von Willebrand factor as a marker of endothelial cell activation following BMT.

Endothelial cell activation may play a role in thrombotic complications of BMT such as hepatic veno-occlusive disease (VOD), right atrial line thrombosis and microangiopathic haemolysis. To assess this, von Willebrand factor antigen (vWF:ag) was measured in 72 patients (25 allografts, 46 autografts and one syngeneic) during the first 6 weeks post-transplant. There was a significant rise in vWF:ag in both allografts and autografts but a greater increase was seen in the allografts. The changes in vWF:ag did not correlate with changes in C reactive protein showing that this was not merely an acute phase response. vWF multimers were normal in a subgroup of uncomplicated transplants showing that there was no large scale endothelial cell disruption. Patients with VOD did not have changes in vWF:ag that were consistently different from uncomplicated controls. Three of four patients who developed line thrombosis had higher levels of vWF:ag compared with control groups; multimeric structure of the vWF was again normal. These results show that there is endothelial cell activation post-BMT and that this is greater in allografts compared with autografts, thus suggesting a possible mechanism for the higher incidence of VOD in this group. There were no useful predictive markers of VOD or thrombosis in individual patients.     

A cytoskeletal actin gene in the mosquito Anopheles gambiae

Five actin genes have been identified in the mosquito Anopheles gambiae , and a constitutively expressed actin gene has been chosen for detailed analysis. We have physically mapped and sequenced this gene and six associated cDNAs, including translated coding regions, as well as the 5 and 3 flanking sequences. Analysis of stage-specific RNA shows this gene to be present in all stages of mosquito development and in an established A. gambiae cell line, thus indicating a cytoskeietal actin. In the sequence of the translated coding region and in pattern of expression, this gene is very similar to the cytoskeietal actin genes of Droso-phila melanogaster , and in sequence, equally similar to the Artemia cytoskeietal actin gene 403 (99.2% identity among the three amino acid sequences). Sequencing of this A. gambiae actin gene (designated actWior its location in chromosome division 1D) and selected cDNAs shows that it possesses three alternative leader sequences; thus the gene appears to have three alternative promoters. These promoters should ultimately prove useful in the production of transgenic constructs for constitutive expression.     

Use of intracavity saline instillation and transvaginal ultrasonography to detect tamoxifen-associated endometrial polyps.

There is sometimes a discrepancy between the apparent thickness of postmenopausal endometria, as determined by transvaginal ultrasonography, and the examination of endometrial biopsies. We describe a case which showed that tamoxifen (20 mg/day over 12 months) decreased impedance to blood flow in the uterine arteries and increased the apparent thickness of the endometrium. Conversely, the analysis of biopsies suggested the presence of an atrophic endometrium. The introduction of sterile saline into the uterine cavity during a repeat ultrasound scan revealed the presence of a large, free-floating endometrial polyp, which was subsequently difficult to see by hysteroscopy.     

Effects of antisense mediated inhibition of cathepsin K on human osteoclasts obtained from peripheral blood.

Cathepsin K is a cystein protease that displays a proteolytic activity against Type I collagen and is abundantly and selectively expressed in osteoclasts where it plays a critical role in bone degradation. Its direct role in bone tissue has been defined by knock-out mice studies and inhibiting strategies in animals models. However, direct proof of cathepsin K function in human osteoclast model in vitro is lacking. The aim of this study is to analyze cathepsin K expression and localization in human osteoclasts obtained from peripheral blood and to examine cathepsin K function in these cells by antisense oligodeoxynucleotide (AS-ODN) strategy. AS-ODN was added to the culture of osteoclast precursors induced to differentiate by RANKL and M-CSF. AS-ODN treatment produced a significant down-regulation of cathepsin K mRNA (>80%) and protein expression, as verified respectively by Real-time PCR and by immunocytochemistry or Western blot. The cathepsin K inhibition caused an impairment of resorption activity as evaluated by a pit formation assay ( p = 0.045) and by electron microscopy, while the acidification process was unaffected. We demonstrated that antisense strategies against cathepsin K are selectively effective to inhibit resorption activity in human osteoclasts, like in animal models.     

Recombinant interleukin 3 induces interleukin 2 receptor expression on early myeloid cells in normal human bone marrow.

Human interleukin 3 (IL-3) is a multipotential cytokine that supports the growth of early hematopoietic progenitors and promotes their response to other, later-acting cytokines. We found that IL-3 was able to induce the expression of interleukin 2 (IL-2) receptor (IL-2R) (CD25) on a subset of early myeloid cells in normal human bone marrow that had been first depleted of mature hematopoietic cells and E-rosette-positive T cells by treatment with soybean lectin and sheep erythrocytes (SBA-E-BM). Immunofluorescence analysis revealed that the CD25+ cells were contained almost entirely within the lymphoblastoid gate of the IL-3-cultured marrow. CD25 was undetectable on freshly isolated marrow and less than 10% CD25+ cells could be detected following liquid culture at 37 degrees C in the presence of 10% human serum, 10% fetal calf serum, or under serum-free conditions. Addition of IL-3 (100 U/ml) significantly increased the expression of CD25 to 37%, 31%, and 24%, respectively. CD25 could also be induced by granulocyte-macrophage colony-stimulating factor (GM-CSF), but no IL-2R was detectable following exposure to granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), interleukin 1 (IL-1), interleukin 4 (IL-4), or IL-2. Expression of CD25 was dependent on the dose of IL-3 or GM-CSF added and was maximal within 24 h of exposure. Two-color immunofluorescence analysis demonstrated that CD25 was not expressed by cells of lymphoid lineage or by mature monocytes, but rather was present on cells that coexpressed CD13, CD33, CD34, MY8, and HLA-DR, and that lacked CD14 or CD11b, thus placing the CD25+ cells at or near the myeloblast stage of differentiation. An identical phenotype was found for CD25+ cells induced by GM-CSF. Cycloheximide completely inhibited the IL-3-induced expression of CD25, indicating the necessity for protein synthesis, and although most of the CD25+ cells were in G0/G1 phase, 25% of the cells were in S or G2M phase, indicating that receptor expression was not cell-cycle dependent. The p75 chain of IL-2R was not detected on the CD25+ cells. IL-3 was also found to directly induce CD25 in greater than 46% of SBA-E-BM enriched for CD34+ cells by panning. Consistent with the expression of only p55 IL-2R, the functional activity of IL-2 on enriched CD34+ cells exposed to IL-3 could not be demonstrated in either granulocyte-macrophage colony-forming unit (CFU-GM) assays or proliferation assays.(ABSTRACT TRUNCATED AT 400 WORDS)