Tryptophan 650 of human granulocyte colony-stimulating factor (G-CSF) receptor, implicated in the activation of JAK2, is also required for G- CSF-mediated activation of signaling complexes of the p21ras route

Granulocyte colony-stimulating factor (G-CSF) induces rapid phosphorylation of JAK kinases as well as activation of the p21ras route through interaction with its specific receptor (G-CSF-R). The cytoplasmic membrane-proximal region of G-CSF-R (amino acids 631 to 684) is necessary for proliferation induction and activation of JAK2. In contrast, activation of Shc and Syp, signaling molecules implicated in the p21ras signaling route, depends on the phosphorylation of tyrosine residues located in the membrane-distal region (amino acids 685 to 813) of G-CSF-R. We investigated whether G-CSF-induced activation of signaling complexes of the p21ras route depends on the function of the membrane-proximal cytoplasmic region of G-CSF-R. A G- CSF-R mutant was constructed in which tryptophan 650 was replaced by arginine and expressed in BAF3 cells (BAF/W650R). In contrast to BAF3 cell transfectants expressing wild-type G-CSF-R, BAF/W650-R cells did not proliferate and did not show activation of JAK2, STAT1, or STAT3 in response to G-CSF. Immunoprecipitations with anti-Shc and anti-Grb2 antisera showed that mutant W650R also failed to activate Syp and Shc. These data indicate that the membrane-proximal cytoplasmic domain of G- CSF-R is not only crucial for proliferative signaling and activation of JAK2 and STATs, but is also required for activation of the p21ras route, which occurs via the membrane-distal region of G-CSF-R.     

Activated protein C decreases plasminogen activator-inhibitor activity in endothelial cell-conditioned medium

Confluent cultures of endothelial cells from human umbilical cord were used to study the effect of activated human protein C (APC) on the production of plasminogen activators, plasminogen activator-inhibitor, and factor VIII-related antigen. Addition of APC to the cells in a serum-free medium did not affect the production of tissue-type plasminogen activator (t-PA) or factor VIII-related antigen; under all measured conditions, no urokinase activity was found. However, less plasminogen activator-inhibitor activity accumulated in the conditioned medium in the presence of APC. This decrease was dose dependent and could be prevented by specific anti-protein C antibodies. No decrease was observed with the zymogen protein C or with diisopropylfluorophosphate-inactivated APC. APC also decreased the t-PA inhibitor activity in endothelial cell-conditioned medium in the absence of cells, which suggests that the effect of APC is at least partly due to a direct effect of APC on the plasminogen activator- inhibitor. High concentrations of thrombin-but not of factor Xa or IXa-- had a similar effect on the t-PA inhibitor activity. The effect of APC on the plasminogen activator-inhibitor provides a new mechanism by which APC may enhance fibrinolysis. The data suggest that activation of the coagulation system may lead to a secondary increase of the fibrinolytic activity by changing the balance between plasminogen activator(s) and its (their) fast-acting inhibitor.     

The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.     

Mobilization of early hematopoietic progenitor cells with BB-10010: a genetically engineered variant of human macrophage inflammatory protein- 1 alpha

BB-10010 is a genetically engineered variant of human macrophage inflammatory protein-1 alpha with improved solution properties. We show here that it mobilizes stem cells into the peripheral blood. We investigated the mobilizing effects of BB-10010 on the numbers of circulating 8-day spleen colony-forming units (CFU-S8), CFU-S12, and progenitors with marrow repopulating ability (MRA). A single subcutaneous dose of BB-10010 caused a twofold increase in circulating numbers of CFU-S8, CFU-S12, and MRA 30 minutes after dosing. We also investigated the effects of granulocyte colony-stimulating factor (G- CSF) and the combination of G-CSF with BB-10010 on progenitor mobilization. Two days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA progenitors by 25.7-, 19.8-, and 27.7-fold. A single administration of BB-10010 after 2 days of G-CSF treatment increased circulating CFU-S8, CFU-S12, and MRA even further to 38-, 33-, and 100- fold. Splenectomy resulted in increased circulating progenitor numbers but did not change the pattern of mobilization. Two days of treatment with G-CSF then increased circulating CFU-S8, CFU-S12, and MRA by 64-, 69-, and 32-fold. A single BB-10010 administration after G-CSF treatment further increased them to 85-, 117-, and 140-fold, respectively, compared with control. We conclude that BB-10010 causes a rapid increase in the number of circulating hematopoietic progenitors and further enhances the numbers induced by pretreatment with G-CSF. BB- 10010 preferentially mobilized the more primitive progenitors with marrow repopulating activity, releasing four times the number achieved with G-CSF alone. Translated into a clinical setting, this improvement in progenitor cell mobilization may enhance the efficiency of harvest and the quality of grafts for peripheral blood stem cell transplantation.     

Natural bioburden levels detected on rigid lumened medical devices before and after cleaning

Controversy exists concerning the degree of microbial contamination associated with the us of rigid lumened medical devices, the efficacy of standard cleaning techniques used to remove pathogenic microorganisms from lumen channels, and whether patients are placed at risk of cross infection because of microbial contamination. In this study the level and types of microorganisms found on rigid lumened medical devices before and after cleaning in a hospital environment were investigated. The bioburden level after clinical use was found to be relatively low, ranging from 10¹ to 10⁴ colony forming units (CFU) per device. After the instruments were cleaned, none of the devices studied contained bioburden levels greater than 10⁴ CFU and 83% had bioburden levels less than or equal to 10² CFU. The bioburden present before cleaning was comprised of organisms derived from the handling of the device, from the hospital environment, and from the patient. The bioburden present after cleaning was comprised of organisms typically derived from the handling of the device and from the hospital environment. The level of bioburden per device was also related to the anatomic site where the device was used, with lower numbers of organisms found on devices exposed to sterile body sites and the respiratory tract.     

Radioiodine treatment with 30 mCi after recombinant human thyrotropin stimulation in thyroid cancer: effectiveness for postsurgical remnants ablation and possible role of iodine content in L-thyroxine in the outcome of ablation

The main steps in the management of differentiated thyroid cancer are thyroidectomy, treatment with iodine-131 ((131)I), and follow-up with whole-body scanning (WBS) and serum thyroglobulin (Tg) determination. Both (131)I treatment and follow-up require maximum stimulation of normal or pathological thyroid remnants by TSH. The use of recombinant human TSH (rhTSH) has been shown to be useful for follow-up, whereas previous reports are not univocal regarding the use of (131)I postsurgical ablation of thyroid remnants, at least when low doses (30 mCi) of (131)I are administered. A possible explanation for the diminished effectiveness of (131)I treatment after rhTSH may be the interference of iodine content of L-thyroxine (L-T4) therapy during the protocol of administration of rhTSH. We have evaluated the effectiveness of stimulation by rhTSH for radioiodine ablation of postsurgical remnants, stopping L-T4 the day before the first injection of rhTSH and restarting L-T4 the day after (131)I. The study included two groups of patients: group 1 included 16 patients with differentiated thyroid cancer (15 papillary cancers and 1 follicular cancer, stages I and II), who were treated with 30 mCi (131)I with the aid of rhTSH, using the standard protocol but stopping L-T4 as stated previously; and group 2 included 24 patients with the same features (histology and stage) of disease treated with 30 mCi in the hypothyroid state after L-T4 withdrawal. In both groups, serum TSH reached a very good stimulation level [76-210 U/liter (mean, 112 +/- 11 SE) and 38-82 U/liter (mean, 51 +/- 3 SE), respectively]. At the first WBS (after (131)I treatment), all patients showed thyroid remnants. Furthermore, two patients of the first group and three patients of the second group showed lymph node metastases. After 1 yr, all patients were studied again and underwent WBS with a tracer dose of (131)I and serum Tg measurement using rhTSH with the same protocol in both groups. The percentage of ablation (undetectable Tg and a negative WBS) was higher, although not reaching statistical significance, in patients treated with rhTSH: 81.2% in patients treated by rhTSH withdrawal and 75.0% in patients treated by L-T4 withdrawal, respectively. No patient experienced symptoms of hypothyroidism during the 4 d of L-T4 interruption, and serum T4 remained in the normal range. Urinary iodine was analyzed in both groups and compared with a control group of patients who received, for diagnostic purposes, rhTSH without stopping L-T4. In the first group, urinary iodine was 47.2 +/- 4.0 microg/liter (mean +/- SE; P = 0.21 vs. the second group, P = 0.019 vs. control group). In the second group, urinary iodine was 38.6 +/- 4.0 microg/liter (mean +/- SE; P < 0.001 vs. control group); urinary iodine in the control group was 76.4 +/- 9.3 microg/liter (mean +/- SE). Our data show that rhTSH, as administered in the protocol stated previously, allows at least the same rate of ablation of thyroid remnants when low doses (30 mCi) of (131)I are used. The possible role of interference of iodine content in L-T4 is not surprising if we consider that the amount of iodine in 30 mCi is negligible (5 microg) compared with the amount of iodine content in a daily dose of T(4) ( approximately 50 microg). The cost of rhTSH seems modest compared with the high cost of complex therapeutic regimens in other areas of oncology and in consideration of the well-being of patients and of the high level of effectiveness of the treatment.     

Ectopic expression of rhombotin-2 causes selective expansion of CD4-CD8- lymphocytes in the thymus and T-cell tumors in transgenic mice

Although the proto-oncogene rhombotin-2 (RBTN-2) is widely expressed in most tissues, it is not expressed in T cells. We investigated the potential for overexpression of RBTN-2 to cause tumors in T cells and other tissues by constructing transgenic mice that expressed RBTN-2 under control of the metallothionein-1 promoter. Despite overexpression of RBTN-2 in all tissues, transgenic mice developed T-cell tumors only, thus indicating that tumorigenesis caused by RBTN-2 is T-cell-specific. Thymic tumors were found between 37 and 71 weeks and were invariably associated with metastasis to nonlymphoid organs. Thymuses from apparently healthy transgenic mice were also examined. In some mice there was an 10-fold increase in the CD4-CD8- thymocyte subset, yet the total number of thymocytes was the same as that in wild-type mice. Thymic homeostasis was maintained by a compensatory reduction in the CD4+CD8+ subset. The expansion of CD4-CD8- thymocytes was associated with increased expression of RBTN-2 and with increased cell proliferation. No differences were found in the proportion of thymocytes undergoing apoptosis in transgenic mice. Furthermore, RBTN-2- induced expansion of CD4-CD8- cells did not block differentiation of these cells. Thymuses with 30% CD4-CD8- cells were essentially monoclonal, indicating that all thymic immunophenotypes were derived from a single clone. Overall, our data are consistent with the following scenario: (1) RBTN-2 expression in T cells causes selective and polyclonal proliferation of CD4-CD8- thymocytes accompanied by a compensatory decrease in other thymocyte subsets; (2) a clone with growth advantage and differentiation potential is selected and populates the thymus; and (3) this clone eventually breaches homeostasis of the thymus, accompanied or followed by metastasis to other organs.