Jack Longley

The mast cell and mast cell disease

Mast cell disease or mastocytosis is a heterogeneous group of clinical disorders characterized by the proliferation and accumulation of mast cells in a variety of tissues, most often the skin. The signs and symptoms of mast cell disease are varied, dependent on the localization of mast cells in different organs and the local and systemic effects of mediators released from these cells. Although mast cell disease is most commonly identified in the skin, involvement of the skeletal, hematopoietic, gastrointestinal, cardiopulmonary, and central nervous systems may be seen. Clinical management of mastocytosis depends most heavily on knowledge of the diverse effects of mast cell mediators on various tissues and organs, the stimuli that can cause their release, and the different methods available for blocking the effects of these mediators.

Detection of specific mRNAs in routinely processed dermatopathology specimens

To determine the effect of different fixatives on the recovery and detection of mRNAs from archival histopathology specimens, biopsies of normal skin were fixed in neutral and alcohol-buffered formalin, acetone, Carnoys fixative, methacarn, and Bouins solution. Tissue was routinely processed, and sections were either mounted for in situ hybridization or deparaffinized for RNA extraction. Extracted mRNA was reverse-transcribed using random hexamers, and the resulting cDNA was amplified by the polymerase chain reaction using primers specific for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and beta-actin. Amplification products of both GAPDH and actin could be detected by gel electrophoresis from tissues processed in all fixatives except Bouins. A parallel study of formalin-fixed, paraffin-embedded archival biopsies accessioned since 1990 gave similar results. Less abundant mRNAs, such as those encoding interleukin-11 or the T-cell receptor beta-chain, could be detected by Southern blotting and hybridization with labeled oligonucleotide probes or by cloning and sequencing. In situ hybridization studies using oligonucleotide probes were most successful with tissue fixed in formalin, including both the experimentally fixed tissues and the archival biopsy samples. Thus, mRNAs may be isolated from and localized in formalin-fixed, paraffin-embedded archival material. Because dermatopathology laboratory archives typically contain samples from a wide spectrum of diseases that can be accessed without Human Investigation Committee approval, these laboratories represent a logical starting point for studying gene regulation and expression in skin.

Type 73 human papillomavirus in esophageal squamous cell carcinoma. A novel association

Human papillomaviruses (HPVs) commonly cause proliferative lesions of squamous epithelia, and infection with certain HPV types carries a high risk of malignant transformation, especially in the uterine cervix but also at other sites, including the esophagus. We used molecular techniques to detect and type HPV in an in situ squamous cell carcinoma in the esophagus of a 39‐year‐old woman.

In situ transcription and detection of CD 1a mRNA in epidermal cells: An alternative to standard in situ hybridization techniques

To develop methods for the investigation of mRNA transcription in rare epidermal cells, we used in situ transcription to study CD1a mRNA in isolated CD1a positive cells. We chose to study this Langerhans cell marker because it is not known which epidermal cells actually produce the CD1a protein and because there is evidence that CD1a mRNA is alternately spliced, a situation which could lead to truncated or alternate protein products in CD1a surface protein negative cells. Disaggregated epidermal cells were resolved into CD1a surface protein positive and negative groups by fluorescence activated cell sorting and cytocentrifuged onto glass slides. A synthetic 52 base, CD1a specific anti-sense oligomer was hybridized to CD1a gene transcripts in these cells, and radiolabeled cDNA synthesized in situ on the oligomerprimed CD1a transcripts. The labeled cDNA fragments were visualized in the cells of origin by autoradiography, and grains per cell were counted. Sixty-eight percent of cells expressing CD1a protein contained CD1a mRNA, as evidenced by grain counts more than two standard deviations above the mean value for similar cells carried through the same procedure with a control oligomer, or the mean value of CD1a surface protein negative cells treated with the CD1a specific oligomer. Thus, it seems likely that the CD1a protein positive epidermal cells use CD1a mRNA to make their own CD1a protein, and that a truncated or masked CD1a protein is not made by CD1a negative neonatal foreskin epidermal cells. In our hands, in situ transcription is simpler and faster than standard methods of in situ hybridization with prelabeled cDNA or RNA probes. Furthermore, it can be applied to the detection of any message of known sequence. The combination of cell sorting and in situ transcription can be used to localize and quantify the expression of specific mRNA by individual cells, allowing the study of rare and difficult-to-obtain cells.