Synthesis, estrogen receptor binding, and tissue distribution of a new iodovinylestradiol derivative (17alpha,20E)-21-[123I]Iodo-11beta-nitrato-19-norp regna-1,3,5 (10),20-tetraene-3,17-diol (E-[123I]NIVE)

We have synthesized and evaluated E-11beta-nitrato-17alpha-iodovinylestradiol (E-NIVE; E-3c) and its 123I-labelled form, as a new potential radioligand for imaging of estrogen receptor (ER)-positive human breast tumors. E-[123I]NIVE was prepared by stereospecific iododestannylation of the E-tri-n-butylstannylvinyl precursor (E-2c), obtained from reaction of 11beta-nitrato-estrone (8) with E-tributylstannylvinyllithium. In competitive binding studies, E-NIVE proved to have high binding affinity for both the rat and the human ER (Ki 280-730 pM), without significant binding to human sex hormone binding globulin. Distribution studies in normal and mammary tumor-bearing rats showed specific ER-mediated uptake of E-[123I]NIVE in the estrogen target tissues, i.e., uterus, ovaries, pituitary, and hypothalamus, but not in the mammary tumors. Selective retention in these target tissues, including tumor tissue, resulted in significant increases over time for the target tissue-to-muscle uptake ratios, but not for the target tissue-to-fat uptake ratios. The tumor-to-fat uptake ratio even appeared constantly below 1. In the primary estrogen target tissues, E-[123I]NIVE displayed high specific ER-mediated uptake and retention, which resulted in moderate target-to-nontarget tissue uptake ratios. In contrast, in tumor tissue, E-[123I]NIVE uptake appeared to be rather low and not ER-specific. As a consequence, E-[123I]NIVE appears to be a less favorable radioligand for ER imaging in breast cancer than the previously studied stereoisomers of 11beta-methoxy-17alpha-[123I]iodovinylestradiol (E- and Z-[123I]MIVE; [123I]E- and [123I]Z-3b).     

Restoration of X-ray and etoposide resistance, Ku-end binding activity and V(D) J recombination to the Chinese hamster sxi-3 mutant by a hamster Ku86 cDNA

Ku is a heterodimeric protein composed of 86 and 70 kDa subunits that binds preferentially to the double-stranded ends of DNA. Recent molecular characterization of ionizing-radiation sensitive (IRs) mutants belonging to the XRCC5 complementation group demonstrated the involvement of Ku in DNA double-strand break (DSB) repair and lymphoid V(D)J recombination. Here, we describe the isolation of a full-length hamster cDNA encoding the large subunit of the Ku heterodimer and demonstrate that the stable expression of this cDNA can functionally restore IR, Ku DNA end-binding activity and V(D)J recombination proficiency in the Chinese hamster IRs sxi-3 mutant. Moreover, we also demonstrate that sxi-3 cells are hypersensitive to etoposide, a DNA topoisomerase II inhibitor, and that resistance to this drug was restored by the Ku86 cDNA. These experiments suggest that a defect in the large subunit of the heterodimeric Ku protein is the sole factor responsible for the known defects of sxi-3 cells and our data of further support the role of Ku in DNA DSB repair and V(D)J recombination.     

The block to HIV-1 envelope glycoprotein-mediated membrane fusion in animal cells expressing human CD4 can be overcome by a human cell component(s)

Membrane fusion mediated by interaction of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein with the human CD4 molecule generally requires that the CD4 be expressed on a human cell. The failure of murine or simian cells expressing human CD4 to form syncytia upon mixing with cells expressing envelope glycoprotein could not be corrected by expression of both molecules at extremely high surface levels using vaccinia virus expression vectors. Video fluorescence microscopic analysis of fluorescent dye transfer between fusing cells indicated that the block occurred at the level of membrane fusion between individual pairs of cells. To gain insight into the basis for this fusion block, we tested the ability of fluorescent probe cells expressing envelope glycoprotein to fuse with transient animal x human hybrid giant cells expressing human CD4. The hybrid giant cells were generated either by low-pH-induced fusion of vaccinia-infected cells or by CD4/HIV-1 envelope glycoprotein-mediated cell fusion. We observed that envelope glycoprotein-expressing probe cells efficiently fused with CD4-expressing animal x human hybrid giant cells, independent of whether the CD4 was originally expressed on the animal or on the human cell. Fusion did not occur with CD4-expressing giant cells derived from animal cells alone. These results indicate that the fusion block is not due to dominant inhibitory components in the animal cell. Rather, they suggest that human cells contain an additional component(s) which, when transferred to the CD4-bearing animal cell, confers the ability to undergo membrane fusion mediated by the HIV-1 envelope glycoprotein.     

Management of lymphoproliferative disorders after cardiac transplantation

We conducted a retrospective study of 516 cardiac recipients who underwent transplantation between April 1983 and April 1992, 19 of whom had development of post-transplantation lymphoproliferative disorders (PTLDs). These 19 patients presented with involvement of lung (5), gastrointestinal tract (5), disseminated disease (6), and adenoids and lymph nodes (3). B-cell proliferations ranging from an atypical hyperplasia to malignant lymphoma developed in 18 patients, and mixed cellularity Hodgkin's disease developed in 1 patient. The 19 patients with PTLD displayed a predominance of both women and cardiomyopathy as the indication for transplantation when compared with two separate control populations. No correlation was found between demographic criteria analyzed and (1) early versus late diagnosis of PTLD after transplantation, (2) the site of PTLD involvement, or (3) the histopathologic category of the PTLD lesion. Patients with gastrointestinal tract and lung PTLD involvement enjoyed an improved survival after both transplantation and PTLD diagnosis when compared with patients with PTLD involvement of all other extranodal sites. We report a high incidence of PTLD involving the lung and gastrointestinal tract in our cohort study. These sites of involvement responded better to a reduction in immunosuppression than did the other extranodal sites of involvement.     

Thoracoscopic pleural tent

Lung volume reduction has been performed in patients with advanced emphysema to relieve dyspnea and improve exercise tolerance. Median sternotomy and video-assisted thoracoscopy have been proposed as equally adequate approaches; however, prolonged postoperative air leakage is the most prevalent complication in all series. For this reason, on the basis of the experience achieved with the median sternotomy approach, buttressing of the suture line with different materials and techniques for space reduction have been proposed. We describe a technique to create a pleural tent after thoracoscopic volume reduction. The thoracoscopic creation of a pleural tent is feasible and results in a duration of postoperative air leaks and hospital stays similar to that achieved with stapler line buttressing.     

Regulation of the proliferative potential of cord blood long-term culture-initiating cells (LTC-IC) by different stromal cell lines: implications for LTC-IC measurement

BACKGROUND AND OBJECTIVE: Long-term culture-initiating cells (LTC-IC) are the best available approximation to an in vitro assay of stem cells in humans although they still represent a heterogeneous population in terms of proliferative capacity and sensitivity to different growth factors. Human umbilical cord blood (CB) is rich in hemopoietic progenitor cells, as measured by clonogenic assays and contains stem cells capable of reconstituting the marrow after ablation in clinical transplantation. We evaluated the influence of culture conditions on the in vitro behavior of LTC-IC from CB. DESIGN AND METHODS: LTC-IC were evaluated in long-term cultures, comparing two types of murine stromal cell lines: M2-10B4 and M2-10B4 transfected with cDNAs for human G-CSF and IL-3. RESULTS: Two and five fold higher numbers of terminally differentiated cells were produced during nine weeks of culture of CB mononuclear or CD34+ cells respectively, in cultures containing a M2-10B4 IL-3 G-CSF cell line compared to cultures containing the parental cell line. Likewise, a higher number of colony-forming cells (CFC) were detected in the supernatant of cultures with the transfected cell line. In contrast, the number of CFC generated within the stromal layer, after 5 or 9 weeks of culture, was significantly higher in cultures on M2-10B4 cells than those on M2-10B4 IL-3 G-CSF. INTERPRETATION AND CONCLUSIONS: Our results show that the proliferative capacity of CB LTC-IC can be strongly influenced by culture conditions and that the frequency of LTC-IC estimated using these cell lines as stromal support is not identical.     

Two regions in the CD80 cytoplasmic tail regulate CD80 redistribution and T cell costimulation

CD28 is a major T cell costimulatory molecule, delivering signals distinct from those of the CD3/TCR complex, which regulate cytokine and cytokine receptor expression, cell proliferation, and cell viability. CD28 needs to be cross-linked to initiate signals, yet both of its ligands, CD80 and CD86, are expressed as monomers. Previously, we determined the cytoplasmic tail of CD80 is required for CD28-mediated costimulation and subcellular relocalization of CD80 in lymphocytes. In this study, we report that Reh B cell transfectants expressing CD80 with mutations in the cytoplasmic tail region either at 275-278 (RRNE-->AAAA, CD80/4A) or serine 284 (S-->A, CD80/SA) can bind ligand similar to transfectants expressing wild-type CD80, yet are unable to costimulate T cell proliferation. These mutant CD80 molecules are expressed on the surface of the Reh cells in small clusters or foci indistinguishable from those of wild-type CD80 molecules. However, mutant CD80 molecules unlike wild-type CD80 cannot be readily induced by ligand into caps. Thus, small clusters of CD80 found on APC are insufficient to initiate CD28-mediated signals, and the formation of CD80 caps appears to be a critical factor regulating the initiation of T cell costimulation. A 30-kDa phosphoprotein that associates with the cytoplasmic tail of CD80 in activated cells may play a role in CD80 redistribution and thus CD28-mediated costimulation. These results indicate two distinct regions of the CD80 cytoplasmic tail regulate its costimulatory function, and both regions are required for CD80 function.     

EMS1 gene expression in primary breast cancer: relationship to cyclin D1 and oestrogen receptor expression and patient survival

The EMS1 and CCND1 genes at chromosome 11q13 are amplified in about 15% of primary breast cancers but appear to confer different phenotypes in ER positive and ER negative tumours. Since there are no published data on EMS1 expression in large series of breast cancers we examined the relationship of EMS1 expression with EMS1 gene copy number and expression of mRNAs for cyclin D1 and ER. In a subset of 129 patients, where matched tumour RNA and DNA was available, EMS1 mRNA overexpression was associated predominantly with gene amplification (P = 0.0061), whereas cyclin D1 mRNA overexpression was not (P = 0.3142). In a more extensive series of 351 breast cancers, there was no correlation between cyclin D1 and EMS1 expression in the EMS1 and cyclin D1 overexpressors (P = 0.3503). Although an association between EMS1 mRNA expression and ER positivity was evident (P = 0.0232), when the samples were divided into quartiles of EMS1 or cyclin D1 mRNA expression, the increase in the proportion of ER positive tumours in the ascending EMS1 mRNA quartiles was not statistically significant (P = 0.0951). In marked contrast there was a significant stepwise increase in ER positivity in ascending quartiles of cyclin D1 mRNA (P = 0.030). A potential explanation for this difference was provided by the observation that in ER positive breast cancer cells oestradiol treatment resulted in increased cyclin D1 gene expression but was without effect on EMS1. The relationship between EMS1 expression and clinical outcome was examined in a subset of 234 patients with median follow-up of 74 months. High EMS1 expression was associated with age > 50 years (P = 0.0001), postmenopausal status (P = 0.0008), lymph node negativity (P = 0.019) and an apparent trend for worse prognosis in the ER negative subgroup. These data demonstrate that overexpression of EMS1 mRNA is largely due to EMS1 gene amplification, is independent of cyclin D1 and ER expression and, in contrast to cyclin D1, is not regulated by oestrogen. Independent overexpression of these genes may confer different phenotypes and disease outcomes in breast cancer as has been inferred from recent studies of EMS1 and CCND1 gene amplification.