Generation of MHC-nonrestricted and restricted oncolytic subsets from human bone marrow.

We analyzed the cytotoxicity and characterized the phenotype of oncolytic bone marrow (BM) lymphocyte subsets generated in vitro by interleukin-2 (IL-2) and stimulator cells (SC). Two irradiated B-lymphoblastoid cell lines (Daudi and EBV-transformed BSM) and fresh human acute myelogenous leukemia (AML) were used as SC. Stimulation with Daudi and IL-2 resulted in a substantial increase in cytotoxic activity (100- to 1000-fold) against a broad range of tumor targets, and total cellular expansion was higher compared to stimulation with IL-2 alone. The most prominent increase was observed in the CD16+ and CD56+/CD3- natural killer (NK) cell subset; however, a significant increase was also observed in CD56+/CD3+ T cells. Functional analysis of Daudi- and IL-2-generated subsets using fluorescence-activated cell sorting (FACS) revealed that most of the lytic activity was mediated by NK cells. Significant potentiation of oncolytic activity and cell growth was also seen in the cultures stimulated with BSM or fresh AML and IL-2. The highest oncolytic activity in the latter cultures was mediated primarily by CD8+, CD3+, and CD56- T cells, although NK cells also participated in cytotoxic activity. The T cell-mediated cytotoxicity was restricted by the major histocompatibility complex (MHC), since most cytotoxicity could be blocked by HLA I antibodies. Additionally, we observed that optimum stimulation of cytotoxicity required effector cell-stimulator cell contact. These data indicate that depending on the tumor used for stimulation, different lymphocyte subsets may be generated in IL-2 cultures. These different approaches may be useful in both specific and nonspecific immunotherapy.     

Generation of NK cell activity from human bone marrow

This study was designed to examine the effect of interleukin 2 (IL 2) on cytotoxic activity of human bone marrow cells and to characterize the IL 2-dependent killer cells and the cell population required for their induction. We have demonstrated that the most aggressive IL 2-dependent killer cells (directed against leukemic and solid cancer targets) exhibited LGL morphology and expressed NK cell-associated antigens NKH1 and CD16, but not T cell-associated antigens CD3, CD4, CD5, or CD8. Similarly, the bone marrow cell population necessary for induction of killer cells with highest cytotoxic activity displayed NK cell surface characteristics, as exemplified by CD16 and Leu-7 antigens. On the contrary, very low or no lytic activity was generated from the bone marrow cell population expressing T cell markers CD3 and CD5. These data indicate that the IL 2-dependent bone marrow-derived killer cells with antitumor activity were activated NK cells. If T cells are involved at all in IL 2-dependent bone marrow killing, their potency is inferior to that of activated NK cells. The clinical applications of these studies are discussed.     

Comparison of recombinant-interleukin-2-activated peripheral blood and tumor-infiltrating lymphocytes of patients with epithelial ovarian carcinoma: Cytotoxicity,growth kinetics and phenotype

Summary We have compared the growth and tumordirected cytotoxic efficacy of recombinant-interleukin-2-(rIL-2)-activated peripheral blood (PBL) and tumor-infiltrating lymphocytes (TIL) from patients with epithelial ovarian carcinoma. These studies demonstrated that TIL and PBL displayed similar levels of cytotoxicity and a broad range of target cell killing, as exemplified by their reactivity against autologous and allogeneic ovarian tumors as well as against tumor cell lines. No specificity of autologous tumor cell killing was manifested by TIL. Even though TIL of some patients showed higher proliferative activity (especially at the later times in rIL-2 culture) this was not a general phenomenon. In fact, in one case TIL did not proliferate at all, and in the other case the PBL proliferated more actively. While the cultures were composed primarily of CD3⁺ lymphocytes, the major cytotoxic cells displayed the CD56⁺ and CD16⁺ phenotype. Addition of OKT3 mAb to rIL-2 cultures resulted in an increased proliferative index, but showed only a minor effect on the cytotoxic potential of cultured lymphocytes. The therapeutic potential of rIL-2-activated TIL and PBL is discussed.Recipient of the Florence Maude Thomas Cancer Research Professorship     

Determination of haplotypes of DNA markers linked to the mucoviscidosis locus: detection of heterozygotes

Linked DNA probes have been used in three families presenting an affected child with cystic fibrosis. The strategy used for the determination of haplotypes associated with parental normal and mutated genes is presented as well as its application to the detection of cystic fibrosis carriers among healthy children.     

HER-2/neu peptide specificity in the recognition of HLA-A2 by natural killer cells

Although natural killer (NK) cells have been described as non-MHC-restricted, new evidence suggests that NK activity can be either up- or down-regulated after interaction with the peptide–MHC-class-I complex expressed on target cells. However, the epitope(s) recognized by NK cells have remained ill-defined. We investigated NK cell recognition of synthetic peptides representing a portion of a self-protein encoded by the HER-2/neu (HER-2) proto-oncogene and presented by HLA-A2. HER-2 nonapeptides C85, E89, and E75 were found partially to protect T2 targets from lysis by freshly isolated and interleukin-2(IL-2)-activated NK cells (either HLA-A2⁺ or A2⁻). This inhibition was not solely due to changes in the level of HLA-A2 expression or conformation of serological HLA-A2 epitopes. Using single-amino-acid variants at position 1 (P1) of two HER-2 peptides, we observed that protection of targets was dependent on the sequence and the side-chain. These results suggest similarities in the mechanism of target recognition by NK and T cells. This information may be important for understanding the mechanisms of tumor escape from immunosurveillance and could help explain the aggressiveness of HER-2-overexpressing tumor cells. Received: 16 March 1999 / Accepted: 3 June 1999     

A novel cell model to study the function of the adrenoleukodystrophy-related protein

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder due to mutations in the ABCD1 (ALD) gene. ALDRP, the closest homolog of ALDP, has been shown to have partial functional redundancy with ALDP and, when overexpressed, can compensate for the loss-of-function of ALDP. In order to characterize the function of ALDRP and to understand the phenomenon of gene redundancy, we have developed a novel system that allows the controlled expression of the ALDRP-EGFP fusion protein (normal or non-functional mutated ALDRP) using the Tet-On system in H4IIEC3 rat hepatoma cells. The generated stable cell lines express negligible levels of endogenous ALDRP and doxycycline dosage-dependent levels of normal or mutated ALDRP. Importantly, the ALDRP-EGFP protein is targeted correctly to peroxisome and is functional. The obtained cell lines will be an indispensable tool in our further studies aimed at the resolution of the function of ALDRP to characterize its potential substrates in a natural context.     

Sebaceous Lipid Profiling of Bat Integumentary Tissues: Quantitative Analysis of Free Fatty Acids,Monoacylglycerides, Squalene,and Sterols

White‐nose syndrome (WNS) is a fungal disease caused by Pseudogymnoascus destructans and is devastating North American bat populations. Sebaceous lipids secreted from host integumentary tissues are implicated in the initial attachment and recognition of host tissues by pathogenic fungi. We are interested in determining if ratios of lipid classes in sebum can be used as biomarkers to diagnose severity of fungal infection in bats. To first establish lipid compositions in bats, we isolated secreted and integral lipid fractions from the hair and wing tissues of three species: big brown bats (Eptesicus fuscus), Eastern red bats (Lasiurus borealis), and evening bats (Nycticeius humeralis). Sterols, FFAs, MAGs, and squalene were derivatized as trimethylsilyl esters, separated by gas chromatography, and identified by mass spectrometry. Ratios of sterol to squalene in different tissues were determined, and cholesterol as a disease biomarker was assessed. Free sterol was the dominant lipid class of bat integument. Squalene/sterol ratio is highest in wing sebum. Secreted wing lipid contained higher proportions of saturated FFAs and MAGs than integral wing or secreted hair lipid. These compounds are targets for investigating responses of P. destructans to specific host lipid compounds and as biomarkers to diagnose WNS.     

Isolation and Characterization of a Primary Proximal Tubular Epithelial Cell Model from Human Kidney by CD10/CD13 Double Labeling

Renal proximal tubular epithelial cells play a central role in renal physiology and are among the cell types most sensitive to ischemia and xenobiotic nephrotoxicity. In order to investigate the molecular and cellular mechanisms underlying the pathophysiology of kidney injuries, a stable and well-characterized primary culture model of proximal tubular cells is required. An existing model of proximal tubular cells is hampered by the cellular heterogeneity of kidney; a method based on cell sorting for specific markers must therefore be developed. In this study, we present a primary culture model based on the mechanical and enzymatic dissociation of healthy tissue obtained from nephrectomy specimens. Renal epithelial cells were sorted using co-labeling for CD10 and CD13, two renal proximal tubular epithelial markers, by flow cytometry. Their purity, phenotypic stability and functional properties were evaluated over several passages. Our results demonstrate that CD10/CD13 double-positive cells constitute a pure, functional and stable proximal tubular epithelial cell population that displays proximal tubule markers and epithelial characteristics over the long term, whereas cells positive for either CD10 or CD13 alone appear to be heterogeneous. In conclusion, this study describes a method for establishing a robust renal proximal tubular epithelial cell model suitable for further experimentation.     

Structural Changes in Senescing Oilseed Rape Leaves at Tissue and Subcellular Levels Monitored by Nuclear Magnetic Resonance Relaxometry through Water Status

Nitrogen use efficiency is relatively low in oilseed rape (Brassica napus) due to weak nitrogen remobilization during leaf senescence. Monitoring the kinetics of water distribution associated with the reorganization of cell structures, therefore, would be valuable to improve the characterization of nutrient recycling in leaf tissues and the associated senescence processes. In this study, nuclear magnetic resonance (NMR) relaxometry was used to describe water distribution and status at the cellular level in different leaf ranks of well-watered plants. It was shown to be able to detect slight variations in the evolution of senescence. The NMR results were linked to physiological characterization of the leaves and to light and electron micrographs. A relationship between cell hydration and leaf senescence was revealed and associated with changes in the NMR signal. The relative intensities and the transverse relaxation times of the NMR signal components associated with vacuole water were positively correlated with senescence, describing water uptake and vacuole and cell enlargement. Moreover, the relative intensity of the NMR signal that we assigned to the chloroplast water decreased during the senescence process, in agreement with the decrease in relative chloroplast volume estimated from micrographs. The results are discussed on the basis of water flux occurring at the cellular level during senescence. One of the main applications of this study would be for plant phenotyping, especially for plants under environmental stress such as nitrogen starvation.The main physiological outcome of leaf senescence is the recycling of organic resources and the provision of nutrients to sink organs such as storage and growing tissues (Buchanan-Wollaston, 1997; Hikosaka, 2005; Krupinska and Humbeck, 2008). In crop plants, senescence progresses from the lower older leaves to the younger top leaves. Macromolecular degradation and the mechanism of reallocation of breakdown products are mediated by the up-regulation of senescence-related genes (Lee et al., 2001) in close relationship with both developmental and environmental conditions (Gombert et al., 2006). This leads to remobilization of carbon and nitrogen (N) compounds mostly from plastidial compartments (Martínez et al., 2008; Guiboileau et al., 2012), involving proteolytic activity in plasts, vacuole, and cytosol (Adam and Clarke, 2002; Otegui et al., 2005), chlorophyll breakdown (Hoertensteiner, 2006), galactolipid recycling (Kaup et al., 2002) in the plastoglobules (Brehelin et al., 2007), and loading of Suc and amino acids into the phloem through appropriate transporters (Wingler et al., 2004; Masclaux-Daubresse et al., 2008). In terms of leaf senescence at the cell level, where chloroplasts are degraded sequentially, relative organelle volume does not seem to be greatly modified, the vacuole remains intact, and in darkness-induced senescence the number of chloroplasts per cell decreases only slightly (Keech et al., 2007). However, major changes in metabolic fluxes and cell water relationships are expected during the senescence program that may be associated with macromolecule catabolism, organic solute synthesis, transport and remobilization, and cell structure reconfiguration such as chloroplast evolution to gerontoplast (Hoertensteiner, 2006; Zhang et al., 2010) through the autophagy process (Wada et al., 2009), accumulation of senescence-associated vacuoles (Otegui et al., 2005), and cell wall degradation (Mohapatra et al., 2010).The senescent leaves of oilseed rape (Brassica napus), a major oleiferous crop, generally fall while still maintaining a high N content (about 2.5%–3% [w/w] of the dry matter; Malagoli et al., 2005). In addition to the environmental impact of this leaking of N out of the plant, the low capacity to remobilize foliar N is associated with a high requirement for N fertilization to meet the potential crop yield (Dreccer et al., 2000). In order to improve the nitrogen use efficiency (NUE), new genotypes are being selected for their ability to maintain high yields under limited N fertilization, mainly via the improvement of N uptake efficiency and N mobilization from the senescing leaves (Hirel et al., 2007). In Arabidopsis (Arabidopsis thaliana) and oilseed rape, N can be remobilized from old to expanding leaves at the vegetative stage during sequential senescence as well as from leaves to seeds at the reproductive stage during monocarpic senescence (Malagoli et al., 2005; Diaz et al., 2008; Lemaitre et al., 2008). Senescence can also be induced by environmental stress such as N starvation (Etienne et al., 2007) or water deficit (Reviron et al., 1992) and propagated from old to mature leaves and delayed in young leaves, suggesting finely tuned high regulation of metabolism at the whole-plant level with consequences for NUE (Desclos et al., 2008). One major challenge to understanding the efficiency of senescence-induced organic resource reallocation and to highlighting major molecular and mechanistic attributes of nutrient recycling is monitoring the kinetics of the structural reorganization of cell structures. This reorganization will provide nutrients remobilized through phloem loading. From a technological and phenotyping point of view, the measurement of N remobilization efficiency has already been addressed in crop species and oilseed rape, as it is a reliable trait to screen for the genetic variability of NUE (Franzaring et al., 2012). However, techniques such as stable isotope feeding are time consuming, destructive, and difficult to adapt to large genotype panels. Therefore, it is important to develop a technique for following changes in water distribution at the cell level in order to understand metabolic reconfigurations occurring throughout senescence.NMR relaxometry has been used in several studies to investigate plant cell structure and functioning (Hills and Duce, 1990; Van As, 1992). The ¹H-NMR signal originates almost entirely from water protons because other ¹H nuclei in the plant produce much less intense signals, as they correspond to molecules that are at a much lower concentration than water. The technique allows the measurement of longitudinal (T1) and transverse (T2) relaxation times and proton spin density. Water proton relaxation times are related to the rotational and translational mobility of water molecules (Van As, 2007). They are also modified by the mobility and structure of the surrounding macromolecules (i.e. starch, proteins, and polysaccharides) through proton exchange (chemical exchange). In plant cells, the water in different cell compartments has different chemical and physical properties and, therefore, different bulk T2 values. Moreover, relaxation times are affected by the exchange of molecules between different compartments that is determined by water diffusion and, therefore, by the compartment size and membrane permeability (Van der Weerd et al., 2002). The slow diffusion process between compartments results in multiexponential behavior of the relaxation signal. The multiexponential relaxation reflects water in cell compartments and, therefore, can be used to study changes in water distribution and properties at a subcellular level and, hence, can be used for the estimation of structural and volume transformations in cell compartments. The T2 relaxation time is more sensitive to small variations in water content and chemical exchange processes than T1 and, therefore, is usually preferred. Indeed, differences in T1 for the different compartments are relatively small, resulting in an averaging effect that results in poor discrimination between water compartments (Van As, 2007).To date, NMR relaxometry has mainly been used for the characterization of fruit and vegetable tissues and has been shown to be effective in providing valuable information about cell organization (Sibgatullin et al., 2007). However, although a number of studies have contributed to the interpretation of the NMR results (Snaar and Van As, 1992; Hills and Nott, 1999; Marigheto et al., 2009), this is still not always straightforward, as the NMR signal depends both on the nature of the plant tissue and on the NMR measurement protocol. The situation is even more complex in the case of leaves, because leaves contain different tissue types characterized by different cell sizes and structures (Teixeira et al., 2005), and only a few studies involving NMR relaxometry in leaves have been reported. Changes in T2 in response to high temperature were investigated in wheat (Triticum aestivum; Maheswari et al., 1999) in order to develop a method for the detection of heat injury. McCain (1995) measured the T2 relaxation time of chloroplast and nonchloroplast water in maple (Acer platanoides) leaves by separating corresponding peaks in an NMR spectrum without taking into account the compartmentalization of nonchloroplast water. Oshita et al. (2006) investigated cell membrane permeability to water in spinach (Spinacia oleracea) leaves by measuring the T1 relaxation time of the leaf protoplasts without consideration of the subcellular structure. Qiao et al. (2005) attempted to associate NMR signal components with different chive (Allium schoenoprasum) cells using combined transverse relaxation and restricted diffusion measurements. Finally, Capitani et al. (2009) recently used a portable unilateral NMR instrument to detect the water status of leaves of herbaceous crops, mesophyllous trees, and natural Mediterranean vegetation under field conditions. Further investigations are necessary to improve leaf characterization by NMR, especially in the attribution of NMR signal components to the tissue and subcellular compartments. Progress in this field would make it possible to use the full potential of noninvasive NMR relaxometry in plant research and phenotyping.Using NMR relaxometry, we describe here the differences in water status that occurred at tissue and cellular levels through different leaf ranks of well-irrigated oilseed rape plants, from the young leaves at the top of the canopy to the senescing older leaves at the bottom of the plant. The aim of the study was to show that changes that occur in the leaves while senescing can be related to changes in water distribution and cell structure. As these changes are directly linked to the modifications in cell compartment organization, especially those occurring in the chloroplast, vacuole, and cell wall due to macromolecule degradation and N and carbon reallocation processes, this study was designed to contribute to the understanding of these physiological processes.     

Substrate specificity overlap and interaction between adrenoleukodystrophy protein (ALDP/ABCD1) and adrenoleukodystrophy-related protein (ALDRP/ABCD2)

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes a peroxisomal member of the ATP-binding cassette (ABC) transporter subfamily D called ALDP. ALDP is supposed to function as a homodimer allowing the entry of CoA-esters of very-long chain fatty acids (VLCFA) into the peroxisome, the unique site of their β-oxidation. ALDP deficiency can be corrected by overexpression of ALDRP, its closest homolog. However, the exact nature of the substrates transported by ALDRP and its relationships with ALDP still remain unclear. To gain insight into the function of ALDRP, we used cell models allowing the induction in a dose-dependent manner of a wild type or a mutated non-functional ALDRP-EGFP fusion protein. We explored the consequences of the changes of ALDRP expression levels on the fatty acid content (saturated, monounsaturated, and polyunsaturated fatty acids) in phospholipids as well as on the levels of β-oxidation of 3 suspected substrates: C26:0, C24:0, and C22:6n-3 (DHA). We found an inverse correlation between the fatty acid content of saturated (C26:0, C24:0) and monounsaturated (C26:1, C24:1) VLCFA and the expression level of ALDRP. Interestingly, we obtained a transdominant-negative effect of the inactive ALDRP-EGFP on ALDP function. This effect is due to a physical interaction between ALDRP and ALDP that we evidenced by proximity ligation assays and coimmunoprecipitation. Finally, the β-oxidation assays demonstrate a role of ALDRP in the metabolism of saturated VLCFA (redundant with that of ALDP) but also a specific involvement of ALDRP in the metabolism of DHA.