Ambiguous Genitalia and Lissencephaly in A 46,XY Neonate with a Novel Variant of Aristaless Gene.

Introduction: Disorders of sexual development can present isolated or as a part of complex genetic syndromes. Case presentation: A newborn with ambiguous genitalia and prenatally diagnosed brain malformations was referred to our hospital. Prenatal ultrasound examination and MRI showed lissencephaly and absence of the corpus callosum. At admission, physical examination revealed microphallus, hypospadia and complete fusion of labioscrotal folds with nonpalpable gonads, normal blood pressure and serum biochemistry. Cortisol level was normal (201 nmol/L), testosterone elevated (14.4 nmol/L), FSH 0.1 IU/L, LH 0.7 IU/L, estradiol 241 pmol/L. Seizures were noted on the 2nd day and the child was started on anticonvulsives. When 17-OHP level results came back elevated (200 nmol/L), ACTH test was performed and the child was started on hydrocortisone and fludrocortisone treatment. Congenital adrenal hyperplasia became unlikely when karyotype result showed normal male karyotype (46, XY, SRY+) with no Mullerian structures seen on ultrasonographic exam. As association of ambiguous genitalia and lissencephaly strongly suggested a mutual genetic background, diagnosis of X-linked lissencephaly with ambiguous genitalia (X-LAG) became apparent. Conclusions: The presented case highlights the importance of looking at the whole clinical picture instead of separate isolated findings with emphasis on patient-centered approach guided by clinical findings and patient history.

Immunocytochemical expression of protein kinase C isoenzymes alpha, delta, epsilon and zeta in differentiating chick chondrocytes in vitro.

In our previous work we have investigated the expression of the serine-threonine kinase protein kinase C (PKC) in the vertebral column of mouse foetuses. In the present work we would verify the expression of four PKC-isoenzymes (alpha, delta, epsilon, zeta) in two distinct phases of the chondrogenesis and the endochondral osteogenesis in vitro. We performed primary cultures of chondrocytes collected from tibiae of 6-day old chick embryos. This cells were cultured for 20 days and than collected on coverslips (stage 1 culture). Other cells of the stage 1 were undergone further differentiation towards the phenotype of osteoblast-like cells (stage 2 culture), in accord to the protocol of Descalzi Cancedda et al. (1992). In stage 1 culture, PKC-epsilon was the most expressed isoform, whereas PKC-alpha exhibited the least intense positivity. In stage 2 culture, PKC-alpha was the most expressed isoform, whereas a marked decrease of PKC-epsilon expression was detected compared to stage 1. No relevant differences were evidenced as regards,the expression of PKC-zeta between the two considered cell culture stages. On these bases, it could be reasonable that these PKC-isoenzymes may be involved at different levels in chondrocytes differentiation as well as in the endochondral ossification process.

In vitro evaluation of the biocompatibility of dental alloys: fibronectin expression patterns and relationships to cellular proliferation rates.

OBJECTIVE: This short-term (72- to 96-hour) in vitro study on fibroblasts evaluated the biocompatibility of 3 single-phase dental alloys by determining cellular proliferation rates and the expression of a glycoprotein, fibronectin, which is involved in cellular adhesion processes. METHOD AND MATERIALS: Flow 2002 fibroblasts were cultured together with 3 single-phase dental alloys of different composition. Proliferation rates were determined by 5-bromodeoxyuridine incorporation. Fibronectin expression was determined by indirect immunofluorescence. RESULTS: At 72 hours, cells cultured with the alloy containing the lowest amount of noble elements (gold, platinum, and palladium) and the highest amount of silver exhibited significantly less proliferation than did controls. At 96 hours, only cultures with the alloy containing the greatest amount of noble elements behaved in a way similar to controls. Fibronectin organization in fibrils and in focal adhesions was correlated to higher cellular proliferation rates. CONCLUSION: Fibronectin organization could be a useful tool to determine the biocompatibility of dental alloys. Among the noble elements, palladium by itself exhibits very good biocompatibility. These indications could be useful for practitioners in the choice of the best alloy for specific clinical applications.

The influence of dental metal alloys on cell proliferation and fibronectin arrangement in human fibroblast cultures.

The biocompatibility of six single-phase dental metal alloys was studied by determining cell proliferation rates correlated to the arrangement of fibronectin (FN) in fibroblast cultures. Immunocytochemical methods were used to detect cell proliferation by 5-bromodeoxyuridine (BrdU) incorporation, and FN organization [i.e. diffuse in the extracellular matrix and organized in fibrils or in focal adhesions (FA)] in human fibroblast cultures. Cell proliferation rates were related to FN arrangement and in particular a higher percentage of cells in the S-phase was related to a predominance of FA. The greatest difference in behaviour compared to that of the controls was detected after 120 and 168 hr: at these times, as well as at previous ones, the alloy with the highest Au content seemed the most biocompatible among those tested, as it behaved in a very similar way to the controls. In contrast, fibroblasts exposed to the other five alloys showed different behaviours from the controls. It is assumed that a correlation exists between FN organization and the percentage of BrdU-positive cells, and that these features vary in the presence of different alloys. The observation of FN arrangement together with cell proliferation rates could be another useful tool in determining the biocompatibility of dental metal alloys.

The presence of implant materials influences fibronectin arrangement and cell growth in fibroblast cultures.

The nature of the bone-implant interface has been much focused in investigating dental implant materials, whereas the relationship between implant and fibroblasts has received much less attention. To evaluate the biocompatibility degree of an implant material, both cell adhesion and cell growth must be tested in the presence of the implant. Four dental implant (A,B,C,D) made in titanium alloy and one of them (C) hydroxyapatite (HA)-coated in fibroblast cultures (48 and 72 h) were tested, by performing immunocytochemical techniques and then by observing fibronectin (FN) arrangement for cell adhesion and counting 5-bromodeoxyuridine (5-BrdU) to evaluate cell proliferation. Different FN arrangements were observed-i.e. organized in fibrils, or in focal adhesion plaques, as well as dispersed in the intercellular space-which varied for the different implants employed at the various culture stages. Equally, the per cent ratio of 5-BrdU positive cells was different, with a more significant increase (p < 0.001) between 48 and 72 h for implant C and the controls. It was observed that the higher percentages of 5-BrdU positive cells were in cultures where FN was organized mainly in focal adhesions, as well as 5-BrdU positive cells increased after 72 h in cultures, which after 48 h presented much FN dispersed in the intercellular space. It may be assumed that a correlation exists between FN arrangement and the percentage of 5-BrdU positive cells and that these two parameters vary in the presence of the different implants. Moreover, the HA-coated implant seems to be the most biocompatible in fibroblast cultures.

Protein kinase C isoenzymes in mouse harderian gland. Differential expression of the alpha- and epsilon-isoforms during pregnancy. Protein kinase C-OC.

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-alpha, -epsilon, -zeta and -eta. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the alpha- and epsilon-isoforms. The 80-kDa native from of PKC-alpha almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C-epsilon appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant in the Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions.(ABSTRACT TRUNCATED AT 250 WORDS)

A simple and rapid staining technique for plastic embedded cartilage and bone.

In this report we describe a simple and rapid staining technique for cartilage and bone embedded in Araldite. Semithin sections of embryonic vertebrae obtained from 15 to 17 day mouse fetuses were stained using an aqueous solution 0.25% with respect to methylene blue, 0.25% with respect to azure A, and 0.5% with respect to Na2 CO3, then counterstained with 1% aqueous pararosaniline chloride (MAP). Results were compared with toluidine blue stained sections. MAP permitted good discrimination of developmental stages of both cells and extracellular matrix within vertebral ossification centers during endochondral ossification. The technique is simple, rapid and applicable to plastic embedded sections, and can be used prior to ultrastructural examination.

Histochemical and ultrastructural investigation on the cytotypes in the mouse Harderian gland.

The presence of melatonin and other biogenic indoleamines in the Harderian gland has been proposed by various authors. In the present work we would have investigated which of the cytotypes of the mouse Harderian gland might be involved in melatonin turnover. For this aim we employed the osmium tetroxide-zinc iodide (ZIO) histochemical technique that has been proposed useful to identify indole monoamines. Harderian glands of male, female and pregnant female mice were studied by light and transmission electron microscopy (TEM). Secretory type B cells were selectively stained by the ZIO-mixture, much more in female than male; type A cells were much more numerous than type B ones and never ZIO-positive. Ultrastructural examinations showed that type B cells contain numerous granules in the whole cytoplasm, in the perinuclear cisterna and around the cytoplasmic vacuoles and vesicles. Myoepithelial cells were sometimes found weakly ZIO-positive. Endothelial cells of capillary blood vessels presented ZIO-precipitates in the cytoplasm, as well as in the perinuclear cisterna. These data may suggest an uptake of melatonin from the blood stream and an involvement of type B secretory cells in melatonin catabolic turnover, or a "in situ" melatonin biosynthesis and endocrine secretion by the same B cells. The higher ZIO-positivity of type B cells in female than male may be related also to the influence of sexual steroid hormones, that characterizes the porphyrin pigment amount involved in melatonin oxidation.

The three-dimensional structure of interdigitating cells.

The in vivo three-dimensional architecture of lymph node and spleen interdigitating cells (IDCs) has been studied in mice by means of a computer reconstruction program and polystyrene models. Tissue fragments treated with an osmium-zinc iodide fixative solution, which gives a brilliant and quite specific impregnation of IDCs, were embedded in Epon and cut into 2 microns thick serial sections. The data was transferred into a "MOP-Videoplan 3D-cell reconstruction" software program and both tridimensional plottings and morphometric measurements were obtained. Then polystyrene models were assembled. Our findings show a cell population of high spatial complexity. IDCs are large cells whose numerous cytoplasmic processes form an extensive three-dimensional network which envelopes lymphoid cells. The most characteristic feature of IDC' architecture is the large, flattened, sheet-like processes which expand for several microns from the cell body, embracing numerous lymphocytes and lymphoblasts. In certain cases surface invaginations create channel-like structures which cross through the IDC peripheral cytoplasm. The impressive extension of the IDC membranous network provides a powerful anatomic strategy which facilitates the interaction between IDCs and T-lymphocytes and creates a unique microenvironment for T-cell activation and proliferation. This study gives new morphological evidence for the strict physical interaction between IDCs and T-lymphocytes and supports the concept that IDC-T lymphocyte aggregation represents a very special microanatomical and functional unit.

A ruthenium red-toluidine blue procedure for staining epoxy sections in the light microscopy.

Ruthenium red (RR) has been widely used as a fixation additive for electron microscopy on the basis of its capacity to retain acid mucopolysaccharide residues of the cell surface coat. Little is known about the properties of this compound as a direct staining agent for epoxy-resin embedded material. In this study, semithin sections of Epon-infiltrated muscle tissue samples have been treated with 1% RR followed by counterstaining with 1% toluidine blue. This 2 step staining procedure has proven to be simple, rapid, and reliable and to give a dramatic improvement in image resolution and contrast. Thus, we believe that histochemical procedures employing RR may find in the future interesting applications for the direct staining of epoxy-resin embedded tissues.

Osmium-zinc iodide reacts with interdigitating cells in the mouse lymph nodes and spleen.

It is known that epidermal Langerhans cells react with osmium-zinc iodide (ZIO) mixtures; therefore they can be visualized by this histochemical method. In the last few years it has been shown that Langerhans cells are closely related to the class of interdigitating cells (IDC) which are antigen presenting cells located in the T-dependent areas of lymph nodes and spleen. In this study the reactivity of murine IDC to ZIO has been assessed. Results demonstrate that ZIO procedure yields to a brilliant and selective staining of IDC. The reactivity pattern is quite similar to that previously observed in epidermal Langerhans cells. This finding gives further support to the concept that Langerhans cells and IDC are closely interrelated cell types.

Visualization of the Langerhans cells in the human vaginal epithelium by the L-dopa histofluorescence method.

In this study we used the technique of L-DOPA histofluorescence for visualizing Langerhans cells in the human vaginal epithelium. Biopsy specimens were incubated with L-DOPA and sectioned by cryostat. The sections were exposed to formaldehyde vapour; during this passage, chemical conversion of L-DOPA into a strongly fluorescent compound occurred. Langerhans cells were clearly visualized in the vaginal epithelium; cell bodies and dendritic processes fluoresced sharply. The method is rapid and specific: it represents an useful tool for demonstrating Langerhans cells in the stratified squamous epithelium of vagina.

Effect of polyethylene glycol 400 on adriamycin induced histamine release.

The activity of polyethylene glycol 400, a widely used drug solvent, was tested on the release of histamine induced by adriamycin in vitro on peritoneal rat mast cells and in vivo in a mouse model. Preincubation of mast cells with high (10 and 5%) concentrations of polyethylene glycol 400 significantly inhibited the important histamine release induced by 100 micrograms/ml of adriamycin; furthermore, polyethylene glycol 400 (3.45 g/kg; 0.345 g/ml) pretreatment almost completely abolished the peritoneal and pericardial mast cell degranulation and the cardiac toxicity caused by an intraperitoneal injection of 15 mg/kg of adriamycin. This effect of polyethylene glycol 400 on adriamycin-induced histamine release could explain the protective action exhibited in vivo on adriamycin treated animals, therefore confirming that adriamycin cardiotoxicity could be related to the release of histamine and other vasoactive substances.

Ethanol potentiates the toxic effects of 1,4-butanediol.

The simultaneous administration of ethanol increases the mortality rate and tissue damage observed in rats after 1,4-butanediol (1,4-BD). A related increase in tissue 1,4-BD concentration supported the hypothesis of an in vivo competition of the two substances for alcohol dehydrogenase. The clinical implications of the results, in light of the recent discovery of the presence of endogenous 1,4-BD in humans are discussed.