Micromanipulation of single cells from tissue imprints is an alternative to laser-assisted microdissection.

The characterization and analysis of single cells by molecular biological methods such as the polymerase chain reaction (PCR) is of increasing interest in biomedical research. Different techniques have been developed to obtain single cells from solid tissue. Currently, the most frequently used technique is laser-assisted microdissection (LAM). However, LAM of tissues cannot exclude contamination of the targeted cells by underlying cell fragments. Moreover, this technique can only be performed if a laser microscope is available. Thus, we developed a method to obtain single cells of fresh solid tissue by the simple technique of tissue imprints. After immunostaining of the imprints, single cells were transferred to a reaction tube using a 27-gauge needle guided by a mechanical micromanipulator. Consequently, we used these cells in a single cell PCR.

C-KIT expression in primary cutaneous T-cell lymphomas.

BACKGROUND: Mutations of the stem cell factor receptor C-KIT play a major pathogenetic role in the development of different malignant diseases like human mastocytosis, myeloproliferative disorders, gastrointestinal stromal tumors, acute myelogenous leukemia, and sinonasal lymphomas. Furthermore, the expression of C-KIT has been described in Hodgkin's disease and nodal CD30+ anaplastic large cell lymphomas (ALCLs). As it is possible to inhibit C-KIT by innovative kinase inhibitors like STI571, it may be an attractive target for new therapeutical approaches. Therefore, we screened more than 50 different types of cutaneous T-cell lymphomas (TCLs) for the presence of C-KIT. Immunohistochemical stainings were performed on paraffin-embedded tissue sections using a polyclonal rabbit anti-human C-KIT antibody. Naphtol-ASD-chloroacetate esterase (NASDCE)-control stainings were performed on every positive sample to distinguish C-KIT-positive lymphoma cells from C-KIT-positive mast cells. RESULTS: We found weak expression of C-KIT in seven of 18 patients with primary cutaneous CD30+ ALCL, two of eight patients with primary cutaneous pleomorphic TCL, six of 18 patients suffering from mycosis fungoides, and three of five patients with Sezary's syndrome. Generally, only a very small population of the lymphoma cells expressed C-KIT. This finding indicates a difference to the systemic variant of CD30+ ALCL. The potential use of C-KIT targeting new therapeutical approaches is therefore discussed critically, because C-KIT expression is very rare in all investigated types of primary cutaneous lymphoma.

[Imiquimod for the topical treatment of focal epithelial hyperplasia (Heck disease) in a child]. / Imiquimod-Creme für die topische Behandlung der fokalen epithelialen Hyperplasie (M. Heck) bei einem Kind.

Focal epithelial hyperplasia (FEH) or Heck disease is a rare skin disease caused by human papilloma viruses (HPV). The case of a 9-year old boy is presented to demonstrate the successful treatment of massive FEH with 5% imiquimod cream. Initially, the patient had noticed several separate papules, which spread and developed into multiple peri- and intraoral papillomatous nodules. The lesions were treated with carbon dioxide laser destruction. However, multiple, skin-coloured papillomatous nodules were found on the tongue, buccal mucosa and lips 1.5 years later. Treatment with imiquimod was initiated, because the patient suffered tremendously from the disease. 5% imiquimod cream was applied 3 times per week. Regression of lesions was obvious after 1 month of treatment. Complete clearance was achieved after 2 additional months of treatment and no recurrence was detected over a follow-up period of 5 months. Our case points out the clinical value of imiquimod for the non-traumatic and almost painless therapy of HPV-induced skin diseases in children.

Mesonephric cell migration induces testis cord formation and Sertoli cell differentiation in the mammalian gonad.

In mammals a single gene on the Y chromosome, Sry, controls testis formation. One of the earliest effects of Sry expression is the induction of somatic cell migration from the mesonephros into the XY gonad. Here we show that mesonephric cells are required for cord formation and male-specific gene expression in XY gonads in a stage-specific manner. Culturing XX gonads with an XY gonad at their surface, as a 'sandwich', resulted in cell migration into the XX tissue. Analysis of sandwich gonads revealed that in the presence of migrating cells, XX gonads organized cord structures and acquired male-specific gene expression patterns. From these results, we conclude that mesonephric cell migration plays a critical role in the formation of testis cords and the differentiation of XY versus XX cell types.

Sry and the testis: molecular pathways of organogenesis.

The gene Sry acts as a switch, initiating pathways leading to the differentiation of a testis rather than an ovary from the indifferent gonad (genital ridge) in mammals. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. Sry must therefore initiate signaling pathways that direct male-specific patterns of proliferation, migration, cell-cell organization, and vascularization. We have identified an increase in male-specific proliferation by 12.0 days post coitum, while proliferation in the female gonad declines. We have also observed male-specific cell migration from the mesonephros into the gonad in a composite organ culture system in which gonads from wild-type mice (CD1) and mesonephroi from a transgenic strain expressing beta-galactosidase in all its cells (ROSA26) were grafted together in vitro at the indifferent stage of gonadogenesis. Migration depends on an active signal that requires the presence of a Y chromosome in the gonadal portion of the graft. The signals that trigger migration operate over considerable distances, suggesting either a long-range diffusible factor or the involvement of a rapid and efficient relay mechanism. Identification of the somatic cells contributed from the mesonephros with cell-specific markers indicated that some of the migrating cells were endothelial, revealing differences in processes of vascularization between male and female gonads. A second distinct population of migrating cells lay in close apposition to endothelial cells, and a third population occupied positions circumscribing areas of condensing Sertoli cells.

Male-specific cell migration into the developing gonad.

BACKGROUND: The gene Sry acts as a developmental switch, initiating a pathway of gene activity that leads to the differentiation of testis rather than ovary from the indifferent gonad (genital ridge) in mammalian embryos. The early events following Sry expression include rapid changes in the topographical organization of cells in the XY gonad. To investigate the contribution of mesonephric cells to this process, gonads from wild-type mice (CD1), and mesonephroi from a transgenic strain ubiquitously expressing beta-galactosidase (ROSA26), were grafted together in vitro. After culture, organs were fixed and stained for beta-galactosidase activity to identify cells contributed from the mesonephros to the male or female gonad. RESULTS: Migration of mesonephric cells occurred into XY but not XX gonads from 11.5-16.5 days post coitum (dpc). Somatic cells contributed from the mesonephros were distinguished by their histological location and by available cell-specific markers. Some of the migrating cells were endothelial; a second population occupied positions circumscribing areas of condensing Sertoli cells; and a third population lay in close apposition to endothelial cells. CONCLUSIONS: OFFgration from the mesonephros to the gonad is male specific at this stage of development and depends on an active signal that requires the presence of a Y chromosome in the gonad. The signals that trigger migration operate over considerable distances and behave as chemoattractants. We suggest that migration of cells into the bipotential gonad may have a critical role in initiating the divergence of development towards the testis pathway.