Biomedical subjects

Nicholas Zavazava

Publications and source records attributed to Nicholas Zavazava.

15 recordsLinked to original sources

PubMed2006

Soluble HLA revisited.

The goal of this editorial is to revisit soluble human leukocyte antigens (sHLA) and to highlight the findings reported by Albitar et al. in this issue on the relation between sHLA levels in Non-Hodgkin's Lymphoma (NHL) and Hodgkin's Disease (HD). We will review key aspects of sHLA including soluble HLA-G, which has received a lot of attention in recent publications. We will then address the role of sHLA in lymphoproliferative diseases and in solid organ tumors. Lastly, we will comment on the results of Albitar et al. and their relevance to clinical application in NHL.

HLA Antigens↗

PubMed2006

Immunogenicity and engraftment of mouse embryonic stem cells in allogeneic recipients.

Embryonic stem cells (ESCs) are pluripotent and therefore able to differentiate both in vitro and in vivo into specialized tissues under appropriate conditions, a property that could be exploited for cellular therapies. However, the immunological nature of these cells in vivo has not been well understood. In vitro, mouse-derived ESCs fail to stimulate T cells, but they abrogate ongoing alloresponses by a process that requires cell-cell contact. We further show that despite a high expression of the NKG2D ligand retinoic acid early inducible-1 by mouse ESCs, they remain resistant to natural killer cell lysis. In vivo, allogeneic mouse ESCs populate the thymus, spleen, and liver of sublethally irradiated allogeneic host mice, inducing apoptosis to T cells and establishing multilineage mixed chimerism that significantly inhibits alloresponses to donor major histocompatibility complex antigens. Immunohistochemical imaging revealed a significant percentage of ESC-derived cells in the splenic marginal zones, but not in the follicles. Taken together, the data presented here reveal that nondifferentiated mouse embryonic stem cells are non-immunogenic and appear to populate lymphoid tissues in vivo, leading to T-cell deletion by apoptosis.

Animals↗

PubMed2006

WOFIE stimulates regulatory T cells: a 2-year follow-up of renal transplant recipients.

BACKGROUND: Initial interruption of immunosuppression for 72 hr was analyzed in renal transplant recipients according to Calne et al.'s "window of opportunity for immunologic engagement" (WOFIE) concept. METHODS: This pilot study was designed as a randomized, open-label, prospective trial of 40 recipients (20 in the WOFIE group, 20 in the control group) of cadaveric kidney transplants who were followed up for 2 years. Immunosuppression comprised tacrolimus (trough levels 5-8 ng/mL), daclizumab (1 mg per kilogram of body weight on day 0 and after 2, 4, 6, and 8 weeks), mycophenolate mofetil (1-2 g/day), and prednisolone (maintenance dose of 10 mg/day). After induction with daclizumab, prednisolone, and mycophenolate mofetil, immunosuppression was interrupted for 72 hr in the WOFIE group. Steroid withdrawal followed in both groups within 12 to 16 weeks posttransplant. RESULTS: Patient and graft survival did not differ significantly between the two cohorts. However, the WOFIE group experienced less acute rejection episodes and developed better graft function. Although all but one of the patients in the WOFIE group successfully discontinued steroid treatment, permanent steroid withdrawal was achieved in only 76.4% of the control group. After daclizumab discontinuation, the WOFIE group demonstrated an increase of CD4CD25 T cells in peripheral blood (P<0.05 vs. control group), which was stable over time and strongly correlated with a significantly higher expression level of Foxp3-mRNA. CONCLUSIONS: Initial interruption of immunosuppression for 72 hr correlates with the induction of regulatory immunologic mechanisms and allows early and reliable minimization of immunosuppressive treatment.

Adolescent↗

PubMed2006

A divalent human leukocyte antigen-B7 fusion-protein up-regulates CD25 and CD69 in alloreactive CD8+ T cells bypassing CD28 costimulation.

BACKGROUND: T cells recognize major histocompatibility complex (MHC) molecules and their cryptic antigenic peptides on antigen-presenting cells and are generally triggered to proliferate, and when sufficient, co-stimulation is available. In soluble form, monomeric MHC molecules can induce apoptosis, anergy, or decreases of the T-cell receptor (TCR). METHODS: A dimeric fusion protein of the human leukocyte antigens (HLA)-B7 was molecularly engineered and expressed in a B-cell line to allow secretion. Alloreactive T cells were generated according to the standard protocol. RESULTS: A dimer of approximately 160 kD was obtained, affinity purified, and used to study T-cell interaction. In immobilized form, this protein efficiently stimulated alloreactive T cells to proliferate and produce interleukin (IL)-2 and interferon (IFN)-gamma in a concentration-dependent manner, up-regulating CD25 and CD69 expression. In contrast, the soluble fusion protein induced T-cell apoptosis. CONCLUSIONS: The dichotomy in T-cell regulation by a divalent MHC fusion protein warrants the use of MHC multimers as custom-designed immune-regulatory molecules both in transplantation and autoimmune disease.

Antigens, CD↗

PubMed2006

Soluble HLA-G molecules induce apoptosis in natural killer cells.

Membrane-bound human leukocyte antigen-G (HLA-G) molecules are primarily expressed by cytotrophoblasts of the fetus. They are thought to protect the fetus from immunologic attack by the maternal immune system and have recently been associated with transplantation graft acceptance. In addition, soluble HLA-G molecules (sHLA-G) have been shown to play a role in the success of pregnancies, but are upregulated in certain cancers. However, the exact mechanism for this regulation has remained elusive. The aim of this study was to examine the mechanism by which sHLA-G interact with natural killer (NK) cells in vitro. sHLA-G effectively blocked NK lysis of target cells via fracticide killing of NK cells by apoptosis. These studies support the protective role of sHLA-G in immunologic reactions by interacting with NK cells, thus providing a regulatory function.

Antibodies↗

PubMed2006

Strategies for developing therapeutic application of human embryonic stem cells.

The ongoing debate on human embryonic stem cells (hESC) is fueled by ethical concerns but also by the legitimate hope that hESC could one day be used for the cure of presently untreatable human diseases. Here we discuss current approaches to and constraints upon hESC differentiation and describe their potential application in clinical medicine.

Cell Differentiation↗

PubMed2005

Recombinant dimeric MHC antigens protect cardiac allografts from rejection and visualize alloreactive T cells.

Monomeric and dimeric soluble major histocompatibility complex (MHC) molecules down-regulate activated T cells in an antigen-specific manner in vitro. This property could be exploited to modulate alloresponses in vivo but has remained difficult to demonstrate. Here, intraperitoneal infusion of a Lewis-derived rat MHC class I molecule, RT1.A(l)-Fc, in Dark Agouti (RT1.A(a)) recipient rats prolonged cardiac graft survival, which led to permanent engraftment. This effect was mediated by T cell impairment of target cell lysis by CD8+ T cells and down-regulation of interferon-gamma production by CD4+ T cells. The binding of the dimeric MHC allowed ex vivo visualization of alloreactive T cells in peripheral blood, splenocytes, and allografts, revealing low frequency of alloreactive CD8+ T cells after establishment of permanent engraftment of cardiac allografts. Thus, these data show the potential of dimeric MHC molecules to promote graft survival and allow visualization of alloreactive T cells.

Animals↗

PubMed2005

Induction of stable mixed chimerism by embryonic stem cells requires functional Fas/FasL engagement.

BACKGROUND: Recent data show the efficacy of embryonic stem cells (ESC) to engraft in allogeneic recipients without host pretreatment. This property is due to their low expression of major histocompatibility complex (MHC) class I antigens and lack of MHC class II expression. Here, we tested the hypothesis that the constitutive FasL expression by ESC is a requirement for their stable engraftment in allogeneic recipients. METHODS: MRL and MRL-lpr/lpr mice (H-2k) were infused allogeneic 129SvJ RW-4 (H-2b) ESC without host preconditioning. The development of mixed chimerism was monitored over 100 days by flow cytometry. RESULTS: Mixed chimerism was detectable by day 7. The amount of donor cells detected varied between 3-5.5% and were lymphoid, but nonmyeloid. Only 50% of lpr/lpr mice engrafted and lost donor cells by day 28 post-ESC infusion. In contrast, >80% wild type mice engrafted and maintained mixed chimerism up to day 100. CONCLUSIONS: These data suggest a critical role for Fas-FasL engagement in ESC engraftment. We conclude that ESC may induce clonal deletion of alloreactive T cells by Fas-induced apoptosis in recipient T cells, protecting them from rejection. The data provide a rationale for improved protocols for the achievement of robust ESC-induced mixed chimerism.

Animals↗

PubMed2005

CTLA-4 is important in maintaining long-term survival of cardiac allografts.

INTRODUCTION: CTLA-4 is a negative regulatory molecule upregulated on activated T cells; however, its role in induction and maintenance of transplant tolerance is not well understood. METHODS: The characteristics and effects of a novel mouse anti-rat CTLA-4 antibody (Ab) (242B58) were examined using fluorescence-activated cell sorter, mixed lymphocyte reaction, enzyme-linked immunospot, signaling studies, and a rat model of cardiac transplant tolerance induced by administration of anti-CD28 Ab and cyclosporine. RESULTS: The anti-CTLA4 Ab was shown to bind to CTLA-4 but not prevent subsequent binding of B7 to CTLA-4. Binding to CTLA-4 did not result in phosphorylation of early cytoplasmic tyrosine kinases, suggesting that this is not a signaling Ab. However, its in vitro function was compatible with antagonization of the effects of CTLA-4, thereby increasing T-cell proliferation and interferon-gamma production in mixed lymphocyte reaction and enzyme-linked immunospot assays, respectively. Administration of 242B58 to animals treated with anti-CD28 Ab and cyclosporine either at the time of transplantation or various time-points up to 33 days posttransplantation did not result in immediate rejection, but rather caused a delayed severe acute allograft rejection at approximately 45 days posttransplant. CONCLUSIONS: Our results seem to be a reflection of the unique properties of the 242B58 Ab, which does not antagonize B7 binding to CTLA-4 and affect its ability to out-compete CD28 for B7 binding. It does, however, seem to interfere with CTLA-4 signaling, suggesting that competition for B7 may be important in induction of tolerance, but signaling through CTLA-4 is more important in maintaining a tolerant phenotype.

Animals↗

PubMed2004

A novel DNA methyltransferase I-derived peptide eluted from soluble HLA-A*0201 induces peptide-specific, tumor-directed cytotoxic T cells.

MHC peptides derived from tumor-associated antigens (TAAs) can serve as the basis for the development of immunotherapeutics to treat human malignancies. Previously, we identified novel HLA-A*0201 (HLA-A2)-restricted peptides recovered from soluble HLA molecules secreted by human tumor cell lines, transfected with truncated genes of HLA-A2 and HLA-B7. Here, 4 candidate peptides eluted from soluble HLA-A2 were selected on the basis of their precursor proteins being TAAs. Peptide p1028 (GLIEKNIEL), derived from DNA methyltransferase I (DNMT-1), which is overexpressed in various human tumors, showed the highest affinity to HLA-A2 and was relatively abundant in the sMHC/peptide complexes of all transfected breast, ovarian and prostate cancer cell lines. Peptide p1028-specific CTLs were generated in vitro and shown to efficiently lyse not only target cells pulsed with the peptide but also HLA-A2-positive breast cancer cell lines MDA-231 and MCF-7. The peptide induced IFN-gamma production in CTLs, which were selectively stained by a p1028 tetramer. Since DNMT-1 is a widely expressed tumor-associated enzyme, the novel DNMT-1-derived, HLA-A2-restricted peptide GLIEKNIEL identified here may provide a suitable candidate for a therapeutic cancer vaccine.

Breast Neoplasms↗

PubMed2003

WOFIE synergizes with calcineurin-inhibitor treatment and early steroid withdrawal in kidney transplantation.

BACKGROUND: According to the window of opportunity for immunologic engagement (WOFIE) concept, as designed by R. Calne, the authors prospectively analyzed the effect of a 72-hr immunosuppressive window on graft function in renal transplant recipients. METHODS: Immunosuppressive therapy comprised tacrolimus (trough levels, 5-8 ng/mL after day 8 posttransplantation), daclizumab (1 mg/kg body weight at day 0 and 2, 4, 6, and 8 weeks posttransplantation), mycophenolate mofetil (MMF) (1-2 g/day), and prednisolone. All patients received an induction therapy including daclizumab, prednisolone, and MMF. WOFIE patients were stopped from immunosuppression for 72 hr posttransplant. Steroids were withdrawn in both groups 12 to 16 weeks after transplantation and dual therapy with MMF and low-dose tacrolimus ensued. RESULTS: Thirty renal transplant recipients (16 in the WOFIE group and 14 in the control group) have been enrolled since May 2000. Patient and graft survival were 93.8% and 87.5%, respectively, in the WOFIE group and 100.0% and 92.9% in the control group, respectively. One patient in the WOFIE group died of cytomegalovirus infection with stable graft function. There were no grafts lost because of acute rejection or primary nonfunction in either group. Patients treated according to the WOFIE protocol revealed less acute rejection episodes during the time of observation (first biopsy-confirmed acute rejection rate 12.5% in the WOFIE group vs. 42.9% in the control cohort). Whereas 92.1% of the WOFIE patients were successfully discontinued from steroids, permanent steroid withdrawal was achieved in only 60% of the control cohort. CONCLUSIONS: Initial interruption of immunosuppression was associated with a decrease of acute graft rejections. Subsequently, the authors postulate a synergistic effect on the immunosuppressive efficiency of calcineurin inhibitors when combined with an initial drug-free window.

Acute Disease↗

PubMed2003

Embryonic stem cells and potency to induce transplantation tolerance.

In the past 40 years, many protocols have been extensively studied for the induction of sustainable transplantation tolerance which might lead to protection of allografts from immunological injury in the clinical setting. Despite the enormous success in rodents, there is still no established protocol available yet for widespread clinical trials. Whilst clonal deletion, clonal anergy and suppression, now coined regulation, have been elucidated as the key immunological elements of tolerance, a better understanding of these mechanisms has so far done little to improve on the survival of organ transplants in humans. Haematopoietic chimaerism, as previously described by Medawar and colleagues [1], remains the most robust tool for tolerance induction. Unfortunately, bone marrow or haematopoietic stem cell transplantation for patients awaiting solid organ transplantation remains a high risk therapy, due to the dangers of graft-versus-host disease. Most recent data, however, indicate the potential of embryonic stem cells (ESC) to offer a possible solution for low risk induction of multilineage mixed chimaerism and tolerance not involving any immunosuppression, due to their unique property of low immunogenicity and high plasticity. Here, what we know about ESC in various species, and the potency and drawbacks of ESC for widespread clinical use, will be discussed.

Animals↗

PubMed2002

HLA-B7 beta-pleated sheet-derived synthetic peptides are immunodominant T-cell epitopes regulating alloresponses.

Chronic rejection of transplanted allografts is the major cause of graft loss after clinical solid organ transplantation. Recent data link the indirect presentation of allopeptides to chronic graft loss; thus, identification of immunodominant epitopes on major histocompatibility complex (MHC) antigens could significantly contribute to establishing novel ways for monitoring and managing chronic rejection. Here, we show that synthetic allo-MHC-derived peptides covering the polymorphic region 56 to 120 of HLA-B7 modulate alloresponses. In particular, the 2 beta-pleated sheet-derived peptides covering residues 91 to 105 and 96 to 120, respectively, but not sequences from the alpha1 helix, were presented by autologous peripheral blood lymphocytes to induce T-cell proliferation. In addition, the 2 beta-pleated sheet-derived peptides and the alpha1-derived peptide residues 60 to 75 abrogated lysis of HLA-B7 target cells by anti-HLA-B7 cytotoxic T lymphocytes (CTLs). Although most residues between 91 and 120 are normally not directly accessible to T cells, our results indicate that peptides derived from the lower surface of the peptide-binding groove of HLA-B7 are immunodominant in HLA-B7 alloresponses. To characterize the binding and stability of allopeptides to T cells, the 62-70 peptide-derived from the 60-75 allopeptide that blocked cytotoxicity of anti-HLA-B7 CTL-was synthesized and coupled with fluorescein isothiocyanate. The peptide specifically labeled anti-B7 CTL, but not anti-HLA-A2 CTL as measured by flow cytometry. Peptide binding to CTL was specific at 4 degrees C and remained stable for 12 hours, whereas it remained stable for less than 2 hours at 37 degrees C. These studies allow the identification of HLA-B7 T-cell epitopes and reveal for the first time a novel, previously unrecognized application of synthetic HLA-derived allopeptides to visualize alloreactive T cells.

Amino Acid Sequence↗

PubMed2002

Analysis of endogenous peptides bound by soluble MHC class I molecules: a novel approach for identifying tumor-specific antigens.

The Human MHC Project aims at comprehensive cataloging of peptides presented within the context of different human leukocyte antigens (HLA) expressed by cells of various tissue origins, both in health and in disease. Of major interest are peptides presented on cancer cells, which include peptides derived from tumor antigens that are of interest for immunotherapy. Here, HLA-restricted tumor-specific antigens were identified by transfecting human breast, ovarian and prostate tumor cell lines with truncated genes of HLA-A2 and HLA-B7. Soluble HLA secreted by these cell lines were purified by affinity chromatography and analyzed by nano-capillary electrospray ionization-tandem mass spectrometry. Typically, a large peptide pool was recovered and sequenced including peptides derived from MAGE-B2 and mucin and other new tumor-derived antigens that may serve as potential candidates for immunotherapy.

Animals↗

PubMed2002

P59(fyn) is upregulated in anergic CD8+ T cells.

The ultimate goal in clinical transplantation is achievement of graft tolerance. Despite long-term immunosuppression, alloantigens on transplants elicit alloresponses that can initiate organ rejection. Acute rejection is mediated by CD8(+) cytotoxic T cells, whereas chronic rejection is a result of many factors including non-immunological events. The aim of this study was to examine the molecular requirements of T cell anergy, a cellular state that is an integral component of tolerance in vivo. In vitro, the tolerant state is usually best represented by T cell anergy, which is defined by loss of the ability of T cells to produce and secrete interleukin-2 upon restimulation. In the literature, molecular changes in anergic CD4(+) T cells have been studied in great detail, but only little is known about functional and biochemical characteristics of anergic CD8(+) T lymphocytes. In this study, we demonstrate, that CD8(+) T cells are rendered anergic by TCR stimulation without costimulation. They exhibit impaired interleukin-2 production and tyrosine-phosphorylation, but markedly upregulated p59(fyn) expression, which could be shown to be an early event during anergization. Anergic CD8(+) T lymphocytes show elevated surface expression of early activation markers as well as costimulatory molecules, especially that of CTLA4. These results, are an important component for the discovery of potential molecular targets, which contribute to the development and maintenance of tolerance.

Abatacept↗