Microencapsulation of dopamine neurons derived from human induced pluripotent stem cells

Background

Dopamine neurons derived from induced pluripotent stem cells have been widely studied for the treatment of Parkinson's disease. However, various difficulties remain to be overcome, such as tumor formation, fragility of dopamine neurons, difficulty in handling large numbers of dopamine neurons, and immune reactions. In this study, human induced pluripotent stem cell-derived precursors of dopamine neurons were encapsulated in agarose microbeads. Dopamine neurons in microbeads could be handled without specific protocols, because the microbeads protected the fragile dopamine neurons from mechanical stress.

Methods

hiPS cells were seeded on a Matrigel-coated dish and cultured to induce differentiation into a dopamine neuronal linage. On day 18 of culture, cells were collected from the culture dishes and seeded into U-bottom 96-well plates to induce cell aggregate formation. After 5 days, cell aggregates were collected from the plates and microencapsulated in agarose microbeads. The microencapsulated aggregates were cultured for an additional 45 days to induce maturation of dopamine neurons.

Results

Approximately 60% of all cells differentiated into tyrosine hydroxylase-positive neurons in agarose microbeads. The cells released dopamine for more than 40 days. In addition, microbeads containing cells could be cryopreserved.

Conclusion

hiPS cells were successfully differentiated into dopamine neurons in agarose microbeads.

General significance

Agarose microencapsulation provides a good supporting environment for the preparation and storage of dopamine neurons.     

Colon-Targeted Delivery of IgY Against <Emphasis Type="Italic">Clostridium difficile</Emphasis> Toxin A and B by Encapsulation in Chitosan-Ca Pectinate Microbeads

This study investigated the use of a newly developed chitosan-Ca pectinate microbead formulation for the colon-targeted delivery of anti-A/B toxin immunoglobulin of egg yolk (IgY) to inhibit toxin binding to colon mucosa cells. The effect of the three components (pectinate, calcium chloride, and chitosan) used for the microbead production was examined with the aim of identifying the optimal levels to improve drug encapsulation efficiency, swelling ratio, and cumulative IgY release rate. The optimized IgY-loaded bead component was pectin 5% (w/v), CaCl₂ 3% (w/v), and chitosan 0.5% (w/v). Formulated beads were spherical with 1.2-mm diameter, and the drug loading was 45%. An in vitro release study revealed that chitosan-Ca pectinate microbeads inhibited IgY release in the upper gastrointestinal tract and significantly improved the site-specific release of IgY in the colon. An in vivo rat study demonstrated that 72.6% of biologically active IgY was released specifically in the colon. These results demonstrated that anti-A/B toxin IgY-loaded chitosan-Ca pectinate oral microbeads improved IgY release behavior in vivo, which could be used as an effective oral delivery platform for the biological treatment of Clostridium difficile infection (CDI).     

Antigen-sensitized CD4+CD62Llow memory/effector T helper 2 cells can induce airway hyperresponsiveness in an antigen free setting

Background

Airway hyperresponsiveness (AHR) is one of the most prominent features of asthma, however, precise mechanisms for its induction have not been fully elucidated. We previously reported that systemic antigen sensitization alone directly induces AHR before development of eosinophilic airway inflammation in a mouse model of allergic airway inflammation, which suggests a critical role of antigen-specific systemic immune response itself in the induction of AHR. In the present study, we examined this possibility by cell transfer experiment, and then analyzed which cell source was essential for this process.

Methods

BALB/c mice were immunized with ovalbumin (OVA) twice. Spleen cells were obtained from the mice and were transferred in naive mice. Four days later, AHR was assessed. We carried out bronchoalveolar lavage (BAL) to analyze inflammation and cytokine production in the lung. Fluorescence and immunohistochemical studies were performed to identify T cells recruiting and proliferating in the lung or in the gut of the recipient. To determine the essential phenotype, spleen cells were column purified by antibody-coated microbeads with negative or positive selection, and transferred. Then, AHR was assessed.

Results

Transfer of spleen cells obtained from OVA-sensitized mice induced a moderate, but significant, AHR without airway antigen challenge in naive mice without airway eosinophilia. Immunization with T helper (Th) 1 elicited antigen (OVA with complete Freund''s adjuvant) did not induce the AHR. Transferred cells distributed among organs, and the cells proliferated in an antigen free setting for at least three days in the lung. This transfer-induced AHR persisted for one week. Interleukin-4 and 5 in the BAL fluid increased in the transferred mice. Immunoglobulin E was not involved in this transfer-induced AHR. Transfer of in vitro polarized CD4⁺ Th2 cells, but not Th1 cells, induced AHR. We finally clarified that CD4⁺CD62L^low memory/effector T cells recruited in the lung and proliferated, thus induced AHR.

Conclusion

These results suggest that antigen-sensitized memory/effector Th2 cells themselves play an important role for induction of basal AHR in an antigen free, eosinophil-independent setting. Therefore, regulation of CD4⁺ T cell-mediated immune response itself could be a critical therapeutic target for allergic asthma.     

Double staining of intracellular cytokine proteins and T-lymphocyte subsets. Evaluation of the method in blood and bronchoalveolar lavage fluid

An immunocytochemical staining method has been developed for simultaneous staining of both cell surface markers (CD4 and CD8) and intracellular cytokine proteins IFN-, IL-4 and IL-5. Cell surface molecules were visualized with alkaline phosphatase, which was developed by Fast Blue BB. Intracellular cytokine proteins were detected by amino-ethyl carbazole. We applied this technique to T cells from T-cell lines and T-cell clones, peripheral blood mononuclear cells and broncho-alveolar lavage fluid cells. Cells were used either unstimulated or stimulated for 4h with 1ng/ml PMA and 1g/ml ionomycin, which proved to be an optimal stimulus taking cytokine staining, cell recovery and cell viability into account. We studied peripheral blood mononuclear cells from healthy subjects and found that without in vitro stimulation on average 0.4% of the cells were IFN- positive cells. In unstimulated broncho-alveolar lavage fluid cells of the 2 allergic asthmatic subjects studied so far we found higher numbers of cytokine-positive cells (up to 22% of the lymphocytes being IL-4⁺ cells). By in vitro stimulation, the numbers of cytokine-positive peripheral blood mononuclear cells from the healthy subjects were increased to maximally 5% IFN-⁺ cells. In stimulated lavage fluid cells from allergic asthmatic subjects maximally 34% of the lymphocytes became IFN-⁺. We conclude that this method allows detection of intracellular cytokine proteins in both CD4⁺ and CD8⁺ T cells without the need for stimulating the cells in vitro. In vitro stimulation may change the cytokine profile detected.     

Clinical Grade Treg: GMP Isolation,Improvement of Purity by CD127pos Depletion,Treg Expansion,and Treg Cryopreservation

Background

Treg based immunotherapy is of great interest to facilitate tolerance in autoimmunity and transplantation. For clinical trials, it is essential to have a clinical grade Treg isolation protocol in accordance with Good Manufacturing Practice (GMP) guidelines. To obtain sufficient Treg for immunotherapy, subsequent ex vivo expansion might be needed.

Methodology/Principal Findings

Treg were isolated from leukapheresis products by CliniMACS based GMP isolation strategies, using anti-CD25, anti-CD8 and anti-CD19 coated microbeads. CliniMACS isolation procedures led to 40–60% pure CD4^posCD25^highFoxP3^pos Treg populations that were anergic and had moderate suppressive activity. Such CliniMACS isolated Treg populations could be expanded with maintenance of suppressive function. Alloantigen stimulated expansion caused an enrichment of alloantigen-specific Treg. Depletion of unwanted CD19^pos cells during CliniMACS Treg isolation proved necessary to prevent B-cell outgrowth during expansion. CD4^posCD127^pos conventional T cells were the major contaminating cell type in CliniMACS isolated Treg populations. Depletion of CD127^pos cells improved the purity of CD4^posCD25^highFoxP3^pos Treg in CliniMACS isolated cell populations to approximately 90%. Expanded CD127^neg CliniMACS isolated Treg populations showed very potent suppressive capacity and high FoxP3 expression. Furthermore, our data show that cryopreservation of CliniMACS isolated Treg is feasible, but that activation after thawing is necessary to restore suppressive potential.

Conclusions/Significance

The feasibility of Treg based therapy is widely accepted, provided that tailor-made clinical grade procedures for isolation and ex vivo cell handling are available. We here provide further support for this approach by showing that a high Treg purity can be reached, and that isolated cells can be cryopreserved and expanded successfully.     

Alginate Microencapsulated Hepatocytes Optimised for Transplantation in Acute Liver Failure

MethodsHuman hepatocyte microbeads (HMBs) were prepared using sterile GMP grade materials. We determined physical stability, cell viability, and hepatocyte metabolic function of HMBs using different polymerisation times and cell densities. The immune activation of peripheral blood mononuclear cells (PBMCs) after co-culture with HMBs was studied. Rats with ALF induced by galactosamine were transplanted intraperitoneally with rat hepatocyte microbeads (RMBs) produced using a similar optimised protocol. Survival rate and biochemical profiles were determined. Retrieved microbeads were evaluated for morphology and functionality.ResultsThe optimised HMBs were of uniform size (583.5±3.3 µm) and mechanically stable using 15 min polymerisation time compared to 10 min and 20 min (p<0.001). 3D confocal microscopy images demonstrated that hepatocytes with similar cell viability were evenly distributed within HMBs. Cell density of 3.5×10⁶ cells/ml provided the highest viability. HMBs incubated in human ascitic fluid showed better cell viability and function than controls. There was no significant activation of PBMCs co-cultured with empty or hepatocyte microbeads, compared to PBMCs alone. Intraperitoneal transplantation of RMBs was safe and significantly improved the severity of liver damage compared to control groups (empty microbeads and medium alone; p<0.01). Retrieved RMBs were intact and free of immune cell adherence and contained viable hepatocytes with preserved function.ConclusionAn optimised protocol to produce GMP grade alginate-encapsulated human hepatocytes has been established. Transplantation of microbeads provided effective metabolic function in ALF. These high quality HMBs should be suitable for use in clinical transplantation.     

The mode of recognition of tumor antigens by noncytolytic-type antitumor T cells: Role of antigen-presenting cells and their surface class I and class II H-2 molecules

Summary The present study investigated the role of antigen-presenting cells (APC) in the activation of noncytolytic T cells against tumor antigens. The noncytolytic-type T cells exerted their antitumor effect by producing -interferon (IFN-) and by activating macrophages as the ultimate effectors. The production of IFN- by these noncytolytic T cells following the stimulation with tumor cells required the participation of Ia⁺ APC, since the depletion of APC from cultures of tumor-immunized spleen cells resulted in almost complete inhibition of the IFN- production. Both L3T4⁺ and Lyt-2⁺ subsets of T cells were capable of producing IFN-, and the requirement of APC for the production of IFN- was the case irrespective of whether noncytolytic T cells were of L3T4⁺ or Lyt-2⁺ phenotype. More importantly, it was demonstrated that the production of IFN- by L3T4⁺ and Lyt-2⁺ T cells was inhibited by addition of the respective anti-class II and anti-class I H-2 antibody to cultures. These results indicate that antitumor L3T4⁺ or Lyt-2⁺ noncytolytic T cells are activated for the IFN- production by recognizing tumor antigens in the context of self-class II or -class I H-2 molecules on APC.This work was supported by a Grant-in-Aid for the Special Project Cancer-Bioscience from the Ministry of Education, Science and Culture, Japan     

Interleukin-7 enhances proliferation responses to T-cell receptor stimulation in naïve CD4+ T cells from human immunodeficiency virus-infected persons

Proliferation responses of naïve CD4⁺ T cells to T-cell receptor and interleukin-7 (IL-7) stimulation were evaluated by using cells from human immunodeficiency virus-positive (HIV⁺) donors. IL-7 enhanced responses to T-cell receptor stimulation, and the magnitude of this enhancement was similar in cells from healthy controls and from HIV⁺ subjects. The overall response to T-cell receptor stimulation alone or in combination with IL-7, however, was diminished among viremic HIV⁺ donors and occurred independent of antigen-presenting cells. Frequencies of CD127⁺ cells were related to the magnitudes of proliferation enhancement that were mediated by IL-7. Thus, IL-7 enhances but does not fully restore the function of naïve CD4⁺ T cells from HIV-infected persons.Interleukin-7 (IL-7) plays an important role in T-cell homeostasis by modulating thymic output (1, 16, 22) and by enhancing the peripheral expansion and survival of both naïve and memory T-cell subsets (12, 18, 20, 25, 26, 31, 32). Under normal circumstances, the homeostatic maintenance of naïve CD4⁺ T cells is regulated by at least two types of signals that include T-cell receptor (TCR) engagement and IL-7 (10, 26, 30). In addition, IL-7 may play an important role in the conversion of effector T cells into long-term memory cells (12, 14).Homeostasis of T cells is dysregulated in human immunodeficiency virus (HIV) infection such that there is a marked depletion of CD4⁺ cells and a progressive loss of naïve CD4 and CD8⁺ T cells (24). Although the mechanisms for these deficiencies are not fully understood, it is possible that impairments in T-cell proliferation and responsiveness to immunomodulatory cytokines could play a role. In HIV disease, IL-7 is increased in plasma (2, 5, 11, 15, 19, 21, 23) and the alpha chain of the IL-7 receptor, CD127, is less frequently expressed among T lymphocytes (2, 5, 11, 21, 23). The ability of patient T cells to respond to IL-7 stimulation may be diminished in HIV disease but may not be related to the density of CD127 expression as it is in T cells from healthy controls (4). Moreover, the responsiveness of T cells, including naïve CD4⁺ lymphocytes, to TCR stimulation is diminished in HIV disease (27-29). Thus, defects in responsiveness to cytokines or TCR stimulation could contribute to the perturbations in T-cell proliferation and survival in HIV disease.In these studies, we examined the responsiveness of naïve CD4⁺ T cells from viremic HIV-positive (HIV⁺) donors (median plasma HIV RNA level, 25,200 copies/ml [range, 1,015 to 1,000,000 copies/ml]; median CD4 cell count, 429 cells/μl [range, 41 to 950 cells/μl]; median age, 38 years [range, 22 to 64 years]; n = 25) and aviremic, highly active antiretroviral therapy (HAART)-treated HIV⁺ donors (plasma HIV RNA level, <400 copies/ml; median CD4 cell count, 309 cells/μl [range, 74 to 918 cells/μl]; median age, 48 years [range, 37 to 55 years]; n = 12) to the combined stimulus of recombinant IL-7 (Cytheris) plus agonistic anti-CD3 antibody. Peripheral blood mononuclear cells (PBMC) were depleted of CD45RO⁺ cells by magnetic bead depletion (>90% purity) and were incubated in medium alone or were stimulated with anti-CD3 antibody, IL-7, or anti-CD3 antibody plus IL-7. CD4⁺CD45RO⁻CD28⁺CD27⁺ cells were assessed for the expression of Ki67 2 days poststimulation by flow cytometric analyses. The addition of IL-7 to anti-CD3 antibody enhanced the induction of Ki67 expression in cells from both HIV⁺ and HIV-negative (HIV⁻) donors (Fig. ​(Fig.11 and Fig. ​Fig.2).2). A diminished response to anti-CD3 antibody was observed among naïve CD4⁺ T cells from viremic HIV⁺ donors. In contrast, cells from aviremic HIV⁺ donors (all receiving antiretroviral therapy) had normal responses to anti-CD3 antibody compared to cells from healthy donors (Fig. ​(Fig.2).2). Importantly, the addition of IL-7 to the cultures significantly improved the responses to above those observed with anti-CD3 alone in HIV⁻ and HIV⁺ donors, regardless of viremia (Wilcoxon signed ranks test; for each comparison, P was <0.04), and the magnitude of that enhancement, although slightly diminished in cells from HIV⁺ donors, was not significantly different between groups of subjects when measured as either the enhancement (n-fold; not shown) or as the change in percent Ki67⁺ cells above the background observed for cells stimulated with anti-CD3 alone (Fig. ​(Fig.3).3). Although IL-7 enhanced responses to TCR stimulation in HIV⁻ subjects, the overall magnitude of the responses among cells from HIV viremic subjects did not reach the levels seen with cells from healthy donors, even in the presence of IL-7 (Fig. ​(Fig.2).2). It should be noted, however, that these functional readouts were not related to clinical indices of plasma HIV RNA level, CD4 cell count, or age when considered as continuous variables, suggesting that the functional perturbations in naïve CD4⁺ T cells are probably undermined by complexities extending beyond HIV replication (not shown). Together, these results suggest that TCR responsiveness is diminished in naïve CD4⁺ T cells from viremic HIV⁺ subjects, whereas responsiveness to IL-7 stimulation is relatively preserved.Open in a separate windowFIG. 1.IL-7 enhances the induction of Ki67 expression in naïve CD4⁺ T cells from healthy controls and HIV⁺ donors. CD45RO-depleted PBMC were incubated with anti-CD3 antibody (100 ng/ml), IL-7 (50 ng/ml), anti-CD3 antibody plus IL-7, or medium alone (RPMI with 10% fetal bovine serum). Cells were gated on CD4⁺CD27⁺CD28⁺ lymphocytes and examined for Ki67 expression by intracellular flow cytometry.Open in a separate windowFIG. 2.IL-7 responsiveness in cells from viremic and aviremic HIV⁺ donors. Plotted values represent the percentages of CD4⁺CD27⁺CD28⁺CD45RO⁻ T cells that expressed Ki67 after a 2-day incubation with anti-CD3 or with anti-CD3 plus IL-7. Percentages of Ki67⁺ cells in cultures without stimulation or with IL-7 only were subtracted from the values shown. Responses of cells from healthy controls (n = 9), HIV⁺ subjects with plasma HIV RNA levels of >400 copies/ml (n = 25), and HIV⁺ subjects on HAART with suppressed viral replication (<400 copies/ml; n = 12) are shown. Statistically significant differences between cells from controls and HIV⁺ donors are indicated. Analyses included Kruskal-Wallis test (P = 0.002) for multigroup comparisons and Mann-Whitney U test for comparison of two groups (*, P < 0.05).Open in a separate windowFIG. 3.IL-7 enhances responses to anti-CD3 antibody stimulation to a similar degree in cells from HIV⁺ and HIV⁻ donors. Naïve CD4⁺ T cells were incubated with IL-7, anti-CD3, anti-CD3 plus IL-7, or medium alone for 2 days. Background division (percent Ki67⁺ cells) in medium alone or IL-7 alone was first subtracted from the responses observed with cells stimulated with anti-CD3 alone or with anti-CD3 plus IL-7, respectively. The magnitude of IL-7 enhancement was then calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7. n = 9, 25, and 12 for healthy controls, viremic subjects, and aviremic subjects, respectively.Previous studies indicate that the frequency of CD127⁺ T cells, particularly memory T-cell subsets, is reduced in patients with HIV disease (5, 11, 21, 23). This could, in part, result from the modulation of receptor expression through increased exposure to IL-7 in vivo and also may reflect accumulation of CD127⁻ effector memory cells (21). We assessed the expression of CD127 in naïve CD4⁺CD45RA⁺CD28⁺CD27⁺ and memory CD4⁺CD45RO⁺ T cells in a subset of patients and asked if the frequencies of CD127⁺ cells were related to the induction of Ki67 expression by anti-CD3 or by anti-CD3 plus IL-7 among naïve CD4⁺ T cells. We reasoned that the ability of IL-7 to enhance responses to TCR stimulation might be limited if CD127 expression was diminished among naïve CD4⁺ T cells from HIV⁺ donors. Alternatively, a defect in functional responses also could be related to increased exposure to IL-7 in vivo, as may be reflected by the absence of CD127 receptor expression on memory T-cell subsets.In agreement with previous studies, our results suggest that CD127 expression is relatively preserved in naïve CD4⁺ T cells from HIV⁺ donors (representative histograms in Fig. ​Fig.4)4) (mean percentage of CD127⁺ cells, 87 and 83 for HIV⁻ donors [n = 5] and HIV⁺ donors [n = 17], respectively; P = 0.96) but is diminished in memory CD4⁺ T cells from HIV⁺ donors (mean percentage of CD127⁺ cells, 83 and 59 for HIV⁻ and HIV⁺ donors, respectively; P = 0.01). The frequencies of CD127⁺ naïve T cells were directly related to the frequencies of CD127⁺ memory T cells (Spearman''s correlations; r = 0.711, P = 0.001; n = 18) in HIV⁺ subjects. This result suggests that a similar mechanism modulates the expression of CD127 in these T-cell subsets, even though the loss of CD127 expression is clearly greater among the memory T cells in HIV disease. Neither CD127 expression among naïve CD4⁺ T cells nor CD127 expression among memory CD4⁺ T cells was related to the functional response of naïve CD4⁺ T cells to anti-CD3 (r = 0.238 and P = 0.36 for naïve CD127 expression; r = 0.293 and P = 0.25 for memory CD127 expression) or to anti-CD3 plus IL-7 (r = 0.32 and P = 0.21 for naïve CD127 expression; r = 0.31 and P = 0.22 for memory CD127 expression). There was a relationship between the percentage of CD127⁺ naïve T cells and the delta Ki67 expression that resulted from the addition of IL-7 to anti-CD3-treated cultures (percentage of Ki67⁺ cells in cultures treated with anti-CD3 plus IL-7 minus the percentage of Ki67⁺ cells in cultures treated with anti-CD3 alone) (Fig. ​(Fig.4).4). This relationship was statistically significant by Pearson''s correlation (r = 0.5, P = 0.041), the use of which was justified based on the normal distribution of the data. Spearman''s analysis, which is independent of data distribution, indicated a similar trend that was not statistically significant (r = 0.41, P = 0.1). The mean fluorescence intensity of CD127 expression on CD4⁺CD45RA⁺CD27⁺CD28⁺ T cells was not significantly related to the delta Ki67 expression induced by IL-7 but also suggested a trend consistent with a direct relationship between these indices (r = 0.45 and P = 0.07 by Pearson''s correlation; r = 0.34 and P = 0.18 by Spearman''s correlation). Despite the relative preservation of IL-7 receptor in naïve CD4⁺ T cells from HIV⁺ donors, the association between the frequencies of CD127⁺ cells and CD4⁺ T-cell proliferation responses to TCR plus IL-7 suggests that subtle IL-7 receptor perturbations might contribute to functional defects of naïve CD4⁺ T cells in HIV-infected persons.Open in a separate windowFIG. 4.CD127 receptor expression is related to enhancement of proliferation by IL-7. (A) Whole blood from a healthy control and an HIV-infected person was examined by flow cytometry for expression of CD127 on CD4⁺CD45RA⁺CD27⁺CD28⁺ (naïve) T cells. The gating strategy for identifying naïve cells involved an initial gate for lymphocyte forward and side scatter (SSC) characteristics (not shown) and then sequential gates for CD4 positive, CD45RA positive and, finally, CD28⁺CD27⁺ double-positive cells. (B) Plotted values indicating the relationship between the delta Ki67 expression in naïve CD4⁺ T cells and the percentage of CD127⁺ naïve T cells that was determined by using freshly isolated whole blood. The delta Ki67 expression was calculated by subtracting the percentage of naïve CD4⁺ cells that expressed Ki67⁺ after anti-CD3 antibody stimulation from the percentage of naïve CD4⁺ cells that expressed Ki67 after stimulation with anti-CD3 plus IL-7.To consider the possibility that antigen-presenting cells could contribute to the diminished response of T-cells to stimulation with TCR plus IL-7, we next asked if defects in TCR-plus-IL-7 stimulation could be detected in purified naïve CD4⁺ T-cell populations. CD4⁺CD45RO⁻ cells were negatively selected by magnetic bead depletion, achieving a purity of >90% as determined by flow cytometric analyses. Purified naïve CD4⁺ T cells were labeled with carboxy fluorescein succinimidyl ester (CFSE) tracking dye and incubated with IL-7, anti-CD3 antibody that was immobilized on a plate, anti-CD3 plus IL-7, or medium alone. The induction of proliferation was measured 7 days later by the dilution of CFSE tracking dye among CD4⁺CD27⁺ cells by calculating the division index (average number of cell divisions of all CD4⁺CD27⁺ cells) and the proliferation index (average number of divisions of CD4⁺CD27⁺ cells that had diluted tracking dye; Flow-Jo analysis software). These purified CD4⁺ T cells proliferated poorly in response to anti-CD3 antibody stimulation alone, providing functional evidence that the samples were free of antigen-presenting cell contamination (Fig. ​(Fig.5A).5A). The combined treatment of anti-CD3 and IL-7 induced cellular expansion, whereas alone, neither stimulus induced cellular proliferation during the 7-day period (Fig. ​(Fig.5A).5A). Responses of cells from HIV⁺ donors were reduced compared to those of cells from healthy donors, confirming that the defects in naïve CD4⁺ T-cell expansion are independent of antigen-presenting cells and not fully corrected by IL-7 (Fig. ​(Fig.5B5B).Open in a separate windowFIG. 5.Diminished responses to TCR plus IL-7 in purified naïve CD4⁺ T cells from HIV⁺ donors. CD4⁺CD45RO⁻ cells were purified from PBMC by negative selection. Cells from HIV⁺ donors (n = 7) and healthy controls (n = 7) were labeled with CSFE and incubated with anti-CD3 immobilized on a plate (5 μg/ml, overnight at 4°C) plus IL-7 (10 ng/ml). CFSE dye dilution was measured among the CD4⁺CD27⁺ cells. (A) Representative histograms showing the dilution of CFSE and CD27 expression among cells incubated with anti-CD3 antibody alone, IL-7 alone, or the combination of anti-CD3 plus IL-7. Placements of quadrant gates were based on an isotype control antibody stain (for CD27 expression) and on cells that had been incubated in medium alone (for CFSE dye dilution). (B) Division indices (average number of cell divisions among CD4⁺CD27⁺ cells) and proliferation indices (average number of cell divisions among CD4⁺CD27⁺ cells that had diluted tracking dye) are shown.IL-7 is a promising candidate for therapeutic and vaccine adjuvant applications in HIV disease. This cytokine may be especially beneficial in circumstances of immune reconstitution, since it normally plays an essential role in T-cell proliferation and survival. Here, we demonstrate that IL-7 efficiently enhances TCR-triggered naïve CD4⁺ T-cell expansion in cells from healthy individuals and from HIV⁺ donors. The mechanism of IL-7 activity is not discerned in these experiments but may involve effects on survival, such as the induction of Bcl-2 (9), or may involve the enhancement of IL-2 or IL-2 receptor expression (6, 8). In any case, our studies provide evidence that IL-7 should provide an effective therapy for the regulation of naïve CD4⁺ T-cell homeostasis and may be useful for vaccine adjuvant applications in HIV disease. The potential of this approach has been illustrated by recent human trials of IL-7 that demonstrated the expansion of naïve T cells in vivo after IL-7 administration to HIV-infected persons (13) and by animal studies, wherein IL-7 administration enhanced T-cell responses to immunization in mice (17).Notably, the depletion studies and purification methods employed here did not necessarily eliminate terminally differentiated effector memory CD4⁺ T cells from our cultures; however, studies of CMV-specific terminally differentiated cells suggested that these cells are primarily CD27⁻ (3), and the use of three markers to identify naïve CD4⁺ T cells, including the ones used here (CD27, CD28, and CD45RO) is estimated to provide 98% assurance that the cells being examined are truly naïve (7). Thus, it is likely that terminally differentiated cells were largely removed from our analyses.Our observations provide confirmation of a significant defect in the responses of naïve CD4⁺ T cells to TCR triggering in HIV disease, and this defect is not fully corrected by IL-7, as shown here, or by IL-2, as we demonstrated previously (27). These deficiencies are reproduced even among naïve CD4⁺ T cells that are purified from professional antigen-presenting cells, indicating that the defects are intrinsic to the T cells and not a consequence of dysfunctional antigen-presenting cells. We propose that functional defects in naïve CD4⁺ T cells from HIV⁺ donors stem primarily from deficiencies in TCR signaling. Further studies that define the nature of naïve CD4⁺ T-cell defects in HIV disease will be required to address the underlying mechanisms.     

Endothelial origin of mesenchymal stem cells

Mesenchymal stem/stromal cells (MSCs) are fibroblastoid cells capable of long-term expansion and skeletogenic differentiation. While MSCs are known to originate from neural crest and mesoderm, immediate mesodermal precursors that give rise to MSCs have not been characterized. Recently, using human embryonic stem cells (hESCs), we demonstrated that mesodermal MSCs arise from APLNR⁺ precursors with angiogenic potential, mesenchymoangioblasts, which can be identified by FGF2-dependent colony-forming assay in serum-free semisolid medium. In this overview we provide additional insights on cellular pathways leading to MSC establishment from mesoderm, with special emphasis on endothelial-mesenchymal transition as a critical step in MSC formation. In addition, we highlight an essential role of FGF2 in induction of angiogenic cells with potential to transform into MSCs (mesenchymoangioblasts) or hematopoietic cells (hemangioblasts) from mesoderm, and discuss correlations of our in vitro findings with the course of angioblast development during embryogenesis.Key words: mesenchymoangioblast, hemangioblast, human embryonic stem cells, endothelial-mesenchymal transition, epithelial-mesenchymal transition, mesenchymal stem cells, endothelial cells, apelin receptor, FGFMesenchymal stem/stromal cells (MSCs) are defined as multipotent fibroblastoid cells that give rise to cells of the skeletal connective tissue including osteoblasts, chondrocytes and adipocytes.^1–4 Although MSCs were described more than 40 years ago and are widely used for cellular therapies, very little knowledge exists regarding the developmental origins of MSCs in the embryo, the hierarchy of MSC progenitors or heterogeneity of MSCs within tissues. It has been demonstrated that during embryonic development, MSCs arise from a two major sources: neural crest and mesoderm.^5–7 Using Cre-recombinase lineage tracing experiments, Takashima et al. identified Sox1⁺ neuroepithelium as pre-cursors of MSCs of neural crest origin. However, direct precursors of mesoderm-derived MSCs were unknown. To identify these precursors, we employed human embryonic stem cells (hESCs) directed toward mesendodermal differentiation in coculture with mouse bone marrow stromal cells OP9,⁸ using the experimental approach depicted in Figure 1. As shown in this differentiation system, mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by expression of apelin receptor (APLNR), otherwise known as angiotensin receptor like-1 receptor. Because we observed a positive selective effect of FGF2 on production of mesenchymal cells from hESCs in OP9 coculture, we decided to test whether FGF2 can induce the formation of colonies with mesenchymal potential from APLNR⁺ mesodermal cells. Indeed, when we isolated APLNR⁺ cells from hESCs differentiated on OP9 for 2 days and placed them in serum-free semisolid medium containing FGF2, we observed the formation of sharply-circumscribed spheroid colonies formed by tightly packed cells with a gene expression profile representative of embryonic mesenchyme originating from lateral plate/extraembryonic mesoderm and CD140a⁺CD146⁺C D90⁺CD56⁺CD166⁺CD31⁻CD43⁻CD45⁻ phenotype typical of mesenchymal cells. Based on cellular composition, we designated these colonies as mesenchymal (MS) colonies and cells forming these colonies as MS colony-forming cells (MS-CFCs). MS colony formation required serum-free medium and was solely dependent on FGF2 as a colony-forming factor. MS colonies were significantly enhanced by PDGF-BB, but suppressed by VEGF, TGFβ1 and Activin A. When transferred to the adherent cultures in serum-free medium with FGF2, individual MS colonies gave rise to multi-potential mesenchymal cell lines with typical phenotype (CD146⁺ CD105⁺ CD73⁺ CD31⁻ CD43/45⁻), differentiation (chondro-, osteo- and adipogenesis) and robust proliferation (>80 doublings) potentials. Using single cell deposition assay, chimeric hESC lines and time-lapse studies we demonstrated the clonality/single cell origin of MS colonies.Open in a separate windowFigure 1Schematic diagram of the experimental approach used to identify precursors and cellular events leading to formation of mesoderm-derived MSCs. hESCs were committed to mesendodermal differentiation through coculture with OP9 for 2 days. APLNR⁺ mesodermal cells were selected using magnetic sorting. In serum-free semisolid medium, APLNR⁺ cells grew into FGF2-dependent compact spheroid colonies composed of mesenchymal cells. MS colonies were formed through establishment of tightly-packed single cell-derived cores (day 3 of clonogenic culture), which expanded into spheroid colonies (days 6 and 12 of clonogenic culture). To evaluate differentiation potential, MS colonies were collected at different stages of clonogenic culture and placed on OP9. The presence of endothelial and mesenchymal cells after coculture of MS colonies with OP9 was evaluated by flow cytometry and immunofluorescence. In addition, colonies at core stage (day 3 of clonogenic culture) and mature colonies (day 12 of clonogenic cultures) were collected for molecular profiling studies. To generate clonal MSC lines, individual mature colonies were plated on the collagen/fibronectin-coated plastic and cultured in presence of FGF2.MS-CFCs could be detected only transiently, with a major peak on day 2 of hESC differentiation and disappeared after 4 days of differentiation. Notably, MS-CFC activity was developed prior to the expression of CD73 and CD105 MSC markers and upregulation of MSC-related genes, i.e., before onset of mesenchymogenesis. APLNR⁺ cells isolated from hESC cultures differentiated for 3 days also formed colonies in response to FGF2; however, the vast majority of these colonies were composed of blood cells and had a morphology similar to the previously described blast (BL) or hemangioblast colonies, which identify a common precursor for hematopoietic and endothelial cells.^9,10To fully evaluate the differentiation potential of MS colonies, we collected these colonies from semisolid cultures and placed them back on OP9 feeders, which are known to support development of a broad range of mesodermal lineage cells, including hematopoietic, vascular and cardiac.^11–13 Using this approach, we confirmed that individual BL colonies possess hemangioblastic potential, i.e., generate both hematopoietic and endothelial cells. When MS colonies were picked from clonogenic cultures and cultured on OP9, we found that the majority of cells differentiated into CD146⁺CD31⁻CD43/CD45⁻ mesenchymal cells as expected. However, we also discovered that MS colonies gave rise to CD31/VE-cadherin⁺CD43/45⁻ endothelial cells, indicating that MS colonies similar to BL colonies possess endothelial potential. The endothelial potential of MS colonies was also confirmed by demonstration of tube formation by MS colonies grown on Matrigel. In contrast, MSC lines derived from MS colonies did not produce any endothelial cells after coculture with OP9 indicating a progressive restriction of differentiation potential following MSC formation. Because single MS-CFC shows potential to form endothelium and MSCs, we designated the MSC precursor identified by this colony-forming assay as mesenchymoangioblast.To define more precisely the cellular events leading to establishing MSCs, we examined the formation of MS colonies using time-lapse cinematography and analyzed the kinetic of their angiogenic potential. As demonstrated by time-lapse studies, APLNR⁺ mesodermal cells placed in semisolid medium possessed a high motility, which was more pronounced before and during the first cell division. Following several divisions, single APLNR⁺ cells formed a core, an immotile structure composed of a small number of tightly packed cells. While APLNR⁺ mesodermal cells lacked endothelial gene expression, molecular profiling of MS colonies at the core stage revealed that these cells acquired angioblastic gene expression profile as indicated by upregulation of FLT1, TEK, CDH5 (VE-cadherin), PECAM1 (CD31), FLI1, SELE (ELAM-1) and ICAM2 endothelial genes. When we collected MS cores (day 3 of clonogenic culture) and placed them on OP9, they formed predominantly VE-cadherin⁺ endothelial clusters, strongly indicating the endothelial nature of the core-forming cells. Subsequently, cells at the periphery of the core underwent endothelial-mesenchymal transition (EndMT) and formed a shell of tightly packed spindle-like cells around the core. When we picked colonies at this stage (day 6 of colony-forming culture) and placed them on OP9, most of the colonies (>70%) grew cell clusters composed of endothelial and mesenchymal cells. In contrast, mature MS colonies collected on day 12 of clonogenic culture formed predominantly clusters of mesenchymal cells, indicating a progressive loss of endothelial potential following colony maturation. Although no CD31 expression was detected in the mesenchymal cells composing mature MS colonies, these cells retained several endothelial traits including surface expression of endothelial tyrosine kinase (TEK or TIE2), FLT1 (VEGFR1) and endomucin. The critical role of EndMT in MS colony formation and MSC development was also congruous with our observation of the suppressive effect of VEGF, a known inhibitor of EndMT,^14,15 on MS colonies. When VEGF was added to MS clonogenic cultures, hESC-derived mesodermal cells were capable of forming angiogenic cores; however, these cores did not transform into mesenchymal cells, indicating that VEGF abrogates MS colony development at the core stage through inhibition of EndoMT. The schematic diagram demonstrating development of mesodermal MSCs is presented in Figure 2.Open in a separate windowFigure 2A model of mesoderm-derived MSC development from hESCs. Coculture with OP9 stromal cells predominantly induces hESC differentiation toward APLNR⁺ mesoderm. APLNR⁺ population contains angiogenic mesodermal precursors with either mesenchymal (mesenchymoangioblast) or hematopoietic (hemangioblast) potentials. Mesenchymoangioblasts and hemangioblasts arise sequentially during differentiation and can be revealed by MS and BL colony formation in response to FGF2. Development of MS and BL colonies in semisolid media proceed through a core stage at which APLNR⁺ cells form clusters of tightly packed cells with angiogenic potential. Subsequently, core-forming cells undergo EndMT giving rise to mesenchymal cells, which form a shell around the core developing into a mature MS colony. VEGF, EndMT inhibitor, blocks MS colony-formation at core stage. The ability of MS-CFCs to generate mesenchymal and endothelial cells can be revealed by coculture of individual colonies with OP9. Similar to MS colonies, BL colonies are formed through establishment of angiogenic core. However, hemangioblast core-forming cells undergo endothelial-hematopoietic transition and grew hematopoietic cells around the core.The close relationship between endothelial and hematopoietic cell development was recognized more than 130 years ago (reviewed by ref. ¹⁶) and confirmed in multiple modern studies.^9,17–22 However, the association between endothelial pre-cursors and MSCs during development was not well established, although cells with endothelial and mural cell potential were identified²³ and the critical role of EndMT in the formation of endocardial cushion²⁴ and testicular cords²⁵ in the embryo was acknowledged. Our hESC-based in vitro studies indicated that formation of mesodermal MSCs proceed through the endothelial stage and likely included at least two successive cycles of cell transitions. Initially APLNR⁺ mesoderm, which consists of fibroblast-like migratory cells, give rise to core structures composed of tightly packed endothelial cells in response to FGF2. Subsequently, endothelial cells forming cores undergo epithelial-mesenchymal transition, i.e., EndMT and form MSCs. The question remains how well this in vitro model reflects in vivo development. Although only sparse data exist regarding MSC precursors in the embryo, development of angiogenic hematopoietic precursors, hemangioblasts was studied more extensively in mammals and birds, and therefore parallels between in vivo and in vitro studies can be drawn. As we demonstrated,⁸ APLNR⁺ mesodermal cells collected from hESCs differentiated on OP9 for 3 days formed disperse BL colonies that identify hemangioblasts in vivo and in vitro.^9,26 Similar to MS colonies, the development of BL colonies required FGF2 and proceeded through angiogenic core formation. However, in contrast to MS cores, BL cores transformed into blood cells, i.e., underwent endothelial-hematopoietic transformation (see Fig. 2). Importantly, in vivo studies identified FGF2 as the essential factor in hemangioblast induction²⁷ analogous to our in vitro observation. In chicken embryo, the activation of FGF signaling leads to aggregation of migrating mesodermal cells adjacent to the endoderm, upregulation of VEGFR2 (KDR) expression, and subsequent formation of angioblasts and hemangioblasts.^28–30 This sequence of events leading to hemangioblast development in vivo considerably resembles what we observed in vitro, and highly suggests accurate recapitulation of embryonic development by our hESC differentiation model. Therefore, searching for an in vivo equivalent of mesenchymonagioblast would be a reasonable next step.In addition to embryonic development, EndMT is also implicated in several pathologies including cancer progression and development of cardiac and renal fibrosis.^31–34 Recently, Olsen group revealed that endothelial cells could be transformed directly into MSCs through overexpression of ALK2 or its activation by TGFβ2 or BMP4,¹⁵ indicating that endothelial cells could be an important source of MSCs in postnatal life. Conversely, the transition from MSCs to endothelial cells, has been also described in reference ³⁵. Based on these observations, a cycle of cell-fate transition from endothelium to MSCs and back to endothelium was proposed as a circuit controlling stem cell state.³⁶ Since multiple parallels could be drawn between EndMT described in adult tissues and during hESC differentiation, one may wonder whether bipotential cells with endothelial and MSC potential similar to embryonic mesenchymoangioblasts are present and constitute an important element of EndMT circuit in adults.In conclusion, the identification of mesenchymoangioblast as a clonogenic precursor of mesoderm-derived MSCs is an important step toward defining pathways of MSC development and specification. In addition, the demonstration of MSC formation from mesoderm through EndMT provides new insights into the mechanisms involved in establishment of MSCs.     

Study of the involvement of osmotic adjustment and H<Superscript>+</Superscript>-ATPase activity in the resistance of <Emphasis Type="Italic">Catharanthus roseus</Emphasis> suspension cells to salt stress

The salt-induced H⁺-ATPase activity and osmotic adjustment responses of Catharanthus roseus (L.) G. Don suspension cultures were studied. Cells were treated with 0, 50 or 100mM NaCl for 7days or were maintained for 8 months with 50 mM NaCl (50T cells). Growth, osmotic potential (), ions content, soluble sugars, proline and total amino acids were determined in the sap of control and salt-treated cells. Salinity reduced cell growth and . The higher decrease in the in salt-treated cells was due to higher accumulation of Na⁺ and Cl^–. The levels of organic solutes, such as soluble sugars, free proline and total amino acids, increased with salt treatment. These results suggest that salt-tolerant cells are able to osmotically adjust. Salinity treatments stimulated H⁺-ATPase activity. Immunodetection of the enzyme showed that the increased activity was due to an increased amount of protein in the plasmalemma. The induction by NaCl, especially at 100 mM NaCl and for 50T cells, could account for the K⁺ and Cl^– uptake but not for higher or lower tolerance.     

IP-FCM: Immunoprecipitation Detected by Flow Cytometry

Immunoprecipitation detected by flow cytometry (IP-FCM) is an efficient method for detecting and quantifying protein-protein interactions. The basic principle extends that of sandwich ELISA, wherein the captured primary analyte can be detected together with other molecules physically associated within multiprotein complexes. The procedure involves covalent coupling of polystyrene latex microbeads with immunoprecipitating monoclonal antibodies (mAb) specific for a protein of interest, incubating these beads with cell lysates, probing captured protein complexes with fluorochrome-conjugated probes, and analyzing bead-associated fluorescence by flow cytometry. IP-FCM is extremely sensitive, allows analysis of proteins in their native (non-denatured) state, and is amenable to either semi-quantitative or quantitative analysis. As additional advantages, IP-FCM requires no genetic engineering or specialized equipment, other than a flow cytometer, and it can be readily adapted for high-throughput applications.Download video file.^{(71M, mov)}     

Caspase 9 Is Essential for Herpes Simplex Virus Type 2-Induced Apoptosis in T Cells

Herpes simplex virus type 2 (HSV-2) induces apoptosis in T cells by a caspase-dependent mechanism. Apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways. Here, we show that the initiator caspase for the intrinsic pathway is activated in T cells following HSV-2 exposure. To determine the respective contributions of intrinsic and extrinsic pathways, we assessed apoptosis in Jurkat cells that are deficient in caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. Our results indicate HSV-2-induced apoptosis in T cells occurs via the intrinsic pathway.Herpes simplex virus (HSV) reactivation in human results in accumulation and persistence of virus-specific CD4⁺ and CD8⁺ cells at the site of reactivation (15, 16). Despite such immune responses, virus reactivation can occur on >75% of days for some individuals (14). Reactivation of the virus in the midst of continued cell-mediated immunity demonstrates the ability of the virus to circumvent the host''s immune system. HSV antigens were readily detected from T cells isolated from human HSV lesions, indicating that T cells are infected with HSV in vivo (1). In vitro, HSV-infected T cells undergo apoptosis (5-7, 10), and we have additionally demonstrated that apoptosis occurs in Jurkat cells, a T-cell leukemia line, and primary CD4⁺ T lymphocytes isolated from human peripheral blood mononuclear cells following exposure to HSV type 2 (HSV-2)-infected human foreskin fibroblasts (5). These results suggest that induction of T-cell death is a mechanism by which the virus limits the effectiveness of local cell-mediated immunity during reactivation.Since T cells are most likely exposed to HSV-2 via infected epithelial cells in vivo, we examined T-cell apoptosis following exposure to infected fibroblasts in vitro. To evaluate whether HSV-2-exposed primary T cells undergo apoptosis by a caspase-dependent mechanism, we examined activation of caspase 3 in human CD4⁺ cells after exposure to HSV-2-infected fibroblasts. Human foreskin fibroblasts (American Type Culture Collection, Manassas, VA) were mock infected or infected with the HSV-2 HG52 strain at a multiplicity of infection of 5. At 6 h postinfection, CD4⁺ cells were exposed to mock-infected or HSV-2-infected fibroblasts at a ratio of approximately 3:1 (lymphocytes to fibroblasts). CD4⁺ cells were isolated to >95% purity using MACS CD4 microbeads (Miltenyi Biotec, Inc., Auburn, CA) immediately prior to experiments with human peripheral mononuclear cells (Memorial Blood Centers, St. Paul, MN) that were stimulated with phytohemagglutinin (Sigma Aldrich, St. Louis, MO) for 48 h and then maintained with interleukin-2 (Invitrogen, Carlsbad, CA) as previously described (5). After 4 h of incubation, CD4⁺ cells were harvested and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 0.3 ng/ml interleukin-2. At 24 h and 48 h postexposure, CD4⁺ cells were probed with a fluorescence-labeled antibody against activated caspase 3 (BD Biosciences, San Jose, CA). Data were collected on a FACSCanto (BD Biosciences) and analyzed with FlowJo software (Tree Star, Ashland, OR). Side scatter and fluorescence were used as dual parameters to clearly gate the cell population with activated caspase 3, and an identical gate was applied to all samples within each experiment. As shown in Fig. ​Fig.1,1, the percentage of cells with activated caspase 3 was 23% in HSV-2-exposed CD4⁺ cells compared with 5% in mock-exposed cells at 24 h postexposure. At 48 h, the percentage of cells with activated caspase 3 increased to 39% in HSV-2-exposed cells compared to 8% in mock-exposed cells. These percentages are comparable to those for Jurkat cells in our previous report (19% for virus-exposed cells versus 5% for mock-exposed cells at 24 h postexposure) (5).Open in a separate windowFIG. 1.Activation of caspase 3 in CD4⁺ cells following exposure to mock- or HSV-2-infected fibroblasts. CD4⁺ cells were isolated from human peripheral mononuclear cells and exposed to mock- or HSV-2-infected fibroblasts. At 24 h and 48 h postexposure, cells were probed with an antibody for activated caspase 3 and analyzed by flow cytometry. (A) A representative flow cytometry analysis from three independent experiments is shown. Side scatter was used as a second parameter to identify the cell populations. An identical gate was applied to all samples within each experiment. Increased percentages of cells with caspase 3 activation are seen following exposure to HSV-2-infected fibroblasts compared to those following exposure to mock-infected fibroblasts. (B) Mean percentages of CD4⁺ cells with caspase 3 activation from three independent experiments at 24 h and 48 h postexposure are shown with standard errors bars.Efficient infection of T cells by HSV upon exposure to HSV-infected human fibroblasts via cell-to-cell spread has been recently demonstrated (1). To delineate the relationship between virus infection and apoptosis, Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above and then coprobed with antibodies for activated caspase 3 and HSV-2 ICP10. As shown in Fig. ​Fig.2,2, two antibodies demonstrated largely mutually exclusive populations in cells exposed to HSV-2-infected fibroblasts. We obtained similar results with antibodies against other HSV-2 antigens, including glycoprotein B, ICP5, and ICP8 (data not shown). The results suggest that infected cells may be inducing apoptosis in uninfected cells via a bystander effect, similar to what has been proposed for HSV-1-infected activated cytotoxic T cells (11). Alternatively, the activation of apoptosis may result in degradation of viral antigens in infected cells.Open in a separate windowFIG. 2.Expression of HSV-2 antigen and caspase 3 activation in Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. Jurkat cells were first exposed to mock- or HSV-2-infected fibroblasts and then coprobed with antibodies for HSV-2 ICP10 and activated caspase 3 at 24 h postexposure. A representative flow cytometry analysis from three independent experiments is shown. Each antibody detected largely mutually exclusive cell populations following exposure to HSV-2-infected fibroblasts.Our results demonstrate an activation of caspase 3 in HSV-2-exposed T cells, suggesting a caspase-mediated apoptosis. Previous studies have supported such a mechanism, as caspase inhibitors block virus-induced cell death (5, 10). Caspase-dependent apoptosis can occur via extrinsic (death receptor) and/or intrinsic (mitochondrial) pathways (3). Here, we sought to determine the respective contribution of each pathway in HSV-2-induced T-cell death.To investigate the contribution of individual apoptosis pathways in HSV-2-exposed T cells, we first evaluated mitochondrial membrane potential in CD4⁺ and Jurkat cells following virus exposure. Mitochondria play a major role in the intrinsic apoptosis pathway (2, 3). T cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above and then probed with chloromethyl-X-rosamine (CMXRos; Invitrogen) at 24 h and 48 h postexposure. CMXRos is a lipophilic cationic fluorescent dye whose staining of cells is dependent on negative mitochondrial membrane potential, and a loss of staining indicates a loss of mitochondrial potential (2, 9). As shown in Fig. ​Fig.3,3, a loss of mitochondrial membrane potential was observed in increased percentages of HSV-2-exposed CD4⁺ and Jurkat cells compared to those for mock-exposed cells. The percentage of cells with a loss of mitochondrial membrane potential reached 49% for CD4⁺ cells and 92% for Jurkat cells at 48 h postexposure. The changes in mitochondrial membrane potential in HSV-2-exposed T cells suggest that the intrinsic apoptosis pathway is activated in these cells. We previously reported that Jurkat and CD4⁺ cells were equally susceptible to apoptosis induced by anti-Fas monoclonal antibody (5). A recent report suggests that activated primary T cells are less susceptible to HSV infection than are Jurkat cells (1). Thus, we believe that a lower percentage of CD4⁺ cells with a loss of mitochondrial membrane potential than Jurkat cells is due to lower susceptibility to HSV infection.Open in a separate windowFIG. 3.A loss of mitochondrial membrane potential in CD4⁺ and Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. CD4⁺ and Jurkat cells were exposed to mock- or HSV-2-infected fibroblasts. At 24 h and 48 h postexposure, cells were probed with chloromethyl-X-rosamine (CMXRos). (A) Increased percentages of CD4⁺ cells with a loss of mitochondrial membrane potential are seen following exposure to HSV-2-infected fibroblasts compared to those seen after mock exposure. Shown are mean percentages of cells with a loss of mitochondrial membrane potential at 24 h and 48 h postexposure, with standard error bars, from three independent experiments. (B) Shown are mean percentages of cells with a loss of mitochondrial membrane potential for Jurkat cells at 24 h and 48 h postexposure, with standard error bars, from three independent experiments.To further define the involvement of intrinsic and extrinsic apoptosis pathways in HSV-2-induced T-cell apoptosis, we investigated whether caspase 8, an initiator protease for the extrinsic pathway, and caspase 9, an initiator protease for the intrinsic pathway, are activated in HSV-2-exposed T cells. CD4⁺ and Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above. Cell lysates were made at 24 h postexposure, and immunoblots were performed with 30 to 50 μg of protein per well, as described previously (5), with anti-caspase 8 and anti-caspase 9 antibodies (Cell Signaling, Danvers, MA). A 24-h time point was chosen for the immunoblots in order to minimize the detection of baseline apoptotic activity that can be seen with primary CD4⁺ cells in cultures with a longer incubation time.As shown in Fig. ​Fig.4,4, CD4⁺ cells that were exposed to HSV-2 had an increase in cleaved, activated fragments of caspase 9. No clear differences between mock-infected and HSV-2-exposed CD4⁺ cells were seen for caspase 8 cleavage. In contrast, increases in both cleaved caspase 8 and cleaved caspase 9 were evident in Jurkat cells following virus exposure. Together, these results indicate that caspase 9 is activated in HSV-2-exposed T cells and suggest activation of the intrinsic apoptosis pathway. Although activation of caspase 8 was seen with Jurkat cells, the contribution of the extrinsic pathway remained in doubt for virus-induced apoptosis in CD4⁺ cells.Open in a separate windowFIG. 4.Detection of cleaved caspase 8 and caspase 9 in CD4⁺ and Jurkat cells following exposure to mock- or HSV-2-infected fibroblasts. Lysates from cells exposed to mock- or HSV-2-infected fibroblasts were assayed for uncleaved and cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from more than three independent experiments are shown. Blots demonstrate increases in cleaved caspase 9 for CD4⁺ and Jurkat cells. An increase in cleaved caspase 8 is also seen for Jurkat cells.To determine the respective contributions of each pathway, we assessed apoptosis in Jurkat cells that are deficient in either caspase 8 or Fas-associating protein with death domain (FADD) for the extrinsic pathway and in cells deficient in caspase 9 for the intrinsic pathway. We chose caspase 3 activation to assess apoptosis in HSV-2-exposed cells, because convergence of extrinsic and intrinsic apoptosis pathways occurs at caspase 3.Jurkat cells deficient in caspase 8 or FADD (American Type Culture Collection) have been previously described (8), and we confirmed the absence of caspase 8 and FADD in respective cell lines (Fig. ​(Fig.5A).5A). We also investigated the parental Jurkat cell A3 subclone and verified that both caspase 8 and FADD were expressed as expected (Fig. ​(Fig.5A).5A). Parental and deficient Jurkat cells were exposed to mock-infected or HSV-2-infected fibroblasts as described above. At 24 h postexposure, cells were probed with fluorescence-labeled antibody against activated caspase 3. As shown in Fig. ​Fig.5B,5B, cells deficient in caspase 8 or FADD underwent apoptosis at levels similar to those of parental cells as determined by caspase 3 activation. The results indicate that neither caspase 8 nor FADD is required for HSV-2-induced apoptosis in Jurkat cells. We additionally evaluated for cleavage products of caspase 8 and caspase 9 in these cells. As shown in Fig. ​Fig.5C,5C, cleaved fragments of caspase 9 were seen in all three cell types following HSV-2 exposure. Interestingly, cleaved fragments of caspase 8 were detected in both parental and FADD-deficient cells, suggesting that FADD is not required for cleavage of caspase 8 following HSV-2 exposure.Open in a separate windowFIG. 5.Loss of caspase 8 or FADD expression does not impede HSV-2-induced apoptosis in Jurkat cells. (A) Immunoblots of I2.1, I9.2, and A3 cell lysates were probed with antibodies for procaspase 8, FADD, and β-actin. FADD is not expressed in I2.1 cells, while caspase 8 is not expressed in I9.2 cells. The parental cell line A3 is a subclone of Jurkat cells that expresses both caspase 8 and FADD. β-Actin expression is shown as a control. (B) Percentages of cells with caspase 3 activation are comparable in cells with caspase 8 or FADD deficiency and in parental A3 cells following exposure to HSV-2. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments. (C) Lysates from cells exposed to mock- or HSV-2-infected fibroblasts were probed for cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from three independent experiments are shown. Blots demonstrate increases in cleaved caspase 8 for I2.1 and A3 cells that were exposed to HSV-2. Increases in cleaved caspase 9 are seen for all three cell types.Jurkat cells deficient in caspase 9 expression and the corresponding caspase 9-reconstituted clones (12, 13) were a gift of Ingo Schmitz (University of Dusseldorf, Dusseldorf, Germany). Immunoblot analysis confirmed the presence or absence of caspase 9 in both cell lines (Fig. ​(Fig.6A).6A). Cells were exposed to mock- or HSV-2-infected fibroblasts and analyzed for activation of caspase 3 as described above. Following exposure to HSV-2, cells deficient in caspase 9 were protected from apoptosis as determined by caspase 3 activation (Fig. ​(Fig.6B).6B). In contrast, cells with reconstituted caspase 9 showed an increased percentage of caspase 3 activation. An evaluation of caspase 8 and caspase 9 cleavage in these cells showed that cleaved fragments were seen only in the cells reconstituted with caspase 9 (Fig. ​(Fig.6C).6C). To confirm that the findings were applicable to the parental Jurkat cell line, we evaluated the effect of a caspase 9 inhibitor, Z-LEHD-fmk (R&D Biosystems, Minneapolis, MN) on Jurkat cells that were exposed to mock- or HSV-2-infected fibroblasts. Jurkat cells were incubated with 100 μM Z-LEHD-fmk or a dimethyl sulfoxide (DMSO) solvent control for 1 h before exposure to fibroblasts, throughout the exposure, and postexposure. At 24 h postexposure, the percentage of virus-exposed cells with activated caspase 3 was inhibited by Z-LEHD-fmk to 3%, identical to the baseline percentage seen with mock-exposed cells with DMSO solvent control (Fig. ​(Fig.6D).6D). Together, these findings indicate that caspase 9 is required for HSV-2-induced apoptosis and cleavage of both caspase 8 and caspase 9 in Jurkat cells.Open in a separate windowFIG. 6.Caspase 9 is required for HSV-2-induced apoptosis in Jurkat cells. (A) Immunoblots of JMR and F9 cell lysates probed with antibodies for procaspase 9 and β-actin. Caspase 9 is not expressed in JMR cells, a subclone of Jurkat cells. Caspase 9 expression is reconstituted in F9 cells, which are JMR cells stably transfected with a plasmid expressing caspase 9. (B) JMR cells with caspase 9 show protection from HSV-2-induced apoptosis, while F9 cells with reconstituted caspase 9 are susceptible to apoptosis induced by the virus. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments. (C) Lysates from JMR and F9 cells were exposed to mock- or HSV-2-infected fibroblasts and then probed for cleaved forms of caspase 8 and caspase 9 by immunoblotting at 24 h postexposure. Representative immunoblots from three independent experiments are shown. Blots demonstrate an increase in cleaved caspase 8 and caspase 9 for F9 cells but not JMR cells. (D) Jurkat cells that were incubated with Z-LEHD-fmk show protection from HSV-2-induced apoptosis compared to results with the DMSO solvent control at 24 h postexposure. Mean percentages of cells with caspase 3 activation, with standard error bars, are shown. The graph represents results from three independent experiments.Our results provide evidence that exposure to HSV-2-infected fibroblasts leads to apoptosis in T cells. It is important to note that our method of activation and maintenance of human peripheral blood mononuclear cells enriches for CD4⁺ cells. Phytohemagglutinin and interleukin-2 nonspecifically activate these cells, and no specific immune response to HSV-2 is expected. Further studies are necessary to determine if our observations with human CD4⁺ cells are applicable to other primary lymphocyte cell types and to virus-specific immune cells. Despite these limitations, the similarity in the pattern of change in apoptotic markers in Jurkat and primary human CD4⁺ cells suggests that apoptosis occurs by a similar mechanism in the two cell types.It is our prediction that the ability of HSV-2 to kill T cells plays a role in mitigating the cell-mediated immune response in vivo. In the present study, we have begun to elucidate the mechanism behind apoptosis in T cells following exposure to HSV-2-infected fibroblasts. Viral antigens could be detected in virus-exposed Jurkat cells, suggesting infection that occurs by cell-to-cell spread. We previously reported that expression of HSV-1 ICP0 and HSV-2 ICP10 results in apoptosis of transfected Jurkat cells (4). Therefore, we predicted that infected cells would be positive for apoptotic markers in the present study. However, viral antigens and activated caspase 3 were largely mutually exclusive in virus-exposed cells. We are currently undertaking studies to further decipher the relationship between viral infection and apoptosis to determine whether apoptosis occurs by a bystander effect in T cells.Our results revealed the requirement of caspase 9 in HSV-2-induced apoptosis of Jurkat cells. The finding that neither caspase 8 nor FADD was required suggests that the extrinsic pathway is not involved in HSV-2-induced apoptosis in Jurkat cells. Consistent with this finding, cleavage of caspase 9 was clearly demonstrated in virus-exposed CD4⁺ cells compared to mock-exposed cells, while cleavage of caspase 8 was not evident. Although similarities between HSV-2-induced apoptosis in CD4⁺ and Jurkat cells predict that similar mechanisms are likely to be responsible in both cell types, the applicability of the finding to other types of T cells remains to be elucidated.In conclusion, the present study demonstrated activation of intrinsic apoptosis pathways in Jurkat and primary human CD4⁺ cells following exposure to HSV-2. Further understanding of virus-induced apoptosis in T cells, which is predicted to be an immune-evasion mechanism during reactivation, is expected to provide means to prevent HSV-2 reactivation in infected individuals.     

Killer cells induced by stimulation with allogeneic tumor cells and subsequent culture with recombinant interleukin-2

Summary Peripheral blood lymphocytes were cultured for 5 days with allogeneic tumor cells (allogeneic mixed lymphocyte/tumor cell culture), and subsequently cultured with recombinant interleukin-2 for 12 days. These cultured cells were found to be cytotoxic to autologous tumor cells. Results of two-color analysis using monoclonal antibodies to cell markers showed that more than 80% of their cultured cells were CD3⁺ cells, and CD4⁺ cells showed a higher distribution than CD8⁺ cells. However, CD8⁺ cells had a much higher killing activity with autologous tumor than did CD4⁺ cells, when estimated by an elimination study using monoclonal antibodies to T cell phenotypes and complement. The cold-target inhibition test showed that the cytotoxicity of these cells for autologous tumor cells was inhibited by unlabeled autologous tumor cells but not by unlabeled stimulator cells. Furthermore, about 40% of the cytotoxicity was suppressed by blocking of HLA class I antigen with a monoclonal antibody on autologous tumor cells. Thus, cytotoxic activity of lymphocytes to autologous tumor restricted by target cell HLA class I antigen is possibly induced by allogeneic tumor-stimulation.     

Localization and identification of nuclear radioactivity in the pituitary gland and genital tract after administering 3H-testosterone, 3H-dihydrotestosterone,or 3H-estradiol to male rhesus monkeys

Summary Target cells for testosterone, dihydrotestosterone, and estradiol in the pituitary gland and genital tract of the male primate were localized by thaw-mount autoradiography, and high performance liquid chromatography was used to identify the metabolites of these steroids in cell nuclei. Castrated rhesus monkeys were injected with ³H-testosterone, ³H-dihydrotestosterone, or ³H-estradiol and killed 60 min later. In the anterior pituitary gland, fewer cells were labeled and less radioactivity was taken up by cell nuclei following the administration of either ³H-testosterone (4% of pars distalis cells and 5 dpm/g DNA) or ³H-dihydrotestosterone (5% of cells and 13 dpm/g DNA) than following the administration of ³H-estradiol (43% of cells and 214 dpm/g DNA). Most of the radioactivity in nuclei was in the form of the unmetabolized parent compound (78–94%). In prostate, seminal vesicles, and penis, ³H-dihydrotestosterone was the predominant form of nuclear radioactivity following both ³H-testosterone (67–90%) and ³H-dihydrostestosterone (94–97%) administration, and both androgens labeled epithelial and smooth muscle cells. In contrast, ³H-estradiol was taken up in unchanged form, by cell nuclei of the genital tract and it labeled connective tissue fibroblasts, but not epithelial cells. Thus, the distributions of target cells for androgens and estrogens were clearly different in all these tissues, and the uptake of testosterone resembled that of its androgenic rather than that of its estrogenic metabolite.     

Immobilization of the recombinant invertase INVB from <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> on Nylon-6

The recombinant invertase INVB (re-INVB) from Zymomonas mobilis was immobilized on microbeads of Nylon-6, by means of covalent bonding. The enzyme was strongly and successfully bound to the support. The activity of the free and immobilized enzyme was determined, using 10% (w/v) sucrose, at a temperature ranging between 15 and 60 °C and a pH ranging between 3.5 and 7. The optimal pH and temperature for the immobilized enzyme were 5.5 and 25 °C, respectively. Immobilization of re-INVB on Nylon-6 showed no significant change in the optimal pH, but a difference in the optimal temperature was evident, as that for the free enzyme was shown to be 40 °C. The values for kinetic parameters were determined as: 984 and 98 mM for of immobilized and free re-INVB, respectively. values for immobilized and free enzymes were 6.1 × 10² and 1.2 × 10⁴ s⁻¹, respectively, and immobilized re-INVB showed of 158.73 μmol h min⁻¹ mg⁻¹. Immobilization of re-INVB on Nylon-6 enhanced the thermostability of the enzyme by 50% at 30 °C and 70% at 40 °C, when compared to the free enzyme. The immobilization system reported here may have future biotechnological applications, owing to the simplicity of the immobilization technique, the strong binding of re-INVB to the support and the effective thermostability of the enzyme.     

PDT effects of m-THPC and ALA, phototoxicity and apoptosis

The purpose of this study was to estimate the efficacy of an endogenous sensitizer (-aminolevulinic acid (or ALA) induced protoporphyrin IX (or PpIX)) and an exogenous sensitizer (meta(tetrahydroxyphenyl)chlorin or m-THPC) on two different cell lines, rat colon adenocarcinoma PROb cells and murine melanoma B16A45 (B16) cells, in apoptosis production. After sensitizer incubation, cells were irradiated with an argon dye laser. LD₅₀ with m-THPC was 2.8 g/ml and 4.7 g/ml under irradiation of 25 J/cm² respectively for PROb and B16 cells. With ALA, LD₅₀ was 150 g/ml and 175 g/ml under 25 J/cm² respectively for PROb and B16 cells. Apoptosis induction by m-THPC or ALA-PDT was detected by DNA gel electrophoresis and quantified using an ELISA assay 24 h after PDT. The maximal apoptosis enrichment factor (MAEF) was reached for 6 g/ml m-THPC at 10 J/cm² for PROb and B16 cells and for 50 g/ml ALA at 25 J/cm² for PROb or B16 cells. Both m-THPC and PpIX are efficient photosensitizers and apoptosis inducers. However, MAEF is obtained by sensitizer or laser doses inducing very different phototoxic effects: MAEF was obtained after m-THPC-PDT with LD₇₈ for PROb cells and LD₃₀ for B16 cells and after ALA-PDT with LD₂₂ for PROb cells and LD₁₈ for B16 cells. However the overall m-THPC/PDT apoptotic induction (under the curve surface analysis) was not different whatever the cell line for 10 and 25 J/cm². On the contrary, ALA-PpIX/PDT apoptotic induction was twice for 25 J/cm² as compared to 50 J/cm² (p < 0.01) for both the PROb and B16 cells. These results indicate that the apoptosis rate in PDT cell killing varies considerably according to cell type and sensitizer.     

Signaling to a B-cell clone by Ek,but not Ak,does not reflect alteration of A k genes

The mouse B-cell clone, CH12.LX (Ia^k, Ly-1⁺, ⁺, ⁺), can be induced to differentiate and secrete antibody in an antigen-specific, H-2-restricted manner. Induction requires two signals. One must be provided by the binding of specific antigen to the membrane IgM; the other is delivered by the binding of E^k-specific T-cell hybridomas to the E^k molecules of CH12.LX (Bishop and Haughton 1986). Previous studies demonstrated that E^k-specific monoclonal antibodies (mAbs) could substitute for T cells in delivering the second differentiative signal (Bishop and Haughton 1986). Although CH12.LX cells present A^k to A^k-restricted or alloreactive T-helper cells, neither T cells nor mAbs specific for A^k induce differentiation (Bishop and Haughton 1986). However, since the A^kspecifc mAbs tested previously were -chain-specific and the Ia epitope specificity of the T cells used was unknown, it is possible that the differentiative signal delivered to the CH12.LX class 11 molecule is chain-specific. Here we report the effects of ten additional Ia^k-specific mAbs upon the differentiation of CH12.LX. In addition, a cl)NA library was prepared from CHI 2.LX cells, clones corresponding to the and chains of the A^k molecule were isolated, and their nucleotide sequences were determined. Finally, the A^k and E^k molecules of CH12.LX and H-2^k spleen cells were compared by two-dimensional gel electrophoresis to examine possible post-translational differences in the Ia^k molecules of CH12.LX.     

Imbalances of T-cell subsets in monoclonal gammopathies

Summary In 42 patients with untreated or treated multiple myeloma (MM) or benign monoclonal gammopathy (BMG) the lymphocytes and T lymphocyte subsets were determined by monoclonal antibodies and other surface markers.In untreated MM, the T cells (1077/l vs 1439/l, P<0.01) and especially the OKT4⁺ lymphocytes (700/l vs 950/l, P<0.05) were significantly reduced compared with a control group. The OKT8⁺ cells were slightly but not significantly decreased.In previously treated MM, the loss of T cells was more pronounced than in the untreated group and was primarily caused by a further reduction of OKT4⁺ cells. Patients with BMG revealed decreased OKT8⁺ lymphocytes (304/l vs 502/l, P<0.001), whereas the OKT4⁺ cells were within the normal range. Therefore, the OKT4/OKT8 ratio was significantly elevated compared with that in untreated MM patients and normal controls (3.31 vs 2.06 vs 2.13; P<0.005).To sum up, in MM the results revealed a reduction of T cells, mainly of OKT4⁺ cells, which is intensified by chemotherapy and persists even after a long therapy-free interval. The different findings of T cell subsets in BMG and MM may be a helpful criterion to differentiate between BMG and MM.     

HIV Infection Is Associated with a Preferential Decline in Less-Differentiated CD56dim CD16+ NK Cells

HIV-1 infection is characterized by loss of CD56^dim CD16⁺ NK cells and increased terminal differentiation on various lymphocyte subsets. We identified a decrease of CD57⁻ and CD57^dim cells but not of CD57^bright cells on CD56^dim CD16⁺ NK cells in chronic HIV infection. Increasing CD57 expression was strongly associated with increasing frequencies of killer immunoglobulin-like receptors (KIRs) and granzyme B-expressing cells but decreasing percentages of cells expressing CD27⁺, HLA-DR⁺, Ki-67⁺, and CD107a. Our data indicate that HIV leads to a decline of less-differentiated cells and suggest that CD57 is a useful marker for terminal differentiation on NK cells.NK cells are effector cells of innate immunity which are pivotal as first-line defense against viral infections, such as HIV infection (14). Large genotypic studies demonstrated a delayed onset of AIDS in HIV-seropositive individuals carrying the activating receptor KIR3DS1 and/or alleles of the inhibiting receptor KIR3DL1 in conjunction with HLA-Bw4-80I (18, 19). Development of NK cells mainly takes place in the bone marrow, from which mature NK cells move out to reside and circulate in peripheral sites (13). Mature NK cells are characterized by granules which harbor granzymes and perforin. These NK cells are fully armed, “ready-to-go” effector cells (17).A number of NK cell abnormalities have been reported in HIV infection (9), including high activation status (2, 10), increased turnover (16), differential expression of activating and inhibitory receptors (20), impaired interaction with dendritic cells (12), and loss of CD56^dim CD16⁺ NK cells (23). CD56^dim CD16⁺ NK cells represent the largest NK cell subset in peripheral blood in healthy individuals. The expression of killer immunoglobulin-like receptors (KIRs) and CD57 are predominant features of this subpopulation (8, 15). CD57 expression on NK cells has been previously associated with replicative senescence on T and NK cells (4), raising the question of how HIV-1 infection alters CD57 expression on CD56^dim CD16⁺ NK cells.To the best of our knowledge, no one has addressed the phenotypic and functional properties of CD56^dim CD16⁺ NK cells that are preferentially lost during HIV infection. Here, we provide evidence that increasing CD57 expression indicates terminal differentiation in healthy individuals, as well in as HIV-infected subjects. We furthermore show that HIV infection is associated with preferential loss of less-differentiated cells, which are characterized by high activation status and turnover.In this study, blood samples from 37 HIV-seropositive individuals and 15 healthy subjects were analyzed; all HIV-infected patients were either antiretroviral therapy naïve or untreated for more than one year. The HIV-positive study cohort comprised 10 patients with a viral load of less than 2,000 copies/ml, 14 patients with a viral load ranging from 2,000/ml to 20,000 copies/ml, and 13 patients with a viral load above 20,000 copies/ml. CD4 T cell counts ranged from 180/μl to 1,355/μl, the average being 457.3/μl.The study was approved by the local ethics commission (Ethikkommission der Medizinischen Hochschule Hannover, Votum No. 3150), and all study participants gave informed written consent for their participation.Flow cytometric analysis was performed on cryopreserved peripheral blood mononuclear cells (PBMCs) as previously described (21, 22). A list of monoclonal antibodies employed in this study is available upon request. For intracellular analysis of granzyme B, perforin, and Ki-67, we used a fixation and permeabilization kit (Invitrogen). At least 1 million events were acquired for each sample, using either a FACSAria or LSR II flow cytometer (BD Biosciences). Data were analyzed with FlowJo (TreeStar). Lymphocytes were defined by forward and side scatter. CD3⁺, CD14⁺, CD19⁺, dead cells, and cell aggregates were removed from analysis based on peridinin chlorophyll protein and Viaprobe staining and gating on a plot of forward-scatter area versus forward-scatter height (Fig. ​(Fig.1A).1A). NK cells and their distinctive subpopulations were defined based on their CD56 and/or CD16 expression. Fluorescence-minus-one (FMO) staining was used to determine threshold values for the expression of specific markers.Open in a separate windowFIG. 1.HIV infection is associated with loss of CD57⁻ and CD57^dim but not CD57^bright CD56^dim CD16⁺ NK cells. (A) Representative gating scheme for identification of NK cells. NK cells were defined as CD3⁻ CD14⁻ CD19⁻ lymphocytes expressing either CD56 or CD16 or both. We divided CD56^dim CD16⁺ NK cells into three subsets based on their level of CD57 expression: CD57⁻, CD57^dim, and CD57^bright cells. Numbers on FACS plots indicate frequency of gated population. SSC-A, side scatter area; FSC-A, forward scatter area; FSC-W, forward scatter width. (B) Comparison of percentages of the CD57⁻, CD57^dim, and CD57^bright subpopulations in control subjects (n = 14) and HIV-seropositive individuals (n = 34) on CD56^dim CD16⁺ NK cells. ns, not significant (P > 0.05); **, P < 0.01; ***, P < 0.001. (C) Frequencies of CD57⁻, CD57^dim, and CD57^bright expressing CD56^dim CD16⁺ NK cells in relation to total NK cells in control subjects (n = 14) and HIV-seropositive individuals (n = 34). (D) Mean frequency of CD56^dim CD16⁺ NK cells in 14 control individuals and in 34 HIV-infected people and the distribution of CD57⁻, CD57^dim, and CD57^bright cells within CD56^dim CD16⁺ NK cells is shown. (E) Relationship between percentage of CD57^dim CD56^dim CD16⁺NK cells and percentage of CD56^neg CD16⁺ NK cells on total NK cells. Horizontal bars in dot plots show the means.NK cells as defined above were sorted from cryopreserved PBMCs on a FACSAria (purities ranged from 91% to 99%). An amount of 10⁵ NK cells was plated per well and stimulated with 10 ng/ml interleukin-15 (IL-15), 100 ng/ml IL-12, and 5 × 10⁴ K562 cells. A CD107a degranulation assay was performed as described previously (1, 12). GraphPad Prism (version 5.0) software was used for statistical evaluation of data. Correlation analysis was performed using the Pearson test. The unpaired t test was performed when two groups were compared, and all t tests were two tailed. Comparison of more than two groups was performed using one-way analysis of variance followed by Tukey''s post-hoc test. P values of less than 0.05 were considered significant.We found that CD57 on NK cells was predominantly expressed on the CD56^dim CD16⁺ population (Fig. ​(Fig.1A).1A). The expression patterns of CD57 allowed us to differentiate between three subfractions within CD56^dim CD16⁺ NK cells, namely, CD57⁻, CD57^dim, and CD57^bright cells. The frequency of the CD57^bright subpopulation on CD56^dim CD16⁺ NK cells was increased compared to the frequency of the CD57^dim subpopulation on CD56^dim CD16⁺ NK cells in HIV-seropositive patients but not in HIV-seronegative control subjects (Fig. ​(Fig.1B).1B). This relative increase was associated with substantial reductions of the CD57⁻ CD56^dim and the CD57^dim CD56^dim NK cell subpopulations of total NK cells in our HIV-seropositive cohort compared to these subpopulations in healthy control subjects (means, 36.6% versus 24.8% [P = 0.0002] and 22.4% versus 15.4% [P = 0.0001]), but the frequencies of CD57^bright CD56^dim NK cells within total NK cells were similar between HIV-infected patients and HIV-seronegative individuals (Fig. ​(Fig.1C).1C). In accordance with previously published data (3, 23), we could confirm that there is a relative loss of CD56^dim CD16⁺ NK cells in HIV infection (mean, 84.3% versus 67.0%, P = 0.0004) (Fig. ​(Fig.1D).1D). Our data indicate that this loss is predominantly due to decreased numbers of CD57⁻ CD56^dim and CD57^dim CD56^dim NK cells, leading to a relative overrepresentation of CD57^bright cells within CD56^dim CD16⁺ NK cells in HIV infection (Fig. ​(Fig.1C).1C). There was no significant correlation between the relative loss of CD57⁻ and CD57^dim NK cells and absolute numbers of CD56^dim CD16⁺ NK cells, but there was a significant inverse correlation between loss of CD57^dim NK cells and increasing percentages of CD56⁻ CD16⁺ cells (Pearson r = −0.54, P = 0.001) (Fig. ​(Fig.1E1E).To determine whether the relative decrease of CD57⁻ and CD57^dim NK cells was associated with parameters of HIV disease progression, we performed correlation analysis of the percentages of CD57⁻ or CD57^dim cells with viral load and CD4 T cell counts. We found no such correlations (Pearson r < 0.2 and P > 0.05 for all) (data not shown). A recent cross-sectional and longitudinal study demonstrated that changes in the NK cell compartment, as shown by down-modulation of Siglec-7 on CD56^dim NK cells, are associated with HIV viremia (5). The longitudinal data in the study indicated that the full restoration of NK cell pathologies required 24 months of antiviral treatment. This suggests that alterations in the NK cell compartment can be driven by HIV viral load but that these changes seem to require a significant amount of time.We next investigated the phenotypic and functional properties of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. For KIR2DL2/DL3/DS2, we detected increasing prevalences of KIR-expressing NK cells with increasing expression of CD57 in both healthy control subjects and HIV-infected blood donors (Fig. ​(Fig.2A).2A). As for KIR3DS1/DL1, we found an increase of KIR⁺-expressing NK cells between CD57⁻ and CD57^bright cells in control individuals and significant differences in percentages of KIR3DS1/DL1-expressing NK cells between CD57⁻ and CD57^dim, as well as between CD57⁻ and CD57^bright, NK cells in our HIV-positive cohort (Fig. ​(Fig.2A).2A). These results suggest that increasing CD57 expression is associated with higher numbers of KIR-expressing NK cells in control subjects and HIV-infected subjects.Open in a separate windowFIG. 2.Phenotypic characterization of the CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells. Representative flow cytometry plots for one control and one HIV-infected subject and summary data for all individuals whose PBMCs were analyzed are shown. CD57⁻, CD57^dim, and CD57^bright NK cells are concatenated to visualize them in a single dot plot. Numbers in contour plots indicate percentages of gated events of the respective subset. (A) Percentages of KIR2DL2/DL3/DS2 and KIR3DS1/DL1-expressing CD57⁻, CD57^dim, and CD57^bright cells were analyzed in control individuals (n = 15) and HIV-infected subjects (n = 37). (B) Numbers of HLA-DR-expressing and CD27-expressing CD57⁻, CD57^dim, and CD57^bright cells in control individuals'' (n = 15) and HIV-infected subjects'' (n = 37) PBMCs were analyzed. Horizontal bars in dot plots show the means. ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We next addressed the question of whether increasing CD57 expression is linked to differential phenotypic properties of NK cells and analyzed the HLA-DR and CD27 expression of the CD57⁻, CD57^dim, and CD57^bright subpopulations on CD56^dim CD16⁺ NK cells. A significantly higher fraction of NK cells expressed HLA-DR in the CD57⁻ than in the CD57^bright subset in both healthy control individuals and HIV-infected subjects (Fig. ​(Fig.2B).2B). A considerably higher portion of NK cells was positive for HLA-DR in HIV-infected individuals than in control subjects (means, 3.2% versus 13.2% [P < 0.0001], 1.8% versus 10.4% [P = 0.001], and 0.9% versus 6.5% [P = 0.005] for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively). We furthermore detected marked differences in frequencies of cells expressing CD27, a member of the tumor necrosis factor (TNF) receptor family (24). CD57⁻ NK cells displayed the highest percentages of CD27⁺ cells, whereas CD57^bright cells were almost all negative for CD27, in both control individuals and HIV-seropositive subjects (Fig. ​(Fig.2B).2B). We thus show that increasing expression of CD57 is associated with differential activation status and differential phenotype.Next, we sought to determine whether CD57 is linked to differential functional phenotypes by assessing the intracellular expression of granzyme B, perforin, and Ki-67. The frequencies of perforin-expressing NK cells did not vary within the different CD57 subsets of CD56^dim CD16⁺ NK cells (Fig. ​(Fig.3A).3A). However, we found that CD57^bright cells displayed the highest frequencies of granzyme B⁺ in both control and HIV-seropositive subjects, whereas CD57⁻ cells exhibited the lowest percentages for granzyme B⁺ cells (Fig. ​(Fig.3A).3A). Conversely, when we studied the expression of Ki-67, we identified the opposite trend: less than 5% of CD57^bright cells in control individuals and less than 10% of CD57^bright cells in HIV-infected study subjects expressed Ki-67 (Fig. ​(Fig.3B).3B). The highest numbers of Ki-67⁺ cells were found in the CD57⁻ population.Open in a separate windowFIG. 3.Functional characterization of CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim CD16⁺ NK cell population. (A) Representative staining results for granzyme B and perforin and summary data for control (n = 14) and HIV-seropositive subjects (n = 36). Numbers in the concatenated contour plots indicate percentages of gated events of the respective subset. B cells were defined as the negative control for granzyme and perforin staining. (B) Percentages of Ki-67⁺ and CD107a⁺ cells on CD57⁻, CD57^dim, and CD57^bright cells within the CD56^dim NK cell population in control (n = 14 and n = 9, respectively) and HIV-seropositive (n = 36 and n = 21, respectively) subjects'' PBMCs were analyzed. Horizontal bars in dot plots show the means. NC, negative control; ns, not significant (P > 0.05); *, P < 0.05; **, P < 0.01; ***, P < 0.001.We also assessed the presence of the degranulation marker CD107a on CD57⁻, CD57^dim, and CD57^bright subpopulations of CD56^dim CD16⁺ NK cells after stimulation with IL-12 and IL-15 and exposure to K562 cells. Similarly to what we had observed for Ki-67 expression, CD57⁻ cells were the most efficient at degranulation when compared with CD57^dim and CD57^bright cells in HIV-infected individuals. Comparison to healthy controls revealed that there was a higher expression of CD107a in HIV-seropositive subjects for each CD57 subset. However, the most effective degranulation occurred in the CD57⁻ and CD57^dim subsets, which are preferentially depleted in HIV infection.We focused our analysis on CD56^dim CD16⁺ NK cells because they constitute the largest NK cell subset in peripheral blood, they are the major NK cell subset expressing CD57 and KIRs, and they are the most prominent subpopulation for cytolytic activity. CD56^dim CD16⁺ cells but not CD56^bright CD16⁻ NK cells were reported to be decreased in HIV-infected subjects (23), which we could confirm in our experiments (data not shown). We did not find CD57 on CD56^bright CD16⁻ NK cells either in healthy or in HIV-infected individuals. CD57 has been described as a marker for replicative senescence, and its expression has been associated with shorter telomeres and diminished proliferative capacities on T and NK cells (4). The presence of this marker on CD56^dim CD16⁺ but not on CD56^bright CD16⁺ NK cells might explain why the latter subset was shown to proliferate more efficiently upon cytokine stimulation (6). We demonstrated that increasing CD57 expression on NK cells was associated with lower numbers of CD27-expressing cells, a marker which is mainly expressed by CD56^bright CD16⁻ NK cells (24). CD56^bright CD16⁻ cells were suggested to be early NK cells, which differentiate from CD34^dim CD45RA⁺ hematopoietic precursor cells with high expression of integrin α₄β₇ (11). These cells can furthermore give rise to CD56^dim CD16⁺ NK cells (7). Our data support this hypothesis, as we show that CD57 can be found on CD56^dim CD16⁺ NK cells but not on CD56^bright NK cells, whereas the opposite is observed for CD27.We demonstrate that differential CD57 expression is associated with distinct functional characteristics. We show for the first time that increasing expression of CD57 on CD56^dim CD16⁺ NK cells is associated with increasing prevalence of KIR⁺ and granzyme B⁺ cells. These cells appear to be more mature and differentiated in terms of KIR and granzyme B expression but less functionally active, as shown by decreased expression of Ki-67 and CD107a. We therefore propose that CD57 is not only a marker for replicative senescence but, in addition, a marker for terminal differentiation on NK cells, which is characterized by increased expression of KIR and higher granzyme B content and “counterbalanced” by decreased degranulation (CD107a) and decreased proliferation (Ki-67).Notably, we observed consistently higher frequencies of granzyme B⁺ cells in all three subsets within CD56^dim CD16⁺ NK cells from HIV-seropositive individuals than in healthy control subjects (means, 52.9% versus 78.7% [P < 0.0001], 65.3% versus 89.6% [P < 0.0001], and 76.5% versus 95.0% [P < 0.0001]for CD57⁻, CD57^dim, and CD57^bright subpopulations, respectively) (Fig. ​(Fig.1C).1C). Furthermore, HIV infection was associated with higher numbers of Ki-67-expressing NK cells (means, 8.4% versus 16.1% [P = 0.0005], 5.3% versus 11.6% [P = 0.0016], and 4.1% versus 6.2% [P = 0.04]) (Fig. ​(Fig.1C).1C). These changes, including the strong increase in HLA-DR-expressing NK cells, probably reflect the systemic immune activation in HIV-infected individuals.In summary, these findings support a view of a differential regulation of NK function and are in concordance with maturation of NK cells with high expression of CD57 on NK cells with a more terminally differentiated phenotype. Our data indicate that high turnover; activation status; and active degranulation as characterized by the expression of Ki-67, HLA-DR, and CD107a are mainly features of CD57⁻ and much less of CD57^dim NK cells. HIV infection is associated with increased activation, proliferation, and cytotoxicity during “early” stages of CD56^dim CD16⁺ NK cell differentiation compared to their occurrence in healthy controls, but those are the very cells that are significantly decreased in chronic HIV infection. A loss of these functionally more active NK cells may be a yet-unappreciated factor in overall NK cell pathology and a further possible explanation for the impairment of NK cells in their contribution to viral control in HIV infection.     

Forced symbiosis between synechocystis spp. PCC 6803 and apo-symbiotic Paramecium bursaria as an experimental model for evolutionary emergence of primitive photosynthetic eukaryotes

Single-cell green paramecia (Paramecium bursaria) is a swimming vehicle that carries several hundred cells of endosymbiotic green algae. Here, a novel model for endo-symbiosis, prepared by introducing and maintaining the cells of cyanobacterium (Synechocystis spp. PCC 6803) in the apo-symbiotic cells of P. bursaria is described.Key words: green paramecia, cyanobacteria, evolution, diagostic PCR, Escherichia coli, food vacuoleSingle-cell Paramecium bursaria, or green paramecia, is often described as a swimming vehicle that carries several hundred cells of endo-symbiotic green algae that are morphologically and genetically almost identical to Chlorella species.¹ Recent bioengineering studies have demonstrated that the high capacity for symbiotic algae inside the host cells can be replaced with various natural and artificial particles such as fluorescent and magnetic microspheres.^2,3This organism has attracted the attention of cell biologists, biochemists and ecologists since P. bursaria serves as an excellent experimental model for studying the nature of endo-symbiosis in which one species propagates inside the cells of other species under the precise control through the chemical communications between the host and symbiont cells, and knowledge on the recognition of the symbiotic partners, exchange of chemicals and regulation of metabolic processes have been documented.^4–6 It is well known that synchronization on the algal cell division is imposed by the hosting paramecia possibly through chemical communication between the partners.⁷ In our recent study focusing on the impacts of the host''s cell cycle and growth status on the life cycle in endo-symbiotic algae, flow-cytometric analysis has revealed that the life cycle of symbiotic algae is largely affected by the growth status of the hosting cells.⁸Occasionally, apo-symbiotic cells of P. bursaria (thus lacking algae) can be found in natural water environments⁹ and also in dark-grown culture of P. bursaria.¹⁰ Interestingly, alga-free cell strains of P. bursaria can be artificially prepared by treating the stocks of green paramecia with cycloheximide¹¹ or some herbicides.^12,13 Some groups have shown that independently cultured apo-symbiotic host cells and ex-symbiotic algae can re-associate and re-establish the symbiotic relationship.^14,15 Due to this experimentally reproducible symbiotic nature, P. bursaria can be the best model for studying the mechanism (and possibly the origins) of endo-symbiosis. In our effort to elucidate the mechanism required for successful symbiosis, our recent report has described a case of symbiosis distortion leading to unregulated growth of symbiotic algae, and the significance and advantage of such material for studying the nature and origin of endo-symbiosis were discussed in reference ³.Above studies suggest that in the evolutional time scale required for emergence of a novel photosynthetic organism, the history of symbiosis in P. bursaria is likely recent; thus, both the symbiont and host organisms are still on the process for developing the mechanism in which partners become highly dependent on each other. From such evolutional points of view, the symbiosis between algae and ciliate in P. bursaria is most likely a fruit of co-evolution between two organisms in which host species developed its tolerance to the presence of photosynthetic symbionts which behave as the source of both the sugars and photosynthesis-associated oxidative stresses.¹⁶Our aim in this study was for experimentally reproducing the conditions mimicking the first contact and development of symbiosis between unicellular ciliate protozoa and photosynthetic bacteria as a novel model for studying the very early evolutional processes for the emergence of photosynthetic eukaryotes, the hypothetical ancestorial organisms of plants.¹⁷ In fact, in our system, the preparation of apo-sympiotic P. bursaria (white cells) after forced algal removal from symbiotic P. bursaria (green cells) allows us loading of any particles of interests both biological and non-biological into the ciliate cells;^2,3 therefore the fate of experimentally loaded particles or organism is of great interest. Here, we describe a novel model endo-symbiotic complex formed between the cells of cyanobacterium (Synechocystis spp. PCC 6803) and the hosting cells derived from alga-removed P. bursaria.P. bursaria strain INA-1 (Fig. 1A; syngen 1, shown to be mating type I as recently tested),¹⁷ which has endosymbiotic green algae (Fig. 1B) was originally collected from the Ongagawa River (Kama-city, Fukuoka Pref., Japan) as described in reference ².Open in a separate windowFigure 1Light microscopic images of P. bursaria and its ex-endosymbiotic algae. (A) Matured cell of P. bursaria harboring the symbiotic green algae. (B) Ex-symbiotic algae isolated from P. bursaria. (C) Alga-free apo-symbiotic host cell of P. bursaria. (D) Cyanobacteria (Synechocystis spp. PCC 6803).Since the cell line was established after single cell isolation, all the cells in the culture were clones sharing identical genetic background. Apo-symbiotic white strain of P. bursaria was prepared from natural green strain (INA-1) as previously described in reference ². These strains were maintained in the lettuce infusion inoculated with the food bacterium Klebsiella pneumoniae 24 h prior to the subculturing of ciliate cells, as described before in reference ⁸. The ciliate culture was initiated with ca. 10–20 cells/ml and propagated to the confluent level (over 1,000 cells/ml) under a light cycle of 12 h light and 12 h dark with ca. 3,500 lux (30 cm from the light source) of fluorescent natural-white light at 23°C.The protocol of Tanaka et al.¹⁸ was employed for preparation of apo-symbiotic white cells. Briefly, the green cells were incubated in the presence of 0.1 µM paraquat for over 24 h under light condition (with a fluorescent white lamp, 3,000 lux at least). Then, a single ciliate lacking algae was separated under a microscope and the cell line of apo-symbiotic paramecia (Fig. 1C) derived from this single cell was propagated in the lettuce infusion inoculated with food bacteria as described above. We found that this herbicide treatment merely enhances the excretion of algae from the ciliate but many portions of resultant ex-symbiotic algae excreted from the ciliates are still alive and capable of growing in vitro.¹⁷As a model organism in order to develop an experimental model for studying the origin of photosynthetic organisms leading to evolution of plants, cyanobacteria would be the best organism since the photosynthetic apparatus in this organism is very similar to the one found in plants.¹⁹ Among cyanobacteria as the material to be loaded to apo-symbiotic P. bursaria cells, Synechocystis sp. PCC 6803 (Fig. 1D) was chosen since it is one of the most highly studied cyanobacteria capable of growth in both autotrophical and heterotrophical manners.²⁰ PCC 6803 cells were propagated in BG-11 medium under a continuous light at ca. 3,000 lux with fluorescent natural-white light at 23°C. Then alga-free apo-symbiotic cells isolated and propagated after forced algal excretion, were used for the introduction of PCC 6803 cells based on the modified procedure used for re-introduction of green algae (re-greening process). Apo-symbiotic P. bursaria cells were incubated with suspension of PCC 6803 cells (10 µl of confluent culture of PCC 6803 into 1 ml of ciliate culture, i.e., 2,000 cells) in lettuce infusion for 30 min in an Eppendorf tube at room temperature. The resultant Synechocystis-fed re-greened cells were collected on a nylon mesh (pore size, 10 µm), and washed out with 10 ml of fresh medium three times.Intact live cells were used for routine observations under a stereomicroscope (SMZ645; Nikon, Tokyo, Japan; Fig. 2A). For obtaining the digital microscopic images with higher resolutions, the P. bursaria cells with and without symbionts were fixed in 3% (w/v) formaldehyde added to the culture medium and fixation was allowed at room temperature for 5 min. Confocal laser scanning microscopic red fluorescent images and differential interference contrast (DIC) images of P. bursaria cells were acquired using a Radiance 2100 microscope (Bio-Rad Laboratories, Hercules, CA; Fig. 2B). The obtained images were processed using Adobe Photoshop software.Open in a separate windowFigure 2Microscopic images of cyanobacteria-loaded cell of P. bursaria. (A) Light microscopic image. (B) Laser scanning confocal microscopic images (Left: nomarski differential interference image, Right: chlorophyll fluorescence image)Diagnostic polymerase chain reactions (PCR) were performed for confirming the presence of PCC 6803 cells in the one-year-old symbiotic culture; following primers and reaction condition were employed. For cyanobacteria specific detection, a set of primers was designed from the Cyanobase.²¹ The PCR products were obtained from positions 1,600,000 to 1,601,783 of the chromosome. The specific primers were Forward primer 5′-GTG GTT TGG GTC AAT GTT-3′ and Reverse primer 5′-CAG GGC CTT GTA AAC TTT-3′. The other standard methods for culture and DNA manipulations of cyanobacteria were described in reference ²².Sysmex flow particle image analyzer FPIA-2100 (Sysmex Co., Kobe, Japan) was used for statistical analysis of algal cell size as previously described in reference ³. PCC 6803 cells were suspended in the sheath medium and used for direct measurement by FPIA-2100.After addition of PCC 6803 suspension to apo-symbiotic cells of P. bursaria, bacterial particles were rapidly taken up by the ciliate through the oral groove. Based on microscopic observation with video, the moment that one bacterium located at the deepest end of oral groove is incorporated in cytosolic space (i.e., constriction and isolation of bacterium-containing membrane-like structure resembling the digestive vacuoles) was recorded, indicating that the ciliate takes up the PCC 6803 cells in a manner similar to take up the food bacterium. However, the fate of food bacteria and cyanobacteria drastically differed. As previously reported, the emerald green fluorescent protein (EmGFP)-labeled cells of E. coli can be used as a model food bacterium and its fate can be visualized by following the changes in fluorescence level and localization.² Based on observation of EmGFP E. coli-loaded apo-symbiotic ciliate, localization of bacteria in several compartments was observed; thus instead of being dispersed throughout the hosting cells, presence of food bacteria are restricted and localized within the food vacuoles (Fig. 3B). The time required for digestion of EmGFP-labeled cells of E. coli was estimated to be 30–60 min (fluorescence could not be observed after 1 h).²Open in a separate windowFigure 3Continuous culturing of Synechocystis spp. PCC 6803 in the host cell. (A) Light microscopic images of wild-type P. bursaria. (B) Laser scanning confocal microscopic image of EmGFP-labeled E. coli-loaded cell of apo-symbiotic P. bursaria.² (C) Light microscopic images of Synechocystis spp. PCC 6803-loaded cell of P. bursaria in the one-year-old symbiotic culture. (D) Statistical analysis of Synechocystis spp. PCC 6803 cells in 1 year-old symbiotic culture using a Symex flow particle image analyzer FPIA -2100.On the other hand, the presence of PCC 6803 cells inside the host ciliate could be observed even after continuous propagation of the ciliate for over 1 year (Fig. 3C), thus indicative of the bacterial growth in the host cells. Initially, number of PCC 6803 cells in the host cells were limited within few particles and restricted within the localized compartments in the ciliate cells, but in the samples after continuous culturing for 1, 3, 6 and 12 months, PCC 6803 cells increased and maintained its population inside the hosting cells and their presence were shown to be dispersed throughout the cytosol of hosting cells.Irie et al.³ tested the loading of various fluorescence-labeled microspheres, green algae and bacteria into the apo-symbiotic P. bursaria. According to their work, there is a tight relationship between the size of particles loaded and the mode of localization within the ciliate. Small nano-particles (sized 50–250 nm in diameter) and polystyrene micro-beads sized around 2–3 µm in diameter are likely packed as groups in localized compartments, thus some tens of particles were gathered in spherical structures (resembling the digestive vacuoles) within the ciliate cells, thus their presence was restricted. Larger micro-beads sized around 4–10 µm in diameter did not gather around each other but likely packed individually, thus showing dispersed localization throughout the cytosol of the ciliate cells.³ While E. coli (sized ca. 2 µm) obeys this size-based localization pattern, PCC 6803 showed dispersed distribution similar to the localization of natural green algae (symbiotic Chlorella species), despite its small size (1.61 µm in diameter, mean size; Fig. 3D).According to Tanaka et al.¹⁸ for discussing the presence or absence of symbiotic microorganisms inside the ciliate, microscopic morphological observation is not sufficient, but instead diagnostic PCR is required. Therefore, the presence and stability of PCC 6803 cells likely propagated within the one-year-old symbiotic complex was examined by diagnostic PCR (Fig. 4). Presence of the genome of PCC 6803 was confirmed in the one-year-old culture of PCC 6803-loaded P. bursaria, supporting our microscopic observation at molecular level.Open in a separate windowFigure 4Diagostic polymerase chain reaction for confirming the presence of Synechocystis spp. PCC 6803 cells in host cell from one-year-old symbiotic culture.We previously proposed two possible biochemical models (models 1 and 2) explaining the co-evolution between Paramecium species and algal symbionts by focusing on the dual roles of algal photosynthesis providing the energy source and the risk of oxidative damage to the hosting cells.¹⁶ The first model is based on the correlation between the (re)greening ability and the tolerance to oxidative stress among the paramecium species, whereas the second model is deduced through discussion on the possible evolutionary selection leading to emergence of Paramecium species tolerant against alga-derived reactive oxygen species through alga-paramecium contacts. These views could be applicable to the events required for the emergence of photosynthetic eukaryotes after incorporation of photosynthetic bacteria as primitive chloroplasts. These views should be critically tested in the future researches using our experimental model system.