. Cytopathological and microbiological results of EBUS were compared to clinical diagnosis 6 months after procedure and performance of EBUS was contrasted to malignant indications. RESULTS: During study period, 333 EBUS bronchoscopies in 315 patients with mediastinal lymphadenopathy were performed. 111 out of 315 (35 %) patients had neither primary signs nor history of a malignant disease, categorised as patients with suspected non-malignant disease. 245 lymph nodes were sampled (median size 15 mm [IQR10 - 19]). Preferred station for TBNA was lymph node station 7 (38 %). Cytopathological findings revealed non-specific inflammation (n = 81; 70 %), carcinoma (n = 7; 6 %), epithelioid cell granulomas (n = 20; 17 %). 7 samples (6 %) were non-representative. Microbiologic testing of lymph nodes identified 3 infections (Mycobacteria tuberculosis [n = 2] and Nocardia nova [n = 1]) relevant to antibiotic therapy. Minor adverse events were observed in 9 out of 115 (8 %) patients. Sensitivity of EBUS-TBNA intervention in suspected non-malignant disease was 76 % and specificity 96 %. CONCLUSIONS: EBUS-TBNA revealed a specific cause for suspected non-malignant lymphadenopathy in one-third of cases and was associated with excellent specificity. Predominant specific causes were granuloma, besides from tumor. In 3 patients pathogen could be isolated by TBNA.

In Vivo Development of Transplant Arteriosclerosis in Humanized Mice Reflects Alloantigen Recognition and Peripheral Treg Phenotype of Lung Transplant Recipients.

Experimentally, regulatory T cells inhibit rejection. In clinical transplantations, however, it is not known whether T cell regulation is the cause for, or an epiphenomenon of, long-term allograft survival. Here, we study naïve and alloantigen-primed T cell responses of clinical lung transplant recipients in humanized mice. The pericardiophrenic artery procured from human lung grafts was implanted into the aorta of NODrag-/- /IL-2rγc-/- mice reconstituted with peripheral blood mononuclear cells (PBMCs) from the respective lung recipient. Naïve or primed allogeneic PBMCs procured 21 days post-lung transplantation with or without enriching for CD4+ CD25high T cells were used. Transplant arteriosclerosis was assessed 28 days later by histology. Mice reconstituted with alloantigen-primed PBMCs showed significantly more severe transplant arteriosclerosis than did mice with naïve PBMCs (p = 0.005). Transplant arteriosclerosis was equally suppressed by enriching for autologous naïve (p = 0.012) or alloantigen-primed regulatory T cells (Tregs) (p = 0.009). Alloantigen priming in clinical lung recipients can be adoptively transferred into a humanized mouse model. Transplant arteriosclerosis elicited by naïve or alloantigen-primed PBMCs can be similarly controlled by potent autologous Tregs. Cellular therapy with expanded autologous Tregs in lung transplantation might be a promising future strategy.

Augmentation of Transient Donor Cell Chimerism and Alloantigen-Specific Regulation of Lung Transplants in Miniature Swine.

Donor alloantigen infusion induces T cell regulation and transplant tolerance in small animals. Here, we study donor splenocyte infusion in a large animal model of pulmonary transplantation. Major histocompatibility complex-mismatched single lung transplantation was performed in 28 minipigs followed by a 28-day course of methylprednisolone and tacrolimus. Some animals received a perioperative donor or third party splenocyte infusion, with or without low-dose irradiation (IRR) before surgery. Graft survival was significantly prolonged in animals receiving both donor splenocytes and IRR compared with controls with either donor splenocytes or IRR only. In animals with donor splenocytes and IRR, increased donor cell chimerism and CD4(+) CD25(high+) T cell frequencies were detected in peripheral blood associated with decreased interferon-γ production of leukocytes. Secondary third-party kidney transplants more than 2 years after pulmonary transplantation were acutely rejected despite maintained tolerance of the lung allografts. As a cellular control, additional animals received third-party splenocytes or donor splenocyte protein extracts. While animals treated with third-party splenocytes showed significant graft survival prolongation, the subcellular antigen infusion showed no such effect. In conclusion, minipigs conditioned with preoperative IRR and donor, or third-party, splenocyte infusions may develop long-term donor-specific pulmonary allograft survival in the presence of high levels of circulating regulatory T cells.

Evaluation of the Effectiveness of Peritoneal Adhesion Prevention Devices in a Rat Model.

BACKGROUND: Abdominal operations are followed by adhesions, a prevalent cause of abdominal pain, and the most frequent cause for bowel obstruction and secondary female infertility. This rat study addresses adhesion prevention capability of Adept(®), Interceed(®), Seprafilm(®), and a novel device, 4DryField(®) PH which is provided as powder and generates its effect as gel. METHODS: Sixty-eight male Lewis rats had cecal abrasion and creation of an equally sized abdominal wall defect, and were grouped randomly: A control group without treatment (n=10); two groups treated with 4DryField(®) PH using premixed gel (n=15) or in-situ gel technique (n=16); one group each was treated with Seprafilm(®) (n=8), Interceed(®) (n=9), or Adept(®) (n=10). Sacrifice was on day 7 to evaluate incidence, quality, and quantity of adhesions, as expressed via adhesion reduction rate (AR). Histologic specimens were evaluated. Statistical analyses used ANOVA and unpaired t-tests. RESULTS: 4DryField(®) PH significantly reduced incidence and severity of adhesions both as premixed gel (AR: 85.2%) and as in-situ made gel (AR: 100%), a comparison between these two application techniques showed no differences in efficacy. Seprafilm(®) did not reduce incidence but severity of adhesions significantly (AR: 53.5%). With Interceed(®) (AR: 3.7%) and Adept(®) (AR: 16.1%) no significant adhesion-reduction was achieved. Except for inflammatory response with Interceed(®), histopathology showed good tissue compatibility of all other devices. CONCLUSION: 4DryField(®) PH and Seprafilm(®) showed significant adhesion prevention capabilities. 4DryField(®) PH achieved the highest adhesion prevention effectiveness without restrictions concerning mode of application and compatibility and, thus, is a promising strategy to prevent abdominal adhesions.

Impairment of the Peritoneal Surface as a Decisive Factor for Intestinal Adhesions in Intraperitoneal Onlay Mesh Surgery - Introducing a New Rat Model.

BACKGROUND: Meshes implanted intraperitoneally are known to cause adhesions potentially resulting in complications such as chronic pain, enterocutaneous fistula, or mesh infection. This study introduces a model for investigation of intestine-to-mesh adhesions and evaluates as to whether missing of visceral peritoneum is causative. METHODS: In 18 rats, rectangular 1.5 x 2 cm patches of an uncoated polypropylene mesh (Ultrapro(®)) were sewn to the inner abdominal wall next to the cecum. Additionally, a meso-suture ensured contact between cecum and mesh. Rats were assigned to 2 groups: in 8 rats the peritoneum was left intact, in 10 the cecum was depleted from peritoneum with abrasion. Sacrifice was on day 7. Macroscopic evaluation used two adhesion scores. Specimens were evaluated microscopically, statistical analyses employed student's t-test. RESULTS: On day 7, rats with mesh implantation combined with locally de-peritonealization by cecal abrasion mostly showed severe cecum-to-mesh agglutination (mean Lauder score 92%, mean total Hoffmann score 90%), whereas meshes of most animals without cecal abrasion only had some coverage with intraabdominal fat (33%, 24%; p = 0.0002). Histological work-up showed adequate wall ingrowth of mesh in all rats. In animals with cecal abrasion, meshes were mostly adhesive with cecal wall. However, when the peritoneum of cecum was unimpaired, abdominal wall above the mesh as well as cecum usually revealed sub-peritoneal tissue and a mono-layer cell coverage as seen in normal peritoneum. CONCLUSION: This study introduces a model mimicking a clinical situation of e.g. hernia repair by intraperitoneally implanted meshes when mesh has contact with normal and with de-peritonealized intestine. The model might be useful for testing mesh types and coatings as well as other devices for their efficacy in adhesion prevention. The high adhesion scores of rats with local de-peritonealization compared with the low scores of animals with intact peritoneum indicate that the integrity of intestinal peritoneum is a decisive factor for adhesion formation.

Epididymitis: ascending infection restricted by segmental boundaries.

STUDY QUESTION: Is the regionalization of epididymitis related to epididymal segmentation? SUMMARY ANSWER: We show for the first time that luminal ascent of bacteria is strictly gated by epididymal segment boundaries, involving ductal constriction adjacent to the infected area. WHAT IS KNOWN ALREADY: The epididymal duct is a continuous, unbranched tube, coiled into segments that are divided by connective tissue septa. Sonographic analysis indicates that swelling associated with epididymitis is predominant in the cauda region. Epididymal segmentation has never been investigated in the context of pathological alterations. STUDY DESIGN, SIZE, AND DURATION: We analyzed segment-specific changes in the epididymal duct in a mouse model and in men. In the mouse epididymitis model (3 days post-infection, injection of bacteria into the lumen of the vas deferens), two Escherichia coli strains were tested: a uropathogenic strain CFT073 (UPEC, n = 7) and a fecal non-pathogenic strain NPEC470 (NPEC, n = 5). Two control groups: phosphate-buffered saline, sham-treated animals (n = 4) and untreated mice (n = 8). In addition, segmentation was verified by ex vivo injection of dye into the interstitial spaces of untreated mouse epididymides. Histological findings were compared with specimens from epididymitis patients (n = 10, age range 14-78, median 60 years) who underwent surgical intervention; control: samples from patients without epididymitis (n = 16, age range 38-87, median 73 years). PARTICIPANTS/MATERIALS, SETTING, AND METHODS: We investigated the ascending infections by detailed histological analysis in correlation with local infection status in a mouse epididymitis model. As a proof of concept, rare patient material from two archives was analyzed: epididymides from patients who underwent surgical intervention for persisting epididymitis, and for control, histologically normal epididymides from men who underwent orchiectomy for therapy of prostatic carcinoma. MAIN RESULTS AND THE ROLE OF CHANCE: Luminal ascent of E. coli in mice was strictly gated by epididymal segment boundaries. In the mouse model, both strains of E. coli were detected exclusively in the distal cauda segment associated with damage of the epithelium and muscle layer. Ductal constriction occurred in the non-infected upstream segments of infected area, putatively blocking further luminal ascent of bacteria in UPEC-infected animals. Corresponding histological and morphological changes were found in epididymitis patients. The caput region was found to be unaffected in patients and the mouse model. LIMITATIONS, REASONS FOR CAUTION: Patient samples represented advanced cases of epididymitis that made surgical intervention necessary. WIDER IMPLICATIONS OF THE FINDINGS: Our data demonstrate the impact of epididymal segmentation, presumably a protective response mechanism against infectious invasion and bacterial ascent, during epididymitis and affirm the importance of rapid intervention. STUDY FUNDING/COMPETING INTERESTS: This work was supported by grants from the State of Hessen (LOEWE-MIBIE) and the DFG (KFO 181). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: No clinical trial involved.

[Tutankhamun: evidence-based paleopathology versus "curse of the pharaoh"]. / Tutanchamun: Evidenzbasierte Paleopathologie vs. "Fluch des Pharao".

AIMS: Several speculations exist regarding possible diseases of the juvenile Pharaoh Tutankhamun. In this review published paleopathological findings and artificial alterations as well as suggestions regarding underlying diseases were characterized. MATERIAL AND METHODS: A selective search of the literature was carried out in the PubMed data base in an arbitrary time interval from 1960 to 2013 (search terms: Tutankhamun, Pharaoh, paleopathology and mummy) and additional supplementary literature. RESULTS: Many artificial changes were a result of embalming and the examinations which have been performed since exhumation in 1922. Evidenced pathologies are craniofacial dysmorphia, bilateral alterations of the feet, malarial disease and an acute traumatic fracture of the knee. The cause of the knee fracture could no longer be reconstructed. Other trauma (e.g. skull fractures) or familial transmission of an eighteenth dynasty syndrome could not be confirmed. CONCLUSION: In addition to many artificial post-mortem alterations, chronic and acute diseases could be verified in Tutankhamun, although the underlying causes are partially unknown.

α-Methylacyl-coenzyme A racemase (AMACR, p504s) is a marker to distinguish malignant melanomas from dysplastic nevi and melanocytic nevi.

Routinely processed skin biopsies are still the mainstay for the diagnosis of melanocytic skin neoplasms (MSNs) and are considered the "gold standard" for individual patient management and clinical trials. The diagnostic challenge of melanocytic lesions of the skin prompts histopathologists to consider new diagnostic tools; among these, immunohistochemistry. We aimed to find putative new immunohistochemical markers, which can supplement the histological criteria used to detect dysplasia. In this immunohistochemical study, we chose a panel of promising biomarkers which could potentially differentiate between different MSN entities. These included α-methylacyl-coenzyme A racemase (AMACR; p504s), which is involved in the degradation of branched chained fatty acid derivates. We analysed a cohort of benign nevi and malignant melanomas. The design of the study included 78 melanocytic skin neoplasms (26 malignant melanomas and 52 benign nevi) in a tissue microarray. Immunohistochemistry of cyclin-dependent kinase inhibitor 2A (p16Ink4a), methylacyl-coenzyme A racemase (AMACR), cyclin D1, and E-cadherin was performed and assessed. We have observed that the p16Ink4a, AMACR, cyclin D1, and E-cadherin showed no exclusive staining for nevi or melanomas. However, a significant overexpression of AMACR was found in malignant melanomas compared to benign nevi. AMACR overexpression was also associated with an increased p16Ink4a staining. Our results suggest AMACR as an immunohistochemical marker for distinguishing malignant melanomas and dysplastic nevi from conventional melanocytic nevi.

[Aberrant reparative tissue remodeling: histopathology and molecular pathology]. / Aberrierender reparativer Gewebeumbau : Histo- und Molekularpathologie.

Not only tumorous infiltrations can lead to destruction of parenchymal organs but also the aberrant proliferation and matrix production of mesenchymal cells and vessels during a dysregulated repair attempt. This fibrogenesis is the result of a complex pathogenesis, which can be investigated in animal models but also in situ to harvest new biomarkers. This article deals particularly with the second method and summarizes molecular pathological findings in various model diseases for aberrant reparative tissue reconstruction. These model diseases include plexiform vasculopathy in pulmonary arterial hypertension (PAH), Quilty lesions in heart transplantation, bronchiolitis obliterans (BO), inflammatory airway remodeling and Epstein-Barr virus (EBV) induced smooth muscle proliferation (posttransplantation smooth muscle tumor, PTSMT).Using in situ molecular pathology, we were able to dismiss an assumed involvement of myofibroblastic cells in vessel reconstruction of the lung in PAH. We could also for the first time perform a comprehensive molecular characterization of the vascular remodeling and prove that plexiform vasculopathy represents a complex-regulated epiphenomenon of excessive pulmonary hypertension. This method also allowed us to describe for the first time the miRNA expression in PAH in a compartment-specific manner and to draw conclusions regarding the damaged overriding regulatory mechanisms. In the same way, we were also able to describe the chimeric character of the complex neoangiogenesis in the donor organ after heart transplantation.After lung transplantation, we identified for the first time a group of tissue-based molecular markers, which can predict later occurrence of BO even in morphologically normal transbronchial biopsies. In addition, we have documented for the first time the molecular characteristics of the morphologically analogous airway reconstruction in lung-transplanted and non-transplanted patients. We could further elucidate the role of matrix metalloproteinases (MMP) and their antagonists in inflammatory airway reconstruction and deduce from this the resulting therapeutic implications. Accordingly, we were able to further clarify the origin, pathogenesis and the malignant potential of EBV-induced PTSMT and for the first time provide an evidence-based therapy recommendation and risk assessment.In summary, this article documents that in situ diagnostics can meet the requirements of the challenging parameters and issues of life sciences. It is to be expected that the technical possibilities will develop analogously to the increasing demands and the in situ method will move further into the focus of molecular pathology.

[Determination of tumor biological parameters in breast cancer: round robin testing for quality assurance]. / Bestimmung tumorbiologischer Parameter beim Mammakarzinom: Ringversuche zur Qualitätssicherung.

Round robin testing for quality assurance in the determination of the breast cancer biomarkers estrogen receptor (ER), progesterone receptor (PR) and epithelial growth factor receptor 2 (HER2) have been carried out in Germany for 13 years. As the first quality assurance trial worldwide tissue microarrays with 20 different breast cancer specimens were used. As a further innovation the challenges were split into a test part representing routine cases and a training part enriched with difficult borderline cases in order to uncover latent weaknesses in the participating laboratories. Certificates are issued based exclusively on the test part. Similar to NordiQC and UKNequas stained slides are assessed externally and the quality of staining and evaluation are considered separately. Since 2010 an additional internet-based trial without assessment of the staining quality is offered for ER and PR. Since the introduction of the round robin trials the numbers of participants (n = 200-250) and the success rates have steadily increased. The breast cancer quality assurance trial ranks first with regard to the number of participants in Germany. It could be demonstrated that regular participation in the round robin test leads to an improvement of staining results of ER, PR and HER2 and hence appears to be mandatory for maintaining quality standards. The use of fully automated immunohistochemical staining procedures has steadily increased and these are now used by approximately 50 % of participants.

[Mesothelioma-30 years after the asbestos ban in Germany]. / Mesotheliome ­ 30 Jahre nach dem Asbestverbot in Deutschland.

In 1993, a total asbestos ban was introduced in Germany. Thirty years later, mesothelioma is still one of the most frequent occupational diseases. Recent data on incidence, mortality, recognized occupational diseases, early detection, and assessment are presented in this article.

Role of human papillomaviruses in persistent and glucocorticoid-resistant juvenile phimosis.

BACKGROUND: In adults, human papillomaviruses (HPV), lichen sclerosus et atrophicus (LSA) and phimosis are considered to be major risk factors for penile cancer. In boys, a possible association between phimosis, LSA and HPV has been suggested. OBJECTIVE: To investigate the role of HPV in the persistence of phimosis in children. PATIENTS AND METHODS: Out of a cohort of 420 boys presenting with foreskin problems, we prospectively sampled the preputial tissue of 82 patients during circumcision: 46 with steroid-naïve and 36 with steroid-resistant phimosis. All foreskins were assessed clinically and histopathologically with regard to appearance, inflammation, oedema, epithelial degeneration and fibrosis. The viral status of the foreskins was determined by immunohistochemistry and highly sensitive PCR, with subsequent subtyping by DNA hybridization (HPV types 6, 11, 16, 18, 31, 33, 35, 39, 42, 44, 45, 51-54, 56, 58, 59, 61, 62, 66-68, 70, 72, 73, 81-84, 90, 91). RESULTS: The foreskins appeared normal in 62 boys and suggestive of LSA in one single case. Small cracks or white scars were present in seven steroid-naïve and 12 steroid-resistant foreskins. LSA was diagnosed microscopically in two of the steroid-naïve and six of the steroid-pretreated group. No evidence of HPV was found in any of the juvenile foreskins. CONCLUSIONS: Our prospective study has provided evidence that HPV is not usually present in the foreskin of boys with persistent phimosis after their first year of life and that topical glucocorticoid treatment failure is not associated with HPV or any specific histopathological changes.

Detection of HHV-6 Virus in specimen of a ductal pancreatic adenocarcinoma with comparison in tumor and normal tissue.

AIMS: The association of human herpesvirus 6 (HHV-6) species with pancreatic cancer is controversially discussed. The aim of this study was to further investigate the postulated association and to identify the basis of HHV-6 DNA positivity reported for pancreatic cancer tissue. METHODS: All samples of patients with pancreatic cancer (cancer and surrounding tissue) were analyzed for presence of HHV-6 DNA by PCR and then selected cases by immunohistochemistry. RESULTS: Sixty eight per cent (68% = 52/77) of all patients were HHV-6 DNA positive in any of the samples, 49% (38/77) were positive in tumor tissue. Specimens of just one patient were HHV-6A DNA positive, all other patients were positive for HHV-6B. Immunohistochemical analysis of HHV-6 DNA positive samples did not reveal any specific HHV-6B protein positive tumor cell. In contrast, supposed immune cells presented intra- and peritumorally expressed HHV-6B-protein. The cause of presence of these cells in the tumor stroma is unknown, as of yet. CONCLUSIONS: HHV-6 DNA-positivity of pancreatic cancer tissue described by us and others is probably not due to the infection of pancreatic cells by HHV-6, but rather due to the migration of HHV-6 positive immune cells into the pancreas. Based on our data, we suppose that there is no direct evidence for HHV-6 as a causative agent of pancreatic cancer, but further in-depth studies (including investigation of immune status of patients) are necessary to make definitive conclusions.

Preparation of large biological samples for high-resolution, hierarchical, synchrotron phase-contrast tomography with multimodal imaging compatibility.

Imaging across different scales is essential for understanding healthy organ morphology and pathophysiological changes. The macro- and microscale three-dimensional morphology of large samples, including intact human organs, is possible with X-ray microtomography (using laboratory or synchrotron sources). Preparation of large samples for high-resolution imaging, however, is challenging due to limitations such as sample shrinkage, insufficient contrast, movement of the sample and bubble formation during mounting or scanning. Here, we describe the preparation, stabilization, dehydration and mounting of large soft-tissue samples for X-ray microtomography. We detail the protocol applied to whole human organs and hierarchical phase-contrast tomography at the European Synchrotron Radiation Facility, yet it is applicable to a range of biological samples, including complete organisms. The protocol enhances the contrast when using X-ray imaging, while preventing sample motion during the scan, even with different sample orientations. Bubbles trapped during mounting and those formed during scanning (in the case of synchrotron X-ray imaging) are mitigated by multiple degassing steps. The sample preparation is also compatible with magnetic resonance imaging, computed tomography and histological observation. The sample preparation and mounting require 24-36 d for a large organ such as a whole human brain or heart. The preparation time varies depending on the composition, size and fragility of the tissue. Use of the protocol enables scanning of intact organs with a diameter of 150 mm with a local voxel size of 1 µm. The protocol requires users with expertise in handling human or animal organs, laboratory operation and X-ray imaging.