Bio

Academic Appointments


Honors & Awards


  • AST Basic Science Investigator Award, American Society of Transplantation (2013)

Professional Education


  • PostDoc, UCSF, Transplantation (1993)
  • PhD, University of California, Davis, Immunology (1989)

Research & Scholarship

Current Research and Scholarly Interests


Our recent studies have challenged the paradigm that NK cells are not active participants in outcomes after solid organ transplantation.. We have demonstrated that recipient-derived NK cells not only infiltrate rejecting liver grafts early after transplantation but also are a significant source of IFNg thus bridging the innate and adaptive immune responses post transplant. In a high responder, DA->Lewis rat orthotopic liver transplant model we demonstrated that depletion of NK cells markedly prolongs graft survival in half of graft recipients and that there is a concomitant increase in expression of NKG2D ligands on hepatocytes (Zhuo et al, 2010). Interestingly, transplantation in the converse direction (Lewis->DA) is a model of spontaneous liver tolerance and we have shown that depletion of NK cells prevents the establishment of tolerance but once tolerance is established, NK cell depletion has no effect. Our studies support a role for NK cells in both graft rejection and tolerance.

NK cells express a wide range of receptors that have activating or stimulatory functions. We examined the activation receptors involved in the interaction of NK cells with dendritic cells (DC) since NK cells have the potential to mediate both maturation and killing of DC and this could alter T cell priming post-transplant. We used RNAi techniques to knock down the activation receptors, NKp46, NKp30 and NKG2D and demonstrated that DC stimulation of NK cells to produce IFNg; is mediated through NKp46 (Wai et al, 2010). NK cell killing of DC is independent of NKp30, NKp46 and NKG2D. We further examined the interactions of NK cells with tumors and determined that NK cell stimulation by tumor requires NKp30 while NK cell cytotoxicity is mediated by both NKp30 and NKG2D. Our studies indicate that NK cells interact with DC and tumors through distinct activating receptors – NKp46 and NKp30/NKG2D respectively. These data are important for the development of therapeutics that will target NK-DC interactions post-transplant and support the feasibility of this approach without compromising the ability of NK cells to kill virally-infected or transformed cells.Ongoing studies, are examining the functional role of NK cell receptors using many state-of-the-art techniques (microscopy, cyTOF, experimental systems).

Emerging studies, including results from our lab, implicate miRNAs in the pathogenesis of disease and recent reports describe the expression of miRNAs in transplant recipients. The functional significance of miRNAs in alloimmune responses and transplantation has not yet been elucidated. Recently we have reported that miRNAs are differentially expressed after transplantation and that miR-182 was significantly increased in rejecting cardiac allografts and in the mononuclear cells that infiltrate the grafts. We further showed that miR-182 targets foxo1 mRNA, a member of the Forkhead box (FOX) protein family of important transcription factors. As miR-182 increases after transplant, there is a concomitant post-transcriptional decrease in FOXO1 protein expression in heart allografts that is localized to both the cardiomyocytes and CD3+ T cells. These studies are the first to demonstrate a mechanistic role for a miRNA in transplantation and thus we are uniquely positioned to move the field forward in our understanding of how miRNAs contribute to alloimmune activation and regulation.

As part of a new large consortium grant (awarded to our group at Stanford), we will be analyzing plasma microRNAs from pediatric recipients of solid organ allografts to develop a non-invasive biomarker for post-transplant lymphoproliferative disorder (PTLD, a serious EBV associated compliaction of transplant). Three inter-related objectives are proposed: 1) to determine a profile of microRNAs that is diagnostic of PTLD, 2) to determine if microRNA expression correlates with specific LMP1 variants, and 3) to determine if microRNAs are predictive of PTLD.

Teaching

2013-14 Courses


Postdoctoral Advisees


Graduate and Fellowship Programs


Publications

Journal Articles


  • PI3K Inhibition Augments the Efficacy of Rapamycin in Suppressing Proliferation of Epstein-Barr Virus (EBV) plus B Cell Lymphomas AMERICAN JOURNAL OF TRANSPLANTATION Furukawa, S., Wei, L., Krams, S. M., Esquivel, C. O., Martinez, O. M. 2013; 13 (8): 2035-2043

    Abstract

    Posttransplant lymphoproliferative disorder (PTLD) continues to be a devastating and potentially life-threatening complication in organ transplant recipients. PTLD is associated with EBV infection and can result in malignant B cell lymphomas. Here we demonstrate that the PI3K/Akt/mTOR pathway is highly activated in EBV+ B cell lymphoma lines derived from patients with PTLD. Treatment with the mTORC1 inhibitor Rapamycin (RAPA) partially inhibited the proliferation of EBV+ B cell lines. Resistance to RAPA treatment correlated with high levels of Akt phosphorylation. An mTORC1/2 inhibitor and a PI3K/mTOR dual inhibitor suppressed Akt phosphorylation and showed a greater anti-proliferative effect on EBV+ B lymphoma lines compared to RAPA. EBV+ B cell lymphoma lines expressed high levels of PI3Kδ. We demonstrate that PI3Kδ is responsible for Akt activation in EBV+ B cell lymphomas, and that selective inhibition of PI3Kδ by either siRNA, or a small molecule inhibitor, augmented the anti-proliferative effect of RAPA on EBV+ B cell lymphomas. These results suggest that PI3Kδ is a novel, potential therapeutic target for the treatment of EBV-associated PTLD and that combined blockade of PI3Kδ and mTOR provides increased efficacy in inhibiting proliferation of EBV+ B cell lymphomas.

    View details for DOI 10.1111/ajt.12328

    View details for Web of Science ID 000322330000013

    View details for PubMedID 23841834

  • Syk-Induced Phosphatidylinositol-3-Kinase Activation in EpsteinBarr Virus Posttransplant Lymphoproliferative Disorder AMERICAN JOURNAL OF TRANSPLANTATION Hatton, O., Lambert, S. L., Phillips, L. K., Vaysberg, M., Natkunam, Y., Esquivel, C. O., Krams, S. M., Martinez, O. M. 2013; 13 (4): 883-890

    Abstract

    Posttransplant lymphoproliferative disorder (PTLD)-associated Epstein-Barr virus (EBV)+ B cell lymphomas are serious complications of solid organ and bone marrow transplantation. The EBV protein LMP2a, a B cell receptor (BCR) mimic, provides survival signals to virally infected cells through Syk tyrosine kinase. Therefore, we explored whether Syk inhibition is a viable therapeutic strategy for EBV-associated PTLD. We have shown that R406, the active metabolite of the Syk inhibitor fostamatinib, induces apoptosis and cell cycle arrest while decreasing downstream phosphatidylinositol-3'-kinase (PI3K)/Akt signaling in EBV+ B cell lymphoma PTLD lines in vitro. However, Syk inhibition did not inhibit or delay the in vivo growth of solid tumors established from EBV-infected B cell lines. Instead, we observed tumor growth in adjacent inguinal lymph nodes exclusively in fostamatinib-treated animals. In contrast, direct inhibition of PI3K/Akt significantly reduced tumor burden in a xenogeneic mouse model of PTLD without evidence of tumor growth in adjacent inguinal lymph nodes. Taken together, our data indicate that Syk activates PI3K/Akt signaling which is required for survival of EBV+ B cell lymphomas. PI3K/Akt signaling may be a promising therapeutic target for PTLD, and other EBV-associated malignancies.

    View details for DOI 10.1111/ajt.12137

    View details for Web of Science ID 000316911900011

    View details for PubMedID 23398911

  • T Cells Contribute to Stroke-Induced Lymphopenia in Rats PLOS ONE Gu, L., Xiong, X., Wei, D., Gao, X., Krams, S., Zhao, H. 2013; 8 (3)

    Abstract

    Stroke-induced immunodepression (SIID) results when T cell and non-T immune cells, such as B cells, NK cells and monocytes, are reduced in the peripheral blood and spleen after stroke. We investigated the hypothesis that T cells are required for the reductions in non-T cell subsets observed in SIID, and further examined a potential correlation between lymphopenia and High-mobility group protein B1 (HMGB1) release, a protein that regulates inflammation and immunodepression. Our results showed that focal ischemia resulted in similar cortical infarct sizes in both wild type (WT) Sprague Dawley (SD) rats and nude rats with a SD genetic background, which excludes the possibility of different infarct sizes affecting SIID. In addition, the numbers of CD68-positive macrophages in the ischemic brain did not differ between WT and nude rats. Numbers of total peripheral blood mononuclear cells (PBMCs) or splenocytes and lymphocyte subsets, including T cells, CD4(+) or CD8(+) T cells, B cells and monocytes in the blood and spleen, were decreased after stroke in WT rats. In nude rats, however, the total number of PBMCs and absolute numbers of NK cells, B cells and monocytes were increased in the peripheral blood after stroke; nude rats are athymic therefore they have few T cells present. Adoptive transfer of WT splenocytes into nude rats before stroke resulted in lymphopenia after stroke similar to WT rats. Moreover, in vitro T cell proliferation stimulated by Concanavalin A was significantly inhibited in WT rats as well as in nude rats receiving WT splenocyte adoptive transfer, suggesting that T cell function is indeed inhibited after stroke. Lastly, we demonstrated that stroke-induced lymphopenia is associated with a reduction in HMGB1 release in the peripheral blood. In conclusion, T cells are required for stroke-induced reductions in non-T immune cells and they are the most crucial lymphocytes for SIID.

    View details for DOI 10.1371/journal.pone.0059602

    View details for Web of Science ID 000316409800104

    View details for PubMedID 23555048

  • Src Kinase and Syk Activation Initiate PI3K Signaling by a Chimeric Latent Membrane Protein 1 in Epstein-Barr Virus (EBV) plus B Cell Lymphomas PLOS ONE Hatton, O., Lambert, S. L., Krams, S. M., Martinez, O. M. 2012; 7 (8)

    Abstract

    The B lymphotrophic ?-herpesvirus EBV is associated with a variety of lymphoid- and epithelial-derived malignancies, including B cell lymphomas in immunocompromised and immunosuppressed individuals. The primary oncogene of EBV, latent membrane protein 1 (LMP1), activates the PI3K/Akt pathway to induce the autocrine growth factor, IL-10, in EBV-infected B cells, but the mechanisms underlying PI3K activation remain incompletely understood. Using small molecule inhibition and siRNA strategies in human B cell lines expressing a chimeric, signaling-inducible LMP1 protein, nerve growth factor receptor (NGFR)-LMP1, we show that NGFR-LMP1 utilizes Syk to activate PI3K/Akt signaling and induce IL-10 production. NGFR-LMP1 signaling induces phosphorylation of BLNK, a marker of Syk activation. Whereas Src kinases are often required for Syk activation, we show here that PI3K/Akt activation and autocrine IL-10 production by NGFR-LMP1 involves the Src family kinase Fyn. Finally, we demonstrate that NGFR-LMP1 induces phosphorylation of c-Cbl in a Syk- and Fyn-dependent fashion. Our results indicate that the EBV protein LMP1, which lacks the canonical ITAM required for Syk activation, can nevertheless activate Syk, and the Src kinase Fyn, resulting in downstream c-Cbl and PI3K/Akt activation. Fyn, Syk, and PI3K/Akt antagonists thus may present potential new therapeutic strategies that target the oncogene LMP1 for treatment of EBV+ B cell lymphomas.

    View details for DOI 10.1371/journal.pone.0042610

    View details for Web of Science ID 000307284100143

    View details for PubMedID 22880054

  • Differential Expression of MicroRNAs During Allograft Rejection AMERICAN JOURNAL OF TRANSPLANTATION Wei, L., Wang, M., Qu, X., MAH, A., Xiong, X., Harris, A. G., Phillips, L. K., Martinez, O. M., Krams, S. M. 2012; 12 (5): 1113-1123

    Abstract

    MicrorRNA are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes. In addition to being involved in many biologic processes, microRNAs are important regulators in innate and adaptive immune responses. Distinct sets of expressed microRNAs are found in different cell types and tissues and aberrant expression of microRNAs is associated with many disease states. MicroRNA expression was examined in a model of heterotopic heart transplantation by microarray analyses and a unique profile was detected in rejecting allogeneic transplants (BALB/c ? C57BL/6) as compared to syngeneic transplants (C57BL/6 ? C57BL/6). The microRNA miR-182 was significantly increased in rejecting cardiac allografts and in mononuclear cells that infiltrate the grafts. Forkhead box (FOX) proteins are a family of important transcription factors and FOXO1 is a target of miR-182. As miR-182 increases after transplant, there is a concomitant posttranscriptional decrease in FOXO1 expression in heart allografts that is localized to both the cardiomyocytes and CD3(+) T cells. The microRNA miR-182 is significantly increased in both peripheral blood mononuclear cells and plasma during graft rejection suggesting potential as a biomarker of graft status. Our results identify microRNAs that may regulate alloimmune responses and graft outcomes.

    View details for DOI 10.1111/j.1600-6143.2011.03958.x

    View details for Web of Science ID 000303235100010

    View details for PubMedID 22300508

  • Changes in natural killer cell subsets in pediatric liver transplant recipients PEDIATRIC TRANSPLANTATION Pham, B., Piard-Ruster, K., Silva, R., Gallo, A., Esquivel, C. O., Martinez, O. M., Krams, S. M. 2012; 16 (2): 176-182

    Abstract

    NK cells are important in the immune response against tumors and virally infected cells. A balance between inhibitory and activating receptors controls the effector functions of NK cells. We examined the fate of circulating NK cells and the expression of the NK cell-activating receptors in pediatric liver transplant recipients. Blood specimens were collected from 38 pediatric liver transplant recipients before transplant, and at one wk, one, three, six, and nine months, and one yr post-transplant. PBMCs were isolated and analyzed for the levels of NK cell activation receptors NKp30, NKp46, and NKG2D in the CD56(dim) CD16(+) and CD56(bright) CD16(+/-) subsets of NK cells. We demonstrated that there is a significant decrease in the percentage of circulating NK cells post-transplant (pretransplant 7.69 ± 1.54 vs. one wk post-transplant 1.73 ± 0.44) in pediatric liver transplant recipients. Interestingly, NKp30 expression is significantly increased, while NKp46 and NKG2D levels remain stable on the NK cells that persist at one wk post-transplant. These data indicate that the numbers and subsets of circulating NK cells are altered in children after liver transplantation.

    View details for DOI 10.1111/j.1399-3046.2012.01653.x

    View details for Web of Science ID 000300709600017

    View details for PubMedID 22360401

  • Syk Activation of Phosphatidylinositol 3-Kinase/Akt Prevents HtrA2-dependent Loss of X-linked Inhibitor of Apoptosis Protein (XIAP) to Promote Survival of Epstein-Barr Virus plus (EBV plus ) B Cell Lymphomas JOURNAL OF BIOLOGICAL CHEMISTRY Hatton, O., Phillips, L. K., Vaysberg, M., Hurwich, J., Krams, S. M., Martinez, O. M. 2011; 286 (43): 37368-37378

    Abstract

    B cell lymphoma survival requires tonic or ligand-independent signals through activation of Syk by the B cell receptor. The Epstein-Barr virus (EBV) protein latent membrane 2a (LMP2a), a mimic of the B cell receptor, provides constitutive survival signals for latently infected cells through Syk activation; however, the precise downstream mechanisms coordinating this survival response in EBV+ B cell lymphomas remain to be elucidated. Herein, we assess the mechanism of Syk survival signaling in EBV+ B cell lymphomas from post-transplant lymphoproliferative disorder (PTLD) to discover virally controlled therapeutic targets involved in lymphomagenesis and tumor progression. Using small molecule inhibition and siRNA strategies, we show that Syk inhibition reduces proliferation and induces apoptosis of PTLD-derived EBV+ B cell lines. Syk inhibition also reduces autocrine IL-10 production. Although Syk inhibition attenuates signaling through both the PI3K/Akt and Erk pathways, only PI3K/Akt inhibition causes apoptosis of PTLD-derived cell lines. Loss of the endogenous caspase inhibitor XIAP is observed after Syk or PI3K/Akt inhibition. The loss of XIAP and apoptosis that results from Syk or PI3K/Akt inhibition is reversed by inhibition of the mitochondrial protease HtrA2. Thus, Syk drives EBV+ B cell lymphoma survival through PI3K/Akt activation, which prevents the HtrA2-dependent loss of XIAP. Syk, Akt, and XIAP antagonists may present potential new therapeutic strategies for PTLD through targeting of EBV-driven survival signals.

    View details for DOI 10.1074/jbc.M111.255125

    View details for Web of Science ID 000296542400037

    View details for PubMedID 21908615

  • The effect of MICA antigens on transplant outcomes: A systematic review. Journal of evidence-based medicine Lu, J., Luo, L., Guo, Y., Long, D., Wei, L., Shan, J., Feng, L., Li, S., Yang, x., Lu, Y., Krams, S., Li, Y. 2011; 4 (2): 106-121

    Abstract

    Background and Objective: Human major histocompatibility complex class I-related gene A (MICA) is reportedly associated with poor transplant outcomes and a high risk of acute and chronic rejection in solid organ transplantation. However, studies on these risks have found conflicting results. In order to identify areas in which additional research is needed, we have undertaken the first systematic review of evidence concerning the risk of anti-MICA antibodies in recipients' sera. Methods: We searched MEDLINE, EMBASE, and the Cochrane Library for original reports of clinical studies involving detection of MICA abs in transplant recipients' sera which used survival rate, acute rejection, and/or chronic rejection as outcome measures. RevMan 5.0.15 was used to calculate relative risk (RR), odds ratios (ORs), and 95% confidence intervals (95%CIs). Results: We found 18 relevant articles, with a total of 6,607 recipients. Follow-up duration ranged from 1 to 15 years. In studies with more than 2 years of follow-up, anti-MICA abs positive in kidney recipients' post-transplant sera was associated with a lower graft survival rate (4 years: RR = 2.04, 95%CI 1.30 to 3.22; 3 years: OR = 3.56, 95%CI 1.47 to 8.62; 2 years: RR = 2.17, 95%CI 1.09 to 4.31) and a higher acute rejection rate (RR = 1.92, 95%CI 1.27 to 2.91), but there was no clear association with chronic rejection. Similar conclusions could not be drawn for heart or liver transplantation due to possible confounding by anti-HLA abs and the small sample sizes of the available studies. Conclusion: Anti-MICA antibodies in recipients' sera may associated with poor graft survival rates and high risk of acute and chronic rejection in solid organ transplantation, but more rigorous studies are needed to confirm or refute this relationship. Current immunosuppressive therapy may fail to suppress the harmful effect of MICA antigens.

    View details for DOI 10.1111/j.1756-5391.2011.01125.x

    View details for PubMedID 23672701

  • Distinct Roles for the NK Cell-Activating Receptors in Mediating Interactions with Dendritic Cells and Tumor Cells JOURNAL OF IMMUNOLOGY Wai, L., Garcia, J. A., Martinez, O. M., Krams, S. M. 2011; 186 (1): 222-229

    Abstract

    NK cells are innate immune cells that are important in tumor immunity, but also have the ability to modulate the adaptive immune system through cytokine production or direct cell-cell interactions. This study investigates the interaction of NK cells with dendritic cells (DCs) and tumor cells, and the role of specific NK cell-activating receptors in this process. Primary rat NK cells and an NK cell line produced IFN-? when cocultured with either DCs or the rat hepatoma cell line McA-RH7777 (McA). This NK cell activation by DCs and McA required cell-cell contact and was dependent on distinct NK-activating receptors. Silencing NK cell expression of NKp46 and NKp30 significantly diminished DC- and McA-mediated NK cell IFN-? production, respectively. NK cells killed immature and mature DCs independently of NKp46, NKp30, and NKG2D; however, cytotoxicity against McA cells was dependent on NKp30 and NKG2D. Thus, we have shown in this study that NKp30 plays dual activating roles in NK-McA tumor interactions by mediating cytokine production and cytotoxicity. More importantly, NK cells are activated by both DCs and hepatoma cells to produce IFN-?, but require distinct NK cell-activating receptors, NKp46 and NKp30, respectively. Our data suggest that therapeutics could be developed specifically to target NK-DC interactions without compromising NK tumor immunity.

    View details for DOI 10.4049/jimmunol.1002597

    View details for Web of Science ID 000285688700029

    View details for PubMedID 21106845

  • Toll-Like Receptor 4 Contributes to Small Intestine Allograft Rejection TRANSPLANTATION Krams, S. M., Wang, M., Castillo, R. O., Ito, T., Phillips, L., Higgins, J., Kambham, N., Esquivel, C. O., Martinez, O. M. 2010; 90 (12): 1272-1277

    Abstract

    Although outcomes for small intestine transplantation (SIT) have improved in recent years, allograft rejection rates remain among the highest of solid organ grafts. The high load of commensal bacteria in the small intestine may contribute through activation of the toll-like receptor (TLR) pathway. In this study, we examine the participation of TLR4 in acute allograft rejection in an orthotopic mouse model of SIT.Wild-type C57Bl/6 (H-2b) or TLR49(-/-) (H-2b) mice were transplanted with syngeneic (C57Bl/6), allogeneic (BALB/c; H-2d), or F1 (BALB/cxC57Bl/6; H-2d/b) vascularized, orthotopic small intestine grafts. Graft recipients were killed on days 2 to 6 posttransplant. Serum cytokines were measured by Luminex, and tissue was obtained for histology and quantitative real-time polymerase chain reaction.BALB/c grafts transplanted into C57Bl/6 recipients exhibited mixed inflammatory infiltrates, destruction of the mucosa, and significant apoptosis. TLR2 and TLR4 transcripts were modestly increased in syngeneic grafts on days 2 and 6 compared with native bowel, whereas TLR2 and TLR4 were significantly increased on days 2 and 6 in allogeneic grafts. Although fully mismatched and F1 grafts were rejected by C57Bl/6 recipients (mean survival time=8.2 and 9.3 days, respectively), graft survival was significantly prolonged in TLR4(-/-) recipients (mean survival time=10.6 and 14.3 days, respectively). Proinflammatory cytokines were markedly reduced in TLR4(-/-) graft recipients.Small intestine graft survival is prolonged in the absence of TLR4, suggesting that gut flora associated with the graft may augment alloimmune responses through TLR4. Thus, the TLR pathway may be a novel therapeutic target for improving SIT allograft survival.

    View details for DOI 10.1097/TP.0b013e3181fdda0d

    View details for Web of Science ID 000285377100006

    View details for PubMedID 21197709

  • Acute Rejection of Small Intestine Allografts Is Associated With Increased Expression of Toll-like Receptors TRANSPLANTATION PROCEEDINGS Castillo, R. O., Wang, M., Ito, T., Higgins, J., Esquivel, C. O., Krams, S. M., Martinez, O. M. 2010; 42 (7): 2676-2678

    Abstract

    Although outcomes after intestinal transplantation have steadily improved owing to advances in immunosuppressive therapy, operative techniques, and postoperative medical management, rejection of the intestinal allograft continues to be a major clinical problem and constitutes the primary reason for graft loss. Although the adaptive immune system has been the major focus of investigation regarding regulation of rejection of the intestinal allograft, the role of the innate immune system has recently become of increased interest. We hypothesized that microbial products of the microflora associated with the intestinal allograft may engage the Toll-like receptor pathway of the innate immune system to potentiate alloimmune responses and rejection of the allograft. To investigate this, we established a murine model for orthotopic intestinal transplantation and allograft rejection. Using this model, we show that the expression of Toll-like receptor 2 is increased 50-fold and the expression of Toll-like receptor 4 is increased 200-fold during rejection of the allograft. We then performed survival studies that showed increased survival of mice, which had the Toll-like receptor knocked out. These preliminary studies suggest an important role for in innate immune system in acute rejection of the small intestinal allografts, and as such represents an emerging and promising area of investigation.

    View details for DOI 10.1016/j.transproceed.2010.05.157

    View details for Web of Science ID 000281942200052

    View details for PubMedID 20832568

  • Identification of the rat NKG2D ligands, RAE1L and RRLT, and their role in allograft rejection EUROPEAN JOURNAL OF IMMUNOLOGY Zhuo, M., Fujiki, M., Wang, M., Piard-Ruster, K., Wai, L., Wei, L., Martinez, O. M., Krams, S. M. 2010; 40 (6): 1748-1757

    Abstract

    NKG2D is a receptor expressed by NK cells and subsets of T lymphocytes. On NK cells, NKG2D functions as a stimulatory receptor that induces effector functions. We cloned and expressed two rat NKG2D ligands, both members of the RAE1 family, RAE1L and RRLT, and demonstrate that these ligands can induce IFN-gamma secretion and cytotoxicity by rat NK cells. To examine changes in expression of NKG2D and the NKG2D ligands RAE1L and RRLT after transplantation, we used a Dark Agouti (DA)-->Lewis rat model of liver transplantation. NKG2D expression was significantly increased in allogeneic liver grafts by day 7 post-transplant. Ligands of NKG2D, absent in normal liver, were readily detected in both syngeneic and allogeneic liver grafts by day 1 post-transplant. By day 7 post-transplant, hepatocyte RAE1L and RRLT expression was significantly and specifically increased in liver allografts. In contrast to acute rejection that develops in the DA-->Lewis model, transplantation of Lewis livers into DA recipients (Lewis-->DA) results in spontaneous tolerance. Interestingly, expression of RAE1L and RRLT is low in Lewis-->DA liver allografts, but significantly increased in DA-->Lewis liver allografts undergoing rejection. In conclusion, our results suggest that expression of NKG2D ligands may be important in allograft rejection.

    View details for DOI 10.1002/eji.200939779

    View details for Web of Science ID 000279077200023

    View details for PubMedID 20306467

  • MicroRNAs as Immune Regulators: Implications for Transplantation AMERICAN JOURNAL OF TRANSPLANTATION Harris, A., Krams, S. M., Martinez, O. M. 2010; 10 (4): 713-719

    Abstract

    The explosion of genetic information from recent advances in sequencing technologies, bioinformatics and genomics highlights the importance of understanding mechanisms involved in gene expression and regulation. Over the last decade, it has become clear that small ribonucleic acids (RNAs) are a central component of the cellular gene regulatory network. MicroRNAs (miRNAs) are a family of endogenous, small, noncoding single-stranded RNA of approximately 22 nucleotides in length that act as posttranscriptional gene regulatory elements. MicroRNAs can inhibit de novo protein synthesis by blocking translation through base-pairing with complementary messenger RNA (mRNA) and also suppress translation by promoting degradation of target mRNA. MicroRNAs are intimately involved in a variety of biologic processes including development, hematopoietic cell differentiation, apoptosis and proliferation. To date, over 800 human miRNAs have been identified, though the biologic function of only a fraction of miRNAs has been elucidated. Here, we discuss how miRNAs are produced, identified and quantitated, and focus on several key miRNAs that govern expression of genes relevant to allograft rejection, tolerance induction and posttransplant infection. Finally, we discuss potential ways in which the miRNA network can be modulated that ultimately may offer new strategies to promote long-term graft survival.

    View details for DOI 10.1111/j.1600-6143.2010.03032.x

    View details for Web of Science ID 000275768300004

    View details for PubMedID 20199506

  • Induced Tolerance to Rat Liver Allografts Involves the Apoptosis of Intragraft T Cells and the Generation of CD4(+)CD25(+)FoxP3(+) T Regulatory Cells LIVER TRANSPLANTATION Fujiki, M., Esquivel, C. O., Martinez, O. M., Strober, S., Uemoto, S., Krams, S. M. 2010; 16 (2): 147-154

    Abstract

    Posttransplant total lymphoid irradiation is a nonmyeloablative regimen that has been extensively studied in rodent models for the induction of tolerance to bone marrow and solid organ allografts. Previous studies of experimental models and clinical transplantation have used total lymphoid irradiation in combination with anti-lymphocyte-depleting reagents and donor cell infusion to promote graft acceptance. In a rat model of orthotopic liver transplantation, we demonstrated that total lymphoid irradiation alone induced long-term graft survival. Apoptotic T cells were detected in markedly higher numbers in the livers of the total lymphoid irradiation-treated group in comparison with the control group of liver allograft recipients. Intragraft CD4(+)CD25(+)FoxP3(+) cells were increased in the total lymphoid irradiation group in the first week post-transplant and remained elevated in the graft and in the spleen. Importantly, the adoptive transfer of splenocytes from recipients that received posttransplant total lymphoid irradiation prolonged the survival of donor heart grafts, but not third-party heart grafts, whereas the depletion of CD4(+)CD25(+) cells from transferred splenocytes abrogated this prolongation. We conclude that posttransplant total lymphoid irradiation significantly increases the apoptosis of T cells in the liver graft and allows the accumulation of CD4(+)CD25(+)FoxP3(+) T regulatory cells, which facilitate the generation of donor-specific tolerance.

    View details for DOI 10.1002/lt.21963

    View details for Web of Science ID 000274437800005

    View details for PubMedID 20104482

  • Activation of the JAK/STAT Pathway in Epstein Barr Virus(+)-Associated Posttransplant Lymphoproliferative Disease: Role of Interferon-gamma AMERICAN JOURNAL OF TRANSPLANTATION Vaysberg, M., Lambert, S. L., Krams, S. M., Martinez, O. M. 2009; 9 (10): 2292-2302

    Abstract

    Epstein Barr virus (EBV) is associated with B-cell lymphomas in posttransplant lymphoproliferative disease (PTLD). Latent membrane protein 1 (LMP1), the major oncogenic protein of EBV, promotes tumorigenesis through activation of NF-kappaB, Erk, p38, JNK and Akt. The Jak/STAT signal transduction pathway is also constitutively active in PTLD-associated EBV(+) B-cell lymphomas. Here we determine the mechanism of Jak/STAT activation in EBV(+) B-cell lymphomas and the role of LMP1 in this process. Immunoprecipitation studies revealed no direct interaction of LMP1 and JAK3, but known associations between JAK3 and common gamma chain, and between LMP1 and TRAF3, were readily detected in EBV(+) B cell lines from patients with PTLD. An inducible LMP1 molecule expressed in EBV(-) BL41 Burkitt's cells demonstrated STAT activation only after prolonged LMP1 signaling. While LMP1 induced IFN-gamma production in BL41 cells, IFN-gamma receptor blockade and IFN-gamma neutralization prior to LMP1 activation markedly decreased STAT1 activation and expression of LMP1-driven IFN-gamma inducible genes. Understanding the mechanisms by which EBV induces cellular signal transduction pathways may facilitate development of new treatments for PTLD.

    View details for DOI 10.1111/j.1600-6143.2009.02781.x

    View details for Web of Science ID 000269801000013

    View details for PubMedID 19656130

  • Decreases in circulating CD4(+)CD25(hi)FOXP3(+) cells and increases in intragraft FOXP3(+) cells accompany allograft rejection in pediatric liver allograft recipients PEDIATRIC TRANSPLANTATION Stenard, F., Nguyen, C., Cox, K., Kambham, N., Umetsu, D. T., Krams, S. M., Esquivel, C. O., Martinez, O. M. 2009; 13 (1): 70-80

    Abstract

    We examined CD4(+)CD25(hi)FOXP3(+) cells Treg in children following liver transplantation and determined the relationship between Treg cell levels in the blood and in the graft. Peripheral blood was obtained from pediatric liver transplant patients at sequential time points: pre-transplant, one month, 3-4 months, 6-7 months, and 11-12 months post-transplant. PBMC were isolated, labeled for CD4, CD25 and FOXP3 expression and analyzed by flow cytometry for CD4(+)CD25(hi)FOXP3(+) cells. Sorted CD4(+)CD25(hi) cells were assessed for functional activity. Pretransplant blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells were not significantly different from post-transplant blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells. However, the blood levels of CD4(+)CD25(hi)FOXP3(+) Treg cells were significantly decreased during acute rejection compared with levels when graft function was stable. Immunohistochemistry revealed that FOXP3(+) cells were increased in the portal region of livers with histopathologic evidence of acute graft rejection compared with livers without evidence of rejection and were localized primarily within the inflammatory infiltrate. These data indicate that Treg cells are found at the site of allograft rejection and may play a role in regulation of alloreactivity. Moreover, monitoring peripheral CD4(+)CD25(hi)FOXP3(+) Treg cell levels may be useful in improving the post-transplant management of pediatric liver allograft recipients.

    View details for DOI 10.1111/j.1399-3046.2008.00917.x

    View details for Web of Science ID 000262285400012

    View details for PubMedID 18331536

  • Tumor-derived Variants of Epstein-Barr Virus Latent Membrane Protein 1 Induce Sustained Erk Activation and c-Fos JOURNAL OF BIOLOGICAL CHEMISTRY Vaysberg, M., Hatton, O., Lambert, S. L., Snow, A. L., Wong, B., Krams, S. M., Martinez, O. M. 2008; 283 (52): 36573-36585

    Abstract

    Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a proven oncogene that is essential for transformation of human B cells by the virus. LMP1 induces constitutive activation of several signal transduction pathways involving nuclear factor kappaB, phosphatidylinositol 3-kinase/Akt, and the mitogen-activated protein kinases (MAPK) p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (Erk). Sequencing of LMP1 isolated from a panel of EBV+ B cell lymphomas identified three different variants of LMP1, each distinct from the B95.8 prototype isoform. All tumor variants of LMP1 as well as the B95.8 LMP1 isoform were able to induce rapid p38 phosphorylation as well as Akt and JNK activation. Additionally all variants showed similar ability to activate nuclear factor kappaB. In contrast, only tumor-derived LMP1 variants induced prolonged Erk activation and c-Fos expression. Sequence analysis revealed only two amino acids, 212 and 366, shared by the tumor variants but distinct from B95.8. Point mutation of either amino acids 212 (glycine to serine) or 366 (serine to threonine) from the B95.8 isoform to the tumor variant version of LMP1 was sufficient for gain of function characterized by sustained activation of Erk and subsequent c-Fos induction and binding to the AP1 site. Our results indicate that the enhanced ability of tumor-derived LMP1 to induce and stabilize the c-Fos oncogene can be localized to two amino acids in the C terminus of LMP1.

    View details for DOI 10.1074/jbc.M802968200

    View details for Web of Science ID 000261840500055

    View details for PubMedID 18986987

  • Epstein-Barr virus, rapamycin, and host immune responses CURRENT OPINION IN ORGAN TRANSPLANTATION Krams, S. M., Martinez, O. M. 2008; 13 (6): 563-568

    Abstract

    To summarize recent advances that contribute to our understanding of the pathobiology of Epstein-Barr virus (EBV)-associated posttransplant lymphoproliferative disease (PTLD), the host immune response to virally infected B cells, and the molecular basis for the effects of mammalian target of rapamycin inhibitors on EBV+ B-cell lymphomas.Cytogenetic and genomic analyses support the concept that the underlying biology of EBV-associated PTLD is complex. Transplant recipients can generate and maintain significant populations of EBV-specific CD8+ memory T cells but the function of these cells may be impaired. EBV invokes multiple strategies to subvert and evade the host immune response. The phosphoinositide-3 kinase/Akt/mammalian target of rapamycin signal transduction pathway is a nexus for growth and survival signals in PTLD-associated EBV+ B-cell lymphomas.Multiple factors influence the development of EBV-associated PTLD including the host immune response to EBV, virally induced effects on the infected cell and the host immune system, and the type and intensity of immunosuppression.

    View details for DOI 10.1097/MOT.0b013e3283186ba9

    View details for Web of Science ID 000261755200001

    View details for PubMedID 19060543

  • Rapamycin, but not cyclosporine or FK506, alters natural killer cell function TRANSPLANTATION Wai, L., Fujiki, M., Takeda, S., Martinez, O. M., Krams, S. M. 2008; 85 (1): 145-149

    Abstract

    Infiltration of natural killer (NK) cells into solid organ allografts is observed in clinical and experimental transplantation. Studies suggest a role for NK cells in acute and chronic rejection of solid organ allografts; however, the effects of immunosuppressive agents on NK cells are not clearly established. Rat NK cell lines were analyzed for proliferation and cytotoxicity in the presence of cyclosporine, FK506, or rapamycin. Lewis recipients of DA liver allografts received immunosuppressive agents after transplantation. NK cells demonstrated robust function both in the absence and presence of cyclosporine and FK506. In contrast, rapamycin significantly inhibited proliferation and cytotoxicity of NK cells. NK cell numbers remained stable in graft recipients treated with cyclosporine and FK506, whereas there was a significant decrease in NK cells in rapamycin-treated recipients. These data indicate that immunosuppressive drugs have differential effects on NK cell function that may impact the immune response of transplant recipients.

    View details for DOI 10.1097/01.tp.0000296817.28053.7b

    View details for Web of Science ID 000252530600023

    View details for PubMedID 18192925

  • Mutations to bid cleavage sites protect hepatocytes from apoptosis after ischemia/reperfusion injury TRANSPLANTATION Riddle-Taylor, E., Nagasaki, K., Lopez, J., Esquivel, C. O., Martinez, O. M., Krams, S. M. 2007; 84 (6): 778-785

    Abstract

    Apoptosis of hepatocytes contributes to many forms of liver pathology and can compromise liver function. Hepatocytes have been shown to require mitochondrial disruption to execute apoptosis, a process that is controlled by members of the Bcl-2 family. Bid is a proapoptotic Bcl-2 family member that is cleaved to its active form, tBid, by caspase 8 and granzyme B. Studies in the Bid-deficient mouse have established that hepatocytes require Bid to undergo apoptosis.We generated aspartic acid to glutamic acid mutations in the rat Bid protein, at the caspase 8 and granzyme B cleavage sites, and utilized recombinant adenoviruses to express this protein in hepatoma cells and in the livers of rats.Cells transduced with recombinant adenoviruses encoding Bid containing mutations to the caspase 8 and granzyme B cleavage sites are significantly protected from both tumor necrosis factor-alpha-induced and cell-mediated apoptosis. Protection occurs through a mechanism that includes decreased Bid cleavage, caspase activation, and mitochondrial membrane damage. Further, after warm ischemia/reperfusion injury, we show that rats expressing cleavage-resistant Bid in the liver display significantly less hepatocyte apoptosis as compared to control rat livers and this results in improved liver function and survival.Our results suggest that reagents that prevent the cleavage of Bid would be an effective strategy to inhibit hepatocyte apoptosis and decrease liver injury.

    View details for DOI 10.1097/01.tp.0000281555.18782.2b

    View details for Web of Science ID 000249841700017

    View details for PubMedID 17893612

  • Rapamycin inhibits proliferation of Epstein-Barr virus-positive B-cell lymphomas through modulation of cell-cycle protein expression TRANSPLANTATION Vaysberg, M., Balatoni, C. E., Nepomuceno, R. R., Krams, S. M., Martinez, O. M. 2007; 83 (8): 1114-1121

    Abstract

    Posttransplant lymphoproliferative disease (PTLD) is a serious complication of solid organ and bone marrow transplantation and is closely associated with Epstein-Barr virus (EBV) infection. We have previously shown that rapamycin (RAPA) directly inhibits the in vitro and in vivo proliferation of EBV-infected B lymphoblastoid cell lines (SLCL), derived from patients with PTLD, by arresting cells in the G1 phase of the cell cycle. The aim of this study is to elucidate the mechanism by which RAPA causes cell cycle arrest in EBV+ B cells.SLCL were cultured without or with RAPA (10 ng/ml) and G1-associated cell cycle proteins were analyzed by immunoblot and densitometric analysis. CDK complexes were immunoprecipitated and incubated with retinoblastoma protein (Rb) substrate. Kinase activity of the complex was determined by Western blot with anti-phospho-Rb antibodies.We show that RAPA decreased both Cyclin D2 and Cyclin D3 protein levels. Furthermore, RAPA decreased the protein levels of cyclin dependent kinase 4 (CDK4) and increased the expression of the CDK inhibitor p27. In contrast, expression of the CDK inhibitor p21 was markedly inhibited by RAPA in the SLCL. Finally, in vitro kinase assays revealed that downstream hyperphosphorylation of Rb by CDK complexes was also decreased by RAPA.The results presented here elucidate key targets of RAPA-induced cell cycle arrest, provide insight into the growth pathways of EBV+ B-cell lymphomas, and demonstrate the potential for RAPA as a therapeutic option in the treatment of PTLD and other EBV+ lymphomas.

    View details for DOI 10.1097/01.tp.0000260142.38619.9c

    View details for Web of Science ID 000246234800017

    View details for PubMedID 17452903

  • EBV can protect latently infected B cell lymphomas from death receptor-induced apoptosis JOURNAL OF IMMUNOLOGY Snow, A. L., Lambert, S. L., Natkunam, Y., Esquivel, C. O., Krams, S. M., Martinez, O. M. 2006; 177 (5): 3283-3293

    Abstract

    The relationship between EBV infection and sensitivity to death receptor (DR)-induced apoptosis is poorly understood. Using EBV- and EBV+ BJAB cells, we provide the first evidence that EBV can protect latently infected B cell lymphomas from apoptosis triggered through Fas or TRAIL receptors. Caspase 8 activation was impaired and cellular FLIP recruitment was enriched in death-inducing signaling complexes formed in EBV-infected BJAB cells relative to parent BJAB cells. Furthermore, latent membrane protein 1 expression alone could reduce caspase activation and confer partial resistance to DR apoptosis in BJAB cells. This protective effect was dependent on C-terminal activating region 2-driven NF-kappaB activation, which in turn up-regulated cellular FLIP expression in latent membrane protein 1+ BJAB cells. Thus, the ability of latent EBV to block DR apoptosis may help to ensure the survival of host cells during B cell differentiation, and contribute to the development of B cell lymphomas, especially in immunocompromised individuals.

    View details for Web of Science ID 000240002800065

    View details for PubMedID 16920969

  • NKp30 is a functional activation receptor on a subset of rat natural killer cells EUROPEAN JOURNAL OF IMMUNOLOGY Hsieh, C. L., Nagasaki, K., Martinez, O. M., Krams, S. M. 2006; 36 (8): 2170-2180

    Abstract

    NKp30 is a stimulatory receptor on human NK cells implicated in tumor immunity, and is capable of promoting or terminating dendritic cell maturation. To gain a better understanding of NKp30 biology, we have investigated the expression and function of rat NKp30 (rNKp30). We generated stable transfectants of rNKp30 in RNK16 cells, a rat NK lymphoma line, and used a novel panel of mAb against rNKp30 to study this receptor. Using agonistic rNKp30 mAb, we demonstrated that rNKp30 mediates robust IFN-gamma production and cytolytic responses from rNKp30-transfected RNK16 cells. We determined by flow cytometry that rNKp30 is expressed by a subset of primary NK cells isolated from the blood and spleen, and to a lesser extent also on liver NK cells. Stimulation of rNKp30 on primary NK cells led to IFN-gamma production. Liver NK cells expressed low levels of NKp30 and had reduced rNKp30-mediated IFN-gamma responses. During an alloimmune response in vivo, the proportion of the rNKp30(+) NK cell subset in the peripheral blood significantly increased, suggesting that rNKp30 may play an important role during alloactivation. Thus, our data demonstrate that NKp30 is indeed expressed in rodents and is a functional stimulatory receptor in a subset of rat NK cells.

    View details for DOI 10.1002/eji.200635982

    View details for Web of Science ID 000239855900017

    View details for PubMedID 16821237

  • EBV+ B lymphoma cell lines from patients with post-transplant lymphoproliferative disease are resistant to TRAIL-induced apoptosis AMERICAN JOURNAL OF TRANSPLANTATION Snow, A. L., Vaysberg, M., Krams, S. M., Martinez, O. M. 2006; 6 (5): 976-985

    Abstract

    Lymphomas associated with post-transplant lymphoproliferative disease (PTLD) represent a significant complication of immunosuppression in transplant recipients. In immunocompetent individuals, EBV-specific cytotoxic T lymphocytes (CTL) prevent the outgrowth of activated B lymphoblasts through apoptosis induction. Soluble versions of TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL) can induce apoptosis in numerous tumor cell types. Given the therapeutic potential of TRAIL, we examined the sensitivity of EBV+ spontaneous lymphoblastoid cell lines (SLCL) derived from patients with PTLD to treatment with soluble TRAIL. Despite abundant expression of TRAIL receptors (TRAIL-R), resistance to TRAIL-induced apoptosis was observed in all SLCL examined. This resistance could not be overcome by concomitant treatment with several pharmacological agents. Unlike BJAB positive control cells, for each SLCL tested, cleavage and activation of caspase 8 was inhibited due to failed recruitment of FADD and caspase 8 to TRAIL receptors upon stimulation. Further indicative of a proximal defect, TRAIL receptor aggregation could not be detected on the cell surface of SLCL following ligand engagement. These results suggest that the use of TRAIL for eliminating PTLD-associated tumors may be of limited clinical utility, and illustrate another mechanism by which EBV+ B lymphoma cells can evade tumor surveillance at the level of death receptor signaling.

    View details for DOI 10.1111/j.1600-6143.2006.01295.x

    View details for Web of Science ID 000236860700014

    View details for PubMedID 16611333

  • IFN-gamma, produced by NK cells that infiltrate liver allografts early after transplantation, links the innate and adaptive immune responses AMERICAN JOURNAL OF TRANSPLANTATION Obara, H., Nagasaki, K., Hsieh, C. L., Ogura, Y., Esquivel, C. O., Martinez, O. M., Krams, S. M. 2005; 5 (9): 2094-2103

    Abstract

    The role of NK cells following solid organ transplantation remains unclear. We examined NK cells in acute allograft rejection using a high responder model (DA-->Lewis) of rat orthotopic liver transplantation. Recipient-derived NK cells infiltrated liver allografts early after transplantation. Since chemokines are important in the trafficking of cells to areas of inflammation, we determined the intragraft expression of chemokines known to attract NK cells. CCL3 was significantly increased in allografts at 6 h post-transplant as compared to syngeneic grafts whereas CCL2 and CXCL10 were elevated in both syngeneic and allogeneic grafts. CXCL10 and CX3CL1 were significantly upregulated in allografts by day 3 post-transplant as compared to syngeneic grafts suggesting a role for these chemokines in the recruitment of effector cells to allografts. Graft-infiltrating NK cells were shown to be a major source of IFN-gamma, and IFN-gamma levels in the serum were markedly increased, specifically in allograft recipients, by day 3 post-transplant. Accordingly, in the absence of NK cells the levels of IFN-gamma were significantly decreased. Furthermore, graft survival was significantly prolonged. These data suggest that IFN-gamma-producing NK cells are an important link between the innate and adaptive immune responses early after transplantation.

    View details for DOI 10.1111/j.1600-6143.2005.00995.x

    View details for Web of Science ID 000231023700003

    View details for PubMedID 16095488

  • Keratins as susceptibility genes for end-stage liver disease GASTROENTEROLOGY Ku, N. O., Lim, J. K., Krams, S. M., Esquivel, C. O., Keeffe, E. B., Wright, T. L., Parry, D. A., Omary, M. B. 2005; 129 (3): 885-893

    Abstract

    Keratins 8 and 18 protect the liver from stress. Keratin 8 and 18 variants in 17 of 467 liver disease explants and 2 of 349 blood bank controls were previously reported in 5 analyzed exonic regions. We asked whether mutations were present in the remaining 10 exons of keratins 8 and 18.Exonic regions were polymerase chain reaction-amplified from genomic DNA, isolated from the above-mentioned 2 cohorts, and analyzed for the presence of mutations. Mutant keratins were also studied biochemically.We identified 10 novel keratin 8 and 18 heterozygous variants in 44 of 467 explants and 11 of 349 controls: keratin 18 deletion (delta64-71), a keratin 8 frameshift that truncates the last 14 amino acids; 8 missense keratin 8 and 18 alterations; and several new polymorphisms. The most common variant, keratin 8 R340H, at the highly conserved R340 was found in 30 of 467 explants and 10 of 349 controls (P = .02) and was confirmed in the diseased livers by generation of an R340H-specific antibody. Germline transmission and variant protein expression were verified. The mutations involved a variety of liver diseases, and some variants had an ethnic background preponderance. Mutations that introduced disulfide bonds (keratin 8 G61C or R453C) decreased keratin solubility, particularly after oxidative stress, whereas others decreased keratin 8 phosphorylation (keratin 8 G433S).The overall frequency of keratin 8 and 18 variants was 12.4% in 467 liver disease explants and 3.7% in 349 blood bank controls (P < .0001). Variants can alter keratin solubility or phosphorylation and may render individuals susceptible to end-stage liver disease, depending on their genetic background and exposure to other insults, such as alcohol or viral infection.

    View details for DOI 10.1053/j.gastro.2005.06.065

    View details for Web of Science ID 000231816500016

    View details for PubMedID 16143128

  • Identification, cloning, and characterization of a novel rat natural killer receptor, RNKp30: A molecule expressed in liver allografts TRANSPLANTATION Hsieh, C. L., Ogura, Y., Obara, H., Ali, U. A., Rodriguez, G. M., Nepomuceno, R. R., Martinez, O. M., Krams, S. M. 2004; 77 (1): 121-128

    Abstract

    As a component of the innate immune system, natural killer (NK) cells may play a significant role in the early events after solid-organ transplantation. Activated NK cells have been shown to infiltrate allografts in transplant models. To better understand NK cells and the role of NK cell receptors in transplantation, we have cloned and begun characterizing a novel rat molecule, rNKp30.RNKp30 cDNA was cloned by 5' rapid amplification of cDNA ends polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR from mononuclear cells infiltrating a rejecting liver allograft. Southern blot analysis was used to determine the rNKp30 gene copy number. RT-PCR and Northern blotting were used to examine rNKp30 RNA expression in NK cells, multiple tissues, and liver grafts. Immunocytochemistry, immunoprecipitation, and Western blot analysis with two anti-rNKp30 polyclonal antibodies, CA680 and CA1071, were performed. Tunicamycin and endoglycosidase treatments determined the extent of rNKp30 glycosylation.RNKp30 is homologous to human and macaque NKp30. It is a single copy gene with five identified single-nucleotide polymorphisms. RNKp30 is expressed by NK cells and is detectable as a single transcript by Northern blot in normal spleen, lymph node, and lung tissues. RNKp30 is a variably N-glycosylated cell surface molecule with a protein backbone of approximately 21 kDa. Elevated transcript expression of rNKp30 is detected in both rejected and spontaneously accepted liver allografts, but not in syngeneic or cyclosporine A-treated allografts.RNKp30 is a glycosylated surface NK cell receptor with limited polymorphism. This putative activation receptor is expressed in liver allografts and may participate in the innate immune response after transplantation.

    View details for DOI 10.1097/01.TP.0000110423.27977.6F

    View details for Web of Science ID 000188329100021

    View details for PubMedID 14724446

  • Rapamycin inhibits the interleukin 10 signal transduction pathway and the growth of Epstein Barr virus B-cell lymphomas CANCER RESEARCH Nepomuceno, R. R., Balatoni, C. E., Natkunam, Y., Snow, A. L., Krams, S. M., Martinez, O. M. 2003; 63 (15): 4472-4480

    Abstract

    EBV-infected B-cell lymphomas are a potentially life-threatening complication in bone marrow and solid organ transplant recipients. Immunosuppressive drugs required to prevent allograft rejection also impair anti-EBV T-cell immunity, thereby increasing the risk of EBV-associated disease. Here we demonstrate that the immunosuppressant rapamycin (RAPA) has a strong antiproliferative effect in vitro on B-cell lines derived from organ transplant recipients with EBV-associated posttransplant lymphoproliferative disorder (PTLD). Furthermore, RAPA significantly inhibits or delays the growth of solid tumors established from EBV-infected B-cell lines in a xenogeneic mouse model of PTLD. RAPA acts via cell cycle arrest, induction of apoptosis, and, most importantly, inhibition of interleukin 10 secretion, a necessary autocrine growth factor. The reduced interleukin 10 production is accompanied by corresponding decreases in the constitutive activation of the growth-promoting transcription factors signal transducer and activator of transcription 1 and 3. Thus, RAPA can limit B-cell lymphoma growth while simultaneously providing immunosuppression to prevent graft rejection in patients who are otherwise at risk for EBV-associated PTLD. Moreover, these findings may have application to other EBV-associated malignancies.

    View details for Web of Science ID 000184562500028

    View details for PubMedID 12907620

  • Keratin 8 and 18 mutations are risk factors for developing liver disease of multiple etiologies PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Ku, N. O., Darling, J. M., Krams, S. M., Esquivel, C. O., Keeffe, E. B., Sibley, R. K., Lee, Y. M., Wright, T. L., Omary, M. B. 2003; 100 (10): 6063-6068

    Abstract

    Keratin 8 and 18 (K8K18) mutations are found in patients with cryptogenic cirrhosis, but the role of keratin mutations in noncryptogenic cirrhosis and the incidence of keratin mutations in the general population are not known. We screened for K8K18 mutations in genomic DNA isolated from 314 liver explants of patients who primarily had noncryptogenic cirrhosis, and from 349 blood bank volunteers. Seven unique K8K18 mutations were found in 11 independent patients with biliary atresia, hepatitis BC, alcohol, primary biliary cirrhosis, and fulminant hepatitis. Seven of the 11 patients had mutations previously described in patients with cryptogenic cirrhosis: K8 Tyr-53 --> His, K8 Gly-61 --> Cys, and K18 His-127 --> Leu. The four remaining patients had mutations at one K8 and three other K18 new sites. Of the 349 blood bank control samples, only one contained the Tyr-53 --> His and one the Gly-61 --> Cys K8 mutations (P < 0.004 when comparing cirrhosis versus control groups). Two additional mutations were found in both the liver disease and blood bank groups and, hence, likely represent polymorphisms. Livers with keratin mutations had cytoplasmic filamentous deposits that were less frequent in livers without the mutations (P = 0.03). Therefore, K8K18 are likely susceptibility genes for developing cryptogenic and noncryptogenic forms of liver disease.

    View details for DOI 10.1073/pnas.0936165100

    View details for Web of Science ID 000182939400089

    View details for PubMedID 12724528

  • Identification of Epstein-Barr virus-specific CD8(+) T lymphocytes in the circulation of pediatric transplant recipients TRANSPLANTATION Falco, D. A., Nepomuceno, R. R., Krams, S. M., Lee, P. P., DAVIS, M. M., Salvatierra, O., Alexander, S. R., Esquivel, C. O., Cox, K. L., Frankel, L. R., Martinez, O. M. 2002; 74 (4): 501-510

    Abstract

    Pediatric transplant recipients are at increased risk for Epstein Barr virus (EBV)-related B cell lymphomas. In healthy individuals, the expansion of EBV-infected B cells is controlled by CD8+ cytotoxic T cells. However, immunosuppressive therapy may compromise antiviral immunity. We identified and determined the frequency of EBV-specific T cells in the peripheral blood of pediatric transplant recipients.HLA-B*0801 and HLA-A*0201 tetramers folded with immunodominant EBV peptides were used to detect EBV-specific CD8+ T cells by flow cytometry in peripheral blood mononuclear cells from 24 pediatric liver and kidney transplant recipients. The expression of CD38 and CD45RO on EBV-specific, tetramer-binding cells was also examined in a subset of patients by immunofluorescent staining and flow cytometry.Tetramer-binding CD8+ T cells were identified in 21 of 24 transplant recipients. EBV-specific CD8+ T cells were detected as early as 4 weeks after transplant in EBV seronegative patients receiving an organ from an EBV seropositive donor. The frequencies (expressed as a percentage of the CD8+ T cells) of the tetramer-binding cells were HLA-B8-RAKFKQLL (BZLF1 lytic antigen peptide) tetramer, range=0.96 to 3.94%; HLA-B8-FLRGRAYGL (EBNA3A latent antigen peptide) tetramer, range=0.03 to 0.59%; and HLA-A2-GLCTLVAML (BMLF1 lytic antigen peptide) tetramer, range=0.06 to 0.76%. The majority of tetramer reactive cells displayed an activated/memory phenotype.Pediatric transplant recipients receiving immunosuppression can generate EBV-specific CD8+ T cells. Phenotypic and functional analysis of tetramer cells may prove useful in defining and monitoring EBV infection in the posttransplant patient.

    View details for Web of Science ID 000177808600012

    View details for PubMedID 12352909

  • CD30 expression identifies the predominant proliferating T lymphocyte population in human alloimmune responses JOURNAL OF IMMUNOLOGY Chan, K. W., Hopke, C. D., Krams, S. M., Martinez, O. M. 2002; 169 (4): 1784-1791

    Abstract

    CD30 is an inducible member of the TNFR superfamily that is expressed on activated T and B cells and some lymphoid malignancies. We have previously shown that human CD30(+) T cells elicited with allogeneic APC are a major source of IFN-gamma and IL-5 production. In the present study we have used alloantigen, as well as anti-CD3 plus anti-CD28 mAb stimulation, to further characterize human CD30(+) T cells with respect to function and the expression of other activation-dependent cell surface molecules, including the related TNFR family members OX-40 and 4-1BB (CD137). Our results indicate that human CD30(+) T cells are a subset of activated T cells that also express CD25 and CD45RO. Moreover, we observed that allogeneic APC consistently induced a greater proportion of CD30(+) cells within the activated T cell population than did stimulation with plate-bound anti-CD3 plus anti-CD28 mAb or stimulation with soluble anti-CD3 plus anti-CD28 and autologous APC. The enhanced induction of CD30 expression by alloantigen was not common to other inducible TNFR family members because anti-CD3 plus anti-CD28 mAbs were far more effective in inducing expression of 4-1BB and OX-40. Furthermore, CD30 expression marked the predominant proliferating T cell population induced by alloantigen as determined by CFSE staining and flow cytometry. These results indicate that CD30, but not 4-1BB or OX-40, is preferentially induced by alloantigen, suggesting that CD30 may be important in human alloimmune responses.

    View details for Web of Science ID 000177365800017

    View details for PubMedID 12165500

  • Constitutive activation of Jak/STAT proteins in Epstein-Barr virus-infected B-cell lines from patients with posttransplant lymphoproliferative disorder. TRANSPLANTATION Nepomuceno, R. R., Snow, A. L., Beatty, P. R., Krams, S. M., Martinez, O. M. 2002; 74 (3): 396-402

    Abstract

    Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4.The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays.All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression.These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL.

    View details for Web of Science ID 000177496000017

    View details for PubMedID 12177620

  • NK cells and transplantation TRANSPLANT IMMUNOLOGY Hsieh, C. L., Obara, H., Ogura, Y., Martinez, O. M., Krams, S. M. 2002; 9 (2-4): 111-114

    Abstract

    The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We have demonstrated that CD8+ T cells are not essential for allograft rejection or for the induction of apoptosis in two experimental models of transplantation. To determine candidate cells types which may play a role in the events leading to graft rejection, the cellular composition of rejecting allografts was determined. We demonstrate that substantial numbers of NK cells, of recipient origin, infiltrate allografts as early as 12 h after transplantation. These NK cells produce cytokines and express cytotoxic mediators such as granzyme B and FasL. It is unknown which NK cell receptors are expressed and activated during transplantation. NK cells express multiple cell surface receptors, including MHC class I binding inhibitory receptors, which deliver a negative signal, and activation receptors, which stimulate cytokine secretion and cytotoxicity of NK cells. To begin to understand NK cell activation in the context of transplantation, we have recently cloned a novel rat immunoglobulin-like surface receptor from a rejecting liver allograft. Sequence analysis demonstrates that this putative activation receptor contains 71% identity to human NKp30 at the DNA level, suggesting that it is the rat homologue (rNKp30). Characterization of NK activation receptors may lead to better understanding of the interactions between the innate and adaptive immune responses in transplantation.

    View details for Web of Science ID 000177317300007

    View details for PubMedID 12180816

  • New approaches to inducing the death of alloreactive lymphocytes CLINICAL AND EXPERIMENTAL IMMUNOLOGY Krams, S. M. 2001; 126 (3): 371-373

    View details for Web of Science ID 000175437200001

    View details for PubMedID 11737049

  • Resistance to Fas-mediated apoptosis in EBV-infected B cell lymphomas is due to defects in the proximal Fas signaling pathway JOURNAL OF IMMUNOLOGY Snow, A. L., Chen, L. J., Nepomuceno, R. R., Krams, S. M., Esquivel, C. O., Martinez, O. M. 2001; 167 (9): 5404-5411

    Abstract

    Post-transplant lymphoproliferative disorder is characterized by the outgrowth of EBV-infected B cell lymphomas in immunosuppressed transplant recipients. Using a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from post-transplant lymphoproliferative disorder patients, we assessed the sensitivity of such lymphomas to Fas-mediated cell death. Treatment with either an agonist anti-Fas mAb or Fas ligand-expressing cells identifies two subsets of SLCL based on their sensitivity or resistance to Fas-driven apoptosis. Fas resistance in these cells cannot be attributed to reduced Fas expression or to mutations in the Fas molecule itself. In addition, all SLCL are sensitive to staurosporine-induced cell death, indicating that there is no global defect in apoptosis. Although all SLCL express comparable levels of Fas signaling molecules including Fas-associated death domain protein, caspase 8, and caspase 3, Fas-resistant SLCL exhibit a block in Fas-signaling before caspase 3 activation. In two SLCL, this block results in impaired assembly of the death-inducing signaling complex, resulting in reduced caspase 8 activation. In a third Fas-resistant SLCL, caspase 3 activation is hindered despite intact death-inducing signaling complex formation and caspase 8 activation. Whereas multiple mechanisms exist by which tumor cells can evade Fas-mediated apoptosis, these studies suggest that the proximal Fas-signaling pathway is impeded in Fas-resistant post-transplant lymphoproliferative disorder-associated EBV(+) B cell lymphomas.

    View details for Web of Science ID 000171858500080

    View details for PubMedID 11673559

  • Apoptotic pathways in primary biliary cirrhosis and autoimmune hepatitis LIVER Fox, C. K., Furtwaengler, A., Nepomuceno, R. R., Martinez, O. M., Krams, S. M. 2001; 21 (4): 272-279

    Abstract

    Autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) are two autoimmune diseases with unknown etiologies that primarily target the liver. In both diseases, liver lesions are accompanied by large infiltrates of mononuclear cells. The purpose of this study was to determine if either the Fas-mediated or the granule-exocytosis pathways contribute to apoptosis in these diseases.To determine the involvement of apoptosis in tissue injury we examined liver tissue for DNA fragmentation and morphological characteristics of apoptosis. The major cytotoxic pathways of activated lymphocytes were compared by quantitating the levels of transcripts for FasL and granzyme B, and expression was confirmed by immunoprecipitation of these molecules.In both diseases, apoptosis was observed. However, the main cell types undergoing apoptosis were hepatocytes in AIH, and biliary epithelial cells in PBC. In AIH the levels of FasL and granzyme B mRNA were increased over the levels detected in normal liver, while in PBC only the levels of granzyme B were elevated. Additionally, in AIH, the ratio of FasL transcripts to granzyme B transcripts was elevated, reflecting a possible increase in the relative contribution of FasL to the progression of the disease. Immunoprecipitation studies further support an increase in FasL protein expression in AIH.These data suggest that both FasL and granzyme B contribute to the apoptosis observed in AIH and PBC. However, FasL appears to play a more prominent role in the induction of hepatocyte apoptosis and tissue destruction in AIH.

    View details for Web of Science ID 000170155900008

    View details for PubMedID 11454191

  • Apoptosis and allograft rejection in the absence of CD8(+) T cells TRANSPLANTATION Ogura, Y., Martinez, O. M., Villanueva, J. C., Tait, J. F., Strauss, H. W., Higgins, J. P., Tanaka, K., Esquivel, C. O., Blankenberg, F. G., Krams, S. M. 2001; 71 (12): 1827-1834

    Abstract

    The requirement for cytotoxic T lymphocytes during allograft rejection is controversial. We previously demonstrated that CD8+ T cells are not necessary for allograft rejection or for the induction of apoptosis in rat small intestinal transplantation. In this study, we examined the mechanisms of apoptosis and rejection after liver transplantation in the absence of CD8+ T cells.Either Lewis or dark agouti rat liver grafts were transplanted into Lewis recipients to create syngeneic and allogeneic combinations. CD8+ T cells were depleted in an additional allogeneic group by treatment with OX-8 mAb on day -1 and day 1 after liver transplant.Apoptosis and rejection were observed in both the CD8+ T cell-depleted allogeneic and allogeneic grafts by hematoxylin and eosin staining, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling staining, and radiolabeled-annexin V in vivo imaging. Granzyme B and FasL were expressed in all allogeneic transplants, including those depleted of CD8+ T cells, indicating that a mononuclear cell other than a CD8+ T cell can be the source of these molecules during allograft rejection. Activation of the caspase cascade was detected in all rejecting allografts. Caspases 3, 8, and 9 were activated at similar significantly elevated levels in both allogeneic and CD8+ T cell-depleted liver grafts.These data indicate that in the absence of CD8+ T cells an alternative pathway, associated with granzyme B and FasL expression and activation of the caspase cascade, can mediate apoptosis and graft rejection.

    View details for Web of Science ID 000169753800020

    View details for PubMedID 11455265

  • Radiolabeled annexin V imaging: Diagnosis of allograft rejection in an experimental rodent model of liver transplantation RADIOLOGY Ogura, Y., Krams, S. M., Martinez, O. M., Kopiwoda, S., Higgins, J. P., Esquivel, C. O., Strauss, H. W., Tait, J. F., Blankenberg, F. G. 2000; 214 (3): 795-800

    Abstract

    To assess the value of imaging rejection-induced apoptosis with technetium 99m and annexin V, a human protein-based radiopharmaceutical used in the diagnosis of acute rejection of a liver transplant, in a well-characterized rodent model of orthotopic liver transplantation.99mTc-radiolabeled annexin V was intravenously administered to six allografted (immunologically mismatched) and five isografted (immunologically matched) recipient rats on days 2, 4, and 7 after orthotopic liver transplantation. Animals were imaged 1 hour after injection of 0.2-2.0 mCi (8.0-74.0 MBq) of radiolabeled annexin V by use of clinical nuclear scintigraphic equipment.All animals in the allografted group demonstrated marked increases of 55% and 97% above the activity in the isografted group in hepatic uptake of annexin V on days 4 and 7, respectively. Severe acute rejection was histologically detected in all allografted livers on day 7. There was no histologic evidence of acute rejection in isografted animals. Dynamic hepatobiliary imaging with 99mTc and mebrofenin, an iminodiacetic acid derivative, demonstrated no correlation with the presence or absence of acute rejection or with annexin V uptake.Noninvasive imaging with radiolabeled annexin V is more sensitive and specific than imaging with 99mTc-mebrofenin in the diagnosis of acute rejection of a liver transplant.

    View details for Web of Science ID 000085478800029

    View details for PubMedID 10715048

  • Involvement of Fas-Fas ligand interactions in graft rejection. International reviews of immunology Martinez, O. M., Krams, S. M. 1999; 18 (5-6): 527-546

    Abstract

    The Fas/Fas ligand (FasL) pathway has been shown to be important in T lymphocyte-mediated cell death and is a key peripheral immunoregulatory mechanism that limits expansion of antigen-activated lymphocytes. The expression of Fas by commonly transplanted organs such as the heart, lung, kidney, and liver suggests that these tissues may be targets of FasL-expressing allospecific cytotoxic T lymphocytes. In this review the current literature examining the Fas/FasL system as a potential cellular effector pathway in tissue injury is discussed. In addition to a deleterious role in destruction of graft tissue, Fas/FasL interactions may have a beneficial role in transplantation. Recent studies suggest that modulation of FasL in target tissue leads to deletion, via apoptosis, of graft infiltrating lymphoid cells. However, an equally compelling series of reports indicate that overexpression of FasL can lead to a heightened immune response. These data are reviewed in the context of strategies to achieve long term allograft survival.

    View details for PubMedID 10672500

  • Posttransplant lymphoproliferative disorders and gastrointestinal manifestations of Epstein-Barr virus infection in children following liver transplantation TRANSPLANTATION Cao, S., Cox, K., Esquivel, C. O., Berquist, W., Concepcion, W., Ojogho, O., Monge, H., Krams, S., Martinez, O., So, S. 1998; 66 (7): 851-856

    Abstract

    Epstein-Barr virus (EBV) infection is common after liver transplantation in children and is associated with the risk of posttransplant lymphoproliferative disorders (PTLD).This retrospective study examined the frequency of gastrointestinal (GI) symptoms and the risk of PTLD in pediatric liver recipients who developed symptomatic EBV infection. We reviewed 172 children who received orthotopic liver transplants between March 1988 to December 1994. Twenty-two cases were retransplants. The mean age at transplantation was 3.7 years (range, 0.1-17 years). The immunosuppressive regimens consisted of induction therapy with Minnesota antilymphocyte globulin/antithymocyte globulin/OKT3 in most cases and maintenance therapy with prednisone and either cyclosporine or tacrolimus (FK506).After 1 year of minimum follow-up, 54 of 172 patients had symptomatic EBV infections (confirmed by serology, histology, or whole blood polymerase chain reaction. At the time of infection, 38.5% (21/54) had either diarrhea or GI bleeding or both. PTLD developed in 11 patients (6.4%). The incidence of PTLD was 42.9% (9/21) when GI bleeding or diarrhea was associated with EBV infections, compared with 6.1% (2/33) when EBV infection was not associated with GI symptoms. Seven of 10 (70%) patients with GI bleeding and 2 of 11 (18.2%) with diarrhea developed PTLD. Of seven patients examined by endoscopy for GI bleeding, two had biopsy-proven PTLD of the GI tract, whereas one of two patients examined by endoscopy for diarrhea had biopsy-proven PTLD.In summary, a high incidence of PTLD was found in patients who developed GI bleeding or diarrhea associated with EBV infection after pediatric liver transplantation. In these patients, endoscopy and biopsy may lead to early diagnosis of PTLD.

    View details for Web of Science ID 000076585400007

    View details for PubMedID 9798693

  • Effect of cyclosporine and tacrolimus on the growth of Epstein-Barr virus-transformed B-cell lines TRANSPLANTATION Beatty, P. R., Krams, S. M., Esquivel, C. O., Martinez, O. M. 1998; 65 (9): 1248-1255

    Abstract

    Transplant patients receiving immunosuppressive drugs are at increased risk for Epstein-Barr virus (EBV)-associated disorders including posttransplant lymphoproliferative disorder. The function of T lymphocytes, which are critical to preventing the expansion of EBV-infected B cells, is inhibited by immunosuppressive drugs. The purpose of this study was to determine whether immunosuppressive drugs have direct effects on EBV-infected B cells.The growth and proliferation of EBV-infected spontaneous lymphoblastoid cell lines (SLCLs), cultured in the presence or absence of cyclosporine (CsA) and tacrolimus (TAC), were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and [3H]thymidine incorporation assays. The effect of CsA and TAC on the viability of SLCLs was determined by cell counts with trypan blue. Apoptosis of SLCLs was induced with an anti-Fas agonist monoclonal antibody in the presence or absence of CsA and TAC and measured by flow cytometry after terminal deoxynucleotidyl transferase end-labeling and propidium iodide staining.CsA and TAC, but not sirolimus, increased the growth of SLCLs. The increased growth in the presence of CsA and TAC was attributable to enhanced cell viability and not increased cell division of SLCLs. In addition, CsA and TAC inhibited Fas-mediated apoptosis of SLCLs.CsA and TAC enhance the survival of EBV-transformed B-cell lines. CsA and TAC promote or augment SLCL growth through protection from cell death but do not affect cell division. The inhibition of cell death by CsA and TAC may contribute to the expansion of EBV-infected cells in immunosuppressed individuals.

    View details for Web of Science ID 000073676600017

    View details for PubMedID 9603175

  • CD30 expression identifies a functional alloreactive human T-lymphocyte subset TRANSPLANTATION Martinez, O. M., Villanueva, J., Abtahi, S., Beatty, P. R., Esquivel, C. O., Krams, S. M. 1998; 65 (9): 1240-1247

    Abstract

    CD30 is a member of the tumor necrosis factor/nerve growth factor receptor family and has been proposed as a marker of specific cytokine-producing subsets in humans. Previous studies have examined the expression of CD30 on established T helper type 1 and T helper type 2 cell clones and the function of CD30+ cells after mitogenic stimulation. In this study, we examined the development and function of CD30+ T cells generated in response to alloantigen.Primary one-way mixed lymphocyte reactions were established, and the expression of CD30 on T lymphocytes was determined by immunofluorescence and flow cytometry. Fluorescence-activated cell sorting was utilized to define the cytokine profile of alloactivated CD30+ cells after restimulation with anti-CD3 monoclonal antibodies or alloantigen. The effect of cyclosporine on the development of CD30+ cells, and on cytokines produced by CD30+ T lymphocytes, in response to alloantigen was determined.CD30+ T lymphocytes could be detected on day 2 of mixed lymphocyte reactions and continued to increase in number and proportion through day 6. Both CD4 and CD8 T cells expressed CD30 after primary alloantigenic stimulation. CD30+ T cells are a subset of alloactivated T cells and are the major source of interferon-gamma and interleukin-5 produced in response to alloantigen. Cyclosporine partially, but not completely, inhibits the development of CD30+ cells, and has a greater effect on interferon-gamma production than on interleukin-5 production.CD30+ T lymphocytes may constitute an important immunoregulatory subset in human allograft rejection.

    View details for Web of Science ID 000073676600016

    View details for PubMedID 9603174

  • CD8(+) cells are not necessary for allograft rejection or the induction of apoptosis in an experimental model of small intestinal transplantation JOURNAL OF IMMUNOLOGY Krams, S. M., Hayashi, M., Fox, C. K., Villanueva, J. C., Whitmer, K. J., BURNS, W., Esquivel, C. O., Martinez, O. M. 1998; 160 (8): 3673-3680

    Abstract

    Allospecific CTL can function as cellular effectors of solid organ graft rejection; however, the specific mechanisms of cell damage remain undetermined. In this study we examined the role of CD8+ T cells in apoptosis and rejection of small intestinal allografts. ACI rat intestinal grafts transplanted into Lewis rat recipients showed apoptosis of epithelial crypt cells on day 3 posttransplant as determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining. By day 7 numerous apoptotic crypt cells were detected in allografts, but were rarely observed in FK506-treated allograft recipients, isografts, or native intestine of allograft recipients. To further investigate the mechanism of rejection, recipient rats were depleted of CD8+ cells by treatment with OX-8 mAbs the day before and the day after transplantation of rat small intestinal allografts. Depletion of CD8+ cells from allograft recipients did not alter the tempo or the histologic features of rejection compared with those in the control (IgG-treated) group. Moreover, there was no difference in the number of apoptotic crypt epithelial cells in the grafts of control and CD8-depleted rats. Reverse transcriptase-PCR analyses determined there were similar levels of transcripts for Fas, Fas ligand, perforin, and granzyme B in control and CD8-depleted allograft recipients. By Western blot it was determined that the levels of Fas ligand protein were increased in the CD8-depleted group compared with those in control and FK506-treated allograft recipients. These data suggest that CD8 cells are not required for tissue injury or apoptotic cell death in small intestine allograft rejection.

    View details for Web of Science ID 000072970400008

    View details for PubMedID 9558067

  • Human hepatocytes produce an isoform of Fas that inhibits apoptosis TRANSPLANTATION Krams, S. M., Fox, C. K., Beatty, P. R., Cao, S., Villanueva, J. C., Esquivel, C. O., Martinez, O. M. 1998; 65 (5): 713-721

    Abstract

    Fas (Apo-1/CD95), a member of the tumor necrosis factor receptor family, can mediate apoptosis when engaged by its ligand or by anti-Fas antibody. Fas is expressed by cells of the immune system and by some nonlymphoid tissues. Numerous studies have suggested that the Fas pathway may play a role in the rejection of allografts. Functional, soluble forms of the Fas receptor are produced by activated peripheral blood mononuclear cells and some transformed cell lines. The purpose of this study was to determine if soluble variants of Fas are produced in the liver and to determine if blockade of the Fas pathway, by liver-derived soluble Fas, inhibits Fas-mediated apoptosis.Liver and purified hepatocyte specimens were analyzed for Fas transcripts by reverse transcriptase-polymerase chain reaction with primers that span the transmembrane region of the molecule. Bile and cell lysates were analyzed for soluble Fas by specific enzyme-linked immunosorbent assay. Lysates were prepared from normal liver and hepatocytes and utilized to block Fas-mediated apoptosis of Jurkat cells as determined by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and flow cytometry.A variant form of Fas is abundantly expressed in normal liver and purified hepatocytes. This variant form of Fas is expressed in all normal liver specimens but only in half of the liver specimens obtained during allograft rejection. The levels of soluble Fas diminish in patients undergoing liver allograft rejection in contrast to patients with stable grafts. Importantly, a soluble form of Fas is produced in the liver by hepatocytes and can specifically inhibit Fas-mediated apoptosis.These data raise the possibility that soluble Fas, produced by hepatocytes, may influence the immune response by blocking Fas-mediated apoptosis and, thus, may have a role in liver transplantation.

    View details for Web of Science ID 000072573800019

    View details for PubMedID 9521208

  • Apoptosis as a mechanism of tissue injury in liver allograft rejection SEMINARS IN LIVER DISEASE Krams, S. M., Martinez, O. M. 1998; 18 (2): 153-167

    Abstract

    Recent studies suggest that apoptosis is an important mechanism of cell death in the rejection of liver allografts and that infiltrating host lymphocytes mediate this process. The first section of this chapter addresses the cells and molecules that initiate the immune response following transplantation of a liver allograft. The recognition of donor alloantigens by infiltrating host lymphocytes stimulates a cascade of immune events which culminate in development of the effector cells that mediate tissue damage. Studies which demonstrate that apoptosis of hepatocytes and bile duct cells accompany allograft rejection are detailed in the second section of this chapter. The final section discusses the potential pathways which lead to apoptosis in liver allograft rejection. The contributions of the granule-exocytosis pathway, the Fas-mediated pathway, and cytokines to the induction of apoptosis in liver allografts are discussed. In addition, the concept that alloreactive graft infiltrating cells are deleted by apoptosis is presented. A further understanding of the mechanisms involved in apoptosis will lead to unique approaches toward the goal of achieving allograft tolerance.

    View details for Web of Science ID 000079131100006

    View details for PubMedID 9606812

  • Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines JOURNAL OF IMMUNOLOGY Beatty, P. R., Krams, S. M., Martinez, O. M. 1997; 158 (9): 4045-4051

    Abstract

    Immunocompromised individuals have an increased incidence of EBV-associated B cell lymphomas. The growth factors responsible for the unrestrained proliferation of these lymphomas have not yet been determined. In this study, spontaneous lymphoblastoid cell lines (SLCL) were derived without the addition of growth factors or virus from four patients with EBV-associated lymphoproliferative disorder. These cell lines were EBV transformed in vivo, and infection was verified through amplification of the viral gp220 gene. SLCL have an activated B cell phenotype (CD19+, CD21+, CD23+, CD38+, and CD40+) and produce IL-6, IL-10, TNF-alpha, and lymphotoxin-alpha. To determine whether these cytokines contribute to autonomous growth, neutralizing Abs for IL-10, IL-6, and TNF-alpha, a soluble TNFR:Fc fusion protein, and soluble IL-10R were used. These experiments established that, of the cytokines produced by SLCL, only IL-10 is an autocrine factor. IL-10 was produced by the majority of cells within each SLCL, and IL-10 secretion was concomitant with SLCL growth. Our findings demonstrate that IL-10 is utilized in the autonomous growth of EBV-related lymphomas and may be crucial in the development of lymphoproliferative disorder.

    View details for Web of Science ID A1997WV76100005

    View details for PubMedID 9126962

  • Elevations in IFN-gamma, IL-5, and IL-10 in patients with the autoimmune disease primary biliary cirrhosis: Association with autoantibodies and soluble CD30 CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY Krams, S. M., Cao, S., Hayashi, M., Villanueva, J. C., Martinez, O. M. 1996; 80 (3): 311-320

    Abstract

    Antimitochondrial antibodies (AMA) which recognize the E2 component of the pyruvate dehydrogenase complex are found in virtually all patients with the autoimmune liver disease primary biliary cirrhosis (PBC). The factors that contribute to elevated AMA and the relationship of the autoantibodies to disease pathogenesis have not been elucidated. Since cytokines are important regulators of antibody production and isotype switching, the association of specific cytokines to antibody production was examined in patients with PBC. Elevations in IL-2, IFN-gamma, IL-4, IL-5, and IL-10 were detected in serum from patients with PBC. However, only IFN-gamma (6012 +/- 1128 pg/ml vs 147 +/- 89 pg/ml, P < 0.0001) and IL-5 (382 +/- 103 pg/ml vs 29 +/- 12 pg/ml, P < 0.001) were significantly elevated compared to normal controls. Moreover, there was a positive correlation in the levels of IFN-gamma, and to a lesser extent IL-5, with the levels of soluble CD30 (sCD30) in the circulation. The elevated levels of sCD30 detected in patients with PBC (194 +/- 29 U/ml vs 39 +/- 9 U/ml in normal controls) suggest that CD30+ cells may produce cytokines, which contribute to the immune abnormalities in patients with PBC.

    View details for Web of Science ID A1996VG74900013

    View details for PubMedID 8811053

  • Liver transplantation at California Pacific Medical Center, San Francisco, California. Clinical transplants Esquivel, C. O., Martinez, O., Krams, S., Lim, J., So, S. K., Concepcion, W., Cox, K. L., Keeffe, E. B. 1994: 163-171

    Abstract

    A number of modifications in patient selection, operative technique, and immunosuppressive management have greatly contributed to the success of the liver transplant program at CPMC. Graft rejection and the timely detection of EBV infection are ongoing problems in hepatic transplantation that are foci of active research in our field. To address these issues, our group is investigating the activity of cytokines and adhesion molecules using sophisticated molecular techniques, and we are developing a sensitive assay for EBV markers in blood. These and other projects currently in progress will continue when we move our liver transplant program to Stanford University Medical Center in January 1995.

    View details for PubMedID 7547535

Conference Proceedings


  • Significance of detecting Epstein-Barr-Specific sequences in the peripheral blood of asymptomatic pediatric liver transplant recipients Krieger, N. R., Martinez, O. M., Krams, S. M., Cox, K., So, S., Esquivel, C. O. JOHN WILEY & SONS INC. 2000: 62-66

    Abstract

    Pediatric allograft recipients are at increased risk for Epstein-Barr virus (EBV)-associated illnesses. The early identification and diagnosis of EBV-associated disorders is critical because disease progression can often be curtailed by modification of immunosuppression. We have previously shown that detection of EBV-specific sequences in the circulation by polymerase chain reaction (PCR) correlated well with the clinical symptoms of EBV infection. The purpose of the current study is to determine the significance of detecting EBV-specific sequences by PCR in asymptomatic pediatric liver transplant recipients. Peripheral-blood DNA was analyzed for the EBV genes, coding from the nuclear antigen 1 (EBNA-1) and the viral capsid antigen (gp220) by PCR. Samples from asymptomatic pediatric liver transplant recipients were analyzed from the immediate postoperative period and at 2- to 4-month intervals thereafter. We followed up 13 of these asymptomatic recipients who tested positive for EBV compared with 7 asymptomatic recipients who tested negative for EBV during the early posttransplantation period. Follow-up ranged from 1.5 to 4 years posttransplantation. Nine patients (69%) initially positive for EBV and asymptomatic ultimately developed symptoms of EBV infection, including fever, lymphadenopathy, rash, respiratory and gastrointestinal symptoms, and/or hepatitis. Five of these patients (56%) went on to develop posttransplant lymphoproliferative disorder based on histological examination of biopsied tissue and immunohistochemical identification of the EBV antigen/DNA in tissue. This is the first report suggesting that detection of EBV-specific sequences in the absence of symptoms may herald impending EBV-associated disorders. Thus, routine monitoring for circulating EBV sequences in asymptomatic recipients may be useful in the early identification of those at risk for developing EBV-associated disease and its ultimate prevention.

    View details for Web of Science ID 000085673100008

    View details for PubMedID 10648579

  • Expression of cytokines and immune mediators during chronic liver allograft rejection Hayashi, M., Martinez, O. M., GARCIAKENNEDY, R., So, S., Esquivel, C. O., Krams, S. M. WILLIAMS & WILKINS. 1995: 1533-1538

    Abstract

    To determine the immune processes involved in chronic liver allograft rejection (CR) we examined in situ cytokine production in tissue from 15 patients with both clinical and histopathological diagnoses of CR. Total RNA was isolated from liver samples, reverse-transcribed and analyzed by RT-PCR for the production of proinflammatory cytokines and immunoregulatory mediators. Transcripts for the Th1-like cytokines IL-2 and IFN-gamma were detected in 53.3% and 46.7% of CR grafts, while they were detected in only 16% and 0% of stable grafts, respectively. The cytotoxic T cell mediator granzyme B was expressed in the majority of liver grafts undergoing CR, but was expressed only in a minority of stable grafts (80% vs. 16%, P < 0.05). The T cell product IL-5 was also significantly upregulated in CR as compared with stable livers (80% vs. 16%, P < 0.01). Other Th2 cytokines--IL-4 and IL-10--and macrophage products--IL-1 beta, IL-6, IL-8, TGF-beta, and TNF-alpha--were not substantially upregulated in CR grafts as compared with stable grafts. PDGF-beta transcripts were detected in the majority of the CR grafts, but were not detected in stable liver grafts (73% vs. 0, P < 0.05). By immunohistochemical staining, we observed that CD3+CD4+, and CD3+CD4- T cells were detected in CR grafts along with CD20+ B cells and CD68+ macrophages. There was, however, a predominant infiltration of CD3+CD4+ lymphocytes. Taken together, these data suggest that infiltrating cells produce proinflammatory and immunoregulatory cytokines that have a role in mediating graft damage in CR.

    View details for Web of Science ID A1995TN23000027

    View details for PubMedID 8545886

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