Catheter malfunction due to spontaneous permcath displacement into medium suprahepatic vein.

One of the last options, when the other possibilities of vascular access present malfunction, is the insertion of a permanent catheter in a central vein, preferentially internal jugular vein. This option is considered when arteriovenous access is impossible. We report a case of malfunction due to a permanent catheter dis-placement solved by vascular interventional radiology.

HCA, an immunoglobulin-like adhesion molecule present on the earliest human hematopoietic precursor cells, is also expressed by stromal cells in blood-forming tissues.

We have previously shown that the HCA/ALCAM (CD166) glycoprotein, a member of the immunoglobulin family that mediates both homophilic and heterophilic cell-cell adhesion, via the CD6 ligand, is expressed at the surface of all of the most primitive CD38(-/lo), Thy-1(+), rho123(lo), CD34(+) hematopoietic cells in human fetal liver and fetal and adult bone marrow. In the present report we show that HCA is also expressed by subsets of stromal cells in the primary hematopoietic sites that sequentially develop in the human embryo and fetus, ie, the paraaortic mesoderm, liver, thymus, and bone marrow. Adult bone marrow stromal cells established in vitro, including those derived from Stro-1(+) progenitors and cells from immortalized cell lines, express HCA. In contrast, no HCA expression could be detected in peripheral lymphoid tissues, fetal spleen, and lymph nodes. HCA membrane molecules purified from marrow stromal cells interact with intact marrow stromal cells, CD34(+) CD38(-) hematopoietic precursors, and CD3(+) CD6(+) peripheral blood lymphocytes. Finally, low but significant levels of CD6 are here for the first time detected at the surface of CD34(+) rho123(med/lo) progenitors in the bone marrow and in mobilized blood from healthy individuals. Altogether, these results indicate that the HCA/ALCAM surface molecule is involved in homophilic or heterophilic (with CD6) adhesive interactions between early hematopoietic progenitors and associated stromal cells in primary blood-forming organs.

HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells.

Recent studies have opened the possibility that quiescent, G0/G1 hematopoietic stem cells (HSC) can be gene transduced; lentiviruses (such as HIV type 1, HIV) encode proteins that permit transport of the viral genome into the nucleus of nondividing cells. We and others have recently demonstrated efficient transduction by using an HIV-1-based vector gene delivery system into various human cell types including human CD34(+) cells or terminally differentiated neurons. Here we compare the transduction efficiency of two vectors, HIV-based and murine leukemia virus (MuLV)-based vectors, on untreated and highly purified human HSC subsets that are virtually all in G0/G1. The HIV vector, but not MuLV vector supernatants, transduced freshly isolated G0/G1 HSC from mobilized peripheral blood. Single-step transduction using replication-defective HIV resulted in HSC that expressed the green fluorescent protein (GFP) transgene while retaining their stem cell phenotype; clonal outgrowths of these GFP+ HSC on bone marrow stromal cells fully retained GFP expression for at least 5 weeks. MuLV-based vectors did not transduce resting HSC, as measured by transgene expression, but did so readily when the HSC were actively cycling after culture in vitro for 3 days in a cytokine cocktail. These results suggest that resting HSC may be transduced by lentiviral-based, but not MuLV, vectors and maintain their primitive phenotype, pluripotentiality, and at least in vitro, transgene expression.

High doses of purified stem cells cause early hematopoietic recovery in syngeneic and allogeneic hosts.

In humans, autologous transplants derived from bone marrow (BM) usually engraft more slowly than transplants derived from mobilized peripheral blood. Allogeneic BM transplants show a further delay in engraftment and have an apparent requirement for donor T cells to facilitate engraftment. In mice, Thy-1.1(lo)Lin-/loSca-1+ hematopoietic stem cells (HSCs) are the principal population in BM which is responsible for engraftment in syngeneic hosts at radioprotective doses, and higher doses of HSCs can radioprotect an allogeneic host in the absence of donor T cells. Using the mouse as a preclinical model, we wished to test to what extent engraftment kinetics was a function of HSC content, and whether at high doses of c-Kit+Thy-1.1(lo)Lin-/loSca-1+ (KTLS) cells rapid allogeneic engraftment could also be achieved. Here we demonstrate that engraftment kinetics varied greatly over the range of KTLS doses tested (100-10,000 cells), with the most rapid engraftment being obtained with a dose of 5,000 or more syngeneic cells. Mobilized splenic KTLS cells and the rhodamine 123(lo) subset of KTLS cells were also able to engraft rapidly. Higher doses of allogeneic cells were needed to produce equivalent engraftment kinetics. This suggests that in mice even fully allogeneic barriers can be traversed with high doses of HSCs, and that in humans it may be possible to obtain rapid engraftment in an allogeneic context with clinically achievable doses of purified HSCs.

Hydroxyurea can be used to increase mouse c-kit+Thy-1. 1(lo)Lin-/loSca-1(+) hematopoietic cell number and frequency in cell cycle in vivo.

The DNA synthesis inhibitor hydroxyurea (HU) was administered to determine whether it induces changes in the cell-cycle status of primitive hematopoietic stem cells (HSCs)/progenitors. Administration of HU to mice leads to bone marrow accumulation of c-kit+Thy-1.1(lo)Lin-/loSca-1(+) (KTLS) cells in S/G2/M phases of the cell cycle. HU is a relatively nontoxic, reversible cell-cycle agent that can lead to approximately a threefold expansion of KTLS cells in vivo and approximately an eightfold increase in the number of KTLS cells in S/G2/M. HSCs in HU-treated mice have undiminished multilineage long-term and short-term clonal reconstitution activity.

Regulation of scavenger receptor expression in smooth muscle cells by protein kinase C: a role for oxidative stress.

Phorbol esters increase scavenger-receptor mRNA expression and receptor activity in smooth muscle cells (SMCs). Our present results demonstrate that activation of protein kinase C (PKC) mediates this increase in receptor expression. This conclusion is based on the findings that (1) phorbol esters induced translocation of PKC-alpha from the cytosol to the membrane fraction; (2) PKC inhibitors blocked the effect of phorbol esters on receptor expression; (3) diacylglycerol, a physiological PKC agonist, enhanced scavenger-receptor activity; and (4) in cotransfected human SMCs, constitutively active PKC-alpha stimulated the expression of a reporter gene under control of the scavenger-receptor promoter. Phorbol ester treatment of SMCs increased intracellular reactive oxygen, and the increase in receptor activity was reduced 30% by the antioxidant N-acetyl cysteine (NAC), suggesting a role for reactive oxygen in phorbol ester-mediated receptor regulation. Furthermore, direct treatment of SMCs with reactive oxygen species increased scavenger-receptor activity. In rabbit SMCs, 100 micromol/L H2O2 alone slightly increased scavenger-receptor mRNA and protein expression. In combination, 100 micromol/L H2O2 and 10 micromol/L vanadate, which promotes formation of OH and enhances the inhibition of protein tyrosine phosphatase by H2O2, increased scavenger-receptor mRNA expression 25-fold in rabbit SMCs and 8-fold in human SMCs. NAC reduced the effect of H2O2 and vanadate by 93%. The increase in SMC scavenger-receptor expression occurs at the level of gene transcription. Receptor mRNA half-life was unchanged after treatment with either phorbol esters or reactive oxygen (approximately 14.5 hours), and induction by phorbol esters increased SMC scavenger-receptor mRNA transcription, as determined by nuclear run-on assay. Multiple cytokines and growth factors that contribute to the generation of reactive oxygen species are present in atherosclerotic lesions. These factors may all contribute to the upregulation of SMC scavenger-receptor activity and therefore to the formation of smooth muscle foam cells.

Magnetic resonance imaging features of chloroma of the shoulder.

A patient with a history of essential thrombocytosis presented with diffuse skeletal pain and restricted motion of the left shoulder. Magnetic resonance imaging (MRI) of the left glenohumeral joint showed a soft tissue mass that displaced the rotator cuff. Biopsy of the mass revealed chloroma. MRI is the method that best characterizes this lesion.

The unexpected G0/G1 cell cycle status of mobilized hematopoietic stem cells from peripheral blood.

Treatment with a combination of cytokines and chemotherapy can effectively stimulate the release of hematopoietic stem cells (HSC) into the peripheral blood (PB), which can then be harvested for transplantation. The cell cycle status of the harvested HSC from mobilized PB (MPB) is of interest because of the impact that cell cycling may have on optimizing the conditions for ex vivo expansion, retrovirus-mediated gene transfer, and the engraftment of transplanted tissues. Therefore, we characterized the cell cycling status of mobilized HSC from mice and humans. The murine HSC, which express the phenotype c-kit+ Thy-1.1lo Lin-/lo Sca-1+, were purified from PB, bone marrow (BM), and spleen after the mice were treated with the mobilizing regimen of granulocyte colony-stimulating factor (G-CSF) or a combination of cyclophosphamide (CTX) and G-CSF. Human HSC (CD34+ Thy-1+ Lin-) and progenitor cells (CD34+ Thy-1-Lin-) were isolated from the BM of untreated healthy volunteers and from MPB of healthy volunteers and patients treated with G-CSF or a combination of CTX and GM-CSF. Cell cycle status was determined by quantitating the amount of DNA in the purified cells after staining with the dye Hoechst 33342. Fluorescence-activated cell sorting analysis of the progenitor cells from the murine and human samples showed an unexpected finding, ie, virtually none of the cells from the MPB was cycling. The G0/G1 status of HSC from MPB was surprising, because a significant proportion of HSC from BM are actively proliferating and, after mobilization, the HSC in the spleen and BM were also actively cycling.

Dominant negative mutations of the scavenger receptor. Native receptor inactivation by expression of truncated variants.

The bovine scavenger receptor was truncated at amino acid 266 or 310 to delete either all or part, respectively, of the collagen-like domain. The truncated receptors were inactive in the binding and internalization of acetyl (Ac) low density lipoprotein (LDL). Coexpression of truncated receptor with the native receptor dramatically reduced the percentage of cells internalizing fluorescently labeled Ac LDL, compared with cells expressing the native receptor alone. The mutant truncated at amino acid 266 was most effective in receptor inactivation, resulting in a 42% or 80% decrease in the percentage of cells expressing active receptor when transfected in a 1:1 or 1:2 molar ratio (native:mutant), respectively, with native receptor. Degradation of 125I-Ac LDL was reduced up to 90% when the native and truncated mutant receptors were coexpressed. Scavenger receptor inhibition was specific because the activity of the LDL receptor was not altered. Transient transfection of the mouse macrophage cell line P388D1 with truncated scavenger receptor resulted in a 65% decrease in the uptake and degradation of Ac LDL but did not decrease the degradation of beta-migrating very low density lipoprotein, which is LDL receptor-mediated. These results demonstrate that expression of truncated bovine scavenger receptor inactivates both the native bovine and murine scavenger receptors, producing a dominant negative phenotype in vitro.

Further characterization of the acetyl LDL (scavenger) receptor expressed by rabbit smooth muscle cells and fibroblasts.

We have previously demonstrated that the acetyl low density lipoprotein (LDL), or scavenger, receptor is expressed by rabbit smooth muscle cells (SMCs) and fibroblasts. Moreover, receptor activity in rabbit fibroblasts was regulated over a wide range by preincubating the cells with secretion products from human platelets or with phorbol esters. The current studies were undertaken to examine the regulation of receptor expression in rabbit SMCs, to characterize further the receptor expressed by rabbit SMCs and fibroblasts, and to determine whether incubating these cells with chemically modified lipoproteins would lead to foam cell formation. Receptor activity was increased fivefold in rabbit SMCs by preincubation with platelet secretion products and nine- to 20-fold by preincubation with phorbol esters or mezerein, a non-phorbol activator of protein kinase C. The phorbol ester-induced increase in receptor activity was due to an increased mass of scavenger receptor as determined by both ligand blots and immunoblots of membrane proteins from these cells. The immunoblotting studies suggest that the SMC scavenger receptor activity is due to type I or type II scavenger receptors. The scavenger receptor expressed by phorbol ester-treated rabbit SMCs and fibroblasts bound chemically modified LDL with an order-of-magnitude higher affinity (Kd 5.1 x 10(-10) M) than did the scavenger receptor expressed by mouse peritoneal macrophages (Kd 4.1 x 10(-9) M), whereas rabbit macrophages bound chemically modified LDL with intermediate affinity (Kd 1.0 x 10(-9) M). Both the phorbol ester-stimulated rabbit fibroblasts and SMCs expressed 10-30% as many receptors per cell as did mouse peritoneal macrophages. Consistent with the difference in the number of receptors, uptake of the chemically modified LDL at 37 degrees C by SMCs led to approximately 25% as much lysosomal degradation and stimulation of cholesterol esterification as in mouse peritoneal macrophages. Incubation of phorbol ester-treated rabbit fibroblasts or SMCs with chemically modified LDL (50 micrograms/ml) for 24 or 48 hours, respectively, resulted in a threefold increase in total cholesterol and a 15-fold increase in cholesteryl ester within the cells. These studies suggest that the scavenger receptor-mediated uptake of modified lipoproteins may contribute to the formation of SMC foam cells in atherosclerotic lesions.

Expression of Tn, sialosyl Tn, and T antigens in human pancreas.

Carbohydrate antigens representing some of the initial steps in mucin O-linked glycosylation were examined in specimens of normal pancreas, chronic pancreatitis, and pancreatic adenocarcinoma. Tn antigen, recognized by Vicia villosa lectin, was expressed by all specimens of normal pancreas (acinar cells) and pancreatic cancers and all but one case of chronic pancreatitis. Sialosyl Tn antigen, recognized by monoclonal antibody TKH2, was expressed in a cancer-associated fashion, being completely absent in normal pancreas but expressed by 56% of chronic pancreatitis and 97% of pancreatic cancers. T antigen, recognized by monoclonal antibody AH9-16, was expressed in 68% of normal pancreas (acinar cells), 67% of chronic pancreatitis, and 48% of pancreatic cancer tissues. These results indicate that normal acinar cells of the pancreas are capable of expressing selected carbohydrate structures associated with the initial steps of mucin glycosylation. The marked expression of sialosyl Tn compared with T antigen in pancreatic cancers suggests that with malignant transformation there is selective usage of glycosyltransferase enzymes involved in mucin oligosaccharide synthesis.

Cell lineage markers in premalignant and malignant colonic mucosa.

Normal colonic epithelial cells consist of several cell types or lineages that are thought to arise from a common stem cell precursor. Neoplastic transformation may occur at different stages in the differentiation of a colonic stem cell to produce tumors that may retain characteristic cell lineage phenotypes. In this study, immunohistochemical techniques were used to identify cell lineage-related markers in fetal, normal, hyperplastic, adenomatous, and cancerous colonic tissue. These markers consisted of secretory component (columnar cells), a purified mucin antigen (mucous or goblet cells), chromogranin A (enteroendocrine cells), lysozyme (Paneth cells), and carcinoembryonic antigen (panepithelial cell marker). Colonic neoplasms, like normal mucosa, predominantly expressed the markers of columnar and goblet cell lineages. Chromogranin A was expressed in a small population of cells in most normal and fetal colonic crypts. Chromogranin A reactive cells were found in 55% of hyperplastic polyps, 31% of adenomatous polyps, and 33% of carcinomas. Lysozyme reactivity was rare in fetal, normal, and hyperplastic specimens, but was present in 86% of adenomas and 40% of carcinomas. Of 42 primary carcinomas, 9% were "pluripotent" and expressed markers of all four cell lineages. In addition to columnar and goblet cell markers, 7% expressed both enteroendocrine and Paneth cell markers, 17% expressed enteroendocrine cell markers, and 24% expressed Paneth cell markers. Two cases (5%) lacked expression of any of the cell lineage markers. The remainder expressed only columnar and goblet cell markers. The markers used in this study appear to identify the major cell lineages of fetal and normal colonic epithelium and can be used to delineate the altered cell lineage phenotypes in premalignant and malignant colonic mucosa.