Peter A Lopez

Pro-tumorigenic effects of miR-31 loss in mesothelioma
Ivanov, Sergey V; Goparaju, Chandra M V; Lopez, Peter; Zavadil, Jiri; Toren-Haritan, Ginat; Rosenwald, Shai; Hoshen, Moshe; Chajut, Ayelet; Cohen, Dalia; Pass, Harvey I
2010 Jul 23;285(30):22809-22817, Journal of biological chemistry
The human genome encodes several hundred microRNA (miRNA) genes that produce small (21-23n) single strand regulatory RNA molecules. Although abnormal expression of miRNAs has been linked to cancer progression, the mechanisms of this dysregulation are poorly understood. Malignant mesothelioma (MM) of pleura is an aggressive and highly lethal cancer resistant to conventional therapies. We and others previously linked loss of the 9p21.3 chromosome in MM with short time to tumor recurrence. In this study, we report that MM cell lines derived from patients with more aggressive disease fail to express miR-31, a microRNA recently linked with suppression of breast cancer metastases. We further demonstrate that this loss is due to homozygous deletion of the miR-31-encoding gene that resides in 9p21.3. Functional assessment of miR-31 activity revealed its ability to inhibit proliferation, migration, invasion, and clonogenicity of MM cells. Re-introduction of miR-31 suppressed the cell cycle and inhibited expression of multiple factors involved in cooperative maintenance of DNA replication and cell cycle progression, including pro-survival phosphatase PPP6C, which was previously associated with chemotherapy and radiation therapy resistance, and maintenance of chromosomal stability. PPP6C, whose mRNA is distinguished with three miR-31-binding sites in its 3'-untranslated region, was consistently down-regulated by miR-31 introduction and up-regulated in clinical MM specimens as compared with matched normal tissues. Taken together, our data suggest that tumor-suppressive propensity of miR-31 can be used for development of new therapies against mesothelioma and other cancers that show loss of the 9p21.3 chromosome
— id: 138201, year: 2010, vol: 285, page: 22809, stat: Journal Article,

Mechanisms of gastrointestinal CD4+ T-cell depletion during acute and early human immunodeficiency virus type 1 infection
Mehandru, Saurabh; Poles, Michael A; Tenner-Racz, Klara; Manuelli, Victoria; Jean-Pierre, Patrick; Lopez, Peter; Shet, Anita; Low, Andrea; Mohri, Hiroshi; Boden, Daniel; Racz, Paul; Markowitz, Martin
2007 Jan;81(2):599-612, Journal of virology
During acute and early human immunodeficiency virus type 1 (HIV-1) infection (AEI) more than 50% of CD4+ T cells are preferentially depleted from the gastrointestinal (GI) lamina propria. To better understand the underlying mechanisms, we studied virological and immunological events within the peripheral blood (PB) and GI tract during AEI. A total of 32 AEI subjects and 18 uninfected controls underwent colonic biopsy. HIV-1 viral DNA and RNA levels were quantified in CD4+ T cells derived from the GI tract and PB by using real-time PCR. The phenotype of infected cells was characterized by using combinations of immunohistochemistry and in situ hybridization. Markers of immunological memory, activation, and proliferation were examined by flow cytometry and immunohistochemistry, and the host-derived cytotoxic cellular response was examined by using immunohistochemistry. GI CD4+ T cells harbored, on average, 13-fold higher HIV-1 viral DNA levels and 10-fold higher HIV-1 RNA levels than PB CD4+ T cells during AEI. HIV-1 RNA was detected in both 'activated' and 'nonactivated' mucosal CD4+ T cells. A significantly higher number of activated and proliferating T cells were detected in the GI tract compared to the PB, and a robust cytotoxic response (HIV-1 specificity not determined) was detected in the GI tract as early as 18 days postinfection. Mucosal CD4+ T-cell depletion is multifactorial. Direct viral infection likely accounts for the earliest loss of CD4+ T cells. Subsequently, ongoing infection of susceptible CD4+ T cells, along with activation-induced cellular death and host cytotoxic cellular response, are responsible for the persistence of the lesion
— id: 73131, year: 2007, vol: 81, page: 599, stat: Journal Article,

In vitro modeling of the HIV-macrophage reservoir
Brown, Amanda; Zhang, Hao; Lopez, Peter; Pardo, Carlos A; Gartner, Suzanne
2006 Nov;80(5):1127-1135, Journal of leukocyte biology
Macrophages are recognized as a putative reservoir for HIV-1, but whether HIV can establish latent infection in this cell type is not known. An in vitro model using long-term cultured primary human monocyte-derived macrophages (MDM) infected with an M-tropic, enhanced green fluorescent protein (EGFP) tagged reporter virus was developed to test the hypothesis that HIV can establish a latent infection of this cell type. The EGFP-IRES-Nef cassette allowed detection of early gene transcription. The expression of GFP+ MDM was followed with time and the GFP- population was purified and analyzed for evidence of latent infection. Interestingly, in MDM cultures propagated for over two months, distinct subpopulations of infected GFP+ cells were observed and quantitated. In particular, infected MDM that displayed a high level of transcription, characterized as the GFP hi group, yet produced low levels of the late viral gene product, p24, increased with time and represented 10% of the GFP+ population in long-term cultures. The high level production of early genes such as Nef, a protein that can facilitate viral immune escape, but low level of structural proteins such as p24 in the GFP hi population suggests that a subset of infected MDM can exhibit an alternative mode of replication. The GFP- MDM population obtained by a two-step purification protocol using flow cytometry and laser ablation contained integrated provirus as assessed by Alu-LTR real-time PCR analyses. A subset of these, were replication competent as shown by their ability to express GFP and/or p24 antigen after reactivation with IL-4
— id: 73130, year: 2006, vol: 80, page: 1127, stat: Journal Article,

Recombinant extracellular domains of tetraspanin proteins are potent inhibitors of the infection of macrophages by human immunodeficiency virus type 1
Ho, Siu-Hong; Martin, Francine; Higginbottom, Adrian; Partridge, Lynda J; Parthasarathy, Varadarajan; Moseley, Gregory W; Lopez, Peter; Cheng-Mayer, Cecilia; Monk, Peter N
2006 Jul;80(13):6487-6496, Journal of virology
Human immunodeficiency virus type 1 (HIV-1) infection of human macrophages can be inhibited by antibodies which bind to the tetraspanin protein CD63, but not by antibodies that bind to other members of the tetraspanin family. This inhibitory response was limited to CCR5 (R5)-tropic virus and was only observed using macrophages, but not T cells. Here, we show that recombinant soluble forms of the large extracellular domain (EC2) of human tetraspanins CD9, CD63, CD81, and CD151 produced as fusion proteins with glutathione S-transferase (GST) can all potently and completely inhibit R5 HIV-1 infection of macrophages with 50% inhibitory concentration values of 0.11 to 1.2 nM. Infection of peripheral blood mononuclear cells could also be partly inhibited, although higher concentrations of EC2 proteins were required. Inhibition was largely coreceptor independent, as macrophage infections by virions pseudotyped with CXCR4 (X4)-tropic HIV-1 or vesicular stomatitis virus (VSV)-G glycoproteins were also inhibited, but was time dependent, since addition prior to or during, but not after, virus inoculation resulted in potent inhibition. Incubation with tetraspanins did not decrease CD4 or HIV-1 coreceptor expression but did block virion uptake. Colocalization of fluorescently labeled tetraspanin EC2 proteins and HIV-1 virions within, and with CD4 and CXCR4 at the cell surfaces of, macrophages could be detected, and internalized tetraspanin EC2 proteins were directed to vesicular compartments that contained internalized dextran and transferrin. Collectively, the data suggest that the mechanism of inhibition of HIV-1 infection by tetraspanins is at the step of virus entry, perhaps via interference with binding and/or the formation of CD4-coreceptor complexes within microdomains that are required for membrane fusion events
— id: 73129, year: 2006, vol: 80, page: 6487, stat: Journal Article,

Lack of Mucosal Immune Reconstitution during Prolonged Treatment of Acute and Early HIV-1 Infection
Mehandru, Saurabh; Poles, Michael A; Tenner-Racz, Klara; Jean-Pierre, Patrick; Manuelli, Victoria; Lopez, Peter; Shet, Anita; Low, Andrea; Mohri, Hiroshi; Boden, Daniel; Racz, Paul; Markowitz, Martin
2006 Dec 5;3(12):e484-e484, PLoS medicine
BACKGROUND: During acute and early HIV-1 infection (AEI), up to 60% of CD4(+) T cells in the lamina propria of the lower gastrointestinal (GI) tract are lost as early as 2-4 wk after infection. Reconstitution in the peripheral blood during therapy with highly active antiretroviral therapy (HAART) is well established. However, the extent of immune reconstitution in the GI tract is unknown. METHODS AND FINDINGS: Fifty-four AEI patients and 18 uninfected control participants underwent colonic biopsy. Forty of the 54 AEI patients were followed after initiation of antiretroviral therapy (18 were studied longitudinally with sequential biopsies over a 3-y period after beginning HAART, and 22 were studied cross sectionally after 1-7 y of uninterrupted therapy). Lymphocyte subsets, markers of immune activation and memory in the peripheral blood and GI tract were determined by flow cytometry and immunohistochemistry. In situ hybridization was performed in order to identify persistent HIV-1 RNA expression. Of the patients studied, 70% maintained, on average, a 50%-60% depletion of lamina propria lymphocytes despite 1-7 y of HAART. Lymphocytes expressing CCR5 and both CCR5 and CXCR4 were persistently and preferentially depleted. Levels of immune activation in the memory cell population, CD45RO(+) HLA-DR(+), returned to levels seen in the uninfected control participants in the peripheral blood, but were elevated in the GI tract of patients with persistent CD4(+) T cell depletion despite therapy. Rare HIV-1 RNA-expressing cells were detected by in situ hybridization. CONCLUSIONS: Apparently suppressive treatment with HAART during acute and early infection does not lead to complete immune reconstitution in the GI mucosa in the majority of patients studied, despite immune reconstitution in the peripheral blood. Though the mechanism remains obscure, the data suggest that there is either viral or immune-mediated accelerated T cell destruction or, possibly, alterations in T cell homing to the GI tract. Although clinically silent over the short term, the long-term consequences of the persistence of this lesion may emerge as the HIV-1-infected population survives longer owing to the benefits of HAART
— id: 73132, year: 2006, vol: 3, page: e484, stat: Journal Article,

The intracellular localization of amyloid beta protein precursor (AbetaPP) intracellular domain associated protein-1 (AIDA-1) is regulated by AbetaPP and alternative splicing
Ghersi, Enrico; Vito, Pasquale; Lopez, Peter; Abdallah, Mona; D'Adamio, Luciano
2004 Feb;6(1):67-78, Journal of Alzheimer's Disease
The Amyloid-beta Protein Precursor (AbetaPP) is a widely expressed transmembrane protein that is extensively processed in intracellular vesicular compartments and on the cell membrane. As a result of two sequential proteolytic cleavages, AbetaPP releases the Amyloid-beta (Abeta) peptide, which accumulates in insoluble plaques in the brain of patients affected by Alzheimer's Disease (AD). Another peptide, a C-terminal fragment named AbetaPP Intracellular Domain (AID), is generated by AbetaPP processing and is released intracellularly. Several functions for AID have been proposed: pro-apoptotic peptide, regulator of calcium homeostasis, molecule involved in transcriptional regulation. Many intracellular proteins, such as Fe65, Jip-1, Shc, Numb and X11alpha, interact with AID and modulate its function by different mechanisms. Here we report the cloning and initial characterization of two isoforms of a novel protein that we named AID Associated protein-1a (AIDA-1a), AIDA-1b and AIDA-1bDeltaAnk. We show that AbetaPP and the AIDA-1 proteins interact in vitro, in living cells and, endogenously, in leukemia cell lines. Transfected AIDA-1a, AIDA-1b and AIDA-1bDeltaAnk localize in different compartments and the intracellular distribution of AIDA-1a can be modified by over-expression of AbetaPP. AIDA-1 proteins are expressed at high levels in the brain; thus, studying their involvement in AbetaPP processing and AID function might give new insights regarding a possible role for these molecules in normal brain development and in the pathogenesis of AD
— id: 73126, year: 2004, vol: 6, page: 67, stat: Journal Article,

alpha-defensins released into stimulated CD8+ T-cell supernatants are likely derived from residual granulocytes within the irradiated allogeneic peripheral blood mononuclear cells used as feeders
Zaharatos, Gerasimos J; He, Tian; Lopez, Peter; Yu, Wenjie; Yu, Jian; Zhang, Linqi
2004 Aug 15;36(5):993-1005, Journal of acquired immune deficiency syndromes. JAIDS
We recently demonstrated the ability of human beta-defensins to inhibit HIV-1 replication in vitro and demonstrated that alpha-defensins account for the great majority of beta-chemokine independent antiretroviral activity in stimulated CD8+ T-cell culture supernatants. In a follow-up study aimed at defining specific subpopulations of CD8+ T-cells that produce alpha-defensins, we have found that in the absence of irradiated allogeneic peripheral blood mononuclear cells (PBMC), stimulated CD8+ T-cell supernatants do not contain alpha-defensins. In our present work, we define residual granulocytes within PBMC fractions as the likely source. In addition, we describe in vitro conditions that promote the internalization of alpha-defensins by cells not natively producing these proteins, thus confounding our ability to define true alpha-defensin producer cells. In light of these findings, alpha-defensins released into stimulated CD8+ T-cell supernatants are unlikely to be derived from the CD8+ T-cells themselves. Moreover, our data imply that under some experimental conditions, a soluble noncytolytic anti-HIV-1 factor other than beta-chemokines is either not produced by CD8+ T-cells or is present in too small quantity to be effective
— id: 73127, year: 2004, vol: 36, page: 993, stat: Journal Article,

Retraction of an interpretation
Zhang, Linqi; Lopez, Peter; He, Tian; Yu, Wenjie; Ho, David D
2004 Jan 23;303(5657):467-467, Science
— id: 107962, year: 2004, vol: 303, page: 467, stat: Journal Article,

Autosomal recessive hypercholesterolemia protein interacts with and regulates the cell surface level of Alzheimer's amyloid beta precursor protein
Noviello, Cristiana; Vito, Pasquale; Lopez, Peter; Abdallah, Mona; D'Adamio, Luciano
2003 Aug 22;278(34):31843-31847, Journal of biological chemistry
The familial Alzheimer's disease gene product amyloid beta protein precursor (A beta PP) is sequentially processed by beta- and gamma-secretases to generate the A beta peptide. Although much is known about the biochemical pathway leading to A beta formation, because extracellular aggregates of A beta peptides are considered the cause of Alzheimer's disease, the biological role of A beta PP processing is only recently being investigated. Cleavage of A beta PP by gamma-secretase releases, together with A beta, a COOH-terminal A beta PP intracellular domain, termed AID. Hoping to gain clues about proteins that regulates A beta PP processing and function, we used the yeast two-hybrid system to identify proteins that interact with the AID region of A beta PP. One of the interactors isolated is the autosomal recessive hypercholesterolemia (ARH) adapter protein. This molecular interaction is confirmed in vitro and in vivo by fluorescence resonance energy transfer and in cell lysates. Moreover, we show that reduction of ARH expression by RNA interference results in increased levels of cell membrane A beta PP. These data assert a physiological role for ARH in A beta PP internalization, transport, and/or processing
— id: 73124, year: 2003, vol: 278, page: 31843, stat: Journal Article,

Relating personal PM and PM-associated elemental carbon exposures to cardiovascular and pulmonary symptoms in a high-risk subpopulation
Kendall, M; Hsu, SI; Lopez, P; Wallace, L; Lippman, M
2002 JUL ;13(4):023-400, Epidemiology
— id: 98250, year: 2002, vol: 13, page: 023, stat: Journal Article,

The gamma-secretase-generated intracellular domain of beta-amyloid precursor protein binds Numb and inhibits Notch signaling
Roncarati, Roberta; Sestan, Nenad; Scheinfeld, Meir H; Berechid, Bridget E; Lopez, Peter A; Meucci, Olimpia; McGlade, Jane C; Rakic, Pasko; D'Adamio, Luciano
2002 May 14;99(10):7102-7107, Proceedings of the National Academy of Sciences of the United States of America
The beta-amyloid precursor protein (APP) and the Notch receptor undergo intramembranous proteolysis by the Presenilin-dependent gamma-secretase. The cleavage of APP by gamma-secretase releases amyloid-beta peptides, which have been implicated in the pathogenesis of Alzheimer's disease, and the APP intracellular domain (AID), for which the function is not yet well understood. A similar gamma-secretase-mediated cleavage of the Notch receptor liberates the Notch intracellular domain (NICD). NICD translocates to the nucleus and activates the transcription of genes that regulate the generation, differentiation, and survival of neuronal cells. Hence, some of the effects of APP signaling and Alzheimer's disease pathology may be mediated by the interaction of APP and Notch. Here, we show that membrane-tethered APP binds to the cytosolic Notch inhibitors Numb and Numb-like in mouse brain lysates. AID also binds Numb and Numb-like, and represses Notch activity when released by APP. Thus, gamma-secretase may have opposing effects on Notch signaling; positive by cleaving Notch and generating NICD, and negative by processing APP and generating AID, which inhibits the function of NICD
— id: 73123, year: 2002, vol: 99, page: 7102, stat: Journal Article,

Jun NH2-terminal kinase (JNK) interacting protein 1 (JIP1) binds the cytoplasmic domain of the Alzheimer's beta-amyloid precursor protein (APP)
Scheinfeld, Meir H; Roncarati, Roberta; Vito, Pasquale; Lopez, Peter A; Abdallah, Mona; D'Adamio, Luciano
2002 Feb 1;277(5):3767-3775, Journal of biological chemistry
The familial Alzheimer's disease gene product amyloid beta precursor protein (APP) is sequentially processed by beta- and gamma-secretases to generate the Abeta peptide. The biochemical pathway leading to Abeta formation has been extensively studied since extracellular aggregates of Abeta peptides are considered the culprit of Alzheimer's disease. Aside from its pathological relevance, the biological role of APP processing is unknown. Cleavage of APP by gamma-secretase releases, together with Abeta, a COOH-terminal APP intracellular domain, termed AID. This peptide has recently been identified in brain tissue of normal control and patients with sporadic Alzheimer's disease. We have previously shown that AID acts as a positive regulator of apoptosis. Nevertheless, the molecular mechanism by which AID regulates this process remains unknown. Hoping to gain clues about the function of APP, we used the yeast two-hybrid system to identify interaction between the AID region of APP and JNK-interacting protein-1 (JIP1). This molecular interaction is confirmed in vitro, in vivo by fluorescence resonance energy transfer (FRET), and in mouse brain lysates. These data provide a link between APP and its processing by gamma-secretase, and stress kinase signaling pathways. These pathways are known regulators of apoptosis and may be involved in the pathogenesis of Alzheimer's disease
— id: 73122, year: 2002, vol: 277, page: 3767, stat: Journal Article,

Contribution of human alpha-defensin 1, 2, and 3 to the anti-HIV-1 activity of CD8 antiviral factor
Zhang, Linqi; Yu, Wenjie; He, Tian; Yu, Jian; Caffrey, Rebecca E; Dalmasso, Enrique A; Fu, Siyu; Pham, Thang; Mei, Jianfeng; Ho, Jaclyn J; Zhang, Wenyong; Lopez, Peter; Ho, David D
2002 Nov 1;298(5595):995-1000, Science
It has been known since 1986 that CD8 T lymphocytes from certain HIV-1-infected individuals who are immunologically stable secrete a soluble factor, termed CAF, that suppresses HIV-1 replication. However, the identity of CAF remained elusive despite an extensive search. By means of a protein-chip technology, we identified a cluster of proteins that were secreted when CD8 T cells from long-term nonprogressors with HIV-1 infection were stimulated. These proteins were identified as alpha-defensin 1, 2, and 3 on the basis of specific antibody recognition and amino acid sequencing. CAF activity was eliminated or neutralized by an antibody specific for human alpha-defensins. Synthetic and purified preparations of alpha-defensins also inhibited the replication of HIV-1 isolates in vitro. Taken together, our results indicate that alpha-defensin 1, 2, and 3 collectively account for much of the anti-HIV-1 activity of CAF that is not attributable to beta-chemokines
— id: 67202, year: 2002, vol: 298, page: 995, stat: Journal Article,

Commercial high speed machines open new opportunities in high throughput flow cytometry (HTFC)
Ashcroft, R G; Lopez, P A
2000 Sep 21;243(1-2):13-24, Journal of immunological methods
Two recent events have opened a new domain of flow cytometry applications which we term high throughput flow cytometry (HTFC). The release of a commercial high speed sorter in 1994 placed HTFC within the reach of anyone who could buy one of the new machines and not just the handful of advanced laboratories worldwide that had custom built their own high speed sorters. The advent in 1999 of HTFC analysis capabilities of 100000 cells/s marks the second stage in this enabling of HTFC. We describe the technical basis of HTFC. The commercial high speed sorters measure cells in dead-times three to six times shorter than conventional machines. They can sort with high yield and high purity at rates from 25000 to 60000 cells/s, depending on their settings, mainly by virtue of their use of high drop creation rates 100000 drops/s or more. Finally, one series can analyse the measured cells at rates exceeding these sort-rates and at least six times faster than conventional sorters could. The performance of the systems made by the three manufacturers can be readily assessed for single laser systems. Comparison becomes difficult for multiple beam machines, due to requirements for multi-beam sampling for each cell and due to the demands of fluorescence compensation between signals from one laser and between signals from two or three lasers. Applications are described in the field of rare cell analysis and isolation as well as from sorting of abundant cell populations
— id: 73121, year: 2000, vol: 243, page: 13, stat: Journal Article,

High-speed sorting using the Cytomation MoFlo
Lopez PA
In living color : protocols in flow cytometry and cell sorting Berlin : Springer, 2000,
— id: 4465, year: 2000, vol: , page: 577, stat: Chapter,

Enkephalin receptors and receptor-mediated signal transduction in cultured human lymphocytes
Heagy, W; Teng, E; Lopez, P; Finberg, R W
1999 Jan 10;191(1):34-48, Cellular immunology
Enkephalins are opioid peptides that bridge the neuroendocrine and immune systems. Using flow cytometry and a fluorescein conjugate of the endogenous pentapeptide methionine-enkephalin (ME), we have identified enkephalin receptors on cultured human lymphocytes. Cell surface recognition sites that bound ME with high affinity and specificity were detected for NALM 6 (pre-B acute lymphoblastic leukemia) and Jurkat (T lymphoma) cells. Brain-like enkephalin receptors were measured for these lymphocytes using conventional radioligand-receptor assays and the highly delta opioid receptor-selective enkephalin analog [3H]DPDPE. Upon activation, the lymphocyte enkephalin receptors transmitted signals that enhanced the accumulation of intracellular cAMP. These studies provide evidence that cultured human lymphocytes of the B (NALM 6 cells) and T (Jurkat cells) lineages express functional enkephalin receptors and suggest that such receptors may be instrumental in the lymphocyte response to opioid peptides and alkaloids
— id: 73146, year: 1999, vol: 191, page: 34, stat: Journal Article,

Fluorescence-activated cell sorting of transfected cells
Adams, P D; Lopez, P; Sellers, W R; Kaelin, W G Jr
1997 ;283:59-72, Methods in enzymology
— id: 73145, year: 1997, vol: 283, page: 59, stat: Journal Article,

Biosafety guidelines for sorting of unfixed cells
Schmid, I; Nicholson, J K; Giorgi, J V; Janossy, G; Kunkl, A; Lopez, P A; Perfetto, S; Seamer, L C; Dean, P N
1997 Jun 1;28(2):99-117, Cytometry
The International Society of Analytical Cytology (ISAC) Biohazard Working Group presents guidelines for sorting of unfixed cells, including known biohazardous samples, using jet-in-air, deflected-droplet cell sorters. There is a risk that personnel operating these instruments could become exposed to droplets and aerosols containing biological agents present in the samples. The following guidelines can aid in the prevention of exposures of laboratory personnel to pathogens contained in the sort samples. The document provides biosafety recommendations for sample handling, operator training and protection, laboratory facility design, and instrument setup and maintenance. In addition, it describes in detail methods for assessment of instrument aerosol containment. Recommendations provided here may also help laboratories to obtain institutional and/or regulatory agency approval for sorting of unfixed and known biohazardous samples
— id: 73144, year: 1997, vol: 28, page: 99, stat: Journal Article,

Fas modulation of apoptosis during negative selection of thymocytes
Castro, J E; Listman, J A; Jacobson, B A; Wang, Y; Lopez, P A; Ju, S; Finn, P W; Perkins, D L
1996 Dec;5(6):617-627, Immunity
A major mechanism maintaining immune tolerance is the deletion of potentially autoreactive thymocytes by apoptosis during development in the thymus. Previous reports suggest that apoptosis is induced by high avidity signals transduced via the T cell receptor; however, the role of signals transduced by other cell surface receptors during thymic selection remains poorly understood. Fas, a member of the TNF receptor family, has been shown to induce apoptosis in mature peripheral T cells; however, the effects of Fas on negative selection of thymocytes have not been previously detected. Using a sensitive terminal deoxynucleotidyl transferase method to detect apoptotic cells, we found that mutant Fas molecules in lpr mice decrease the sensitivity of thymocytes to T cell receptor-mediated apoptosis and that blockade of Fas-Fas ligand interactions in vivo can inhibit antigen-induced apoptosis of thymocytes in non-lpr mice. Thus, we have shown that Fas, in conjunction with antigen-specific signals, can modulate apoptosis during negative selection of thymocytes
— id: 73143, year: 1996, vol: 5, page: 617, stat: Journal Article,

In situ fluorescence labeling of sheep lung microvascular endothelium
Abdi, K; Rogers, R A; Li, X; Lopez, P; Rawn, J; Mentzer, S J
1995 Apr;31(4):310-315, In vitro cellular & developmental biology. Animal
Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro
— id: 73142, year: 1995, vol: 31, page: 310, stat: Journal Article,

Functional isolation and characterization of human hematopoietic stem cells
Berardi, A C; Wang, A; Levine, J D; Lopez, P; Scadden, D T
1995 Jan 6;267(5194):104-108, Science
Hematopoietic cells differentiate in steps marked by the acquisition or loss of specific phenotypic characteristics. Human bone marrow cells that were responsive to the early-acting cytokines Kit ligand and interleukin-3 were forced to a metabolic death. The subfraction remaining represented 1 in 10(5) bone marrow mononuclear cells, were determined to be quiescent by cell cycle analysis, and had a stem cell immunophenotype. The cells were highly enriched for long-term culture-initiating cells, were capable of secondary colony formation, and produced both myeloid and lymphoid progeny. Thus, this technically simple strategy led to the efficient purification of cells with characteristics of hematopoietic stem cells
— id: 73141, year: 1995, vol: 267, page: 104, stat: Journal Article,

A simple fluorescence method for surface antigen phenotyping of lymphocytes undergoing DNA fragmentation
Hardin, J A; Sherr, D H; DeMaria, M; Lopez, P A
1992 Sep 18;154(1):99-107, Journal of immunological methods
Apoptosis, a metabolically active process of programmed cell death characterized by DNA fragmentation, is believed to play an important role in development of lymphocyte repertoires and in embryogenesis. Studies of this phenomenon would be greatly facilitated by the development of a simple assay capable of identifying and isolating intact apoptotic cells. A rapid fluorescence assay which identifies relatively small, intact cells containing fragmented DNA is described in this report. Thymocytes in which DNA fragmentation is induced by culture with or without dexamethasone are readily identified by their bright blue fluorescence after a 15 min treatment with Hoechst 33342, a DNA binding fluorochrome which diffuses through cell membranes. Since Hoechst 33342 staining does not require destruction of the cell membrane, it is possible to directly phenotype cell surface antigen expression on Hoechst 33342bright lymphocytes by conventional immunofluorescence techniques and to evaluate membrane integrity of Hoechst 33342bright cells by dye exclusion criteria. The advantages of this system are that it: (1) is rapid and simple, (2) quantitates the percentage of cells fragmenting their DNA and presumably undergoing apoptosis, (3) permits standard immunofluorescence staining of cell surface markers to identify even minor cell subsets of presumably apoptotic cells within heterogeneous populations, (4) provides the tools (fluorescence activated cell sorting) for purifying intact cells containing fragmented DNA for further biochemical studies, and (5) provides a means for identifying cells which exclude vital dyes and in which DNA fragmentation will eventually result in cell death
— id: 73125, year: 1992, vol: 154, page: 99, stat: Journal Article,

A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells
Rodewald, H R; Moingeon, P; Lucich, J L; Dosiou, C; Lopez, P; Reinherz, E L
1992 Apr 3;69(1):139-150, Cell
We have identified a dominant fetal thymocyte population at day 14.5 of gestation in the mouse that lacks CD4 and CD8 but expresses Fc gamma RII/III several days prior to acquisition of the T cell receptor (TCR) in vivo. If maintained in a thymic microenvironment, this population of CD4-CD8-TCR-Fc gamma RII/III+ thymocytes differentiates first into CD4+CD8+TCRlowFc gamma RII/III- thymocytes and subsequently CD4+CD8-TCRhighFc gamma RII/III- and CD4-CD8+TCRhighFc gamma RII/III- mature Ti alpha-beta lineage T cells. However, if removed from the thymus, the CD4-CD8-TCR-Fc gamma RII/III+ thymocyte population selectively generates functional natural killer (NK) cells in vivo as well as in vitro. These findings show that a cellular pool of Fc gamma RII/III+ precursors gives rise to T and NK lineages in a microenvironment-dependent manner. Moreover, they suggest a hitherto unrecognized role for Fc receptors on primitive T cells
— id: 73128, year: 1992, vol: 69, page: 139, stat: Journal Article,

Inhibition of immune functions by antiviral drugs
Heagy, W; Crumpacker, C; Lopez, P A; Finberg, R W
1991 Jun;87(6):1916-1924, Journal of clinical investigation
Immune functions were evaluated in vitro for PBMC isolated from healthy donors and cultured with the antiviral agents, 3'-azido-3'-deoxythymidine (AZT), ribavirin, ganciclovir, 2'3'-dideoxyinosine (ddI), or acyclovir. To identify methods for assessing the effects of antiviral drugs on immune cells, the PBMC response to mitogens, Con A, or phytohemagglutinin was evaluated from measurements of [3H]thymidine and [14C]-leucine incorporation, cell growth, cellular RNA, DNA, and protein levels, and the PBMC proliferative cycle (i.e., progression from G0----G1----S----G2 + M). At clinically relevant concentrations, AZT, ribavirin, or ganciclovir diminished PBMC responsiveness to mitogen. The numbers of proliferating cells in G1, S, and G2 + M phases of the cell cycle, DNA content, and [3H]thymidine uptake were decreased in cultures treated with AZT, ribavirin, or ganciclovir. AZT or ribavirin but not ganciclovir reduced RNA and protein in the cultures and inhibited cell growth. Whereas AZT, ribavirin, or ganciclovir were antiproliferative, ddI or acyclovir had little, if any, effect on PBMC mitogenesis. The inhibitory effects of antivirals on immune cells may contribute to the immune deterioration observed in patients following prolonged use of the drugs
— id: 73133, year: 1991, vol: 87, page: 1916, stat: Journal Article,

Assembly and function of the T cell antigen receptor. Requirement of either the lysine or arginine residues in the transmembrane region of the alpha chain
Blumberg, R S; Alarcon, B; Sancho, J; McDermott, F V; Lopez, P; Breitmeyer, J; Terhorst, C
1990 Aug 15;265(23):14036-14043, Journal of biological chemistry
The T cell receptor (TCR) for antigen consists, on the majority of peripheral lymphocytes, of an immunoglobulin-like, disulfide-linked heterodimeric glycoprotein: the alpha and beta chain. These proteins are noncovalently linked to at least four nonvariant proteins which comprise the CD3 complex: CD3 gamma, delta, epsilon, and zeta. Whereas the TCR alpha and beta proteins have positively charged residues in the transmembrane region, all the CD3 proteins have similarly placed negatively charged amino acid residues. It has been suggested that these basic and acidic amino acid residues may play an important role in TCR.CD3 complex assembly and/or function. In this paper, the structural and functional role of the lysine and arginine residues of the TCR alpha chain was addressed using oligonucleotide mediated site directed mutagenesis. The Arg256 and Lys261 residues of the TCR alpha cDNA of the HPB-ALL cell line were mutated to either Gly256 and/or Ile261. The altered cDNAs were transfected into a TCR alpha negative recipient mutant cell line of REX, clone 20A. Metabolic labeling of the T cell transfectants showed that mutation of either the Arg256 or Lys261 amino acid residues had no effect on the ability of the TCR alpha chain to form either a heterodimer with the TCR beta chain or a complex with the CD3 gamma, delta, and epsilon proteins. Consequently, the Arg256 to Gly256 and Lys261 to Ile261 mutations did not prevent the formation of a mature, functional TCR.CD3 complex on the cell surface as determined by immunofluorescence, cell surface radioiodination, and the ability of the transfectants to mobilize intracellular calcium after stimulation with a mitogenic anti-CD3 epsilon monoclonal antibody. In contrast, a mutant cDNA in which both the Arg256 and Lys261 residues were mutated to Gly256 and Ile261, respectively, failed to reconstitute the cell surface expression of the TCR.CD3 complex and, consequently, the ability to respond to mitogenic stimuli. In the absence of both the Arg256 and Lys261 residues, TCR alpha beta heterodimer formation was not observed. Cotransfection studies in COS cells showed that the failure of assembly of a heterodimer was likely due to an inability of the mutated TCR alpha chain to form a subcomplex with either the CD3 gamma, delta, epsilon, or zeta proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
— id: 73135, year: 1990, vol: 265, page: 14036, stat: Journal Article,

Characterization of functional GTP binding proteins in Jurkat T cell mutants lacking either CD3-Ti or CD2 surface receptors
Moingeon, P; Jin, Y J; Stebbins, C C; Lopez, P A; Alcover, A; Reinherz, E L
1990 Jul;128(2):578-588, Cellular immunology
G proteins are membrane-bound molecules involved in coupling of surface receptors with signal transduction effector systems in multiple cell types including T lymphocytes. Given that mature T cells which lack antigen receptors (CDl-Ti) are refractory to stimulation through CD2 or other accessory molecules, T cell receptor components likely play a critical role in coupling surface receptors with signal transduction effectors. It has recently been proposed that modulation of T cell receptor components with MAbs results in a physical loss or functional inactivation of G protein(s). In view of the importance of the T cell activation process, we herein examined G proteins in untreated or antibody-modulated Jurkat T cells as well as in genetic variants lacking either CD3-Ti or CD2 surface receptors. 43- and 41-kDa G protein alpha chains are ADP ribosylated with cholera (CTX) and pertussis (PTX) toxins, respectively, in wild type and receptor minus cell populations. In the wild type Jurkat cell line as well as in CD3- and CD2- variants, AlF4- can activate the G protein(s) presumably associated with phospholipase C to generate polyphosphoinositide turnover as well as an increase in cytoplasmic free calcium ions. Furthermore, G protein(s) linked to adenylylcyclase, a pathway which inhibits T lymphocyte activation, can be directly activated with CTX in the absence of CD3-Ti or CD2 on the membrane. Importantly, AlF4- can also induce polyphosphoinositide turnover in Jurkat cells whose T cell receptor proteins have been modulated with anti-CD3 MAb. These data provide functional and biochemical evidence that at least certain G proteins are intact in the absence of surface expression of CD3-Ti or CD2 molecules and imply that CD3-Ti desensitization is not singularly due to G protein loss
— id: 73134, year: 1990, vol: 128, page: 578, stat: Journal Article,

Dissection of the human CD2 intracellular domain. Identification of a segment required for signal transduction and interleukin 2 production
Chang, H C; Moingeon, P; Lopez, P; Krasnow, H; Stebbins, C; Reinherz, E L
1989 Jun 1;169(6):2073-2083, Journal of experimental medicine
To evaluate those residues in the 117 amino acids of the CD2 cytoplasmic domain required for transduction of T lymphocyte activation signals, a full-length human CD2 cDNA and a series of deletion and substitution mutants were inserted into the ovalbumin-specific, I-Ad-restricted murine T cell hybridoma 3DO54.8 using a retroviral system. The resulting cells express surface CD2 protein and unlike the parental murine line, are reactive with murine anti-human CD2 antibodies. Anti-T11(2) plus anti-T11(3) antibody stimulation of cells expressing a full-length CD2 cDNA results in a characteristic rise in cytosolic-free calcium [( Ca2+]i), and subsequent IL-2 secretion that accompany CD2 stimulation in human T lymphocytes. Transfectants expressing CD2 delta C98 and CD2 delta C77, partially deleted CD2 molecules containing the entire extracellular and transmembrane CD2 segments but only 98 and 77 amino acids of the cytoplasmic domain, respectively, are also activated by anti-CD2 mAbs. In contrast, clones expressing more severely truncated CD2 structures, CD2 delta C43 and CD2 delta C18, are not stimulated. These data show that the cytoplasmic domain plays an essential role in transduction of activation signals via CD2, and that the segment between amino acid residues 253 and 278 is necessary for activation. This region contains two tandem repeats of the sequence PPPGHR, thought to form part of a putative cationic site. Disruption of the latter by site-directed mutagenesis does not affect IL-2 gene induction, suggesting that only one of the repeats is required for activating this function of the CD2 molecule
— id: 73137, year: 1989, vol: 169, page: 2073, stat: Journal Article,

Phosphatidylinositl-anchored antigens defined by non-lineage mAb
Selvaraj P; Low MG; Lopez P; Springer TA
Leucocyte typing IV : white cell differentation antigens Oxford : Oxford University Press, 1989,
— id: 4464, year: 1989, vol: , page: 734, stat: Chapter,

Interdependence of CD3-Ti and CD2 activation pathways in human T lymphocytes
Alcover, A; Alberini, C; Acuto, O; Clayton, L K; Transy, C; Spagnoli, G C; Moingeon, P; Lopez, P; Reinherz, E L
1988 Jul;7(7):1973-1977, EMBO journal
Human T lymphocytes can be activated through either the antigen/MHC receptor complex T3-Ti (CD3-Ti) or the T11 (CD2) molecule to proliferate via an IL-2 dependent mechanism. To investigate the relationship of these pathways to one another, we generated and characterized Jurkat mutants which selectively express either surface CD3-Ti or CD2. Here we show that CD3-Ti- mutants fail to be stimulated by either pathway to increase phosphoinositide turnover, mobilize calcium or induce the IL-2 gene. The activation capacity of these mutants via CD2 as well as CD3-Ti can be restored following reconstitution of surface CD3-Ti expression upon appropriate DNA transfer (e.g. Ti beta subunit cDNA into Ti beta- Jurkat variants). Collectively, these results demonstrate that CD3-Ti and CD2 pathways are interdependent and that phosphoinositide turnover is linked to the CD3-Ti complex
— id: 73138, year: 1988, vol: 7, page: 1973, stat: Journal Article,

A protocol for Papanicolaou staining of cytologic specimens following flow analysis
Berkan, T K; Reeder, J E; Lopez, P A Jr; Gorman, K M; Wheeless, L L Jr
1986 Jan;7(1):101-103, Cytometry
A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman 'Metricel' filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired
— id: 73136, year: 1986, vol: 7, page: 101, stat: Journal Article,

Multidimensional slit-scan prescreening system: preliminary results of a single blind clinical study
Wheeless, L L; Patten, S F; Berkan, T K; Brooks, C L; Gorman, K M; Lesh, S R; Lopez, P A; Wood, J C
1984 Jan;5(1):1-8, Cytometry
A multidimensional slit-scan flow system was developed to serve as an automated prescreening instrument for gynecological cytology. A 2-year single blind clinical study was carried out to evaluate system performance. Cellular material was collected by scraping the uterine cervix and stained in suspension with acridine orange. Seven hundred and forty specimens (701 patients) including 156 abnormal specimens representing a broad spectrum of abnormality were analyzed. Approximately 50,000 cells were analyzed for each specimen. The system false-positive rate was 17.6% while the false-negative rate was 2.8%. All misclassified abnormals were specimens with cellular changes consistent with a slight dysplasia of nonkeratinizing type. The instrument in its present configuration appeared sensitive to the entire spectrum of abnormality existing in the female genital tract and it classified as abnormal any specimen containing on the order of 0.1% (or greater) abnormal cells
— id: 73139, year: 1984, vol: 5, page: 1, stat: Journal Article,

Syringing as a method of cell dispersal. II. Effect on abnormal cells
Lopez, P A; Cambier, M A; Wheeless, L L Jr
1981 Sep;3(3):235-238, Analytical & quantitative cytology
The generation of turbulent shear force using a constant pressure syringing device has been demonstrated to be a simple, effective method for the dispersal of intermediate and superficial squamous cells. This paper reports results of an evaluation of the effects of syringing on the dispersal of abnormal cells. Gynecologic cell samples obtained from preinvasive and invasive lesions were syringed. Overall cell loss, as well as degree of dispersion, was evaluated. Doublet, triplet and larger abnormal cell groupings remained in most syringed aliquots regardless of pressure. Although some cell loss was observed in the majority of cases, the percentage of single abnormal cells was increased in 96% of the syringed aliquots
— id: 73140, year: 1981, vol: 3, page: 235, stat: Journal Article,