Determinants and effects of sinus worm Skrjabingylus nasicola (Nematoda: Metastrongyloidae) infestation in invasive American mink Neovison vison in Germany.

Skrjabingylus nasicola (Leuckart, 1842) are geographically widespread nematodes that parasitize the nasal and frontal sinus cavities of smaller mustelids. As most prior work was solely based on the analysis of bone injuries of museum skull, little is known about the determinants and effects of infestation in the host species. Working on fresh skulls, we aimed to analyze infestation patterns in American mink (Neovison vison Schreber, 1777) from nine study areas in northern Germany and to identify factors that explained infestation prevalence and intensity in the host species. The prevalence (46.7-62.9 %) and infestation intensity values (4.5-10.89 nematodes) reported here were relatively large, especially compared to other American mink populations in Europe. Considering mink diet, our study sites probably harbored a larger number of infested paratenic hosts and climate did not have a substantial negative influence on survival of S. nasicola larvae. We did not observe any significant sex-age differences in either prevalence or intensity of S. nasicola infestation. We did not find a negative impact of an infestation on the host animals' body weight, confirming prior results that the parasite is not a significant mortality factor in mustelids. Our study suggests that this holds even outside the native distributional range where the host's defenses might not be optimally adapted to an autochthonous parasite.

Mitotic stability of fragile X mutations in differentiated cells indicates early post-conceptional trinucleotide repeat expansion.

We demonstrate here that somatic variation of CGG repeat length is based on a mosaic of cells with different but stable FMR-1 alleles and does not reflect permanent mitotic instability. The length of a particular allele in an individual cell was maintained in progeny cells establishing a clone. The mutation patterns of multiple repeats in the DNA of fetal tissues were identical and did not significantly change during proliferation in vitro. It is proposed that genotype mosaicism and expansion to full mutation are generated post-conceptionally by the same molecular mechanism in a particular window of early development.

Recruitment of old genes to new functions: evidences obtained by comparing the orthologues of human XLMR genes in mouse and chicken.

Gene mapping data indicate that the human X chromosome is enriched in genes that affect both, higher cognitive efficiency and reproductive success. This raises the question whether these functions are ancient, or whether conserved X-linked genes were recruited to new functions. We have studied three X-linked mental retardation (XLMR) genes by RNA in situ hybridization in mouse and in chicken, in which these genes are autosomal: Rho guanine nucleotide exchange factor 6 (ARHGEF6), oligophrenin (OPHN1), and p21 activated kinase 3 (PAK3). In the mouse these genes are specifically expressed in telencephalic regions. Their orthologues in the chicken gave patterns of similar specificity in ancient parts of the brain, i.e. cerebellum and mesencephalon, but were not expressed in the telencephalon. Also in the testes, specific expression was only found in mouse, not in chicken. These data are interpreted such that certain genes on the X chromosome gained novel functions during evolution.

Fumarate hydratase enzyme activity in lymphoblastoid cells and fibroblasts of individuals in families with hereditary leiomyomatosis and renal cell cancer.

BACKGROUND: Hereditary leiomyomatosis and renal cell cancer (HLRCC) is the autosomal dominant heritable syndrome with predisposition to development of renal cell carcinoma and smooth muscle tumours of the skin and uterus. OBJECTIVE: To measure the fumarate hydratase (FH) enzyme activity in lymphoblastoid cell lines and fibroblast cell lines of individuals with HLRCC and other familial renal cancer syndromes. METHODS: FH enzyme activity was determined in the whole cell, cytosolic, and mitochondrial fractions in 50 lymphoblastoid and 16 fibroblast cell lines including cell lines from individuals with HLRCC with 16 different mutations. RESULTS: Lymphoblastoid cell lines (n = 20) and fibroblast cell lines (n = 11) from individuals with HLRCC had lower FH enzyme activity than cells from normal controls (p<0.05). The enzyme activity in lymphoblastoid cell lines from three individuals with mutations in R190 was not significantly different from individuals with other missense mutations. The cytosolic and mitochondrial FH activity of cell lines from individuals with HLRCC was reduced compared with those from control cell lines (p<0.05). There was no significant difference in enzyme activity between control cell lines (n = 4) and cell lines from affected individuals with other hereditary renal cancer syndromes (n = 22). CONCLUSIONS: FH enzyme activity testing provides a useful diagnostic method for confirmation of clinical diagnosis and screening of at-risk family members.

Increase of FMRP expression, raised levels of FMR1 mRNA, and clonal selection in proliferating cells with unmethylated fragile X repeat expansions: a clue to the sex bias in the transmission of full mutations?

Fragile X syndrome is a triplet repeat disorder caused by expansions of a CGG repeat in the fragile X mental retardation gene (FMR1) to more than 220 triplets (full mutation) that usually coincide with hypermethylation and transcriptional silencing. The disease phenotype results from deficiency or loss of FMR1 protein (FMRP) and occurs in both sexes. The underlying full mutations arise exclusively on transmission from a mother who carries a premutation allele (60-200 CGGs). While the absolute requirement of female transmission could result from different mechanisms, current evidence favours selection or contraction processes acting at gametogenesis of pre- and full mutation males. To address these questions experimentally, we used a model system of cultured fibroblasts from a male who presented heterogeneous unmethylated expansions in the pre- and full mutation size range. On continual cell proliferation to 30 doublings we re-examined the behaviour of the expanded repeats on Southern blots and also determined the expression of the FMR1 gene by FMRP immunocytochemistry, western analysis, and RT-PCR. With increasing population doublings, expansion patterns changed and showed accumulation of shorter alleles. The FMRP levels were below normal but increased continuously while the cells that were immunoreactive for FMRP accumulated. The level of FMR1 mRNA was raised with even higher levels of mRNA measured at higher passages. Current results support the theory of a selection advantage of FMRP positive over FMRP deficient cells. During extensive proliferation of spermatogonia in fragile X males, this selection mechanism would eventually replace all full mutations by shorter alleles allowing more efficient FMRP translation. At the proliferation of oogonia of carrier females, the same mechanism would, in theory, favour transmission of any expanded FMR1 allele on inactive X chromosomes.

Wavelength dependency of photodynamic effects after sensitization with 5-aminolevulinic acid in vitro and in vivo.

A promising new therapeutic modality for skin cancer, administration of the heme precursor 5-aminolevulinic acid followed by light irradiation, is known as photodynamic therapy. Photofrin, the only clinically approved sensitizer, has an absorption maximum at 630 nm, the wavelength used in most experimental and clinical trials with 5-aminolevulinic acid. We investigated photodynamic efficacy of irradiation with coherent light at wavelengths ranging from 622 to 649 nm in vitro and in vivo as well as the content and distribution of intracellular porphyrin after administration of 5-aminolevulinic acid. HaCaT immortalized human keratinocytes were sensitized with 30 micrograms/ml 5-aminolevulinic acid for 24 h in vitro. By cell viability determined with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, the best cell-killing effects were observed after irradiation at 635 nm. Using an amelanotic melanoma (A-Mel-3) grown subcutaneously in Syrian Golden hamsters, we confirmed these results in vivo: tumor growth was markedly delayed in animals treated with 100 mg/kg 5-aminolevulinic acid intravenously and irradiated with coherent light at 635 nm as compared to animals irradiated at 630 nm. This photodynamic effect is probably mediated by large amounts of the photosensitizing porphyrin, protoporphyrin IX, localized in cell membranes as visualized by confocal laser scan microscopy and as determined by high pressure liquid chromatography in vitro. The results suggest that irradiation at 635 nm with a coherent light source is more effective than irradiation at 630 nm for photodynamic therapy with 5-aminolevulinic acid.

Multiple congenital anomalies/mental retardation (MCA/MR) syndrome due to interstitial deletion 1q.

A severely retarded male infant was found to have a previously undescribed multiple congenital anomalies/mental retardation (MCA/MR) syndrome including microdolichocephaly, prominence of metopic suture, coarse scalp hair, epicanthus, anteverted nostrils, micrognathia, posteriorly angulated malformed auricles, preaxial hexadactyly, clinodactyly, camptodactyly, hypospadias, cryptorchidism, inguinal hernias, agenesis of left kidney, and pyloric stenosis. This syndrome was due to an interstitial del(1)(q25.2q31.2) associated with a paracentric inv(1)(q31.2q44).

Fragile X: carrier detection in pregnancy.

The expression of the fragile X in 9 pregnant women (4 obligate carriers and 5 carriers at risk) was not significantly different from the expression observed either before or after pregnancy in the same individuals. Hence, carrier diagnosis during pregnancy seems to be as reliable as in non-pregnant women.

How can the frequency of false-negative findings in prenatal diagnoses of fra(X) be reduced: experience with first trimester chorionic villi sampling.

We report on 12 prenatal diagnoses performed between weeks 10 and 13 on normal women with a well-documented family history of the Martin-Bell syndrome. Seven were obligate and three were potential carriers. One male and 2 female fetuses were found to be fragile X [fra(X)]-positive. The diagnoses were confirmed in fibroblasts or lymphocytes after interruption or postnatally. In one fra(X)-negative female fetus, the analysis of linked DNA markers indicated that most probably she was a heterozygote. Reexamination after birth gave a fra(X)-positive result. Hence this was a case of a false-negative prenatal fra(X) result. The occurrence of false-negative cytogenetic results represents a common problem that limits the sensitivity of prenatal diagnostics in the Martin-Bell syndrome. A study of linked DNA markers can improve the reliability of negative cytogenetic results in first trimester prenatal diagnosis. In case of doubt, the chromosomes could be reexamined after fetal blood sampling.

The dup(3q) syndrome: report of eight cases and review of the literature.

Clinical and cytogenetic examinations were performed on eight unrelated infants with duplication of part of the long arm of chromosome 3. A review of published cases shows a clinical syndrome characterized by statomotoric retardation, shortened life span, and a multiple congenital anomalies (MCA) syndrome of abnormal head configuration, hypertrichosis, hypertelorism, ocular anomalies, anteverted nostrils, long philtrum, maxillary prognathia, down-turned corners of the mouth, highly arched or cleft plate, micrognathia, malformed auricles, short, webbed neck, clinodactyly, simian crease, talipes, and congenital heart disease. The dup(3q) syndrome is a clinically easily recognizable entity.

Proliferation of tumor spheroids after shock-wave treatment.

Multicellular tumor spheroids (MCTS) grown from the bladder cancer cell line RT112 and from the prostate cancer cell line PCA were exposed to 200 or 800 electromagnetically generated focused ultrasound shock waves. RT112 cells showed a distinct but transient decrease in proliferation whereas the effect of PCA cells was less pronounced. Flow-cytometric measurements of DNA content and Ki67 expression revealed no significant changes in the cell cycle distribution. The capacity of RT112 cells exposed to 800 shock waves to re-grow as MCTS was markedly decreased, indicating an alteration of intercellular adhesion.

Intravesical instillation of 5-aminolevulinic acid: the fluorescent metabolite is limited to urothelial cells.

OBJECTIVES: For photodynamic therapy 5-aminolevulinic acid is an attractive compound, since it is a physiologic endogenous substance, and its application in excess results in the accumulation of the metabolite protoporphyrin IX, a very effective photosensitizer. The topical application of 5-aminolevulinic acid in the urinary bladder led to pronounced fluorescence in neoplasias when excited with violet laser light during cystoscopy. The aim of this study was the determination of the transmural distribution of protoporphyrin IX in order to estimate potential efficacy and side effects of a therapeutic application of 5-aminolevulinic acid. METHODS: 5-Aminolevulinic acid was instilled prior to cystoscopy and biopsies were taken of lesions that were either fluorescing or nonfluorescing. Fluorescence distribution was analyzed by fluorescence microscopy on cryostat sections prepared from 72 biopsy specimens. In addition, multicellular spheroids grown from tumor cells and fibroblasts were exposed to 5-aminolevulinic acid and analyzed accordingly. RESULTS: Biopsy preparations showed that the fluorescence of protoporphyrin IX was limited to normal and neoplastic urothelial cells. Clinical findings were supported by the in vitro data, which showed negative protoporphyrin IX fluorescence in fibroblasts. CONCLUSIONS: Thus, 5-aminolevulinic acid might be superior in selective accumulation to conventional sensitizers known to localize also in endothelial cells of the tumor stroma. The data appear to hold great promise for 5-aminolevulinic acid in photodynamic diagnosis and therapy in bladder cancer, as phototoxicity will be limited to mucosal lesions. Bladder shrinkage due to photodamage of subepithelial structures even in case of high light doses is not expected.

Fluorescence photodetection of neoplastic urothelial lesions following intravesical instillation of 5-aminolevulinic acid.

OBJECTIVES: Tiny papillary tumors and flat urothelial lesions such as dysplasia or carcinoma in situ can easily be missed during routine cystoscopy. Various methods for in vivo detection of fluorescing agents (preferentially localized in malignant tissue) have been developed. Most of them are based on systemically administered synthetic porphyrin compounds and require sensitive detection devices and image processing units for fluorescence visualization. The usefulness of intracellularly accumulated endogenous protoporphyrin IX (PPIX), induced by 5-aminolevulinic acid (ALA), for diagnosis of early bladder cancer and the correlation with cystoscopic, microscopic, and fluorescence findings was investigated. METHODS: ALA was instilled intravesically in 68 patients, followed by fluorescence cystoscopy with violet light from a krypton ion laser that produced fluorescence excitation. There were 299 biopsies obtained from fluorescing and nonfluorescing areas of the bladder. RESULTS: ALA-induced fluorescence could be easily observed with the naked eye during cystoscopy under violet light illumination. All tumor lesions were sharply marked with brightly shining red fluorescence. Correlation of fluorescence and microscopic findings gave a sensitivity of 100% and a specificity of 68.5%. There were 26 malignant or precancerous lesions that were missed during routine cystoscopy but were detected only by ALA-induced fluorescence. CONCLUSIONS: Labeling of urothelial lesions by PPIX fluorescence induced by intravesically instilled ALA seems to be a promising diagnostic procedure for malignant lesions that are difficult to visualize with standard cystoscopy.

Cellular fluorescence of the endogenous photosensitizer protoporphyrin IX following exposure to 5-aminolevulinic acid.

Supplying 5-aminolevulinic acid (ALA), a precursor in the biosynthetic pathway to heme from an external source leads to an accumulation of the endogenous fluorescent photosensitizer protoporphyrin IX (PPIX). Following instillation of ALA in the urinary bladder neoplastic tissue can be discerned by fluorescence cystoscopy or treated by illumination with light of an appropriate wavelength. In order to provide a biological rationale for the clinical findings, we have analyzed the capacity of three different cell lines to accumulate PPIX by flow cytometry. Three different urothelial cell lines, normal fibroblasts and endothelial cells were exposed to ALA under varying conditions. Urothelial cell lines J82 and RT4, derived from malignancies of the bladder displayed fluorescence intensities 9- and 16-fold, respectively, above the fluorescence level of the normal urothelial cell line HCV29. Human umbilical cord endothelial cells fluoresced moderately while the fibroblast cell line N1 exhibited a fluorescence level comparable to those of the cancer cells. Fluorescence increased with increasing cell density and was also dependent on the growth of cells as monolayers or multicellular spheroids. Increasing ALA concentrations led to saturation of fluorescence after 4 h of incubation at cell type-specific fluorescence levels obtained at different ALA concentrations. Continuous incubation in medium containing serum resulted in a linear rise of fluorescence during the first 4 h, which was followed by a saturation period (8-24 h) and a renewed rise. In the case of serum depletion, fluorescence intensities were significantly higher and increased linearly during the entire 48 h incubation period.(ABSTRACT TRUNCATED AT 250 WORDS)

Human follicular and papillary thyroid carcinoma cells interact differently with human venous endothelial cells.

Follicular thyroid carcinomas (FTC) characteristically spread via blood vessels, while papillary thyroid carcinomas (PTC) predominantly metastasize to lymph nodes. This different behavior of cancer cells originating from one organ was investigated by layering multicellular tumor spheroids (MCTS) consisting of various kinds of human thyroid cells onto confluent monolayers of human venous endothelial cells (HEC). The MCTS and HEC were cocultured in an incubation chamber fixed under a microscope, and the behavior of the cells was investigated. In this way significant differences between FTC, PTC, and follicular adenoma cells (FTA) were observed regarding their in vitro behavior upon interaction with HEC. FTC cells required 20 min for adhesion and another hour until they migrated out of a spheroid, whereas PTC- and FTA-MCTS were adhesive after 2 h or later, and their cells did not start migration until 5 h of incubation. Furthermore, one FTC-spheroid triggered about 100 endothelial cells to enter the replication cycle, while no spheroid consisting of either PTC or FTA cells induced more than 20 endothelial cells to start proliferation. During these processes, the cells of the MCTS and the endothelial cells contacted each other directly and remained viable. The results show that FTC cells interact faster and more intensively with human endothelial cells than PTC and FTA cells. Thus the study suggests that an enhanced capability of the FTC cells to interact with venous endothelial cells might favor the clinically observed hematogenous spreading of follicular thyroid carcinomas.

Expression of proliferating cell nuclear antigen (PCNA) and Ki-67 in dysplasia in inflammatory bowel disease.

OBJECTIVE: Previous studies have revealed large variations in the interobserver agreement of dysplasia grading in inflammatory bowel disease. Therefore, we investigated the diagnostic value of two novel monoclonal antibodies (MIB 1 against Ki-67 and PC 10 against PCNA) in the detection of dysplasia. METHODS: A total of 62 biopsies were investigated and histologically classified as follows: 13 probably positive for dysplasia; 15 low-grade dysplasia; five high-grade dysplasia; and 15 inflammation without dysplasia and 14 normal controls. The percentage of positive Ki-67- or PCNA-stained nuclei (= labelling index) was determined in relation to the distribution throughout the mucosa. RESULTS: In all biopsies PCNA-labelling index exceeded that of Ki-67-labelling index. In the superficial half of the crypt PCNA- and Ki-67-labelling indices in the biopsies with 'indefinite for dysplasia, probably positive' or low-grade dysplasia exceeded that of normal tissue (P < 0.001). However, an unequivocal discrimination between biopsies with 'indefinite for dysplasia, probably positive' or low-grade dysplasia and inflammation was not possible. PCNA- and Ki-67-labelling indices were significantly higher in high-grade than in low-grade dysplasia (PCNA 81.4% vs. 44.3%, Ki-67 54.8% vs 30.9%, P < 0.001). Most interestingly, labelling indices of both markers were significantly (P < 0.0001) higher in biopsies with high-grade dysplasia than with active inflammation in the superficial half of the crypt. CONCLUSION: PCNA and Ki-67 are useful adjuncts in the diagnosis of high-grade dysplasia, because high-grade dysplasia can easily be distinguished from low-grade dysplasia or active inflammation if the distribution of the positive-stained cells within the mucosa is taken into account. Lower unspecific binding and lower influence on proliferation activity by inflammation prompts us to prefer Ki-67 (MIB 1).

Shock wave induced endothelial damage--in situ analysis by confocal laser scanning microscopy.

For more than a decade, extracorporal shock wave lithotripsy has been a standard clinical method for the treatment of urinary stones. However, side effects that are likely to be correlated to vessel damage can often be observed using noninvasive diagnostic techniques, e.g., magnetic resonance imaging. To avoid side effects it is useful to understand the interaction between shock waves and the vascular system. In particular, this is important in view of new applications like gallstone lithothripsy. In the present study, we exposed human umbilical vessels to electromagnetically generated ultrasound shock waves to analyze subsequent alterations of their endothelial layer. Following en face preparation and fluorescent staining, the endothelium was examined in a confocal laser scanning microscope. Endothelial cells of the shock wave exposed vessels revealed permeabilization of plasma membranes and mitochondrial alterations as potentially lethal damage. An increase in the number of stress fibres may indicate functional changes possibly influencing vessel wall permeability.

Flow cytometric analysis of cell suspensions exposed to shock waves in the presence of the radical sensitive dye hydroethidine.

The occurrence of intracellularly and extracellularly generated free radicals during shock wave exposure on an experimental Siemens lithotripter was tested with the radical sensitive dyes hydroethidine and dichlorofluorescin (DCFH). DCFH, a nonfluorescent compound, is oxidised to dichlorfluorescein (DCF) by hydrogen peroxide in the presence of peroxidase. DCF green fluorescence intensity was used for fluorescence spectrometric measurement of hydrogen peroxide generated during shock-wave treatment of cell-free dye solutions. The fluorescence intensity of ethidium, the oxidised form of hydroethidine, was used for the flow-cytometric measurement of intracellular oxidising reagents present in RT4 tumour cells during shock-wave exposure. Changes in membrane permeability, which influence the intracellular content of ethidium, were controlled by counterstaining the cells with propidium iodide, an indicator for membrane integrity. We observed no increase in intracellular ethidium fluorescence intensity after shock-wave treatment of single cell suspensions and therefore no indication for shock-wave-induced intracellular free radicals.

The combined effects of high-energy shock waves and ionising radiation on a human bladder cancer cell line.

The effects of high-energy shock waves (HESW) generated by an experimental Siemens lithotripter in combination with 137Cs gamma-rays were examined in vitro. Proliferation after treatment of immobilised pellets of either single cells or multicellular spheroids of the bladder cancer cell line RT4 was determined using colony-forming assays and cell cycle analysis. Surviving and cell cycle fractions were calculated for each shock wave and radiation application mode separately, and for sequential combination in different successions for the purpose of characterizing the interaction of both treatment modalities. Combination of HESW and ionising radiation turned out to act additively or slightly supra-additively on both biologic models.