Impact of collaborative actions of Leishmania (Viannia) braziliensis subpopulations on the infection profile.

This study focuses on the role of the population structure of Leishmania spp. on the adaptive capacity of the parasite. Herein, we investigate the contribution of subpopulations of the L. (V.) braziliensis Thor strain (Thor03, Thor10 and Thor22) in the profile of murine macrophages infection. Infection assays were performed with binary combinations of these subpopulations at stationary phases. The initial interaction time showed major effects on the combination assays, as demonstrated by the significant increase in the infection rate at 5 h. Based on the endocytic index (EI), Thor10 (EI = 563.6) and Thor03 (EI = 497) showed a higher infection load compared to Thor22 (EI = 227.3). However, the EI decreased in Thor03 after 48 h (EI = 447) and 72 h (EI = 388.3) of infection, and showed changes in the infection level in all Thor10/Thor22 combinations. Assays with CellTrace CFSE-labelled Thor22 promastigotes indicated an increase (~1.5 fold) in infection by this subpopulation in the presence of Thor10 when compared to the infection profile of Thor03/Thor22 combinations in the same proportions. In addition, the potential of these subpopulations, alone or in binary combinations, to modulate the expression of cytokines and nitric oxide (NO) in vitro was investigated. Lower NO and tumour necrosis factor-α production levels were observed for all Thor10/Thor22 combinations at 24 h compared to these subpopulations alone. In contrast, Thor03/Thor22 combination assays increased IL-10 production at this time. Collectively, these results provide in vitro evidence on the potential of L. (V.) braziliensis population structure to play a relevant role in a host infection by this parasite.

Expansion of plasmablasts and loss of memory B cells in peripheral blood from COVID-19 patients with pneumonia.

Studies on the interactions between SARS-CoV-2 and humoral immunity are fundamental to elaborate effective therapies including vaccines. We used polychromatic flow cytometry, coupled with unsupervised data analysis and principal component analysis (PCA), to interrogate B cells in untreated patients with COVID-19 pneumonia. COVID-19 patients displayed normal plasma levels of the main immunoglobulin classes, of antibodies against common antigens or against antigens present in common vaccines. However, we found a decreased number of total and naïve B cells, along with decreased percentages and numbers of memory switched and unswitched B cells. On the contrary, IgM+ and IgM- plasmablasts were significantly increased. In vitro cell activation revealed that B lymphocytes showed a normal proliferation index and number of dividing cells per cycle. PCA indicated that B-cell number, naive and memory B cells but not plasmablasts clustered with patients who were discharged, while plasma IgM level, C-reactive protein, D-dimer, and SOFA score with those who died. In patients with pneumonia, the derangement of the B-cell compartment could be one of the causes of the immunological failure to control SARS-Cov2, have a relevant influence on several pathways, organs and systems, and must be considered to develop vaccine strategies.

IFN-γ+ CD4+T cell-driven prophylactic potential of recombinant LDBPK_252400 hypothetical protein of Leishmania donovani against visceral leishmaniasis.

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease causing high morbidity and mortality in developing countries. Vaccination is considered the most effective and powerful tool for blocking transmission and control of diseases. However, no vaccine is available so far in the market for humans. In the present study, we characterized the hypothetical protein LDBPK_252400 of Leishmania donovani (LdHyP) and explored its prophylactic behavior as a potential vaccine candidate against VL. We found reduced hepato-splenomegaly along with more than 50% parasite reduction in spleen and liver after vaccination in mice. Protection in vaccinated mice after the antigen challenge correlated with the stimulation of antigen specific IFN-γ expressing CD4+T cell (~4.6 fold) and CD8+T cells (~2.1 fold) in vaccinated mice in compared to infected mice, even after 2-3 months of immunization. Importantly, antigen-mediated humoral immunity correlated with high antigen specific IgG2/IgG1 responses in vaccinated mice. In vitro re-stimulation of splenocytes with LdHyP enhances the expression of TNF-α, IFN-γ, IL-12 and IL-10 cytokines along with lower IL-4 cytokine and IL-10/IFN-γ ratio in vaccinated mice. Importantly, we observed ~3.5 fold high NO production through activated macrophages validates antigen mediated cellular immunity induction, which is critical in controlling infection progression. These findings suggest that immunization with LdHyP mount a very robust immunity (from IL-10 towards TFN-γ mediated responses) against L. donovani infection and could be explored further as a putative vaccine candidate against VL.

Improvements in Flow Cytometry-Based Cytotoxicity Assay.

The flow cytometry-based assay has been increasingly used to assess the cell-mediated cytotoxicity since the 1980s due to its advantages over the conventional radioactive 51 Cr release assay (CRA), such as higher sensitivity at the single-cell level and nonradioactivity. The basic principle of this assay is the usage of two dyes, one nontoxic dye for labeling targets or effector cells to distinguish one from another, one viability dye for discrimination of dead from live cells. Due to the problem of spontaneous release or leakage of the nontoxic dye, the concern about the cross-staining has not yet been clearly elucidated. In this study, carboxyfluorescein diacetate succinimidyl ester (CFSE) was utilized to label target cells and Hoechst 33258 was used as the viability dye. We confirmed that no cross-staining occurred between the effector and target cells after 4 h of coculture. We also found that the cytotoxicity would be overestimated if effector cells instead of target cells were labeled due to the exclusion of viable targets in effector-target conjugates. Using EDTA at the end of culture or labeling targets can solve this problem. Furthermore, the gating strategy could be improved by plotting CFSE against forward scatter (FSC) to discriminate some early apoptotic events. Due to the loss of target cells lysed by effector cells, counting beads are normally preferable in this assay. Here, we found an alternative to the use of beads in standardizing the flow cytometry-based assay. Instead of using beads, sample acquisition in a fixed time was shown to have the same effect in specific lysis evaluation as the beads application but have a greater stability than the latter. With a good quality control, the acquisition time for each sample could be shortened to 15 s, thus making this work to be done efficiently, especially in the case of larger sample sizes. Collectively, the findings in this study can improve the flow cytometric cytotoxicity assay to be carried out in a more accurate, efficient, and cost-effective way. © 2020 International Society for Advancement of Cytometry.

Viability assessment using fluorescent markers and ultrastructure of human biopsied embryos vitrified in open and closed systems.

RESEARCH QUESTION: Are there any differences in viability and ultrastructure amongst embryos biopsied on Day 5 versus Day 3 following vitrification in open and closed systems and compared to fresh embryos? DESIGN: One hundred human embryos (40 blastocysts biopsied on Day 5 and subsequently vitrified in open or closed systems and 60 Day 3 biopsied embryos that developed to blastocysts but were rejected for transfer following preimplantation genetic testing for monogenic/single gene defects and for aneuploidies were either treated fresh [n = 20] or vitrified [n = 40] in open or closed systems) and following warming and culture for 4 h were subjected to viability staining with carboxyfluorescein-diacetate succinimidylester/propidium iodide or processed for transmission electron microscopy. RESULTS: No statistically significant differences were observed in the viability of human biopsied embryos following vitrification in open and closed systems. Compared to fresh embryos, vitrified ones had a higher incidence of damage (propidium iodide-stained cells) irrespective of the vitrification method (P = 0.005). These damaged cells were more prominent in Day 5 biopsied blastocysts and mainly located at the position of cutting. Characteristic lipofuscin droplets (representative of apoptosis) and a higher number of vacuoles and distension of mitochondria were also more evident in vitrified embryos, although this was not statistically assessed. CONCLUSIONS: Vitrification in open and closed systems does not adversely affect the viability and ultrastructure of Day 5 and Day 3 biopsied embryos as revealed by the minimal yet statistically significant cell damage observed. This damage may be compensated by the embryos, which in their attempt to fully recover following vitrification, potentially enable 'rescue' processes to eliminate it.

Tracking Dye-Independent Approach to Identify and Isolate In Vitro Expanded T Cells.

T cell proliferation is routinely identified in vitro using tracking dyes or through detecting intracellular upregulation of the nuclear protein, Ki-67. However, labeling with tracking dyes is cumbersome, associated with cellular toxicity, while Ki-67 cannot be used to identify and isolate viable T cells, and both techniques are incompatible with MACS technology. Here, we introduce a simple tool to identify and isolate in vitro T cell expansion that is tracking dye-independent and allows for sorting of viable T cells. We show that CD71, a transferrin receptor, and CD98, a heterodimer glycoprotein involved in both integrin signaling and amino-acid transport, are both highly upregulated on proliferating T cells upon in vitro stimulation, and that CD71 expression is maximal on the more recent progeny T cells, while CD98 upregulation remains stable across different generations of progeny T cells. Moreover, we demonstrate that the upregulation of CD71 and CD98 identifies CFSElow T cells and provides further proof of the antigen-specificity of T cells identified by CD71 and CD98 dual upregulation based on tetramer staining. We further show that CD71 can be used to enrich for in vitro expanding T cells using MACS technology. In conclusion, we show that CD71 and CD98 can be used to identify and isolate expanded T cells following in vitro stimulation and that CD71 is an MACS-compatible alternative to tracking dyes or Ki-67 detection. © 2019 International Society for Advancement of Cytometry.

LongShengZhi Capsule reduces carrageenan-induced thrombosis by reducing activation of platelets and endothelial cells.

Formation of thrombosis is associated with activation of platelets and endothelial cells. The effect of LongShengZhi Capsule (LSZ), a traditional Chinese medicine used for treatment of vascular diseases, on thrombosis was investigated in this study. BALB/c mice were induced thrombosis by injection of carrageenan while receiving pre or simultaneous LSZ treatment. We also compared the therapeutic effects of LSZ and clopidogrel on formed thrombi. LSZ inhibited carrageenan-induced thrombi in mouse tissue vessels. In addition, LSZ but not clopidogrel reduced formed thrombi with a short time window. The reduction of thrombi by LSZ was associated with reduced serum P-selectin, reduced expression of TNF-α and P-selectin and activated matrix metalloproteinase 2 expression in tissues. In vitro, LSZ decreased thrombin-induced human platelet clot retraction which was associated with inactivation of AKT and ERK1/2. LSZ also reduced adhesion of platelets or THP-1 monocytes to human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein or lipopolysaccharide. The anti-adherent actions of LSZ was attributed to reduction of oxidative stress, expression of platelet receptors (P2Y12, PAR4 and CD36) and AKT activity in platelets. LSZ also reduced adhesion molecules or tissue factor but activated tissue factor pathway inhibitor expression in HUVECs. Taken together, our study demonstrates the antithrombotic properties of LSZ by reducing activation of platelets and endothelial cells, and suggests its potential application in clinics.

IL-10-producing regulatory B cells exhibit functional defects and play a protective role in severe endotoxic shock.

Dysregulated host immune homeostasis in sepsis is life-threatening even after a successfully treated bacterial infection. Lipopolysaccharide (LPS) is an endotoxin that is a major contributor to the aberrant immune responses and endotoxic shock in gram-negative bacterial sepsis. However, the current knowledge of the role of B cells in endotoxic shock is limited. Here, we report that CD1d expression in B cells and the percentage of CD5+CD1dhi regulatory B (Breg) cells decreased in a mouse model of endotoxic shock. Interestingly, IL-10 but not FasL expression in CD5+CD1dhi Breg cells in response to endotoxin was dramatically reduced in severe septic shock mice, and the regulatory function of CD5+CD1dhi Breg cells in vitro to control the Th1 response was also diminished. Adoptive transfer of CD5+CD1dhi Breg cells from healthy WT mice but not IL-10 deficient mice downregulated the IFN-γ secretion in CD4+ T cells and conferred protection against severe endotoxic shock in vivo. Our findings demonstrate the change and notable therapeutic potential of IL-10-producing Breg cells in endotoxic shock.

Detection and Quantification of Extracellular Vesicles via FACS: Membrane Labeling Matters!

: The field of extracellular vesicle (EV) research is challenged by the lack of standardized protocols to identify and specifically distinguish between exosomes and ectosomes, which are released via exocytosis or plasma membrane shedding, respectively. Using sequential centrifugation, we separated EV subpopulations from supernatants of COLO 357 pancreas carcinoma cells based on size and mass. After 10,000× g centrifugation, we reconstituted high-speed (hs) EVs from the pellet, directly labeled them with the membrane dye carboxyfluorescein diacetate succinimidyl ester (CFSE), and performed flow cytometry based analysis. The aim was to optimize the conditions for EV labeling and detection and hence to obtain a maximum yield of intact hsEVs. We found that, for sufficient labeling of EVs, minimal temperature variations and short incubation times correlated with EV stability. Furthermore, threshold adjustment significantly improved the sensitivity of the flow cytometer for the detection of CFSE labeled hsEVs. When cells were CFSE labeled, we observed a transition of fluorescence onto EVs that were reconstituted from the pellet but not onto those that remained in the supernatant after hs centrifugation, suggesting the indirect labeling of EVs based on the way of biogenesis as a specific method for the distinction of exosomes and ectosomes. Protocol standardization is of major importance for the use of EVs as diagnostic markers in liquid biopsies.

Enhancement of natural killer activity and IFN-γ production in an IL-12-dependent manner by a Brassica rapa L.

Certain food components possess immunomodulatory effects. The aim of this study was to elucidate the mechanism of the immunostimulatory activity of Brassica rapa L. We demonstrated an enhancement of natural killer (NK) activity and interferon (IFN)-γ production in mice that were orally administered an insoluble fraction of B. rapa L. The insoluble fraction of B. rapa L. significantly induced IFN-γ production in mouse spleen cells in an interleukin (IL)-12-dependent manner, and NK1.1+ cells were the main cells responsible for producing IFN-γ. Additionally, the results suggested that the active compounds in the insoluble fraction were recognized by Toll-like receptor (TLR) 2, TLR4, and C-type lectin receptors on dendritic cells, and they activated signaling cascades such as MAPK, NF-κB, and Syk. These findings suggest that B. rapa L. is a potentially promising immuno-improving material, and it might be useful for preventing immunological disorders such as infections and cancers by activating innate immunity.

Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures.

Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

Quantitating ADCC against adherent cells: Impedance-based detection is superior to release, membrane permeability, or caspase activation assays in resolving antibody dose response.

Monoclonal antibody-based immunotherapeutics will dominate Pharma's next generation of blockbuster drugs, and Fc-associated functions, including antibody dependent cellular cytotoxicity (ADCC) are among the highly desired activities mediated by these antibodies. Therefore, quantitative evaluation of ADCC is required during drug development. Our objective was to find the most suitable and reliable nonradioactive method for quantitative analysis of in vitro ADCC against adherent cells, which often serve as models for solid tumors. The test system was comprised the HER2 positive JIMT-1 cells targeted by the specific therapeutic antibodies trastuzumab (Herceptin® ) and pertuzumab (Perjeta® ). These cells are resistant to the direct biological effects of these antibodies, and, therefore, allow the isolated assessment of ADCC. We compared fluorescein diacetate (FDA) and carboxyfluorescein diacetate succinimidyl ester (CFSE) release as a fluorescent alternative to 51 Cr release; propidium iodide (PI) uptake revealing increased membrane permeability; the PanToxiLux assay measuring ADCC induced pro-apoptotic protease activity in flow cytometry; and an impedance-based real time cell adhesion test. We found that release assays are compromised by high spontaneous release of the label. PI uptake could not differentiate well between spontaneous NK activity and specific ADCC. The PanToxiLux assay, besides allowing for shorter assay times, offers improvement over the previous approaches in distinguishing spontaneous and antibody mediated NK action, but, probably owed to the prolonged detached state of adherent target cells, only at highly saturating antibody concentrations. In the case of adherent target cells, impedance-based cell analysis attains functional information exclusively on the target cells without having to label them for distinguishing from effectors or assay readout. It also allows continuous monitoring for days, and specifically detects target cell detachment, as the final functional consequence of ADCC. The sensitivity of this method even allows for quantitating the additivity and saturability of ADCC as a function of antibody concentration. We conclude that impedance-based assays are the most sensitive for quantitatively assessing in vitro ADCC on adherent target cells. © 2017 International Society for Advancement of Cytometry.

Identification of HLA-A*11:01-restricted Mycobacterium tuberculosis CD8(+) T cell epitopes.

New vaccines are needed to combat Mycobacterium tuberculosis (MTB) infections. The currently employed Bacillus Calmette-Guérin vaccine is becoming ineffective, due in part to the emergence of multidrug-resistant tuberculosis (MDR-TB) strains and the reduced immune capacity in cases of HIV coinfection. CD8(+) T cells play an important role in the protective immunity against MTB infections, and the identification of immunogenic CD8(+) T cell epitopes specific for MTB is essential for the design of peptide-based vaccines. To identify CD8(+) T cell epitopes of MTB proteins, we screened a set of 94 MTB antigens for HLA class I A*11:01-binding motifs. HLA-A*11:01 is one of the most prevalent HLA molecules in Southeast Asians, and definition of T cell epitopes it can restrict would provide significant coverage for the Asian population. Peptides that bound with high affinity to purified HLA molecules were subsequently evaluated in functional assays to detect interferon-γ release and CD8(+) T cell proliferation in active pulmonary TB patients. We identified six novel epitopes, each derived from a unique MTB antigen, which were recognized by CD8(+) T cells from active pulmonary TB patients. In addition, a significant level of epitope-specific T cells could be detected ex vivo in peripheral blood mononuclear cells from active TB patients by an HLA-A*11:01 dextramer carrying the peptide Rv3130c194-204 (from the MTB triacylglycerol synthase Tgs1), which was the most frequently recognized epitope in our peptide library. In conclusion, this study identified six dominant CD8(+) T cell epitopes that may be considered potential targets for subunit vaccines or diagnostic strategies against TB.

Interaction of Lymphocytes with Mesenchymal Stem Cells.

We studied the interaction of neural stem cells and dental pulp-derived mesenchymal stem cells with lymphocytes from autologous and heterologous donors. Flow cytometry analysis with the use of CFSE-labeled lymphocytes demonstrated an increase in the content of proliferating CD8, CD16 and CD56 cells, but not CD4 cells in cultures of HLA-DR-negative mesenchymal stromal cells from the dental pulp co-cultured with lymphocytes. In neural cultures expressing HLA-DR, all subpopulations of T cells and NK cells were activated. No differences between the autologous and heterologous cultures were revealed.

Evaluation of lymphocytes inactivation by extracorporeal photopheresis using tetrazolium salt based-assay.

Extracorporeal photopheresis (ECP) is accepted as a second-line therapy for the treatment of acute and chronic steroid-refractory graft versus host disease (GvHD), cutaneous T-cell lymphoma and solid organ transplantation. ECP should be validated: we compared in parallel apoptosis and proliferation analysis of patient lymphocytes treated with 8-MOP ECP using respectively Annexin V/7-aminoactinomycin D (7-AAD) and CFSE with a tetrazolium salt (WST-1) method. Using WST-1 assay we found a significant decrement (p < 0.01) of metabolic activity at 4 days between ECP-treated and untreated cells. This finding was confirmed by the significant decrease of cell proliferation and increase of cell death observed by CFSE and 7AAD-Annexin V, respectively. Accordingly, once validated against a reference method, WST-1 could represent a rapid and easy assay for routinely quality control of ECP.

Biological quality control for extracorporeal photochemotherapy: Assessing mononuclear cell apoptosis levels in ECP bags of chronic GvHD patients.

Extracorporeal photochemotherapy (ECP) is a treatment approved by the FDA for cutaneous T-cell lymphoma, and it is currently used off-label for graft-versus-host disease (GvHD) and other conditions. In agreement with good practices for the therapeutic use of human cells, quality control has to be performed to validate the ECP procedure with the off-line technique. Since no gold-standard biological test is available, we assessed the apoptosis generated in the ECP bag using a flow cytometric analysis. Thirty-one ECP procedures performed on 13 patients with chronic GvHD were studied by monitoring the induction of mononuclear cell (MNC) apoptosis using annexin V/propidium iodide double staining; residual lymphocyte proliferation to standard mitogens was also measured in 17 of the procedures. The kinetics of apoptosis was analyzed at different times in MNCs untreated or treated with 8-methoxy-psoralen plus ultraviolet A; the variation (ΔAPOPTOSIS ) after 24 h revealed the efficacy of the treatment. In 88.6% of the 31 ECP procedures, ΔAPOPTOSIS was >15% (the "alerting" threshold for ΔAPOPTOSIS was set at 15% on the basis of our data); in the remainder (19.4%), the increment in apoptosis was lower. In four procedures, the proliferation assay was useful for assessing the effect of ECP on the apheretic bag. In conclusion, both flow cytometric assays enabled a biologically significant result to be obtained. In our opinion, the apoptosis test-being faster and easier than the proliferation test-could be a reliable way to validate ECP procedures.

Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3).

BACKGROUND: The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance. OBJECTIVE: Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy. METHODS: In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13). RESULTS: Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells. CONCLUSION: In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.

The role of p21 Waf1/Cip1 in large airway epithelium in smokers with and without COPD.

Airway epithelium alterations, including squamous cell metaplasia, characterize smokers with and without chronic obstructive pulmonary disease (COPD). The p21 regulates cell apoptosis and differentiation and its role in COPD is largely unknown. Molecules regulating apoptosis (cytoplasmic p21, caspase-3), cell cycle (nuclear p21), proliferation (Ki67/PCNA), and metaplasia (survivin) in central airways from smokers (S), smokers-COPD (s-COPD) and non-smokers (Controls) were studied. The role of cigarette smoke extracts (CSE) in p21, survivin, apoptosis (caspase-3 and annexin-V binding) and proliferation was assessed in a bronchial epithelial cell line (16HBE). Immunohistochemistry, image analysis in surgical samples and flow-cytometry and carboxyfluorescein succinimidyl ester proliferative assay in 16HBE with/without CSE were applied. Cytoplasmic and nuclear p21, survivin, and Ki67 expression significantly increased in large airway epithelium in S and in s-COPD in comparison to Controls. Caspase-3 was similar in all the studied groups. p21 correlated with epithelial metaplasia, PCNA, and Ki67 expression. CSE increased cytoplasmic p21 and survivin expression but not apoptosis and inhibited the cell proliferation in 16HBE. In large airway epithelium of smokers with and without COPD, the cytoplasmic p21 inhibits cell apoptosis, promotes cell proliferation and correlates with squamous cell metaplasia thus representing a potential pre-oncogenic hallmark.

Human umbilical cord blood mesenchymal stem cells reduce colitis in mice by activating NOD2 signaling to COX2.

BACKGROUND & AIMS: Decreased levels or function of nucleotide-binding oligomerization domain 2 (NOD2) are associated with Crohn's disease. NOD2 regulates intestinal inflammation, and also is expressed by human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs), to regulate their differentiation. We investigated whether NOD2 is required for the anti-inflammatory activities of MSCs in mice with colitis. METHODS: Colitis was induced in mice by administration of dextran sulfate sodium or trinitrobenzene sulfonic acid. Mice then were given intraperitoneal injections of NOD2-activated hUCB-MSCs; colon tissues and mesenteric lymph nodes were collected for histologic analyses. A bromodeoxyuridine assay was used to determine the ability of hUCB-MSCs to inhibit proliferation of human mononuclear cells in culture. RESULTS: Administration of hUCB-MSCs reduced the severity of colitis in mice. The anti-inflammatory effects of hUCB-MSCs were greatly increased by activation of NOD2 by its ligand, muramyl dipeptide (MDP). Administration of NOD2-activated hUCB-MSCs increased anti-inflammatory responses in colons of mice, such as production of interleukin (IL)-10 and infiltration by T regulatory cells, and reduced production of inflammatory cytokines. Proliferation of mononuclear cells was inhibited significantly by co-culture with hUCB-MSCs that had been stimulated with MDP. MDP induced prolonged production of prostaglandin (PG)E2 in hUCB-MSCs via the NOD2-RIP2 pathway, which suppressed proliferation of mononuclear cells derived from hUCB. PGE2 produced by hUCB-MSCs in response to MDP increased production of IL-10 and T regulatory cells. In mice, production of PGE2 by MSCs and subsequent production of IL-10 were required to reduce the severity of colitis. CONCLUSIONS: Activation of NOD2 is required for the ability of hUCB-MSCs to reduce the severity of colitis in mice. NOD2 signaling increases the ability of these cells to suppress mononuclear cell proliferation by inducing production of PGE2.

Suppressive activity and altered conventional phenotype markers/mediators of regulatory T cells in patients with self-limiting hepatitis E.

Hepatitis E virus (HEV) is a major cause of self-limiting acute viral hepatitis in several developing countries. Elevated levels of peripheral CD4(+) CD25(+) Foxp3(+) , CD4(+) CD25(-) Foxp3(+) and rise in IL-10 in hepatitis E have been associated with the involvement of regulatory T cells (Treg). The functional role of the same is yet elusive. In the current study, we have assessed (i) Foxp3 expression by real-time PCR and by flow cytometry, (ii) the levels of antigen-specific IL-10 and TGF-ß by ELISA, (iii) functional analysis of Treg cells and (iv) expression of Treg-associated conventional phenotypes by flow cytometry in 54 acute patients, 44 recovered individuals from hepatitis E and in 33 healthy controls. Foxp3 mRNA elevation in the acute compared with recovered group and elevation in Foxp3(+) cells in both patient groups were significantly elevated. The levels of IL-10 and TGF-ß in the acute patients and TGF-ß in the recovered individuals were elevated. Significantly higher expression of CTLA-4, PD1, GITR, CD95, CD103 and CD73 on Treg and T effector (Teff) cells was detected in the patient groups. Treg cells of acute patients and recovered individuals exhibited suppressive activity indicating that the Treg cells of hepatitis E patients are functional. The suppressive capacity of Treg cells in acute hepatitis E patients was significantly higher compared with the recovered individuals. Based on our findings, the suppressive functionality of these key markers associated with hepatitis E Treg function need further exploration to get a better understanding of the mechanisms of Treg-mediated suppression.