Thymic nurse cell multicellular complexes in HY-TCR transgenic mice demonstrate their association with MHC restriction

This study examines thymic nurse cell (TNC) function during T-cell development. It has been suggested that TNCs function in the removal of nonfunctional and/or apoptotic thymocytes and do not participate in major histocompatibility complex restriction. We analyzed TNCs isolated from both normal C57BL/6 mice and C57BL/6 TgN (TCRHY) mice (HY-TCR transgenic mice). Using confocal microscopic analyses of TNCs isolated from C57BL/6 animals, we showed that 75%-78% of the enclosed thymocyte subset was viable, and 87%-90% of these cells expressed both CD4 and CD8. CD4 and CD8 also were expressed on TNC thymocytes isolated from both male and female HY-TCR transgenic mice. The transgenic female thymus was shown to have 17 times more TNCs per milligram of thymus than the transgenic male thymus. TNCs from HY-TCR transgenic females were 8-10 microm larger than transgenic male TNCs, and the female TNCs contained five times more thymocytes within intracytoplasmic vacuoles, with less than 4% apoptosis. However, more than 42% of the thymocytes within transgenic male TNCs were apoptotic. The large number and size of TNCs containing viable thymocytes in the female transgenic thymus suggest that TNC function is not limited to the removal of apoptotic thymocytes. We believe that the selective uptake of viable double-positive thymocytes by TNCs in C57BL/6 and HY-TCR transgenic female mice provides evidence that this interaction occurs during the process of major histocompatibility complex restriction.     

Circulating macrophages as well as developing thymocytes are enclosed within thymic nurse cells.

Both thymic nurse cells (TNCs) and macrophages have been reported to function as antigen-presenting cells during the process of MHC restriction. Negative selection, which results in the apoptosis of potentially autoreactive thymocytes, is believed to be associated with both macrophages and TNCs in the cortex. Both cell types have also been reported to ingest thymocytes undergoing positive and negative selection. However, macrophages ingest apoptotic thymocytes, while TNCs have been shown to internalize viable cells. A subset of the TNC-engulfed population is allowed to mature and is released, while the remaining fraction becomes apoptotic and is absorbed within the TNC cytoplasm through lysosomal activity. A recent report described a subset of rat TNCs that contain macrophages as well as thymocytes within their cytoplasm. We examined freshly isolated TNCs from C57BL/6 mice and found that, of the TNC population recovered, 1.7% contained macrophages within its cytoplasm. There also were macrophages tightly bound but not internalized into the multicellular structure at a rate of 2.9%. The total association of macrophages with TNCs was approximately 4.6%. This unique association of macrophages with TNCs was also observed in vitro when freshly isolated thymocytes (containing macrophages) were added to cultures of cells from the TNC cell line tsTNC-1. The macrophage-TNC interaction was found to be dynamic, with macrophages moving rapidly into and out of TNCs containing cytoplasmic thymocytes. Macrophages within TNCs showed a close association with cytoplasmic thymocytes. We then labeled peritoneal macrophages with CFDA SE, a cell tracking dye, and returned them to the mouse peritoneum. Within 1 h, labeled macrophages were detectable in the thymus. This is the first investigation to show a direct interaction between peripheral macrophages and TNCs. These results suggest that TNCs and macrophages work together as antigen-presenting cells.     

The immortalization of thymic nurse cells by SV40 virus

Thymic nurse cells (TNCs) are stromal elements that contain between 20 and 200 T cells within their cytoplasm. Because of this unique feature they are believed to play a role in thymocyte development. Unfortunately, it has been difficult to obtain pure TNCs in quantities sufficient for extensive evaluation of their thymic function. As a result, only a limited amount of information is available that characterizes TNCs or the T cell population(s) found within their cytoplasm. We have now used SV40 to infect and immortalize TNCs from C57BL/6 mice. SV40-transformed TNCs were found to specifically bind and internalize cells from an immature thymocyte line isolated in our laboratory. These results describe a method of obtaining pure populations of TNCs for future studies of their thymic function, and suggest that binding to specific subpopulations of lymphoblasts may be necessary for internalization.     

Less-told stories about corporate globalization: transnational corporations and CSR as the politics of (ir)responsibility in Africa

In the wake of transformations being ushered by globalization, figures suggest that there is a rise in the power of transnational corporations (TNCs), raising important questions about the exercise of such power and/or how to hold them accountable for it. Concomitantly, corporate social responsibility (CSR) discourse has emerged as a new discursive formation; a new meta-narrative that is propagated by TNCs. It seeks to portray the actions of TNCs as oriented by such values as “responsibility,” “sustainability,” “development.” Situated within what is emerging as “an anthropological imperative to critique” the actions of corporations, this article takes a critical approach to such a meta-narrative. It argues that not only do TNCs behave irresponsibly in contexts outside the Global North where they can easily get away with doing so, but also that the CSR discourse of responsibility helps to occlude these often damaging actions by TNCs. Drawing from an overview of the often untold or less-known stories of damaging actions by TNCs in Africa in the recent past, this article illustrates the disturbing co-existence of socially irresponsible actions amidst a forceful tendency to circulate a feel-good CSR discourse of responsibility.     

Questionable Thymic Nurse Cell

Since their discovery in 1980, thymic nurse cells (TNCs) have been controversial. Questions pertaining to the existence of the TNC as a “unit” cell with thymocytes completely enclosed within its cytoplasm were the focus of initial debates. Early skeptics proposed the multicellular complex to be an artifact of the procedures used to isolate TNCs from the thymus. Since that time, TNCs have been found in fish, frogs, tadpoles, chickens, sheep, pigs, rats, mice, and humans. Their evolutionary conservation throughout the animal kingdom relieved most speculations about the existence of TNCs and at the same time demonstrated their apparent importance to the thymus and T-cell development. In this review we will discuss and debate reports that describe (i) the organization or structure of TNCs, (ii) the thymocyte subset(s) found within the cytoplasm of TNCs and their uptake and release, and (iii) the function of this fascinating multicellular interaction that occurs during the process of T-cell development. Discussions about the future of the field and experimental approaches that will lead to answers to remaining questions are also presented.     

The characterization of invertebrate troponin C

TNCs from lobster, mussel, and squid migrated with rabbit TNC at an apparent mol. wt of 18,000. Electrophoretic mobilities in the presence or absence of Ca2+ were compared: the electrophoretic mobility of rabbit TNC was greater in the presence of Ca2+ than it its absence, but all invertebrate TNCs tested migrated identically, whether Ca2+ was present or not. The Ca2+-binding capacity of invertebrate TNCs was only one Ca2+ ion per molecule. The alpha-helix contents in the presence or absence of Ca2+ were compared: rabbit TNC changed by a value of 16% and invertebrate TNCs by 4%. Antibodies to loligo TNC did not cross-react with rabbit TNC, but did interact with their molluscan TNCs.     

Questionable thymic nurse cell.

Since their discovery in 1980, thymic nurse cells (TNCs) have been controversial. Questions pertaining to the existence of the TNC as a "unit" cell with thymocytes completely enclosed within its cytoplasm were the focus of initial debates. Early skeptics proposed the multicellular complex to be an artifact of the procedures used to isolate TNCs from the thymus. Since that time, TNCs have been found in fish, frogs, tadpoles, chickens, sheep, pigs, rats, mice, and humans. Their evolutionary conservation throughout the animal kingdom relieved most speculations about the existence of TNCs and at the same time demonstrated their apparent importance to the thymus and T-cell development. In this review we will discuss and debate reports that describe (i) the organization or structure of TNCs, (ii) the thymocyte subset(s) found within the cytoplasm of TNCs and their uptake and release, and (iii) the function of this fascinating multicellular interaction that occurs during the process of T-cell development. Discussions about the future of the field and experimental approaches that will lead to answers to remaining questions are also presented.     

Thymic nurse cells exclusively bind and internalize CD4+CD8+ thymocytes.

Thymic nurse cells (TNC) contain 20-200 thymocytes within specialized vacuoles in their cytoplasm. The purpose of the uptake of thymocytes by TNCs is unknown. TNCs also have the capacity to present self-antigens, which implies that they may serve a function in the process of thymic education. We have recently reported the development of thymic nurse cell lines that have the ability to bind and internalize T cells. Here, we use one of these TNC lines to identify the thymocyte subpopulation(s) involved in this internalization process. TNCs exposed to freshly isolated thymocytes bind and internalize CD4 and CD8 expressing thymocytes (CD4+CD8+ or double positives) exclusively. More specifically, a subset of the double-positive thymocyte population displayed binding capacity. These double-positive cells express cell surface alpha beta type T cell antigen receptor (TCR), as well as CD3 epsilon. Binding was not inhibited in the presence of antibodies against CD3, CD4, CD8, Class I antigens, or Class II antigens. These results describe two significant events in T cell development. First, TNCs exclusively bind and internalize a subset of alpha beta TCR expressing double-positive T cells. Also, binding is facilitated through a mechanism other than TCR recognition of major histocompatibility complex antigens. This suggests that thymocyte internalization may be independent of the process used by TNCs to present self-antigen.     

Lysosomal-mediated degradation of apoptotic thymocytes within thymic nurse cells

A thymic epithelial cell line (tsTNC-1) that maintains the ability to selectively bind and internalize immature alphabetaTCR(lo)CD4(+)CD8(+) thymocytes in vitro was used in long-term coincubation experiments to determine the ultimate fate of thymocytes that remained within intracytoplasmic vacuoles of thymic nurse cells (TNCs). In an earlier report, a subset of the population released from the TNC interaction was shown to mature to the alphabetaTCR(hi)CD69(hi) stage of development, while thymocytes that bided within the TNC cytoplasm died through the process of apoptosis. Here, we show the presence of both apoptotic and nonapoptotic thymocytes within the cytoplasm of freshly isolated TNCs as well as in tsTNC-1 cells in culture. A microscopic analysis revealed total degradation of the cytoplasmic apoptotic thymocyte population that remained in tsTNC-1 cells after an 8- to 10-h incubation period. A quantitative analysis showed an increase of cytoplasmic thymocyte degradation over time to almost 80% after 9 h of incubation. However, in the presence of bafilomycin A1, which is used to inhibit acidification of lysosomal vesicles, degradation of apoptotic thymocytes never reached 10%. These data suggest that lysosomes within TNCs play a role in the degradation of apoptotic thymocytes. We examined tsTNC-1 cells before the addition of thymocytes to cultures and found lysosomes to be clustered around the nucleus in the cytoplasm of TNCs. Shortly after the internalization event, apoptotic thymocytes move to the area of the cytoplasm containing lysosomes. Using the confocal microscope, we obtained evidence that shows the degradation event to be facilitated through the fusion of lysosomes with the specialized vacuoles within TNCs containing apoptotic cells.     

Hyperspectral Imaging for Mapping of Total Nitrogen Spatial Distribution in Pepper Plant

Visible/near-infrared (Vis/NIR) hyperspectral imaging was employed to determine the spatial distribution of total nitrogen in pepper plant. Hyperspectral images of samples (leaves, stems, and roots of pepper plants) were acquired and their total nitrogen contents (TNCs) were measured using Dumas combustion method. Mean spectra of all samples were extracted from regions of interest (ROIs) in hyperspectral images. Random frog (RF) algorithm was implemented to select important wavelengths which carried effective information for predicting the TNCs in leaf, stem, root, and whole-plant (leaf-stem-root), respectively. Based on full spectra and the selected important wavelengths, the quantitative relationships between spectral data and the corresponding TNCs in organs (leaf, stem, and root) and whole-plant (leaf-stem-root) were separately developed using partial least-squares regression (PLSR). As a result, the PLSR model built by the important wavelengths for predicting TNCs in whole-plant (leaf-stem-root) offered a promising result of correlation coefficient (R) for prediction (R_P = 0.876) and root mean square error (RMSE) for prediction (RMSEP = 0.426%). Finally, the TNC of each pixel within ROI of the sample was estimated to generate the spatial distribution map of TNC in pepper plant. The achievements of the research indicated that hyperspectral imaging is promising and presents a powerful potential to determine nitrogen contents spatial distribution in pepper plant.     

In vitro behavior of thymic nurse cell-like complexes from mechanically and enzymatically dissociated frog tadpole thymuses

Cellular complexes, analogous by virtue of their external appearance, size, and number of seemingly internalized thymocytes to thymic nurse cells (TNCs) of endothermic vertebrates, were seen in short-term cultures (6-8 days) of mechanically and enzymatically dissociated thymuses of leopard frog tadpoles. Most TNC-like complexes from mechanically disrupted thymuses were covered with many thymocytes that morphologically resembled the "internalized" thymocytes. With time in culture, most complexes remained spherical and lost their externally adherent and "internalized" thymocytes. Some complexes, however, adhered to the glass substratum by means of macrophage-like cells. After one typically appearing TNC from a mechanically dissociated thymus had released its "internalized" thymocytes and spread completely over the glass substratum, it could be seen to consist actually of 9-10 stromal cells with the appearance of epithelial cells, macrophages, and dendritic cells. TNC-like structures from enzymatically dissociated thymuses had few, if any, attached thymocytes. Although these structures closely resembled murine TNCs initially, they displayed abnormal transformations within a few days of culture. Our observations led us to question the assumption that all TNCs from mechanically as well as enzymatically isolated TNCs from vertebrate thymuses are single cells. Rather, some if not all of the so-called TNC may actually be entities composed of several stromal cell types that enclose thymocytes. We suggest that this configuration seen in vitro may reflect the architecture of the compartmentalized reticular stromal cell meshwork that characterizes the intact thymus.     

Optimization of ex vivo hematopoietic stem cell expansion in intermittent dynamic cultures

For the ex vivo expansion of CD34⁺ cells, culture conditions were optimized using cytokine cocktails and media change methods. In addition, static, orbital-shake, and stirred cultures were compared. After cultivation, total cell expansion, immunophenotypes, clonogenic ability, and metabolite concentration in media were analyzed. Optimized media change methods enhanced the number of total nucleated cells (TNCs) by 600-fold (from 10⁴ to 6 × 10⁶ cells) in static cultures. Furthermore, intermittent orbital-shake cultures gave the highest fold increase of TNCs and CD34⁺/CD38⁻ cells. These results imply that proliferation of CD34⁺ cells in intermittent shake cultures was more efficient than that in static cultures under optimized culture conditions.     

The circadecadal rhythm of oscillation of umbilical cord blood parameters correlates with geomagnetic activity – An analysis of long-term measurements (1999–2011)

Recently, we have shown that the contents of total nucleated cells (TNCs) and CD34⁺ hematopoietic stem and progenitor cells (CD34⁺ HSPCs) as well as the cord blood volume (CBV) in umbilical cord blood (UCB) show a circadecadal (~10 years) rhythm of oscillation. This observation was based on an analysis of 17,936 cord blood donations collected during 1999–2011. The aim of the present study was to investigate whether this circadecadal rhythm of oscillation in TNCs, CD34⁺ HSPCs and CBV is related to geomagnetic activity. For the analysis, the yearly averages of TNCs, CD34⁺ HSPCs and CBV in UCB were correlated with geomagnetic activity (Dcx index). Our analysis revealed that (i) all three UCB parameters were statistically significantly correlated with the level of geomagnetic activity, (ii) CBV showed a linear correlation with the Dcx index (r = 0.5290), (iii) the number of TNCs and CD34⁺ HSPCs were quadratic inversely correlated with the Dcx index (r = ?0.5343 and r = ?0.7749, respectively). Furthermore, (iv) CBV and the number of TNCs were not statistically significantly correlated with the number of either modest or intense geomagnetic storms per year, but (v) the number of CD34⁺ HSPCs was statistically significantly correlated with the number of modest (r = 0.9253) as well as intense (r = 0.8683) geomagnetic storms per year. In conclusion, our study suggests that UCB parameters correlate with the state of the geomagnetic field (GMF) modulated by solar activity. Possible biophysical mechanisms underlying this observation, as well as the outcome of these findings, are discussed.     

Is there a glycosaminoglycan-related heterogeneity of the thymic epithelium?

We determined the synthesis and secretion of glycosaminoglycans by three distinct preparations of mouse cultured thymic epithelial cells. These comprised primary cultures of thymic nurse cells (TNCs), which are normally located within the cortex of the thymic lobules, as well as two murine thymic epithelial cells, bearing a mixed, yet distinct, cortico-medullary phenotype. We first identified and measured the relative proportions of the various glycosaminoglycans in the three epithelial cells. Non-sulfated glycosaminoglycans are preponderantly secreted by the TNCs, while the sulfated glycans (particularly heparan sulfate) are relatively more abundant on the cell surface. The three types of epithelial cells differ markedly in their heparan sulfate composition, mainly due to different patterns of N- and O-sulfation. In addition, the cells differ in the synthesis and secretion of other glycosaminoglycans. Thus, TNCs secrete high amounts of dermatan sulfate + chondroitin sulfate to the culture medium. IT-76M1 cells secrete high proportions of heparan sulfate while 2BH4 cells show a more equilibrated proportion of dermatan sulfate/chondroitin sulfate and heparan sulfate. The three epithelial cells also differ in their capacity to produce hyaluronic acid and 2BH4 cells are distinguished by their high rate of synthesis of this glycosaminoglycan. In conclusion, our results show that distinct thymic epithelial cells can synthesize different types of glycosaminoglycans. Although it remains to be definitely determined whether these differences reflect the in vivo situation, our data provide new clues for further understanding of how glycosaminoglycan-mediated interactions behave in the thymus.     

Long-term effects of cryopreservation on clinically prepared hematopoietic progenitor cell products

Background aimsThe question of how long hematopoietic progenitor cells (HPCs) destined for clinical applications withstand long-term cryopreservation remains unanswered. To increase our basic understanding about the stability of HPC products over time, this study focused on characterizing long-term effects of cryopreservation on clinically prepared HPC products.MethodsCryovials (n = 233) frozen for an average of 6.3 ± 14.2 years (range, 0.003–14.6 years) from HPC products (n = 170) representing 75 individual patients were thawed and evaluated for total nucleated cells (TNCs), cell viability, viable CD34+ (vCD34+) cells and colony-forming cells (CFCs). TNCs were determined by use of an automated cell counter, and cell viability was measured with the use of trypan blue exclusion. Viable CD34 analysis was performed by means of flow cytometry and function by a CFC assay.ResultsSignificant losses in TNCs, cell viability, vCD34+ cells and CFC occurred on cryopreservation. However, once frozen, viable TNCs, vCD34+ cells and CFC recoveries did not significantly change over time. The only parameter demonstrating a change over time was cell viability, which decreased as the length of time that an HPC product was stored frozen increased. A significant negative correlation (correlation coefficient = −0.165) was determined between pre-freeze percent granulocyte content and post-thaw percent viability (n = 170; P = 0.032). However, a significant positive correlation was observed between percent viability at thaw and pre-freeze lymphocyte concentration.ConclusionsOnce frozen, HPC products were stable for up to 14.6 years at <−150°C. Post-thaw viability was found to correlate negatively with pre-freeze granulocyte content and positively with pre-freeze lymphocyte content.     

The human and mouse orthologous LIM-only proteins respectively encoded in chromosome 6 and 17 show a different expression pattern

Thymocytes interact with various subpopulations of thymic epithelial cells (TECs) at different stages of their development. To identify new molecules specifically expressed in TECs and/or thymic nurse cells (TNCs), we used representational difference analysis. We identified a LIM protein located on mouse chromosome 17 (m17TLP) and belonging to the family of the LIM-only proteins (LIMo). We found a new splice variant in addition to the two described A and B isoforms. The three alternative species of m17TLP are found strictly in the thymic stroma. This protein is expressed on a subpopulation of TECs and TNCs. Strikingly, we found that the human ortholog of m17TLP, located on chromosome 6 (h6LIMo), is expressed in most tissues, but not in skeletal muscle. We have identified four human splice variants of h6LIMo which differ in their carboxy-terminal regions. The sequence comprising the genomic structure suggests that CRP2 is the closest known relative of m17TLP. Although the human and mouse nucleotide sequences are 88-97% homologous, this homology is reduced to 47% in the promoter regions, which strongly suggests that their differential expression is related to their promoter regulatory activity.     

Theoretical estimation of the calcium-binding constants for proteins from the troponin C superfamily based on a secondary structure prediction method. II. Applications

Ca2+-binding properties of the following proteins, classified as members of the troponin C (TNC) superfamily have been discussed: TNCs, calmodulins (CaMs), vitamin D-dependent calcium-binding proteins (CaBPs), myosin light chains (LCs), S-100 chains, parvalbumins (PVs), oncomodulin (OCM), sarcoplasmic calcium binding proteins (SCPs), calcineurin B (CB) and calcium vector protein (CaVP). Assuming the most probable domain pairing, the Ca2+-binding constants of these proteins have been predicted from their sequences using the method presented in the preceding paper. The results are critically compared with the available experimental data. For some proteins (TNCs, CaMs, CaBPs, LCs, CB and CaVP) our predictions are consistent with the experimental results. For the others, substantial discrepancies between the predicted and measured KCa values are observed. They result from some structural peculiarities of those proteins: a unique, three-domain organization in the case of PVs and OCM, unusual sequences of binding loops in the case of S-100 and a lack of a standard helix-loop-helix organization of Ca2+-binding domains in the case of SCPs.     

In situ characterization in freeze-fractured mouse thymuses of lymphoepithelial complexes ultrastructurally similar to isolated thymic nurse cells

Scanning and transmission electron microscopy of the cracked surfaces of cryofractured pre-fixed C57BL/Ka mouse thymus reveals the existence of cell complexes, distinct from the surrounding cell organization, in which groups of lymphocytes are delimited by large cytoplasmic sheets or envelopes. These complexes, located in the subcapsular and cortical regions, display morphological features similar to that of the thymic nurse cells (TNCs), which can be isolated from the mouse or human thymus enzymatically dissociated. They can be considered as dynamic systems able to modify their three-dimensional organization, namely with regard to intrathymic cellular traffic involved in T-lymphocyte maturation.     

Safety and tolerability of intrathecal delivery of autologous bone marrow nucleated cells in children with cerebral palsy: an open-label phase I trial

Background aimsCerebral palsy (CP) is related to severe perinatal hypoxia with permanent brain damage in nearly 50% of surviving preterm infants. Cell therapy is a potential therapeutic option for CP by several mechanisms, including immunomodulation through cytokine and growth factor secretion.MethodsIn this phase I open-label clinical trial, 18 pediatric patients with CP were included to assess the safety of autologous bone marrow–derived total nucleated cell (TNC) intrathecal and intravenous injection after stimulation with granulocyte colony-stimulating factor. Motor, cognitive, communication, personal-social and adaptive areas were evaluated at baseline and 1 and 6 months after the procedure through the use of the Battelle Developmental Inventory. Magnetic resonance imaging was performed at baseline and 6 months after therapy. This study was registered in ClinicaTrials.gov (NCT01019733).ResultsA median of 13.12 × 10⁸ TNCs (range, 4.83–53.87) including 10.02 × 10⁶ CD34+ cells (range, 1.02–29.9) in a volume of 7 mL (range, 4–10.5) was infused intrathecally. The remaining cells from the bone marrow aspirate were administered intravenously; 6.01 × 10⁸ TNCs (range, 1.36–17.85), with 3.39 × 10⁶ cells being CD34+. Early adverse effects included headache, vomiting, fever and stiff neck occurred in three patients. No serious complications were documented. An overall 4.7-month increase in developmental age according to the Battelle Developmental Inventory, including all areas of evaluation, was observed (±SD 2.63). No MRI changes at 6 months of follow-up were found.ConclusionsSubarachnoid placement of autologous bone marrow–derived TNC in children with CP is a safe procedure. The results suggest a possible increase in neurological function.