Systematic approach to diagnosis and management of biphasic insulin allergy with local anti-inflammatory agents.

In this report, we present a patient with a rarely encountered form of biphasic insulin allergy refractory to a conventional desensitization procedure. We describe a systematic approach to this clinical problem with a set of insulin preparations containing antihistamine and/or corticosteroid to verify the type of hypersensitivity reaction and identify treatment options. The approach taken here may be of use for patients with similar conditions who require insulin for adequate diabetic management but who would otherwise be forced to discontinue insulin because of allergic reactions.

Rate of precipitation of calcium phosphate on heated surfaces.

Fouling of a heated stainless steel surface by calcium phosphate precipitation has been studied in an annular flow apparatus, instrumented to provide a constant heat flux while measuring local metal-surface temperatures. Models of the heat and mass-transfer boundary layers are used to estimate interfacial temperatures and concentrations, from which the heterogeneous reaction rate is inferred. The analysis indicates that the reaction rate is a function of both chemical kinetics and mass transfer limitations.

Detection of the Philadelphia chromosome in acute lymphoblastic leukemia by pulsed-field gel electrophoresis.

The Philadelphia (Ph1) chromosome is an acquired abnormality in the malignant cells of 10% to 25% of patients with acute lymphoblastic leukemia (ALL). Unlike chronic myelogenous leukemia (CML), where the molecular detection of the Ph1 chromosome is relatively straightforward using conventional Southern hybridization analysis, the detection of the Ph1 chromosome in ALL is complicated by the existence of several molecular subtypes, and the fact that translocation breakpoints are dispersed over a large genomic area. To circumvent these difficulties, we investigated pulsed-field gel electrophoresis (PFGE) to determine if this method could be used directly on clinical samples to detect the Ph1 chromosome in ALL. We report that, in a study of seven patients with Ph1-positive ALL, we could easily detect the Ph1 using only a single PFGE analysis, regardless of the Ph1 subtype, and we could confirm that the translocations occur either within or very near the BCR gene in all seven. We conclude that PFGE is a useful technique for the detection of the Ph1 in ALL, which ultimately may find wide applicability in the detection of other chromosomal abnormalities in other malignancies.

Transcription factor GATA-3 is required for development of the T-cell lineage.

THE zinc-finger transcription factor GATA-3 is expressed in haematopoietic cells and in the developing kidney and nervous system. Within the haematopoietic lineages, expression of GATA-3 is restricted to thymocytes and T cells. Functionally important GATA-3 binding sites have been identified in multiple T-cell-specific genes. Mice containing homozygous null mutations of the GATA-3 gene die on embryonic day 12, precluding a detailed assessment of the role of GATA-3 in haematopoietic development. Here we have used murine embryonic stem (ES) cells containing homozygous mutations in the GATA-3 gene (GATA-3(-/-)) in conjunction with the RAG-2(-/-) (ref. 10) and C57BL/6 complementation systems to study the role of GATA-3 in mammalian haematopoiesis. Our results show that GATA-3(-/-) ES cells can contribute to the development of the mature erythroid, myelomonocytic and B-cell lineages, but fail to give rise to thymocytes or mature peripheral T cells. The differentiation of GATA-3(-/-) T cells is blocked at or before the earliest double-negative (CD4-/CD8-) stage of thymocyte development, such that the GATA-3(-/-) ES cells are unable to contribute measurably to the double-negative thymocyte population. These findings suggest that GATA-3 is an essential and specific regulator of early thymocyte development.

PU.1 exhibits partial functional redundancy with Spi-B, but not with Ets-1 or Elf-1.

Previously it was shown that the Ets proteins, PU.1 and Spi-B, exhibit functional redundancy in B lymphocytes. To investigate the possibility that PU.1 or Spi-B or both share overlapping roles with Ets-1 or Elf-1, PU.1(+/-)Ets-1(-/-), PU.1(+/-)Elf-1(-/-), and Spi-B(-/-)Ets-1(-/-) animals were generated. No blood cell defects were observed in these animals except those previously reported for Ets-1(-/-) mice. Therefore, no genetic overlap was detected between PU.1 or Spi-B with Ets-1 or Elf-1. In contrast, the results confirmed functional redundancy for PU.1 and Spi-B in that PU.1(+/-)Spi-B(-/-) bone marrow progenitors yielded smaller colonies in methylcellulose cultures than did wild-type, PU.1(+/-) or Spi-B(-/-) progenitors. In addition, PU.1(+/-)Spi-B(+/+), PU.1(+/-)Spi-B(+/-), and PU.1(+/-) Spi-B(-/-) mice displayed extramedullary splenic hematopoiesis. In summary, PU.1 and Spi-B regulate common target genes required for proliferation of hematopoietic progenitors or their committed descendants, whereas Ets-1 or Elf-1 do not appear to regulate shared target genes with PU.1 or Spi-B.

The LKLF transcription factor is required for normal tunica media formation and blood vessel stabilization during murine embryogenesis.

The transcriptional programs that regulate blood vessel formation are largely unknown. In this paper, we examine the role of the zinc finger transcription factor LKLF in murine blood vessel morphogenesis and homeostasis. By in situ hybridization and immunohistochemistry, we show that LKLF is expressed as early as embryonic day 9.5 (E9.5) in vascular endothelial cells throughout the developing mouse embryo. To better understand the function of LKLF, we used homologous recombination in embryonic stem (ES) cells to generate LKLF-deficient (LKLF-/-) mice. Both angiogenesis and vasculogenesis were normal in the LKLF-/- mice. However, LKLF-/- embryos died between E12.5 and E14.5 from severe intra-embryonic and intra-amniotic hemorrhaging. This bleeding disorder was associated with specific defects in blood vessel morphology. Umbilical veins and arteries in the LKLF-/- embryos displayed an abnormally thin tunica media and aneurysmal dilatation before rupturing into the amniotic cavity. Similarly, vascular smooth muscle cells in the aortae from the LKLF-/- animals displayed a cuboidal morphology and failed to organize into a compact tunica media. Consistent with these findings, electron microscopic analyses demonstrated endothelial cell necrosis, significant reductions in the number of vessel-wall pericytes and differentiating smooth muscle cells, and decreased deposition of extracellular matrix in the LKLF-/- vessels. Despite these defects, in situ hybridization demonstrated normal expression of platelet-derived growth factor B, Tie1, Tie2, transforming growth factor beta, and heparin-binding epidermal growth factor in the vasculature of the LKLF-/- embryos. Therefore, LKLF defines a novel transcriptional pathway in which endothelial cells regulate the assembly of the vascular tunica media and concomitant vessel wall stabilization during mammalian embryogenesis.

Expression of the IFN alpha receptor in hairy cell leukaemia.

The expression of IFN alpha receptors on the surface of hairy cell leukaemia cells was studied using affinity crosslinking methods, flow cytometry with a recently produced anti-IFN alpha receptor monoclonal antibody (IFNaR3), and binding techniques. IFN alpha receptors were detected by flow cytometry and affinity crosslinking in eight cases showing a normal receptor structure with 125I-IFN alpha 2-receptor complexes with molecular weights of 210, 130 and 110 kD. The hairy cell leukaemia cells from one patient did not express IFN alpha receptors as determined by flow cytometry, affinity crosslinking and binding studies. Northern blot analysis showed expression of mRNA for the gene coding for the putative IFN alpha receptor in all cases studied, including the patient with lack of IFN alpha receptor expression on hairy cells. This patient was refractory to IFN alpha 2 treatment. Our data suggest that lack of expression of IFN alpha receptors in rare cases may be associated with refractoriness to IFN alpha 2 therapy.

Expression pattern of the Ets-related transcription factor Elf-1.

Members of the Ets family of winged helix-loop-helix transcription factors play important roles in the development and function of multiple mammalian cell lineages. Elf-1 is an Ets-related transcription factor that is expressed at high levels in T cells and is known to regulate the expression of several T cell genes, including the granulocyte-macrophage colony stimulating factor (GM-CSF) gene, the interleukin-2 receptor alpha subunit (IL-2Ralpha) gene, and the CD4 gene. In the studies described in this report, we have characterized the pattern of expression of Elf-1 in the developing mouse embryo and in adult mouse tissues as well as in multiple immortalized human and murine cell lines. Elf-1 is expressed at high levels throughout thymocyte development, with equivalent levels of Elf-1 expression seen in all subsets of maturing thymocytes and T cells. Somewhat surprisingly, however, Elf-1 is also expressed at high levels in epithelial cells lining the oral cavity, the lung, the CNS, and the gastrointestinal and urinary tracts as well as in the skin of the developing mouse embryo and at lower levels in the adult mouse testis and liver. Western blot analyses of a large number of immortalized cell lines demonstrated high-level Elf-1 expression in T and B lymphocyte and macrophage cell lines as well as in two prostate carcinoma cell lines. Low-level expression was observed in fibroblasts, embryonic stem cells, and myoblasts. Taken together, our data suggest that in addition to its role in regulating T cell development and function, Elf-1 may regulate gene expression in the B cell and myelomonocytic lineages, as well as in multiple epithelial cell types during murine embryonic development.