Abnormal myotonic dystrophy protein kinase levels produce only mild myopathy in mice.

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

Intermediate filaments in malignant melanomas. Identification and use as marker in surgical pathology.

Intermediate-sized filaments have been studied in human malignant melanomas and in normal melanocytes by immunofluorescence microscopy with antibodies directed against keratin, vimentin, desmin, neurofilament protein, and glial filament protein. Both human melanotic and amelanotic tumor cells and tumor metastases as well as normal melanocytes in human skin and in the rat eye contain exclusively intermediate filaments of the vimentin type. No reaction was seen with antibodies to keratin, desmin, neurofilaments, or glial filaments. These latter four antisera, however, gave strong reactions in epidermis and other epithelial tissues, muscle, or neural tissues, respectively. The results favor a mesenchymal character of melanocytes, although a neuroectodermal origin in an early developmental stage is possible. The finding that melanomas contain exclusively vimentin intermediate filaments may prove useful in differential diagnosis of melanomas from other tumor types.

Constitutive and inducible expression of SKALP/elafin provides anti-elastase defense in human epithelia.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a serine proteinase inhibitor first discovered in keratinocytes from hyperproliferative human epidermis. In addition to the proteinase inhibiting domain which is directed against polymorphonuclear leukocyte (PMN) derived enzymes such as elastase and proteinase 3, SKALP contains multiple transglutaminase (TGase) substrate domains which enable crosslinking to extracellular and cell envelope proteins. Here we show that SKALP is constitutively expressed in several epithelia that are continuously subjected to inflammatory stimuli, such as the oral cavity and the vagina where it co-localizes with type 1 TGase. All epithelia from sterile body cavities are negative for SKALP. In general, stratified squamous epithelia are positive, whereas pseudostratified epithelia, simple/glandular epithelia and normal epidermis are negative. SKALP was found in fetal tissues of the oral cavity from 17 wk gestation onwards where it continued to be expressed up to adult life. Remarkably, in fetal epidermis SKALP was found from week 28 onwards, but was downregulated to undetectable levels in neonatal skin within three months, suggesting a role during pregnancy in feto-maternal interactions or in the early maturation phase of the epidermis. Immunoelectron microscopy revealed the presence of SKALP in secretory vesicles including the lamellar granules. In culture models for epidermal keratinocytes we found that expression of the endogenous SKALP gene provided protection against cell detachment caused by purified elastase or activated PMNs. Addition of exogenous recombinant SKALP fully protected the keratinocytes against PMN-dependent detachment whereas superoxide dismutase and catalase were only marginally effective. These findings strongly suggest that the constitutive expression of SKALP in squamous epithelia, and the inducible expression in epidermis participate in the control of epithelial integrity, by inhibiting PMN derived proteinases.

Monoclonal antibody against human ovarian tumor-associated antigens.

Mouse monoclonal antibodies (OV-TL 3) were raised against human ovarian tumor-associated antigens for diagnostic purposes. A cloned hybridoma cell line was obtained by fusion of murine myeloma cells with spleen lymphocytes from BALB/c mice immunized with a tumor cell suspension prepared from an ovarian endometrioid carcinoma. The antibodies were initially screened for their ability to bind on frozen sections of human ovarian carcinoma tissue and a negative reaction on gastric carcinoma tissue by indirect immunofluorescence. The reactivity of the selected OV-TL 3 clone (IgG1 subclass) was studied on normal and neoplastic tissues as well as on a cell line derived from the original tumor cell suspension used for immunization. OV-TL 3 antibodies stained frozen sections of human ovarian carcinomas of the following histological types: serous, mucinous, endometrioid, and clear cell. No reaction was found with breast cancers or other nongynecological tumors. No differences in staining pattern were observed between primary and metastatic ovarian carcinomas. OV-TL 3 antibodies brightly stained ovarian carcinoma cell clusters in ascitic fluids and left unstained mesothelial cells and peripheral blood cells. The OV-TL 3-defined antigen also remained strongly expressed on a cell line derived from the endometrioid ovarian carcinoma originally used for generation of OV-TL 3 clone. Reactivity was weak and irregular in a few ovarian cysts, while traces of fluorescence were sometimes detected in epithelial cells lining the female genital tract. In only 3 specimens of 15 endometrium carcinomas was weak focal reactivity with OV-TL 3 antibodies observed. The results of the immunofluorescence study were confirmed by the more sensitive avidin-biotin method and by 125I-labeled OV-TL 3 antibodies. Thus OV-TL 3 recognizes a common antigen for most ovarian carcinomas and may be a useful tool for rapid diagnosis of ovarian carcinomas.

Design of immunoliposomes directed against human ovarian carcinoma.

Factors (protein/lipid ratio, pH of incubation medium, incubation time, anchor molecule density in the bilayer) affecting the covalent binding of anti-ovarian carcinoma Fab' to liposomes containing the anchor molecule MPB-PE (N-(4-(p-maleimidophenyl)butyryl)phosphatidylethanolamine) were explored. Standard experimental conditions were chosen and information on the relevant physicochemical parameters of the liposome dispersions was collected (mean particle diameter, size distribution, charge). The reproducibility of standard immunoliposomes prepared in subsequent batches in terms of Fab' binding, particle size and charge was established. In addition, preservation of immunoreactivity, no marker loss, and no aggregation/fusion was found for the standard immunoliposomes over a period of at least 3 weeks at 4 degrees C. In vitro up to 35,000 immunoliposomes were estimated to bind per human ovarian carcinoma cell. Internalization of the immunoliposomes could not be demonstrated. Electron micrographs showed binding of specific immunoliposomes to human ovarian carcinoma cells growing intraperitoneally in athymic nude mice.

Developmental expression of the cell adhesion molecule-like protein tyrosine phosphatases LAR, RPTPdelta and RPTPsigma in the mouse.

Using RNA in situ hybridization we compared the expression patterns of the cell adhesion molecule-like receptor-type protein tyrosine phosphatases LAR, RPTP sigma and RPTP sigma during mouse development. We found that LAR is expressed in basal lamina-associated epithelial tissues of (neuro)ectodermal, neural crest/ectomesenchyme and endodermal origin. RPTP sigma is found in (neuro)ectodermal, neural crest-derived systems and in mesoderm-derived tissues. The expression pattern of RPTP sigma largely parallels that of RPTP sigma, in concordance with their proposed evolutionary history

Postnatal centralization of muscle fibre nuclei in centronuclear myopathy.

Postnatal centralization of muscle fibre nuclei, which were previously located subsarcolemmally, is described in a case of centronuclear myopathy (CNM) in a male patient with generalized muscle weakness since birth. A muscle biopsy was taken at the age of 11 months; no particular abnormalities were observed at this stage apart from an unusual variation in fibre size. A distinctly below average muscle fibre diameter, increased endomysial connective tissue, and features typical for CNM were found in a biopsy taken 9 yr later. Immunohistochemical studies using antibodies to desmin, vimentin, laminin and type IV collagen revealed altered staining patterns compared with normal fibres. The abnormalities in the patterns of cytoskeletal proteins point to a defective regulation of the composition and organization of the cytoskeletal network during development, paralleled by abnormalities in the extracellular matrix.

Abnormal expression of intermediate filament proteins in X-linked myotubular myopathy is not reproduced in vitro.

Expression patterns of the intermediate filament proteins (IFPs) desmin and vimentin, in biopsy material taken from a 1 day old boy with fatal neonatal X-linked myotubular myopathy (XLMTM) were compared with the expression of these proteins in cultured myotubes, from the same patient. Immunohistochemical studies revealed the persistence of high levels of desmin in virtually all, and vimentin in most, of the myofibres within the patient's biopsy. Analysis of intermediate filament expression in differentiating, cultured muscle cells did not reveal overt differences between XLMTM cultures and cultures of control muscle. Titin distribution patterns indicated a normal process of myofibrillogenesis in XLMTM myotubes. We conclude that the failure to properly regulate IFP-expression is not intrinsic to XLMTM muscle fibres. The possibility that this failure is due to a defective external, possibly neural factor, is discussed.

Cellular response against exoerythrocytic forms of Plasmodium berghei in rats.

Rats were infected with Plasmodium berghei sporozoites, and 47, 51, and 57 hr later exoerythrocytic parasites were examined by electron microscopy. At 47 hr, approximately 30% of nearly mature exoerythrocytic parasites were degenerating and were surrounded by a cellular infiltrate of Kupffer cells, monocytes, monocyte-derived macrophages, and neutrophils. Neutrophils appeared to be actively ingesting electron-dense fuzzy parasite material which was normally present in the parasitophorous vacuole. By 51 hr other mononuclear cells penetrated with filopodia between the host hepatocyte and exoerythrocytic parasite, and directly into the exoerythrocytic parasite. Exoerythrocytic parasites that formed merozoites at 51 hr lacked any notable cellular infiltration.

Retrograde seeding of endometrial epithelial cells by uterine-tubal flushing.

OBJECTIVE: To estimate the amount of endometrial epithelial cells in peritoneal fluid (PF) after uterine-tubal flushing (40 mL) throughout the menstrual cycle. DESIGN: We cultured the cell pellet of flush medium present in the peritoneal cavity. SETTING: University Hospital Nijmegen, The Netherlands. PATIENTS: Ninety-two women with various infertility-related factors. INCLUSION CRITERIA: (1) ovulatory cycle, (2) patent tubes, and (3) no adhesions. INTERVENTIONS: None MAIN OUTCOME MEASURE(S): The number of developing epithelial cell colonies were counted after 7 days. We started to record the amount of flush medium recovered during the study. RESULTS: The amount of flush medium recovered was positively correlated with the presence of endometriosis (P = 0.017). Endometrial epithelial cells were identified in 85 flush media (92%). The number of epithelial cell colonies varied from 0 to 100 or more and was higher when flushing was performed during the early follicular phase (P less than 0.01). High estradiol-17 beta and progesterone levels in culture medium did not change the number of developing cell colonies. Methylene blue significantly reduced the number of cell colonies (P = 0.002). CONCLUSIONS: Uterine-tubal flushing results in varying numbers of endometrial epithelial cells in PF. Methylene blue adversely affects the growth potential of these cells.

Endometrial epithelial cells in peritoneal fluid during the early follicular phase.

Peritoneal fluid (PF) was obtained during the early follicular phase in 24 women at laparoscopy as part of infertility investigation. The cells present in PF were pelleted and cultured. Developing endometrial epithelial cell colonies were identified in 19 women (79%). Identification of these cell colonies was facilitated using the monoclonal antibody BW 495/36 as specific marker. The number of endometrial epithelial cell colonies showed a large variation (1 to 200 or more PF sample). No significant distinction in incidence and number of cell colonies was found between women with minimal (n = 11) and without endometriosis (n = 12). A significant correlation with number of cell colonies was found in women with infertility and no mechanical and male infertility factors. These data indicate that retrograde transport of viable endometrial cells during menstruation occurs in most women with patent tubes. Implications of the results for the relation between retrograde menstruation, endometriosis, and infertility are discussed.

Immunophenotyping of congenital myopathies: disorganization of sarcomeric, cytoskeletal and extracellular matrix proteins.

We have studied the expression and distribution patterns of the intermediate filament proteins desmin and vimentin, the sarcomere components titin, nebulin and myosin, the basement membrane constituents collagen type IV and laminin, and the reticular layer component collagen type VI in skeletal muscle of patients with "classic" congenital myopathies (CM), using indirect immunofluorescence assays. In all biopsy specimens obtained from patients with central core disease (CCD), nemaline myopathy (NM), X-linked myotubular myopathy (XLMTM) and centronuclear myopathy (CNM), disease-specific desmin disturbances were observed. Vimentin was present in immature fibres in severe neonatal NM, and as sarcoplasmic aggregates in one case of CNM, while the amounts of vimentin and embryonic myosin, observed in XLMTM, decreased with age of the patients. Abnormal expression of myosin isoforms was found in several CM biopsies, although the organization of myosin and other sarcomere components was rarely disturbed. Basement membrane and reticular layer proteins were often prominently increased in severe cases of CM. We conclude that (i) desmin is a marker for individual types of CM and might be used for diagnostic purposes; (ii) the expression patterns of the differentiation markers desmin, vimentin and embryonic myosin in XLMTM, point either to a postnatal muscle fibre maturation or to a variable time-point of maturational arrest in individual patients; (iii) the correlation between the distribution patterns of extracellular matrix proteins and clinical presentation points to a role of these proteins in pathophysiology of CM.

Increased expression of a 68-kDa protein in the corpus cavernosum of some men with erectile dysfunction.

Erectile dysfunction (ED) may be caused by abnormalities of intracavernous penile structures. In order to investigate whether specific proteins could be identified that might be related to ED, the composition of structural proteins in cavernous tissues of patients with ED was compared to that of normal cavernous tissues by gel electrophoresis. Increased expression of a 68-kDa nonionic detergent extraction-resistant protein was demonstrated in tissues of more than half of the patients with vasculogenic ED, whereas only one out of nine normal cavernous tissues showed the same phenomenon. Increased expression was not related to a specific type of vascular insufficiency, aging, or diabetic constituency. Histochemical and immunochemical studies revealed that the increased amount of the 68-kDa protein is not merely the result of a surplus of nervous, smooth muscle, or elastic tissues. Furthermore, antibodies specific for 68-kDa neurofilament and 62- to 67.5-kDa tropoelastin did not recognize the 68-kDa protein on Western blots. The possibility that the 68-kDa protein may help us understand the etiology of certain cases of erectile dysfunction is discussed.

A scanning electron microscopic study of the sporogonic development of Plasmodium falciparum in Anopheles stephensi.

The full development of Plasmodium falciparum in Anopheles stephensi mosquitoes was studied by scanning electron microscopy. Ookinetic development was described from in vitro cultures. Growing oocysts beneath the basal lamina of the midgut wall mechanically stretch this lamina until it is torn and displaced by day 7. In young oocysts the wall appears smooth. In older oocysts wrinkles in the wall are visible after routine fixation. Osmium tetroxide postfixation greatly reduced the occurrence of these wrinkles. Intracapsular development of sporozoites was visualized after mechanical manipulation of the oocysts during sample preparation. In contrast to P. berghei, no ectopic development was seen in P. falciparum in the mosquito midgut. The mechanism of sporozoite escape from the oocyst appears to be similar to that described for rodent malaria. Fracturing of salivary glands provided the first view by scanning electron microscopy of sporozoites located in proximal and distal gland cells and in the draining duct.

Distribution and features of melanocytes during inner ear development in pigmented and albino rats.

In this developmental study, the distribution and features of melanocytes in the inner ear of pigmented and albino rats was investigated with the use of an antibody, which specifically reacts with a melanocyte differentiation antigen present in the membranes of (pre)melanosomes. Melanocyte precursors could be traced from 13 days post conception onwards and the course was followed to their targets in the inner ear. Melanocytes which settle in the modiolus appeared to reach their target along another pathway than strial and vestibular melanocytes. No difference was observed in the melanocyte distribution between pigmented and albino rats. The integration of melanocytes into the stria vascularis was associated with an increased rate of melanosome production in both strains, but in the albinos far fewer melanosomes were produced. After the stria had reached maturity, melanosome production was arrested and melanosomes were subject to lysosomal digestion. In the stria of the pigmented rats, cells with aggregations of disintegrating melanosomes appeared and persisted into adulthood. In the adult, the majority of the intermediate cells contained only a few scattered melanosomes, while melanosomes could only rarely be detected in the albinos. These observations indicate that there is a close relationship between melanosome production and the process of interdigitation of melanocytes with the marginal cells. It seems unlikely that melanosomes or melanin make any important contribution to the function of the adult stria vascularis. Outside the stria, the features of melanocytes in both strains were similar to skin melanocytes.

Biological effects of high energy shock waves in mouse skeletal muscle: correlation between 31P magnetic resonance spectroscopic and microscopic alterations.

To investigate the in vivo effects of electromagnetically generated high energy shock waves (HESW) on skeletal muscle, we used in vivo 31P nuclear magnetic resonance (NMR) spectroscopy (MRS) measurements and correlated the results with microscopical studies. Mouse skeletal muscle (calf muscle) was exposed to 200 or 800 HESW (Pmax: 37.5 MPa, Pmin: 5.2 MPa, tr: 30-120 ns, tw: 340 ns, frequency: 1.25 Hz). In the 31P MRS spectra, transient alterations were observed. A prominent increase of inorganic phosphate (Pi) peaks was found, as well as the appearance of Pi with different chemical shifts, reflecting the presence of different pH values (5.4-7.1) in cellular or tissue compartments. Within 20-96 h after exposure, pH values and Pi levels returned to normal. The changes were more pronounced in the animals treated with 800 HESW as compared to 200 HESW. Light and electron microscopy demonstrated focal degenerations of muscle fibers. This process consisted of disorganization of myofilaments and structural changes in sarcoplasmic organelles and was progressive in time. The (ultra)structural changes were not present in all myofibers (i.e., between affected degenerating fibers unaffected intact fibers were seen). Several ultrastructural abnormalities were also found in capillaries even up to severe dilatation and disruption, as well as in the peripheral nerves. The degeneration of the preexisting myofibers was predominantly confined to type 1 fibers and was followed by a regeneration of the muscle tissue by proliferation of myoblasts. A notable amount of myotubes still showed vacuolization. We conclude that in vivo HESW exposure of skeletal muscle tissue results in a degeneration of myofibers. The cellular effects are present in foci and associated with changes in the 31P NMR spectra. The NMR spectroscopy technique provides us with a noninvasive method to evaluate in a longitudinal way the biological effects of HESW.

Multiparameter analysis of four human renal cell carcinoma xenografts in nude mice.

Four human renal cell carcinoma xenografts (RC2, RC14, RC43, NC65), maintained in nude mice for several years, were investigated in a multi - disciplinary study, using (immuno) histochemical, biochemical and ultrastructural techniques. Histological, cellular, nuclear and biological characteristics were investigated. All tumors showed histologically recognizable features of human renal cell carcinomas, although marked differences between the four tumors were seen, both at the histological and ultrastructural level. Flowcytometric analysis of tumor cell suspensions allowed DNA quantification as well as the detection of subpopulations. Immunohistochemical staining procedures using tissue specific antibodies against intermediate filament proteins revealed two populations of tumor cells. Most tumor cells in three of the xenografts coexpressed cytokeratins and vimentin, while in RC43 most of the tumor cells expressed only vimentin. Northern blot analysis showed a higher expression of vimentin mRNA in all tumors as compared to normal kidney tissue. RC43 showed a three-fold higher level of vimentin mRNA than the other xenografts. Growth potential in the human tumor cloning system was evaluated by temporal growth pattern analysis. These experiments showed that the xenografts resemble human primary renal cell tumors in different ways, and reflect different characteristics that can be present in human renal cell carcinoma.

Multidisciplinary evaluation of rat renal cell carcinoma.

The rat renal cell carcinoma system as described by deVere White and Olsson in 1980 is used widely as a model for its human counterpart. The tumor arose spontaneously in a male Wistar Lewis rat and its behaviour has been shown to be stable during multiple passages. We have compared this tumor with the human renal cell carcinoma using a multidisciplinary approach. Light microscopy and electron microscopy showed a great resemblance of this rat tumor to a human renal cell carcinoma of the clear cell type with the ultrastructural presence of desmosomes. With the use of tissue specific antibodies against intermediate filament proteins, it could be shown that their expression is comparable to human renal cell carcinoma, i.e. coexpression of vimentin and different cytokeratins in the tumor cells. The cells could also be shown to contain cytokeratin 18. An aneuploid cell population in the tumor, expressing both vimentin and keratin, could be characterized by DNA flow cytometry in double labeling experiments. Comparison of normal and malignant rat kidney tissue by Northern blot analysis revealed increased levels of vimentin mRNA. In conclusion, this tumor model seems to have several histological and biological properties in common with the human renal tumor.

Immunocytochemical markerprofile of endometriotic epithelial, endometrial epithelial, and mesothelial cells: a comparative study.

A comparative immunocytochemical study was performed on endometriotic epithelial versus endometrial epithelial and normal mesothelial cells in order to obtain further evidence for either the endometrial implantation or mesothelial metaplasia theory. The three cell types could not be distinguished by keratin subtyping, using monoclonal antibodies (MAbs) to keratins 5, 7, 8, 14, 18, and 19. The epithelial markers HMFG-2 and BW 495/36, and a newly developed MAb NEND-3 (against endometrial cells) discriminated between generally negatively reacting mesothelial cells and positively reacting endometrial and endometriotic epithelial cells. The MAb NEND-3 appeared to be specific for endometrial (and endometriotic) epithelial cells since no reactivity with other epithelial cell types was found. Typing with MAbs against ovarian carcinoma related antigens (OV-TL 3, OV-TL 10 and OC 125) did not permit sufficient distinction. The marker profile of cultured endometrial, endometriotic and mesothelial cells confirmed the immunocytochemical findings on frozen sections. Although the data are consistent with the endometrial implantation theory, mesothelial metaplasia can not be excluded with regard to the histogenesis of endometriosis since metaplastic mesothelium may express different antigen markers.

An experimental model for tympanosclerosis. A preliminary report.

This study deals with an animal model where tympanosclerosis could be evoked with high reproducibility during the course of a sterile otitis media, induced by Eustachian tube obstruction. The histopathological features of this induced lesion were very similar to those reported in human specimens. It was concluded that this process is most probably triggered by a mechanical deformation of the tympanic membrane.