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This paper presents the energy savings in refurbishment of the buildings using Life Cycle Assessment at material level by comparing three insulation materials that offer higher thermal performance and greater environmental sustainability. The comparison is made on products having the same insulation performance (U-value). After the enumeration of intervention practices for energy saving in buildings, the Life Cycle Assessment methodology in refurbishment of the buildings is applied at material level using Life Cycle Inventory to transform material volume input into impact on the environment as output. The Life Cycle Assessment (LCA) was carried out according to the requirements of the Environmental Product Declaration (EPD). After assessing the building performance before and after isolation, the “embodied energy”, which comes from the materials manufacturing as phases of the refurbishment of the buildings, was determinate and compared with the operational energy of the building. This LCA methodology in refurbishment of the buildings at material level was evaluated for Expanded Polystyrene (EPS), Extruded Polystyrene (XPS) and Rigid polyurethane (PUR) insulation products. This methodology has been used on a building in Galati, Romania (School No. 9). A spider diagram was used to represent the criteria in a consistent, graphical way in relative units. The extreme values for a given criteria indicate the poor performance of the insulation material. Finally, the assessment of the building performance before and after using the insulation and the determination of the “embodied energy” compared to the operational energy of the building resulted in a 55% energy saving in the use stage of the building after refurbishment.

Immune activation needs to be tightly regulated to control immune-mediated tissue damage. Inhibitory pathways serve to terminate an immune response and resolve inflammation. Persistent exposure to antigens can drive development of adaptive regulatory cells. Similarly exposure of activated T cells to the recombinant ILT3-Fc molecule during priming triggers the differentiation of CD8 T suppressor cells and the induction of CD4 T helper anergy. Ts express high levels of immunoregulatory signature genes together with low levels of microRNA which control their function. Analysis of microRNA contained by exosomes from cultures in which T cells were alloactivated in the presence or absence of ILT3.Fc, demonstrated that this agent inhibits the release of inflammatory microRNA. The source of such inflammatory microRNA was found to reside in alloactivated CD4 T cells, since exosomes from MLC primed CD4 T cells were shown to diminish the suppressive activity of ILT3-Fc-induced CD8(+) Ts at high effector to suppressor T cell ratios. This indicates that inflammatory exosomes can swing the balance between effector and regulatory T cells in favor of immunity. These data suggest that isolation and characterization of micro-RNA containing exosomes in patients' circulation may be of use for treatment, prevention and monitoring of immune activation.

Presensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allogaft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. Our data reinforce the concept that these two types of assays should be used in parallel for pre-and post-transplantation monitoring of anti-HLA antibodies in recipients of solid organ allografts.

Alloantigen specific CD8 T suppressor cells can be generated in vitro either by multiple stimulations of CD3 T cells with allogeneic APC or by single stimulation in primary MLC containing recombinant ILT3.Fc protein. The aim of the present study was to determine whether multiple MLC stimulation induced in CD8(+) CD28(-) T suppressor cells molecular changes that are similar to those observed in CD8 T suppressor cells from primary MLC containing ILT3.Fc protein. Our study demonstrates that the characteristic signatures of CD8 T suppressor cells, generated by either of these methods are the same consisting of up-regulation of the BCL6 transcriptional repressor and down-regulation of inflammatory microRNAs, miR-21, miR-30b, miR-146a, and miR-155 expression. In conclusion microRNAs which are increased under inflammatory conditions in activated CD4 and CD8 T cells with helper or cytotoxic function show low levels of expression in CD8 T cells which have acquired antigen-specific suppressor activity.

Antigen-specific CD8 suppressor T cells (CD8 + Ts) are adaptive regulatory T cells that are induced in vivo and in vitro by chronic antigenic stimulation of human T cells. CD8 + Ts induce the upregulation of the inhibitory receptors ILT3 and ILT4 on monocytes and dendritic cells rendering these antigen presenting cells (APCs) tolerogenic. Tolerogenic APCs induce CD4 + T helper anergy and elicit the differentiation of CD4 + and CD8 + T regulatory/suppressor cells. Overexpression of membrane ILT3 in APC results in inhibition of NF-κB activation, transcription of inflammatory cytokines and costimulatory molecules. Soluble ILT3-Fc which contains only the extracellular, Ig-like domain linked to mutated IgG1 Fc, is strongly immunosuppressive. ILT3-Fc, induces the differentiation of human CD8 + Ts which inhibit CD4 + Th and CD8 + CTL effector function both in vitro and in vivo. The acquisition of Ts' function by primed CD8 + T cells treated with ILT3-Fc was demonstrated to be the effect of the significant upregulation of BCL6, a transcriptional repressor of IL-2, IFN-gamma, IL-5 and granzyme B. The upregulated expression of BCL6, SOCS1 and DUSP10 is integral to the signature of ILT3-Fc-induced CD8 + Ts. These genes are known inhibitors of cytokine production and TCR signaling and are targeted by miRNAs which are suppressed by ILT3-Fc. ILT3-Fc induces tolerance to allogeneic human islets and reverses rejection after its onset in a humanized NOD/SCID mouse model. Based on these findings we postulate that ILT3-Fc may become an important new agent for treatment of autoimmunity and transplant rejection.

The involvement of humoral response in allograft rejection has been suggested by both immunologic and histochemistry studies. In the present study, we explored the role of alloantibodies in a large cohort of heart allograft recipients followed for 15 years. Sequential samples of sera were obtained from 950 recipients of heart allografts before and after transplantation at the time when protocol endomyocardial biopsies were performed. The presence of anti–human leukocyte antigen (HLA) antibodies was investigated using complement mediated cytotoxicity and solid phase assay (SPA). Our data reveal an inverse correlation between the development of alloantibodies after transplantation and heart allograft survival. The 15-year graft survival was highest in patients who never developed alloantibodies (70%) or who displayed them only before transplantation (71%); graft survival in recipients who showed antibodies both before and after transplantation (56%), or only after transplantation (47%), was lower. The deleterious effect of antibodies on graft survival started 8 years after transplantation, suggesting that the production of de novo antibodies may have been triggered by some later event. We found that patients with de novo antibodies appearing more than 1 year after transplantation had the poorest survival. Furthermore, the development of de novo antibodies was preceded in 76% of these patients by cellular rejection grade 3 or higher, according to the International Society for Heart Transplantation (ISHT) grading criteria. Development of antibody-mediated rejection (AMR) had a significant negative impact on graft survival (16% in AMR + vs 63% in AMR – patients, p = 0.0008). Of the 23 patients with AMR, 21 displayed cytotoxic donor-specific antibodies (DSA) at the time of diagnosis, and in 18 of these cases SPA showed that they were directed against the donors' HLA. The data demonstrate that the detection of alloantibodies permits a better definition of AMR in heart allograft recipients. Identification of patients at risk for developing AMR is of great importance for early treatment of rejection episodes.

Similar to helper and cytotoxic T cells, CD8(+) T suppressor cells (Ts) acquire antigen specificity via direct interaction with antigen-presenting cells (APC). They induce the upregulation of the inhibitory receptor immunoglobulin-like transcript (ILT)3 on professional and nonprofessional APC, rendering these cells tolerogenic and able to induce the differentiation of further waves of regulatory and suppressor T cells. This review sums up evidence that ILT3 is the centerpiece of CD8(+) Ts-driven suppression and acts as a master switch in the regulation of CD8(+) and CD4(+) T-cell responses to antigens in transplantation, autoimmunity, allergy, and cancer.

We explored the possibility that antigen-specific human CD8 + T cells, which display cytotoxic or suppressor function, can recognize the same peptide epitope. Using the human leukocyte antigen-A0201 restricted immunodominant cytomegalovirus epitope pp65-NLVPMVATV for pulsing either mature/immunogenic or ILT3 high ILT4 high tolerogenic dendritic cells (DC), we generated cytotoxic and suppressor CD8 + T-cell lines, respectively. Our data indicate that modulating the functional state of DC is crucial to the development of tolerogenic or immunogeneic peptide-specific vaccines.

The interleukin-2 receptor alpha chain (IL-2Ra, CD25) plays a major part in shaping the dynamics of T cell populations following immune activation, due to its role in T cell proliferation and survival. Strategies to blunt the effector responses in transplantation have been developed by devising pharmaceutical agents to block the IL-2 pathways. However, such strategies could adversely affect the CD25(+)FOXP3(+)T regulatory (T reg) populations which also rely on intereukin-2 signaling for survival. The present study shows that a cohort of heart allograft recipients treated with Daclizumab (a humanized anti-CD25 antibody) display FOXP3 expression patterns consistent with functional T regulatory cell populations. High levels of FOXP3 were observed to correlate with lower incidence of and recovery from acute rejection, as well as lower levels of anti-donor HLA antibody production. Therefore, T reg populations appear fully functional in patients treated with Daclizumab, even when 5 doses were administered. By comparison, patients treated with fewer doses or no Daclizumab had a higher incidence of acute rejection, antibody production and graft failure. Therefore, our data indicates that Daclizumab treatment does not interfere with the generation of regulatory T cells and has a beneficial effect on heart allograft survival.

The role of humoral immunity in causing antibody-mediated rejection (AMR) of organ allografts has been extensively documented. For this reason, negative complement-dependent cytotoxicity (CDC) cross-matches between recipient sera and donor T and B lymphocytes have become a mandatory requirement for cadaveric kidney transplantation. However, the significance of donor-specific antibodies (DSAs) detectable only by flow cytometry (FC) or solid phase assays (SPA) but not CDC is still controversial. We have performed a retrospective analysis of FC cross-matching results in 80 consecutive cadaver kidney allograft recipients. Antibodies against HLA class I and class II antigens were measured by CDC and SPA in sequential samples of sera obtained prior to transplantation. The preoperative cross-match was performed by CDC using magnetically sorted T and B cells from donor spleen. Sera obtained from each patient before and at the time of transplantation were included in the final cross-match. The sample of serum obtained at the time of transplantation was cross-matched retrospectively by FC and analyzed for anti-HLA antibody specificity on high resolution SPA. The actuarial kidney allograft survival at one year was 98%. Two of these eighty patients lost the graft, one due to AMR, the other for reasons unrelated to DSAs. Donor-specific antibodies were detected by FC in 17 of 80 patients, yet only 6 of 17 had an early episode of AMR. This episode was successfully reversed by desensitization therapy using intravenous immunoglobin (IVIG) and plasmapheresis. Flow cytomery cross-matching showed 95% specificity but only 35% sensitivity for prediction of AMR (p = 0.002). There was a significant correlation between high panel reactive antibodies (PRA) and positive FC cross-matching (p = 0 .0001), as well as high PRA and AMR (p = 0.0004 by CDC and 0.0011 by Luminex). Reversible AMR occurred 12-30 days post-transplantation in 8 patients. Of these 8 patients, 3 had no detectable DSAs in spite of C4d positivity, 4 had C4d deposition in conjunction with anti-HLA antibodies, and 1 patient had DSAs (anti-MICA) yet no C4d deposition. We conclude that early initiation of desensitization protocols can prevent transplant failure and that retrospective FC cross-matches may facilitate the diagnosis of AMR. Extensive analysis of patients' sera using a comprehensive set of tests may contribute to early treatment and better understanding of the mechanism underlying humoral rejection.

Immunoglobulin-like transcript (ILT)-3 is a transmembrane receptor, which belongs to the immunoglobulin superfamily. In previous studies, we showed that allospecific CD8+CD28- T suppressor cells (Ts) induce the expression of ILT3 in human endothelial cells (EC) rendering them tolerogenic. Using a polymerase chain reaction (PCR)-based approach, we now demonstrate by cell fractionation and sequencing studies that ILT3 precursor RNA is expressed and retained in nuclei of resting EC. Ts interaction with EC or exposure of EC to interleukin-10 (IL-10) and interferon alpha (IFN-alpha) triggers processing of ILT3 pre-mRNA. Western blot analysis showed that the expression of the mature ILT3 transcript is accompanied by production of ILT3 protein.

Human CD8+ FOXP3+ T suppressor cells (TS) were previously shown to induce the expression of the inhibitory receptors, Immunoglobulin-like transcript (ILT) 3 and ILT4 on dendritic and endothelial cells, rendering them tolerogenic to allogeneic T cells. We have demonstrated the importance of CD8+ TS in a rat model of heart allo-transplantation. Tolerance was induced in ACI recipients by multiple transfusions of UVB-irradiated blood from Lewis heart donors. CD8+ T cells from tolerant ACI rats expressed FOXP3, transferred tolerance to naive secondary hosts and induced the upregulation of the inhibitory receptor, paired immunoglobulin-like receptor (PIR)-B, an ILT4 orthologue, in Lewis dendritic cells (DC) and heart endothelial cells (EC). When long-term surviving Lewis heart allografts with PIR-B+ EC were retransplanted from a primary to a secondary ACI recipient they did not elicit rejection. This study focuses attention on the need to develop agents that act directly on graft EC in order to achieve tolerance.

The direct interaction between antigen specific CD8 + CD28 − T suppressor cells (T S ) with dendritic cells (DC) results in the tolerization of DC by inducing the upregulation of immunologlobulin like transcript 3 (ILT3) and ILT4. We show here that such tolerogenic DC anergize alloreactive CD4 + CD45RO + CD25 + T cells converting them into regulatory T cells (T R ), which in turn, continue the cascade of suppression by tolerizing other DC. Interleukin 10 (IL-10) and interferon-α (IFN-α) also induce ILT3 and ILT4 upregulation in DC, rendering them tolerogenic. This implies a common mechanism of DC-mediated suppression. This finding and the observation that in organ allograft recipients quiescence is associated with the presence in the circulation of donor-specific T S and T R emphasize the importance of the cross talk between tolerogenic DC and T cells in suppression of the immune response.

Introduction Acute rejection currently accounts for 11% of the deaths of allograft recipients within the first six months of transplantation. Though endomyocardial biopsy is the most common method of detecting rejection, the procedure is highly invasive and expensive. MicroRNAs are RNA sequences that regulate gene expression and immune function and can be detected through tests of sera in blood. Previous studies have identified a correlation between microRNAs and acute rejection. This study hopes to address the usage of certain microRNAs as possible non-invasive biomarkers of acute cardiac cellular rejection. Methods We measured the expression of six different microRNAs (miR-146a, miR-155, miR-21, miR-30b, miR-30c, and miR-9) in 4 patients with rejection and in 16 healthy individuals. Using a Student’s t -test, miR-21 demonstrated a statistically significant difference in expression and was selected to test for expression in 12 heart allograft patients who did and did not experience acute cellular rejection, as defined by an ISHLT endomyocardial biopsy grade of 2R/3A on the day of serum collection. Chi-square analysis was used to compare expression levels between the two groups of patients and determine the accuracy of miR-21 levels in distinguishing between rejection and quiescence. Results Analysis of microRNA-21 levels showed significant up-regulation in patients with acute cellular rejection compared to patients without rejection. Patients with a minimum of 40% up-regulation were considered to be predictive for rejection. Rejection was predicted with 80% sensitivity, 100% specificity, a positive predictive value of 100% and negative predictive value of 98%, demonstrating high accuracy in determining rejection (P = 0.0001). Conclusions The study has shown that differential expression of microRNA-21 occurs in heart allograft patients experiencing rejection. Although further research will be done to determine the exact mechanism through which miR-21 affects rejection, it shows great promise as a non-invasive biomarker of cardiac transplant rejection.

Aim Pre-sensitization against a broad array of HLA is associated with prolonged waiting times and inferior kidney allograft survival. Although the use of solid phase assay (SPA) for the detection and characterization of anti-HLA antibodies provides greater sensitivity than complement-dependent lymphocytotoxicity (CDC) assay, it often detects donor specific antibodies (DSA) which turn out to be clinically irrelevant. In this study, our aim is to determine whether kidney deceased donor recipients with preformed DSA detectable in serum obtained prior to transplant are at an increased risk of graft failure. Methods We studied 640 patients who received a kidney transplant from a deceased donor between January 1, 2009 and December 31, 2014 at Columbia University Medical Center. Sera collected from the recipients prior to transplantation were tested for DSA by CDC and SPA with single antigen coated beads. All patients were transplanted with negative CDC crossmatch. Results We analyzed the actuarial graft survival in patients with a primary allograft ( n = 525) and in patients with a secondary allograft ( n = 115). Kidney allograft survival was significantly higher in primary allograft recipients (85%) than in secondary allograft recipients (71%) at 6 years. ( P = 0.02) Forty-six out of 525 (8.7%) primary kidney allograft recipients and sixty-two out of 115 (53.9%) secondary kidney allograft recipients had SPA-detectable DSA in sera collected prior to transplantation. Six years following transplantation of primary kidney allografts the actuarial graft survival was 83% and 85%, respectively, regardless of whether DSA were detectable in recipients’ pre-transplantation sera. ( P = 0.39) In recipients of secondary kidney allografts with and without SPA-detectable DSA, the actuarial graft survival was 67% and 77% at 6 years, respectively. ( P = 0.12) Conclusions The presence of SPA-detectable DSA in recipients of kidney allografts from deceased donors is associated with lower long term (6 year) actuarial graft survival although the difference does not reach statistical significance.

Aim The aim of this study was to evaluate the prognostic value of the C1q-SPA assay in recipients of kidney from deceased donors. Methods The study population consisted of 123 recipients transplanted at Columbia University Medical Center between January 1 and December 31, 2012. None of the patients showed DSA that could be detected by complement dependent lymphocytotoxicity. Sera obtained from each patient prior to transplantation were evaluated for donor-specific anti-HLA IgG antibody (DSA) by Luminex single-antigen bead (SAB) testing (SAB-IgG). SAB-IgG positive sera were further tested by Luminex SAB-based assay detecting C1q-binding HLA antibodies (SAB-C1q). Results The one year kidney allograft survival was 96.6% in this patient population. Four recipients had an episode of AMR although 27 showed DSA detectable by SAB-IgG. C1q testing shows DSA in 7 sera among the SAB-IgG positive results (7/27, 26%), 3 of which were in patients with AMR and 4 in patients without AMR. Conclusions There was a positive correlation between the presence of C1q detectable DSA in pre-transplantation sera and development of AMR. However, the correlation between SAB-IgG, SAB-C1q and rejection was weaker, suggesting the need of further evaluation of methods that can predict the risk of AMR, without affecting the patients’ chances of receiving a transplant.

Aim To determine whether the expansion of γδ T cells observed in pediatric heart allograft recipients associates with transplant outcomes. Methods We studied a population of 58 pediatric heart transplant recipients under the auspices of the Institutional Review Board. We performed Ab determinations by solid phase assays (SPA-IgG, “LSAB”). Flow cytometry staining was performed to determine the percentage of lymphocytic lineage markers: CD3, CD4, CD8, CD19, CD16& 56, CD45, TCRγδ, TCRαβ. Results Pediatric heart allograft recipients often require total or partial thymectomy at the time of their first heart operation, which leads to lifelong alterations in their T cell repertoire. While the prevailing population of circulating T cells in non-thymectomized children carries the TCRαβ TCR (96 ± 3%), we observed a significant expansion of TCRγδ T cells by Flow Cytometry in 37 of 58 patients studied. Within the population with high circulating fraction of gd T’s (8–20% of lymphocytes, n = 37), 23 patients (23/37, or 62%) developed anti-HLA antibodies detected by SPA-IgG (LSAB), while 14 did not. In patients with normal TCRγδ fractions ( Conclusion Thymectomy in pediatric heart recipients which results in the expansion of γδ T cells is associated with lesser risk of alloantibody development. The finding that patient with low γδ T cells are more likely to produce anti HLA Abs suggests that thymectomy results in a breakdown of immune regulation. Th2 impairment may result in insufficient cytokine production to fully support antibody production in these patients. Although mechanistic studies are needed to understand the causation relationships underpinning this phenomenon, the association alone suggests that monitoring of γδ T cell expansion may constitute a useful tool for Ab development prediction in pediatric heart transplant patients.

Aim Exosomes may contribute to cell-cell communication at the level of immunological synapses or at a distance, by delivering mRNA and miRNA modifying the recipient cell protein production and gene expression. The present study aims to determine whether the effect of ILT3Fc on allostimulated CD8 T cells is accompanied by changes in the miRNA content of exosomes generated during allostimulation. Methods Exosomes were isolated from culture supernatants of MLC in which CD3 T cells had been stimulated with allogeneic APC in the presence or absence of ILT3Fc. The identity and relative quantity of the exosomal miRNA content was determined by RT-PCR. The inhibitory or inflammatory properties of these exosomes were evaluated by 24h pretreatment of sorted CD8 Ts from MLC containing ILT3Fc. The CD8 T cells were then tested for the effect on the proliferation of naive, syngeneic T cells primed in response to the same stimulating APC. Results CD8 Ts primed in the presence of ILT3Fc inhibit syngeneic CD4 T cell responses by >80%. When CD8 Ts are pretreated with inflammatory exosomes, and are used in MLC at lower than 1:1 ratios to respoding Th cells, they lose inhibitory activity. These exosomes contain miRNA-21, -30b, -146a, -155, -395, and Let7a, known to be increased in T cells during activation. In contrast, exosomes obtained from T cells primed in the presence of ILT3Fc, do not abolish CD8 Ts inhibitory potential, suggesting that ILT3Fc blocks the release into the medium or alters the RNA content of inflammatory exosomes. Conclusions Micro RNA contained by exosomes released from alloactivated T cells enhance T helper activity even in the presence of T suppressor cells. This suggests the potential use of exosomal inflammatory miRNA as a monitor of cellular immune responses against the graft. Furthermore, such exosomes may be of use in eliciting immune T cell responses against tumors or microbial agents.

Aim To establish the impact of anti-HLA antibodies formed prior and/or following heart transplantation in a cohort of 137 pediatric recipients, transplanted between January 2000 and December 2011. Methods Anti-HLA class I and class II antibodies were monitored by traditional CDC methods using donor and panel T and B lymphocytes and SPA for detection of DSA. Endomyocardial biopsies were performed by protocol and whenever rejection was suspected. Results The allograft survival was 89% at 10 years in patients who had no lymphocytotoxic antibodies either before or after transplantation, yet dropped to 55% in patients who developed anti-HLA class I antibodies (p Conclusions All AMR episodes occurring in patients from this study population have documented evidence of complement binding antibodies and C4d deposition in the graft, reinforcing the conclusion that an integrated study of pathologic, immunologic and functional changes is required for timely and effective treatment of AMR.

Aim Since ILT3Fc induces the upregulation of BCL6, DUSP10 and SOCS1 via downregulation of miRNA in T suppressor cells, we investigated the possibility that this molecule also inhibits TCR signaling and nuclear localization of transcription factors. Methods Signaling studies were performed using Jurkat cells stimulated with CD3/CD28 monoclonal antibodies or PMA for various periods of time in the presence or absence of ILT3Fc. Western blots were used to analyze total cell lysates or nuclear fractions, using antibodies recognizing TCR signaling proteins or TCR-activated transcription factors. Results The Jurkat cell line is similar to activated human T lymphocytes and shows high binding of ILT3Fc (>90%), representing an ideal tool for studying signaling events downstream of the ILT3 ligand. We found that micro RNA gene promoters can be activated by anti-CD3/CD28 mAbs in Jurkat cells and are inhibited by treatment with ILT3Fc. Mutations at the AP1 binding sites in the miR-21 and miR-155 gene promoters abolished reporter gene activity. ILT3Fc consistently inhibited PMA-induced reporter activities, demonstrating that AP1 is crucial to the inhibitory effect of ILT3Fc. Nuclear fractions of cell extracts obtained from activated Jurkat cells were probed for various subunits of the AP-1 protein complexes. ILT3Fc inhibited and delayed TCR signaling events such as cFOS and FOSB synthesis and translocation into the nucleus. Conclusions These results indicate that the inhibitory activity of ILT3Fc is mediated through its effect of TCR signaling during the very early stages of T cell activation. This finding that ILT3Fc regulates miRNA transcriptionally by targeting TCR/AP1 signaling pathways indicates that this agent raises the threshold of TCR triggering inducing anergy. This phenomenon in conjunction with the capacity of ILT3Fc to inhibit several micro RNA explains the strong immunomodulatory activity of ILT3Fc, a reliable inducer of regulatory T cells and transplantation tolerance.