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Showing papers by "Paul G. Richardson published in 2000"


Journal ArticleDOI
01 Nov 2000-Blood
TL;DR: Clinical activity of Thal against MM that is refractory to conventional therapy is demonstrated and mechanisms of anti-tumor activity of thalidomide and its potent analogs (immunomodulatory drugs [IMiDs]) are delineated.

922 citations


Journal Article
TL;DR: A double-labeling protocol combining intracellular cytokeratin with epithelial cell adhesion molecule (Ep-CAM) or disialo-ganglioside (GD2) antigen (small cell lung carcinoma, neuroblastoma, melanoma antigen) was developed and examples of doubly labeled cultured cells and cancer cells from breast and small cell lung cancer patients are shown.
Abstract: An automated rare event detection system (Rare Event Imaging System) is described for the recognition of cancer cells that appear at low frequencies (1 in 1 million) in peripheral blood (PB) or bone marrow (BM). The instrumentation includes an automated fluorescence microscope (Nikon Microphot-FXA) with a cooled charge coupled device camera and a 60-MHz Pentium personal computer. Main features of the system are rapid analysis of large microscopic fields, including a total cell count, detection of fluorescently labeled cells, and a display of digitally stored images of the detected cells. Furthermore, the X,Y coordinates of each identified object are stored and can be recalled for morphological analysis of the cell using higher magnification or different fluorescent filter sets. The preparation of the blood or BM samples for automated analysis consists of lysis of the RBCs, attachment of sample cells onto adhesion slides, fixation, and fluorescent labeling with anticytokeratin antibodies. Cytokeratin-positive cells, however, were detected in 17% of the samples from healthy blood donors using this procedure (mean number, approximately 7/10(6) mononuclear cells in positive samples). To improve the specificity of the rare event detection, a double-labeling protocol combining intracellular cytokeratin with epithelial cell adhesion molecule (Ep-CAM) (breast, ovarian, colon, and lung carcinoma antigen) or disialo-ganglioside (GD2) antigen (small cell lung carcinoma, neuroblastoma, melanoma antigen) was developed. Examples of doubly labeled cultured cells and cancer cells from breast and small cell lung cancer patients are shown. Using the double-labeling protocol, no "positive" cells were seen in samples of healthy blood donors. Automated rare event detection (cytokeratin single-staining) was applied to 355 PB, BM, and stem cell (SC) samples from breast cancer patients before autologous BM transplantation. Cytokeratin-positive cells were found in 52% of BM, 35% of PB, and 27% of SC samples at frequencies of 1-1020 positive cells/10(6) mononuclear cells, thereby establishing the efficacy of the technique in the detection of rare cancer cells in hematopoietic tissue samples of cancer patients.

87 citations


Journal ArticleDOI
TL;DR: Using mouse BAC clones spanning an imprinted interval of proximal mouse chromosome 7 and the genomic sequence of the related interval of human chromosome 19q13.4, a novel mouse gene is identified, Usp29 (ubiquitin-specific processing protease 29), near two known imprinted genes, Peg3 and Zim1, which may represent another imprinted gene involved in the ubiquitination pathway.
Abstract: Although the two alleles of most mammalian autosomal genes are functionally equivalent, the maternal and paternal copies of a subset of genes are distinguished by genomic imprinting, a mechanism by which one allele is epigenetically modified and repressed, depending upon parental origin. The epigenetic modification that determines imprinting is yet to be defined, but several lines of evidence strongly suggest that differences in DNA methylation and chromatin structure may be part of the molecular mechanism (Tilghman 1999). About 30 different imprinted genes have been isolated from human and mouse DNA, and more imprinted genes are predicted to be present in mammalian genomes (Barlow 1997; Morrison and Reeve 1998). The imprinted genes that have been described to date are clustered in discrete chromosomal regions, and the clustering of those genes is conserved in mammals (Barlow 1995; Bartolomei and Tilghman 1997). These data have been interpreted to imply that genome imprinting is a long-range mechanism that controls allele-specific transcription of multiple genes within a defined chromosomal region (Nicholls 1994; Dittrich et al. 1996; Tilghman et al. 1998, 1999). Imprinted genes exhibit several unique features in addition to parent-of-origin-specific monoallelic expression and differential methylation. The known imprinted genes generally are expressed at early stages of development, and many encode proteins with functions related to embryonic growth (Tilghman 1999). Recent studies also suggest the involvement of imprinted genes in controlling the parental caring behavior of mammals (Lefebvre et al. 1998; Li et al. 1999). Several imprinted genes such as UBE3A (Rougeulle et al. 1998), IGF2 (Moore et al. 1997), ZNF127 (Jong et al. 1999), and IGF2R (Wutz et al. 1997) display bidirectional transcription, producing both sense and antisense transcripts. A number of imprinted genes including H19 and IPW are expressed without any coding capability; the final products of these genes are RNAs rather than proteins (Bartolomei and Tilghman 1997). Imprinted genes also tend to be comosed of relatively small numbers of exons and to contain smaller introns than nonimprinted genes (Hurst et al. 1996), although there are some notable exceptions (e.g., KvLQT1; Lee et al. 1997). Early mouse genetic studies with translocation mutant mice predicted the presence of nine different imprinted domains distributed onto seven chromosomes (Beechey and Cattanach 1996). The chromosomal locations of most imprinted genes, in fact, are consistent with this initial prediction (Imprinting Map; http://www.mgu.har.mrc.ac.uk/imprinting/imptables.html#impmaps). Mouse chromosome 7 (Mmu7) was predicted in these early studies to contain three different imprinted domains, located in centromeric, central, and distal regions (Searle and Beechey 1990). The central and distal regions of Mmu7 are syntenically homologous to intervals of human chromosome 15q13-11 and 11p15, respectively, that contain genes associated with two imprinting genetic disorders—Prader–Willi/Angelman (Nicholls et al. 1998) and Beckwith–Wiedemann syndromes (Reik and Maher 1997). Each of these domains contains several imprinted genes whose order, sequence, and imprinting status are highly conserved in humans and mice. Until recently, only one imprinted gene, Peg3 (paternally expressed gene 3), had been localized to the centromeric imprinted domain of Mmu7 (Kuroiwa et al. 1996) and the homologous region of human chromosome 19q13.4 (Kim et al. 1997a). We exploited the well-developed physical map surrounding human PEG3 and the known conservation of this interval in humans and mice to identify a second mouse-imprinted gene, Zim1 (imprinted zinc-finger gene 1), located downstream of Peg3 (Kim et al. 1999). More recent studies identified a related and homologously positioned human zinc-finger gene, ZIM2, and showed that the gene shares seven 5′-exons with PEG3, a feature that distinguishes this pair of human genes from their closest murine counterparts (Kim et al. 2000). The significant divergence of Zim1 and ZIM2 gene organization and amino acid sequence suggests that the region surrounding PEG3 has undergone significant changes during the course of mammalian evolution. Through analysis of clone contigs spanning homologous segments of Mmu7 and sequenced regions of human 19q13.4, we have isolated and characterized another novel mouse gene, Usp29 (ubiquitin-specific processing protease 29) and have identified its human counterpart. Our analyses show that Usp29 is also imprinted and is expressed mainly from the paternal allele during embryogenesis and in adult brain. The sequence of a full-length 7.6-kb mouse cDNA clone and human coding sequences indicate that Usp29 encodes a novel ubiquitin-specific processing protease, and together with Ube3a may represent a second imprinted gene involved in the ubiquitination pathway.

79 citations


Journal ArticleDOI
TL;DR: It is demonstrated that patients undergoing autologous transplantation for breast cancer experience a prolonged period of T cell dysfunction, in contrast to B, NK, and DC recover more rapidly, which carries significant implications for the design of post-transplant immunotherapy.
Abstract: The present study examines the nature of humoral and cellular immune reconstitution in 28 patients with advanced breast cancer following high-dose chemotherapy with stem cell rescue. Patients underwent testing of T, B, NK and dendritic cell function at serial time points until 1 year post transplant or until the time of disease progression. Abnormalities in T cell phenotype and function were observed following high-dose chemotherapy that persisted for at least 6-12 months. The vast majority of patients experienced an inversion of the CD4/CD8 ratio and demonstrated an anergic response to candida antigen. Mean T cell proliferation in response to PHA and to co-culture with allogeneic monocytes was significantly compromised. In contrast, mean IgG and IgA levels were normal 6 months post transplant and NK cell yields and function were transiently elevated following high-dose chemotherapy. Dendritic cells generated from peripheral blood progenitors displayed a characteristic phenotype and were potent inducers of allogeneic T cell proliferation in the post-transplant period. The study demonstrates that patients undergoing autologous transplantation for breast cancer experience a prolonged period of T cell dysfunction. In contrast, B, NK, and DC recover more rapidly. These findings carry significant implications for the design of post-transplant immunotherapy.

36 citations


Journal ArticleDOI
TL;DR: It is concluded that the role of HDCT for HRPBC or MBC patients has not yet been fully defined and that longer follow-up of the ongoing randomized trials is necessary to clarify this important question.

29 citations


Journal ArticleDOI
TL;DR: The sequence of alkylating agent treatment may substantially influence response and Mechanistic studies indicate significant changes in glutathione S1 transferase, a known mechanism for broadly based resistance to AAs.
Abstract: Introduction: A preliminary analysis of our double high-dose chemotherapy with stem cell rescue (HD-SCR) clinical trial for breast cancer, and preclinical cross-resistant studies, suggested that melphalan (M) adversely affected response to subsequent chemotherapy, i.e., that the sequence of alkylating agents (AAs) might affect response. We, therefore, constructed and examined preclinical models to determine whether prior exposure to M, in fact, adversely affected response to other therapy. Purpose: The purpose of the study was to determine whether the sequence of AAs, specifically the prior use of M, adversely affected response to subsequent treatment. Methods: The methods employed were the following: (1) Human tumor cell lines rendered resistant by in vitro sequential exposure to five different AAs were developed. The resistant cell lines were examined for cross-resistance to alkylating and other agents. (2) In vivo studies in the p388 mouse leukemia for resistance and cross-resistance among the AAs. (3) In vivo studies of the effect of sequence of AAs on response in mice bearing EMT6 breast cancer. (4) The double transplant model was developed in the mouse and the sequence of high-dose AAs was studied. (5) Biochemical and reverse transcriptase-polymerase chain reaction (RT-PCR) studies of the various resistant tumor cell lines. Results: (1) The in vitro human tumor cells resistant to M were cross-resistant in 57% of tests to other AAs. In contrast, resistance for other AAs crossed to other agents in only 10 to 20% of tests. (2) The in vivo studies of p388 indicated that resistance to M commonly crossed to other AAs and many non-AAs. (3) The results for the mouse breast cancer (EMT6) studies of the sequence of AAs again indicated that M employed first markedly reduced responsiveness to subsequent treatment, particularly with AAs. (4) The double transplant model: again, M first markedly reduced response to other agents. (5) The in vitro resistant human tumor cell lines, particularly the breast cancer cell line MCF7, were found to contain high concentrations of glutathione S1 transferase gamma, which is consistent with that mechanism being responsible for resistance. Conclusion: The sequence of alkylating agent treatment may substantially influence response. Melphalan, particularly, produces resistance that commonly crosses to the other AAs. Mechanistic studies indicate significant changes in glutathione S1 transferase, a known mechanism for broadly based resistance to AAs.

9 citations


01 Mar 2000
TL;DR: It is reported that “in vivo purging” with rituximab (Rituxan) during stem-cell collection is safe and does not adversely affect engraftment.
Abstract: We previously reported that “in vivo purging” with rituximab (Rituxan) during stem-cell collection is safe and does not adversely affect engraftment. We now report on our transplant experience with rituximab. From June 1998 to December

1 citations