Aleksandra Klimczak

Human TH17 cells are long-lived effector memory cells.

Human TH17 cells function as long-lived effector memory cells in the context of chronic disease. Forever Seventeen “It” is that quality possessed by some which draws all others with its magnetic force. Novelist Elinor Glyn In teen television drama parlance, T helper 17 (TH17) cells have been immunology’s “it girl” for the past few years. Discovered in 2007, this beguiling subset of TH cells is associated with autoimmune disease and long-term antitumor immunity, and the presence of these cells in tumors positively correlates with patient survival. Although mouse TH17 cells are believed to be short-lived, studies of advanced human cancers suggest that human TH17 cells persist, but little is known about the nature of these cells in the context of human disease. Now, Kryczek et al. define the detailed phenotype and functional aspects of human TH17 cells in diverse diseased human tissues and find that these cells resemble terminally differentiated, long-lived memory T cells with a twist. The authors used a well-defined human system to study the defining features and behavior of TH17 cells in the pathological microenvironments of human graft-versus-host disease, ulcerative colitis, and cancer. The fraction of TH17 cells was increased in these tissues during the chronic phases of the diseases. Furthermore, the TH17 cells not only displayed characteristics in common with terminally differentiated memory T cells, but also sported some genetic and functional signatures of their own. For example, disease-associated human TH17 cells wore the standard phenotypic markers of terminal differentiation and, in adoptive transfer experiments, drove persistent antitumor immunity. However, these versatile human T cells also displayed a high capacity for proliferative self-renewal, cell-type plasticity, and enriched expression of antiapoptotic genes. The cell membrane signaling protein Notch and the transcriptional regulatory protein hypoxia-inducible factor–1α (HIF-1α) were shown to jointly regulate the expression and function of the Bcl-2 family of antiapoptosis proteins. An intricate understanding of human TH17 cell biology in disease contexts should reveal new “it” molecules—therapeutic targets that permit TH17-directed treatment in patients with autoimmune diseases and advanced tumors. T helper 17 (TH17) cells have been shown to contribute to multiple disease systems. However, the functional phenotype and survival pattern of TH17 cells as well as the underlying mechanisms that control TH17 cells have been poorly investigated in humans, significantly hampering the clinical targeting of these cells. Here, we studied human TH17 cells in the pathological microenvironments of graft-versus-host disease, ulcerative colitis, and cancer; TH17 cell numbers were increased in the chronic phase of these diseases. Human TH17 cells phenotypically resembled terminally differentiated memory T cells but were distinct from central memory, exhausted, and senescent T cells. Despite their phenotypic markers of terminal differentiation, TH17 cells mediated and promoted long-term antitumor immunity in in vivo adoptive transfer experiments. Furthermore, TH17 cells had a high capacity for proliferative self-renewal, potent persistence, and apoptotic resistance in vivo, as well as plasticity—converting into other types of TH cells. These cells expressed a relatively specific gene signature including abundant antiapoptotic genes. We found that hypoxia-inducible factor–1α and Notch collaboratively controlled key antiapoptosis Bcl-2 family gene expression and function in TH17 cells. Together, these data indicate that human TH17 cells may be a long-lived proliferating effector memory T cell population with unique genetic and functional characteristics. Targeting TH17-associated signaling pathway would be therapeutically meaningful for treating patients with autoimmune disease and advanced tumor.

Mesenchymal Stromal Cells and Tissue-Specific Progenitor Cells: Their Role in Tissue Homeostasis

Multipotent mesenchymal stromal/stem cells (MSCs) reside in many human organs and comprise heterogeneous population of cells with self-renewal ability. These cells can be isolated from different tissues, and their morphology, immunophenotype, and differentiation potential are dependent on their tissue of origin. Each organ contains specific population of stromal cells which maintain regeneration process of the tissue where they reside, but some of them have much more wide plasticity and differentiate into multiple cells lineage. MSCs isolated from adult human tissues are ideal candidates for tissue regeneration and tissue engineering. However, MSCs do not only contribute to structurally tissue repair but also MSC possess strong immunomodulatory and anti-inflammatory properties and may influence in tissue repair by modulation of local environment. This paper is presenting an overview of the current knowledge of biology of tissue-resident mesenchymal stromal and progenitor cells (originated from bone marrow, liver, skeletal muscle, skin, heart, and lung) associated with tissue regeneration and tissue homeostasis.

IL-6 production in ovarian carcinoma is associated with histiotype and biological characteristics of the tumour and influences local immunity.

The presence of interleukin (IL)-6 in peritoneal carcinomatous fluid (PCF) and its effect on immune cells composition in PCF in patients with advanced ovarian carcinoma was studied. In 21 out of 30 ovarian carcinoma patients, PCF IL-6 levels were found to exceed those seen in PCFs of patients with gastrointestinal cancer. IL-6 activity was higher in serous/mucinous than in endometrioid and undifferentiated ovarian carcinoma PCF (P = 0.05). Ovarian carcinoma PCF IL-6 activities were correlated with serum C-reactive protein levels (r = 0.65, P = 0.0000, n = 25). Ovarian carcinoma PCF leucocyte profile differed from that in blood with respect to: (i) lower percentage of NK and CD8+ and (ii) higher percentage of B and CD45RO+, CD14+ and HLA-DR+ cells. The proportions of CD45RO+ in blood were correlated with IL-6 levels in PCF. Corresponding to PCF ovarian carcinoma tumours were stained for the presence of Ki-67 antigen and p53. The highest proportions of Ki-67+ cells and cells showing accumulation of p53 were seen in undifferentiated tumours. A low grade of p53 staining was seen in tumours associated with high IL-6 levels in PCF. It was evident that IL-6 production (i) depended on the histiotype of the tumour, (ii) influenced the local immune system in favour of accumulation of B, and T memory cells, and (iii) was higher in patients lacking p53 accumulation.

Applications of bilateral vascularized femoral bone marrow transplantation for chimerism induction across the major histocompatibility (MHC) barrier Part II

Bilateral vascularized bone marrow transplant (VBMT) model was designed to induce chimerism across the major histocompatibility (MHC) barrier under combined αβ T-cell receptor monoclonal antibody and cyclosporine A (αβ-TCRmAb/CsA) protocol. Seventeen transplants were performed between BN(RT1n) donors and Lewis(RTIl) recipients. Group I, isograft controls; Group II, allografts rejection controls; Group III, allografts under 7-day protocol of αβ-TCRmAb/CsA. Donor bilateral femoral bones were bilaterally anastomosed to the abdominal aorta and inferior vena cava of recipient. At day 7 posttransplantation, all bone flaps were viable. Groups I and III survived without signs of rejection. In Group III, peak level of chimerism in peripheral blood was evaluated at day 21 (24.2%), at day 63 declined to 1.5%, and was maintained at this level thereafter. Donor-derived cells were present in the bone marrow of recipients at 28.2% at day 21 posttransplant. Histology confirmed viability of bone marrow cells in isograft during the entire follow-up and up to 35 days in treatment Group III. Bilateral VBMT induced donor-specific chimerism across the MHC barrier under the immunomodulatory protocol of αβ-TCRmAb/CsA.

Enrichment of normal progenitors in counter‐flow centrifugal elutriation (CCE) fractions of fresh chronic myeloid leukemia leukapheresis products

Abstract: Objectives : The aim of this study was to assess the suitability of a technique based on counter‐flow centrifugal elutriation (CCE), which should allow one to enrich chronic myeloid leukemia (CML) patients unstimulated native leukapheresis product (nLP) in CD34 + HLADR – cells and BCR‐ABL negative cells. Methods : Six newly diagnosed CML patients were subjected to leukapheresis, and the products were subfractionated with the use of CCE. nLP and all fractions were studied for the presence of CD34 + cells and a proportion of BCR‐ABL fluorescence in situ hybridization (FISH) + cells. Results : CCE fractions with a high flow rate contained the highest proportion of CD34 + cells [mean (SEM) 6.89% (3.88)]. However, CD34 + cells present in low‐rate CCE fractions showed a higher proportion of HLADR – [49.6% (13.5 in 70u2003mLu2003min −1 ) and 21.5% (11.6 in 110u2003mLu2003min −1 )] than those in 170u2003mLu2003min −1 [3.2%u2003(2.5)] and ‘rotor off’[3.4% (1.9)]. This was associated with lower proportions of BCR‐ABL FISH + [8.1% (4.8) and 1.9 (1.7)] and smaller BCR‐ABL to ABL transcript ratios [0.58 ( 17 ) and 0.26 (0.08) in 70u2003and 110u2003mLu2003min −1 ] fractions as compared to 140 and 170u2003mLu2003min −1 fractions [21.6% (5.2) and 31.6% (15.3) for BCR‐ABL FISH + cells and 0.75 (0.16) and 0.90 (0.24) for BCR ‐ ABL / ABL ]. Fractions with the lowest proportions of BCR ‐ ABL ‐positive cells and the lowest BCR ‐ ABL / ABL transcript ratios (110u2003mLu2003min −1 ) contained from 1.3u2003×u200310 6 to 82.7u2003×u200310 6 (median: 3.97u2003×u200310 6 ) CD34 + cells. Conclusions : In the present study we have shown that CCE may be used effectively to obtain nLP fractions enriched in normal hematopoietic progenitors.

Chimerism-Based Experimental Models for Tolerance Induction in Vascularized Composite Allografts: Cleveland Clinic Research Experience

The preclinical experimental models of vascularized composite allografts (VCAs) have been rapidly developed for the assessment of immunomodulatory protocols for clinical application. Recently, researchers have focused on immunomodulatory protocols which overcome the immunologic barrier between the allogeneic donor and recipient and may lead to tolerance induction. In order to test the feasibility of chimerism induction, experimental VCAs have been performed in different models including rodents, large animals, and nonhuman primates. These models differ in the complexity of transplanted tissue and in their responses to immunomodulatory protocols. In most applications, VCA contains multiple-tissue components; however, each individual component of CTA possesses unique immunologic characteristics that ultimately contribute to the chimerism induction and successful outcome of the VCA. Heterogenic character and complexity of tissue components in different VCA models determine the quality and robustness of donor-specific chimerism. As introduced in experimental studies, variable immunomodulatory options have been studied to achieve tolerance to VCA in rodents and large animal models allowing for widespread application in clinic. In this paper, based on our own experience, we have analyzed the current knowledge of tolerance-inducing strategies via chimerism induction in VCA experimental models in the context of immunomodulatory protocols and VCA complexity and their relevance and applicability to clinical practice.

Accumulation of CD45RO+ cells in peritoneal carcinomatous fluid favours survival of ovarian carcinoma patients

Abstract. In 44 patients with advanced ovarian carcinoma (OC) a fraction of CD45RO+ lymphocytes in the blood and peritoneal carcinomatous fluid (PCF) was investigated. Thirty-one patients received cisplatinum with cyclophosphamide ± doxorubicin. This group was followed from 2.2 to 9 years (mean: 45 months). In 23 out of 31 patients, the percentage of CD45RO+ lymphocytes was higher in the PCF than in the blood samples. Patients with these higher lymphocyte levels experienced longer survival than those who did not show any excess of CD45RO+ lymphocytes in PCF (P=0.02). This was further verified by the use multivariate Cox analysis which included an assessment of the percentage of CD45RO+ lymphocytes in PCF, age, FIGO status, histology, treatment (CAP or CP) and residual disease (RD) post-surgery. This analysis revealed that two factors had an independent power of prediction: RD (P=0.02) and the percentage of CD45RO+ cells in PCF (P=0.04). Therefore, CD45RO+ lymphocytes were studied in further detail in a group of 20 patients. This study revealed that PCF CD45RO+ lymphocytes were characterized by: (1) a higher proportion of cells co-expressing activation markers (HLA-DR, CD28) and higher levels of mRNA for CXC chemokines (IP-10, IL-8) and for IL-10, but with lower levels for IL-2; (2) higher levels of Ki67, bcl-2 and p53 mRNA as compared to those in blood. In conclusion, in the present study it was found that an accumulation of activated CD45RO+ cells in PCF had a beneficial effect on the survival of patients receiving platinum-based chemotherapy.

Immunomodulatory Effects of Different Cellular Therapies of Bone Marrow Origin on Chimerism Induction and Maintenance Across MHC Barriers in a Face Allotransplantation Model

Many more patients would benefit from vascularized composite allotransplantation if less toxic and safer immunosuppressive protocols will become available. Tolerance induction protocols with donor cells co-transplantation are one of the promising pathways to reduce maintenance immunosupressive regimens. We investigated the role of donor bone marrow cells (BMC), mesenchymal stromal cells (MSC) and in vivo created chimeric cells (CC) used as supportive therapies in a fully MHC-mismatched rat face transplantation model. Twenty-four fully MHC-mismatched hemiface transplantations were performed between ACI (RT1a) donors and Lewis (RT1l) recipients under combined seven-day immunosuppressive regimen of anti-αβ-T-cell receptor (TCR) monoclonal antibody and cyclosporin A. We studied four experimental groups—group 1: no cellular therapy; group 2: supportive therapy with BMC; group 3: supportive therapy with MSC; group 4: supportive therapy with CC generated in a primary chimera. We evaluated clinical and histological rejection grades, transplanted cells migration, donor-specific chimerism in the peripheral blood and bone marrow compartments, and CD4+/CD25+ T-cell levels. Face allograft rejection was observed at 26.8xa0±xa00.6xa0days post-transplant (PT) in the absence of cellular therapy, at 34.5xa0±xa01.1xa0days for group 2, 29.3xa0±xa00.8xa0days for group 3, and 30.3xa0±xa01.38 PT for group 4. The longest survival was observed in allografts supported by co-transplantation of BMC. All support in cellular therapies delayed face allograft rejection by chimerism induction and/or immunomodulatory properties of co-transplanted cells. Survival time was comparable between groups, however, further studies, with different cell dosages, delivery routes and delivery times are required.

Pulse-Modulated Radio-Frequency Alternating-Current-Driven Atmospheric-Pressure Glow Discharge for Continuous-Flow Synthesis of Silver Nanoparticles and Evaluation of Their Cytotoxicity toward Human Melanoma Cells

An innovative and environmentally friendly method for the synthesis of size-controlled silver nanoparticles (AgNPs) is presented. Pectin-stabilized AgNPs were synthesized in a plasma-reaction system in which pulse-modulated radio-frequency atmospheric-pressure glow discharge (pm-rf-APGD) was operated in contact with a flowing liquid electrode. The use of pm-rf-APGD allows for better control of the size of AgNPs and their stability and monodispersity. AgNPs synthesized under defined operating conditions exhibited average sizes of 41.62 ± 12.08 nm and 10.38 ± 4.56 nm, as determined by dynamic light scattering and transmission electron microscopy (TEM), respectively. Energy-dispersive X-ray spectroscopy (EDS) confirmed that the nanoparticles were composed of metallic Ag. Furthermore, the ξ-potential of the AgNPs was shown to be −43.11 ± 0.96 mV, which will facilitate their application in biological systems. Between 70% and 90% of the cancerous cells of the human melanoma Hs 294T cell line underwent necrosis following treatment with the synthesized AgNPs. Furthermore, optical emission spectrometry (OES) identified reactive species, such as NO, NH, N2, O, and H, as pm-rf-APGD produced compounds that may be involved in the reduction of the Ag(I) ions.

Expression and activity of multidrug resistance proteins in mature endothelial cells and their precursors: A challenging correlation

Active cellular transporters of harmful agents—multidrug resistance (mdr) proteins—are present in tumor, stem and endothelial cells, among others. While mdr proteins are broadly studied in tumor cells, their role in non-tumor cells and the significance of their action not connected with removal of harmful xenobiotics is less extensively documented. Proper assessment of mdr proteins expression is difficult. Mdr mRNA presence is most often evaluated but that does not necessarily correlate with the protein level. The protein expression itself is difficult to determine; usually cells with mdr overexpression are studied, not cells under physiological conditions, in which a low expression level of mdr protein is often insufficient for detection in vitro. Various methods are used to identify mdr mRNA and protein expression, together with functional tests demonstrating their biological drug transporting activities. Data comparing different methods of investigating expression of mdr mRNAs and their corresponding proteins are still scarce. In this article we present the results of a study concerning mdr mRNA and protein expression. Our goal was to search for the best method to investigate the expression level and functional activity of five selected mdr proteins—MDR1, BCRP, MRP1, MRP4 and MRP5—in established in vitro cell lines of human endothelial cells (ECs) and their progenitors. Endothelial cells demonstrated mdr presence at the mRNA level, which was not always confirmed at the protein level or in functional tests. Therefore, several different assays had to be applied for evaluation of mdr proteins expression and functions in endothelial cells. Among them functional tests seemed to be the most conclusive, although not very specific.