Pseudoxanthoma elasticum (PXE) is a heritable disorder of connective tissue, affecting mainly skin, eye and the cardiovascular system. PXE is characterized by dystrophic mineralization of elastic fibres. The condition is caused by loss of function mutations in ABCC6. We generated Abcc6 deficient mice (Abcc6-/-) by conventional gene targeting. As shown by light and electron microscopy Abcc6-/- mice spontaneously developed calcification of elastic fibres in blood vessel walls and in Bruch's membrane in the eye. No clear abnormalities were seen in the dermal extracellular matrix. Calcification of blood vessels was most prominent in small arteries in the cortex of the kidney, but in old mice, it occurred also in other organs and in the aorta and vena cava. Newly developed monoclonal antibodies against mouse Abcc6 localized the protein to the basolateral membranes of hepatocytes and the basal membrane in renal proximal tubules, but failed to show the protein at the pathogenic sites. Abcc6-/- mice developed a 25% reduction in plasma HDL cholesterol and an increase in plasma creatinine levels, which may be due to impaired kidney function. No changes in serum mineral balance were found. We conclude that the phenotype of the Abcc6-/- mouse shares calcification of elastic fibres with human PXE pathology, which makes this model a useful tool to further investigate the aetiology of PXE. Our data support the hypothesis that PXE is in fact a systemic disease.

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SUMMARYA number of cell types situated along interfaces of various tissues and organs such as the peritoneum and the intestine have been shown to secrete inflammatory cytokines in a polarized fashion. Retinal pigment epithelial (RPE) cells are positioned at the interface between the vascularized choroid and the avascular retina, forming part of the blood-retina barrier. These cells are potent producers of inflammatory cytokines and are therefore considered to play an important role in the pathogenesis of ocular inflammation. Whether cytokine secretion by these cells also follows a vectorial pattern is not yet known, and was therefore the subject of this study. Monolayers of human RPE cells (primary cultures and the ARPE-19 cell line) cultured on transwell filters were stimulated to produce IL-6 and IL-8 by adding IL-1b (100 U/ml) to either the upper or the lower compartment. After stimulation, the human RPE cell lines showed polarized secretion of IL-6 and IL-8 towards the basal side, irrespective of the side of stimulation. The ARPE-19 cell line also secreted IL-6 and IL-8 in a polarized fashion towards the basal side after basal stimulation; polarized secretion was, however, not apparent after apical stimulation. The observation that human RPE cells secrete IL-6 and IL-8 in a polarized fashion towards the choroid may represent a mechanism to prevent damage to the adjacent fragile retinal tissue.

BACKGROUND The risk of recurrent oncological disease due to the reintroduction of cancer cells via autotransplantation of cryopreserved ovarian tissue is unknown. METHODS A systematic review of literature derived from MEDLINE, EMBASE and the Cochrane Library was conducted. Studies on follow-up after autotransplantation; detection of cancer cells in ovarian tissue from oncological patients by histology, polymerase chain reaction or xenotransplantation; and epidemiological data on ovarian metastases were included. RESULTS A total of 289 studies were included. Metastases were repeatedly detected in ovarian tissue obtained for cryopreservation purposes from patients with leukaemia, as well as in one patient with Ewing sarcoma. No metastases were detected in ovarian tissue from lymphoma and breast cancer patients who had their ovarian tissue cryopreserved. Clinical studies indicated that one should be concerned about autotransplantation safety in patients with colorectal, gastric and endometrial cancer. For patients with low-stage cervical carcinoma, clinical data were relatively reassuring, but studies focused on the detection of metastases were scarce. Oncological recurrence has been described in one survivor of cervical cancer and one survivor of breast cancer who had their ovarian tissue autotransplanted, although these recurrences may not be related to the transplantation. CONCLUSIONS It is advisable to refrain from ovarian tissue autotransplantation in survivors of leukaemia. With survivors of all other malignancies, current knowledge regarding the safety of autotransplantation should be discussed. The most reassuring data regarding autotransplantation safety were found for lymphoma patients.

D. fragilis and G. lamblia were the most frequently encountered parasites in our study population. Improved diagnostic tests are essential tools to study the prevalence and pathogenesis of D. fragilis.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro-and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. In most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor. Int.

Pseudoxanthoma elasticum (PXE) is a hereditary disease characterized by progressive dystrophic mineralization of the elastic fibres. PXE patients frequently present with skin lesions and visual acuity loss. Recently, we and others showed that PXE is caused by mutations in the ABCC6/MRP6 gene. However, the molecular pathology of PXE is complicated by yet unknown factors causing the variable clinical expression of the disease. In addition, the presence of ABCC6/MRP6 pseudogenes and multiple ABCC6/MRP6-associated deletions complicate interpretation of molecular genetic studies. In this study, we present the mutation spectrum of ABCC6/MRP6 in 59 PXE patients from the Netherlands. We detected 17 different mutations in 65 alleles. The majority of mutations occurred in the NBF1 (nucleotide binding fold) domain, in the eighth cytoplasmatic loop between the 15th and 16th transmembrane regions, and in NBF2 of the predicted ABCC6/MRP6 protein. The R1141X mutation was by far the most common mutation identified in 19 (32.2%) patients. The second most frequent mutation, an intragenic deletion from exon 23 to exon 29 in ABCC6/MRP6, was detected in 11 (18.6%) of the patients. Our data include 11 novel ABCC6/MRP6 mutations, as well as additional segregation data relevant to the molecular pathology of PXE in a limited number of patients and families. The consequences of our data for the molecular pathology of PXE are discussed.

Dientamoeba fragilis is a globally occurring parasite that has been recognized as a causative agent of gastrointestinal symptoms. A single-round PCR was developed to detect D. fragilis DNA directly from human stool samples. The genetic diversity of D. fragilis from 93 patients and 6 asymptomatic carriers was examined by PCR followed by restriction fragment length polymorphism and sequencing of part of the small-subunit rRNA gene. The data show that D. fragilis sequences can be studied directly from fecal specimens despite the absence of a cyst stage and without the need for prior culturing. In addition, the results suggest strongly that D. fragilis shows remarkably little variation in its small-subunit rRNA gene.Dientamoeba fragilis is a protozoan parasite found in the mucosal crypts of the large intestines of humans. Originally D. fragilis was considered an ameba, but based on ultrastructural characteristics (2), antibody data (8), and phylogenetic data originating from 16S-like rRNA gene sequences, it has been established that it is a trichomonad (20), with no identified cyst stage. Most recent literature accepts that D. fragilis is an important enteric pathogen (7,10,18), with an estimated incidence of symptomatic infection of between 4 and 91% (11,21,25,26). Symptoms include abdominal pain, bloating, and diarrhea.Because of the lack of a cyst stage, diagnosis can be performed only on freshly passed stool or by the use of fixatives and permanent stains. In addition, day-to-day shedding is highly variable, which imposes the need for multiple sampling (24). These features have likely led to an underestimation of the prevalence of D. fragilis, which is reported to vary between 0.2% and more than 19% depending upon the population studied (3,9,15,16,26).Despite the relatively high prevalence of D. fragilis and its apparent role in patients presenting with gastrointestinal complaints, surprisingly little is known about its pathogenicity, route of transmission, epidemiology, and genetics. Because only some infected persons experience symptoms, it is possible that D. fragilis is a heterogeneous species with nonpathogenic and pathogenic variants with similar morphology but different pathogenicities. This has been suggested for other lumendwelling protozoa such as Giardia duodenalis (13) and demonstrated for Entamoeba histolytica/Entamoeba dispar (4, 6). In a first report on variability in D. fragilis, the 16S-like ribosomal subunit DNA sequence of cultured D. fragilis from a small number of patients was analyzed by restriction fragment length polymorphism (RFLP) (14). Two of the 11 isolates gave a different restriction fragment pattern, indicating that there was genetic diversity in these D. fragilis cultures.The cumbersome and time-consuming techniques to maintain D. fragilis in culture in combination with the rapid disappearance of trophozoites from fresh feces have probably precluded more extensive and detailed studies of the genetic variability in D. fragilis. In addition, culture is not suitable for molecular ana...