Localization, etiology and impact of calcium phosphate deposits in renal allografts.

Hypercalcemia, hypophosphatemia and renal phosphate wasting are common after kidney transplantation. Animal data suggest that these alterations in mineral metabolism may contribute to calcium phosphate (CaPhos) deposition in the kidney and renal dysfunction. We tested the hypothesis that CaPhos deposition is highly prevalent in the early posttransplant period and is related to a disturbed mineral metabolism. For this purpose, biomarkers of mineral metabolism and renal calcium and phosphorus handling were prospectively assessed in 201 renal transplant recipients. CaPhos deposits were observed in 4.6, 30.4 and 24.7% of protocol biopsies obtained at the time of engraftment, and 3 and 12 months thereafter, respectively. In multivariate logistic regression analysis, high calcium and low serum phosphorus levels were independently associated with renal CaPhos deposition at month 3. The extent of CaPhos deposition correlated significantly with the severity of mineral metabolism disturbances. Renal function after a mean follow-up of 33 months was similar in patients with and without CaPhos deposition at month 3. In conclusion, our data demonstrate that CaPhos deposition is highly prevalent in the early posttransplant period and suggest that a disordered mineral metabolism is implicated in its pathogenesis. The clinical relevance of CaPhos deposition remains to be established.

Quantification of material nonlinearity in relation to microdamage density using nonlinear reverberation spectroscopy: Experimental and theoretical study.

High amplitude vibrations induce amplitude dependence of the characteristic resonance parameters (i.e., resonance frequency and damping factor) in materials with microscopic damage features as a result of the nonlinear constitutive relation at the damage location. This paper displays and quantifies results of the nonlinear resonance technique, both in time (signal reverberation) and in frequency (sweep) domains, as a function of sample crack density. The reverberation spectroscopy technique is applied to carbon fiber reinforced plastic (CFRP) composites exposed to increasing thermal loading. Considerable gain in sensitivity and consistent interpretation of the results for nonlinear signatures in comparison with the linear characteristics are obtained. The amount of induced damage is quantified by analyzing light optical microscopy images of several cross-sections of the CFRP samples using histogram equalization and grayscale thresholding. The obtained measure of crack density is compared to the global macroscopic nonlinearity of the sample and explicitly confirms that the increase in nonlinearity is linked to an increased network of cracks. A change from 1% to 3% in crack density corresponds to a tenfold increase in the signature of nonlinearity. Numerical simulations based on a uniform distribution of a hysteretic nonlinear constitutive relation within the sample support the results.

Tacticity in chiral phononic crystals.

The study of vibrational properties in engineered periodic structures relies on the early intuitions of Haüy and Boscovich, who regarded crystals as ensembles of periodically arranged point masses interacting via attractive and repulsive forces. Contrary to electromagnetism, where mechanical properties do not couple to the wave propagation mechanism, in elasticity this paradigm inevitably leads to low stiffness and high-density materials. Recent works transcend the Haüy-Boscovich perception, proposing shaped atoms with finite size, which relaxes the link between their mass and inertia, to achieve unusual dynamic behavior at lower frequencies, leaving the stiffness unaltered. Here, we introduce the concept of tacticity in spin-spin-coupled chiral phononic crystals. This additional layer of architecture has a remarkable effect on their dispersive behavior and allows to successfully realize material variants with equal mass density and stiffness but radically different dynamic properties.

Bilirubin conjugates in bile of man and rat in the normal state and in liver disease.

Conjugates of bilirubin were studied in normal bile of man and rat, and in bile of liver patients. In general human bile was obtained by duodenal intubation. In addition T-tube bile was examined in patients operated on for mechanical obstruction. The bile pigment compositions of duodenal and T-tube bile were similar in two patients where comparison was possible. Obstruction of the bile duct in rats was used as an animal model for obstructive jaundice. Diazotized ethyl anthranilate was used for determination of total conjugated bile pigment and for thin-layer chromatography (t.l.c.) analysis of the derived azopigments. The available t.l.c. procedures are versatile and allow rapid and quantitative analysis. A variety of conjugated azopigments can be distinguished. With chloroform, negligible amounts of unconjugated bilirubin are extracted from bile of man. Therefore, the percentage of monoconjugated bile pigments present in the initial bile sample can be calculated from the percentage of azodipyrrole found after diazotization. Normal bile from man and rat yields similar azopigment patterns. The dominant component is azopigment-delta (azodipyrrole beta-D-monoglucuronoside). Small amounts of azopigments with complex conjugating structures (gamma-azopigments) are present in both cases. Human bile further yields small amounts of azopigments containing xylose or glucose (called azopigments-alpha(2) and -alpha(3), respectively). Monoconjugated bilirubin (estimated from the percentage of azodipyrrole) amounts of 22% of total bile pigments in human bile and to 39% in murine bile. In both, the bulk of bile pigment is bilirubin diglucuronoside. From bile of patients with acquired liver diseases a new azopigment group (beta-azopigment) was derived. The gamma-azopigment group was increased; the delta-azopigment group (containing azodipyrrole beta-D-monoglucuronoside) was decreased. No differentiation was possible between intra- and extrahepatic cholestasis. The percentage of beta-azopigment showed a positive correlation with serum bilirubin concentration (r = 0.6). Recovery of the diseases was accompanied by normalization of the azopigment patterns. In rats, hydrostatic or mechanical obstruction induced increases in beta- and gamma-azopigments and a decrease in delta-azopigment similar to the changes observed in bile of liver patients. Complete normalization was obtained 6 hr after relieving the hydrostatic obstruction (duration 15-21 hr). In contrast, with man after surgery for extrahepatic obstruction, T-tube bile was not normalized when the T-tube was withdrawn (10 days after operation). Hydrostatic obstruction in rats provides an easy model when postobstructive bile pigment composition and parameters have to be investigated. The present investigations stress the importance of the physiopathological state when studying bilirubin conjugation. Hindrance to bile secretion induced heterogeneity of bilirubin conjugates and stimulated the formation of complex structures.

Severe alport phenotype in a woman with two missense mutations in the same COL4A5 gene and preponderant inactivation of the X chromosome carrying the normal allele.

The X-linked form of Alport disease, caused by mutations in the COL4A5 or the COL4A6 gene, usually leads to terminal renal failure in males, while affected females have a more variable and moderate phenotype. We detected in a female patient, with a severe Alport phenotype, two new missense mutations. One mutation (G289V) occurred in exon 15 and converted a glycine in a collagenous domain of COL4A5 to a valine. The second mutation, located in exon 46, substituted a cysteine proximal to the NC1 domain of COL4A5 for an arginine. In white blood cells and kidney both mutations were present on > 90% of the mRNA, while at the genomic level the patient was heterozygous for both mutations. The two mutations therefore occurred in the same COL4A5 allele. No mutation was found in the COL4A5 promoter region by sequencing nor was a major rearrangement of the normal allele detected. A skewed pattern of X inactivation was demonstrated in DNA isolated from the patient's kidney and white blood cells: > 90% of the X chromosomes with the normal COL4A5 allele was inactivated. It is suggested that this skewed inactivation pattern is responsible for the absence of detectable normal COL4A5 mRNA and hence the severe phenotype in this woman.

TuBaFrost 3: regulatory and ethical issues on the exchange of residual tissue for research across Europe.

The regulatory regimes for research with residual tissue and accompanying data differ widely between countries in the European Union (EU): from specific consent to opt-out or even no consent at all. This could greatly hamper research where the exchange of tissue and accompanying data has become the gold standard, like in TubaFrost. Instead of adhering to international guidelines, which have a democratic deficit, or an attempt for a new set of possible harmonising rules, TubaFrost chose to create a coordinating rule: if tissue may legitimately be used for a certain kind of research in the country where it was taken and under whose jurisdiction the patient falls, it may also be used for such research in the country where it is sent to in the context of a scientific program even if in that other country other regulations would apply for research with residual tissue taken from patients under their jurisdiction. This coordinating rule has a sound basis in EU law in general and will solve the problems related to diverging national regulatory regimes in the case of cross national research with residual tissue.

TuBaFrost 6: virtual microscopy in virtual tumour banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated Virtual Microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting bio-repositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

TuBaFrost 1: Uniting local frozen tumour banks into a European network: an overview.

TuBaFrost is the consortium responsible for the creation of a virtual European human frozen tumour tissue bank: a collection of high quality frozen residual, accurately classified tumour tissue samples, which are stored in European cancer centres and universities. This virtual tissue bank, searchable on the internet, has rules for access and use, and a code of conduct to comply with the various legal and ethical regulations in European countries. The easy accessibility and the European scale of the bank will result in the availability of a large number of samples even of rarer tumour types. Standardisation of collection, storage and quality control throughout the network is achieved minimising inter-institutional variability. A website providing access to upload, search and request samples is a key tool of the tissue bank. The search engine makes use of virtual microscopy. An overview of the development of the European virtual frozen tissue bank infrastructure is described in this paper. The various key aspects are described in more detail in a series of articles to appear in this Journal.

TuBaFrost 2: Standardising tissue collection and quality control procedures for a European virtual frozen tissue bank network.

Tumour Bank Networking presents a great challenge for oncological research as in order to carry out large-scale, multi-centre studies with minimal intrinsic bias, each tumour bank in the network must have some fundamental similarities and be using the same standardised and validated procedures. The European Human Frozen Tumour Tissue Bank (TuBaFrost) has responded to this need by the promotion of an integrated platform of tumour banks in Europe. The operational framework for TuBaFrost has drawn upon the best practice of standard workflows and operating procedures employed by members of the TuBaFrost project and key initiatives worldwide.

TuBaFrost 4: access rules and incentives for a European tumour bank.

When designing infrastructure for a networked virtual tumour bank (samples remain at the collector institutes and sample data are collected in a searchable central database), it is apparent that this can only function properly after developing an adequate set of rules for use and access. These rules must include sufficient incentives for the tissue sample collectors to remain active within the network and maintain sufficient sample levels in the local bank. These requirements resulted in a key TuBaFrost rule, stating that the custodianship of the samples remains under the authority of the local collector. As a consequence, the samples and the decision to issue the samples to a requestor are not transferred to a large organisation but instead remain with the collector, thus allowing autonomous negotiation between collector and requestor, potential co-authorship in publications or compensation for collection and processing costs. Furthermore, it realises a streamlined cost effective network, ensuring tissue visibility and accessibility thereby improving the availability of large amounts of samples of highly specific or rare tumour types as well as providing contact opportunities for collaboration between scientists with cutting edge technology and tissue collectors. With this general purpose in mind, the rules and responsibilities for collectors, requestors and central office were generated.

TuBaFrost 5: multifunctional central database application for a European tumor bank.

Developing a tissue bank database has become more than just logically arranging data in tables combined with a search engine. Current demand for high quality samples and data, and the ever-changing legal and ethical regulations mean that the application must reflect TuBaFrost rules and protocols for the collection, exchange and use of tissue. To ensure continuation and extension of the TuBaFrost European tissue bank, the custodianship of the samples, and hence the decision over whether to issue samples to requestors, remains with the local collecting centre. The database application described in this article has been developed to facilitate this open structure virtual tissue bank model serving a large group. It encompasses many key tasks, without the requirement for personnel, hence minimising operational costs. The Internet-accessible database application enables search, selection and request submission for requestors, whereas collectors can upload and edit their collection. Communication between requestor and involved collectors is started with automatically generated e-mails.

TuBaFrost: European virtual tumor tissue banking.

TuBaFrost is a consortium responsible for the task to create a virtual European human frozen tumor tissue bank, composed of high quality frozen tumor tissue collections with corresponding accurate diagnosis stored in European cancer centers and universities, searchable on the Internet, providing rules for access and use and a code of conduct to comply with the various legal and ethical regulations in European countries. Such infrastructure would enlarge tissue availability and accessibility in large amounts of specified or even rare tumor samples. Design of an infrastructure for European residual tissue banking with the described characteristics, clear focus points emerge that can be broken down in dedicated subjects: (1) standardization and quality assurance (QA) to avoid inter-institute quality variation; (2) law and ethics enabling exchange of tissue samples possible between institutes in the different European countries, where law and ethics are characterized by a strong variability; (3) rules for access, with sufficient incentives for collectors; (4) central database application containing innovations on search and selection procedures; (5) support when needed with histology images; and (6) Internet access to search and upload, with in addition a solid website giving proper information on the procedures, intentions and activities not only to the scientific community, but also to the general public. One consortium decision, part of the incentives for collectors, had major impact on the infrastructure; custodianship over the tissues as well as the tissues stay with the collector institute. Resulting in specimens that are not given to an organization, taking decisions on participation of requests, but instead the local collected tissues stay very easy to access by the collector and allows autonomous negotiation between collector and requestor on cooperation, coauthorship in publication or compensation in costs. Thereby, improving availability of large amounts of high quality samples of a highly specified or rare tumor types and contact opportunities for cooperation with other institutes.

Virtual microscopy in virtual tumor banking.

Many systems have already been designed and successfully used for sharing histology images over large distances, without transfer of the original glass slides. Rapid evolution was seen when digital images could be transferred over the Internet. Nowadays, sophisticated virtual microscope systems can be acquired, with the capability to quickly scan large batches of glass slides at high magnification and compress and store the large images on disc, which subsequently can be consulted through the Internet. The images are stored on an image server, which can give simple, easy to transfer pictures to the user specifying a certain magnification on any position in the scan. This offers new opportunities in histology review, overcoming the necessity of the dynamic telepathology systems to have compatible software systems and microscopes and in addition, an adequate connection of sufficient bandwidth. Consulting the images now only requires an Internet connection and a computer with a high quality monitor. A system of complete pathology review supporting biorepositories is described, based on the implementation of this technique in the European Human Frozen Tumor Tissue Bank (TuBaFrost).

Cytogenetic characterization of tenosynovial giant cell tumors (nodular tenosynovitis).

Chromosome investigation in six localized forms of tenosynovial giant cell tumors, also known as modular tenosynovitis, revealed an identical translocation between chromosomes 1 and 2, t(1;2)(p11;q35-36) in three tumors, a variant translocation t(1;5)(p11;q22) in a fourth case, and a t(2;16)(q33;q24) in a fifth case. One case showed a normal karyotype. Although morphologically rather uniform, these benign tumors appear to be cytogenetically heterogeneous, but the chromosome changes seem to cluster in 2 regions, 1p11 and 16q24.

Completion thyroidectomy after the unexpected diagnosis of thyroid cancer.

The optimal surgical management of well-differentiated thyroid cancer (DTC) remains a controversial topic. Preoperative and peroperative investigations quite frequently fail to detect thyroid cancer in cold nodules, and only postoperative histological examination reveals malignancy. In these cases many physicians perform a completion thyroidectomy. Others recommend a conservative approach with close follow-up because of the increased risk of complications after re-operation. In our department, routine management includes completion thyroidectomy once the histopathological report concludes that there is carcinoma, except in cases of papillary carcinoma measuring less than 1 cm. The aim of our study was to determine the incidence of contralateral malignancy and of complications after completion thyroidectomy. We reviewed the records of 29 patients--25 women and 4 men-- who all underwent completion thyroidectomy because of an unexpected diagnosis of DTC. Residual malignancy was found in 12 patients (41.4%) after completion thyroidectomy. In ten patients (34.5%) the malignancy was localised in the contralateral lobe and two patients (6.9%) had lymph node metastases. Postoperative transient hypocalcaemia (< 8.0 mg/dl) occurred in five patients (17.2%) and permanent hypocalcaemia (lasting longer than 6 months) was a feature in two patients. One patient suffered transient laryngeal nerve injury occurred in one patient and there were no permanent lesions. In conclusion, we found residual DTC in 41.4% of patients undergoing reintervention. Because of the rather low re-operation rate, we prefer to perform a completion thyroidectomy to remove potential occult malignancy and to allow for postoperative 131I-treatment in all patients with a diagnosis of malignancy in their thyroid lobectomy specimen, with the exception of papillary carcinoma < 1 cm.

Secretion of prostatic binding protein by rat ventral prostate: influence of age and androgen.

Rat ventral prostate of adult male rats contain a large amount of prostatic binding protein (PBP). Immunological evidence indicates that this protein is a specific secretion product of this gland. The amount and concentration of PBP in ventral prostate show marked changes as a function of age. PBP is low but detectable (0.009 and 0.002 U/mg prostate) in 5- and 10-day-old rats and increases thereafter in a biphasic way to adult levels (0.619 U/mg prostate). After castration of PBP drops to 0.054 U/mg prostate after 10 days and 0.030 U/mg prostate after 21 days. The concentration of PBP returns to precastration levels after 2 weeks of androgen treatment. Estradiol and progesterone are ineffective in this respect. The antiandrogen, cyproterone acetate, counteracts the stimulatory effect of testosterone propionate.

Morphometric analysis of human bone biopsies: a quantitative structural comparison of histological sections and micro-computed tomography.

As a part of the European Union BIOMED I study "Assessment of Bone Quality in Osteoporosis," the trabecular structure of transiliac bone biopsies was assessed by conventional histomorphometry and by three-dimensional microcomputed tomography (microCT). Sixty-three cylindrical human transiliac bone specimens were obtained post mortem from 27 women and 36 men (age 23-92 years) and evaluated morphometrically in this study. For the conventional histomorphometric analysis, a pair of four undecalcified sections were prepared from two regions in the mediolateral plane of the bone cores. The sections were stained with the von Kossa technique to obtain high-contrast two-dimensional images. Prior to the histologic sectioning the samples were measured with a desktop microCT, providing a 14 microm nominal resolution. The morphometric parameters computed by both methods in two or three dimensions, respectively, were bone volume density (BV/TV), bone surface density (BS/TV), trabecular thickness (Tb.Th), and trabecular separation (Tb.Sp). Results showed highly significant (p < 0.0001) correlations between conventional histology and microCT for BV/TV (r = 0.93), BS/TV (r = 0.91), Tb.Th (r = 0.84), and Tb.Sp (r = 0.91). The mean percentage differences between histology and microCT ranged from 2.5% (BV/TV) to 6.1% (Tb.Th). The high correlations and the low differences in terms of absolute numbers between conventional histomorphometric and microtomographic analysis are very promising for the use of micro-tomographic imaging. MicroCT is a nondestructive, fast, and very precise procedure that allows the measurement of cancellous and compact bone in unprocessed biopsies or small bones, as well as a fully automatic determination of three-dimensional morphometric indices.

Neuromuscular choristoma (hamartoma) with smooth and striated muscle component: case report with immunohistochemical and ultrastructural analysis.

Benign peripheral nerve tumors with a mesenchymal component are rare and are represented principally by the neuromuscular choristoma. We describe an 11-month-old male infant who presented with a mass arising from the left brachial plexus. The lesion was bound firmly to the involved nerves and consisted histologically and ultrastructurally of bundles of well-differentiated smooth muscle cells intermingled with striated muscle fibers and unmyelinated nerve fibers. In the 13 previously published cases of neuromuscular choristoma, no smooth muscle component was observed. This unique peripheral nerve choristoma with not only striated but also smooth muscle closely resembled neuromuscular choristoma and was considered a variant of it.

Cutaneous sclerosing perineurioma with cryptic NF2 gene deletion.

Sclerosing perineurioma is a recently described variant of perineurioma that characteristically occurs in the fingers and palms of young adults. We report a cutaneous sclerosing perineurioma with preservation of the axons and Schwann cells in the center of the whorls of perineurial cells, a feature that previously was reported to be typically absent in these lesions. Additionally, clonal chromosomal abnormalities of chromosome 10 and a cryptic deletion of the 5'BCR and NF2 loci on chromosome 22 were present. These findings further support the hypothesis that a gene on chromosome 22 may play a role in the pathogenesis of perineurioma. The NF2 gene is a logical candidate because of its involvement in other nerve sheath tumors.