Immunohistochemical investigation of the coma blister and its pathogenesis

The erythematous patches and vesicles that are observed in coma patients, usually from an overdose of medication, are known as coma blisters. However, it is unknown whether the degenerated sweat gland is a necrosis or apoptosis. We immunohistochemically examined such skin lesions to investigate the characteristics and pathogenesis of the coma blister. Skin lesions were obtained from a forensic autopsy case, a woman in her thirties, of caffeine intoxication. Those lesions were observed in the left femoral, the lower left thigh, and the right knee. Histologically, the skin lesions showed that the keratinocytes had necrosed and the epidermis was thin in some areas. Eccrine sweat gland degeneration was observed. Obvious inflammatory cell infiltrations were not detected. Immunohistochemically, we stained each skin lesion against CD3, CD8, CD45RO, cytokeratin, 70 kD heat shock protein, ubiquitin, 150 kD oxygen regulated protein, and caspase-cleaved keratin 18 neo-epitope M30. They were also stained with an in situ apoptosis detection kit. Degenerated sweat glands featured CD45RO and M30 immunoreactivity. Immunohistochemical staining for CD45RO, CK-L, and M30 might be useful to observe sweat gland degeneration in the coma blister. Therefore, the apoptosis might be related to coma blisters and sweat gland degenerations

Cyclic AMP Responsive Element Binding Proteins Are Involved in ‘Emergency’ Granulopoiesis through the Upregulation of CCAAT/Enhancer Binding Protein β

In contrast to the definitive role of the transcription factor, CCAAT/Enhancer binding protein α (C/EBPα), in steady-state granulopoiesis, previous findings have suggested that granulopoiesis during emergency situations, such as infection, is dependent on C/EBPβ. In this study, a novel lentivirus-based reporter system was developed to elucidate the molecular switch required for C/EBPβ-dependency. The results demonstrated that two cyclic AMP responsive elements (CREs) in the proximal promoter region of C/EBPβ were involved in the positive regulation of C/EBPβ transcription during granulocyte-macrophage colony-stimulating factor (GM-CSF)–induced differentiation of bone marrow cells. In addition, the transcripts of CRE binding (CREB) family proteins were readily detected in hematopoietic stem/progenitor cells. CREB was upregulated, phosphorylated and bound to the CREs in response to GM-CSF stimulation. Retroviral transduction of a dominant negative CREB mutant reduced C/EBPβ mRNA levels and significantly impaired the proliferation/differentiation of granulocyte precursors, while a constitutively active form of CREB facilitated C/EBPβ transcription. These data suggest that CREB proteins are involved in the regulation of granulopoiesis via C/EBPβ upregulation

Lentivirus-based reporter system.

<p>A. Expression of C/EBPβ mRNA in c-kit⁺ bone marrow cells with or without GM-CSF stimulation <i>in vivo</i>. *:<i>P</i><0.05, n = 3. Data are representative of two independent experiments. B. Schematic illustration of the lentivirus vector for reporter assays. SIN, self-inactivated long terminal repeat; PGK prom, phosphoglycerate kinase promoter; Ars I, sea urchin arylsulfatase insulator sequence. C. Elongation factor-1 (EF-1) promoter activity in bone marrow cells revealed by the lentivirus-based reporter system. Bone marrow cells were analyzed by flow cytometry after two days incubation with GM-CSF following viral infection. Shaded histogram, promoter-less control; open histogram, EF-1 promoter.</p

Involvement of CREB-C/EBPβ pathway in candidemia-induced “emergency granulopoiesis.”

<p>A. Expression of C/EBPβ mRNA in c-kit⁺ bone marrow cells with or without candidemia. *:<i>P</i><0.05, n = 3. B. Western blotting analysis of c-kit+ bone marrow cells with or without candidemia. C. Chromatin immunoprecipitation of CREB using c-kit+ bone marrow cells during candidemia induced emergency granulopoiesis. Data are representative of two independent experiments.</p

Flow cytometric analysis of the C/EBPβ proximal promoter region using the lentivirus-based reporter system.

<p>A. Schematic illustration of the lentivirus vector. SIN, self-inactivated long terminal repeat; PGK prom, phosphoglycerate kinase promoter; Ars I, sea urchin arylsulfatase insulator sequence. B and C. Bone marrow cells were transduced with the lentivirus vector containing the indicated fragment and were analyzed for Thy1.1 and d2EGFP expression after 48 hours stimulation with GM-CSF. D and E. Effects of the mutated CREs at −110 and −65 bp on the activity of the promoter fragment (−243 to +16 bp). Shaded histogram, promoter-less control; open histogram, promoter fragment. Wt, wild type; mut, mutated. *:<i>P</i><0.05, **:<i>P<0.01</i> (n = 3). Data are representative of three independent experiments.</p