Bari Nazario - Academia.edu (original) (raw)
Papers by Bari Nazario
Transplant International, 1998
The availibility of a method to measure the effects of drugs on immune reactivity should be helpf... more The availibility of a method to measure the effects of drugs on immune reactivity should be helpful in optimizing treatment after organ transplantation. Since cyclosporine A (CSA) interferes with activation of T cells and cytokine synthesis, production of IL-2 and IFN-y might constitute a marker of this drug's effects. We measured the capacity for mitogen-stimulated production of these cytokines in whole blood by using immunostaining of intracellular and membrane antigens, followed by flow cytometry. The percentage of CD4' T cells producing IL-2 or IFN-y was strongly reduced in 20 transplant patients compared with 24 healthy controls. The capacity for IL-2 production of CD4+ and CD8' cells correlated inversely with CSA blood levels (P values 0.0087 and 0.0396, respectively). IFN-y production by CD4' T cells showed a negative correlation with the prednisolone dose (P = 0.0175) and, for the CD8' subset, with CSA trough levels (P = 0.0023). These data show that inhibition of T cell cytokine synthesis by CSA and prednisolone can be quantified.
The Journal of Immunology
Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by disc... more Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated sp...
The Journal of Immunology
The survival of heterotopic heart allografts was determined in mongrel dogs treated with total ly... more The survival of heterotopic heart allografts was determined in mongrel dogs treated with total lymphoid irradiation (TLI) alone or in combination with other immunosuppressive agents. TLI alone (total dose, 1800 rad) minimally prolonged graft survival as compared with untreated controls. However, marked synergy was observed when TLI was combined with a 10-day post-transplant course of rabbit anti-dog thymocyte globulin (ATG). Approximately 40% of recipients given TLI and ATG showed specific unresponsiveness, as judged by the lack of rejection on serial biopsies for more than 1 yr and the prompt rejection of third party hearts. The addition of post-transplant azathioprine (90 to 180 days) to the TLI and ATG regimen increased the mortality of recipients and reduced the fraction of dogs showing specific unresponsiveness. Infusion of donor bone marrow cells at the time of heart transplantation failed to induce specific unresponsiveness in recipients given TLI alone or TLI in combination ...
The Journal of Immunology
C57BL/Ka bone marrow and spleen cells fractionated by density gradients were transplanted to leth... more C57BL/Ka bone marrow and spleen cells fractionated by density gradients were transplanted to lethally irradiated (800 rad) BALB/c recipients. Unfractionated bone marrow and spleen cell mixtures (1:1), or high density fractions of these cells induced acute lethal graft-vs-host disease (GVHD). In contrast, low and middle density fractions of bone marrow and spleen cell mixtures reconstituted the irradiated hosts, and the majority survived for at least 100 days. The latter cells contained sufficient hemopoietic cells for reconstitution, but were deficient in inducing GVHD. Examination of the T cell subsets in the low density fractions showed a reduction of typical alpha beta TCR+ CD4+ or CD8+ cells and little change in the proportion of alpha beta TCR+ CD4- CD8- cells. BALB/c mice injected with the BCL1 B cell leukemia/lymphoma were lethally irradiated and transplanted with unfractionated BALB/c or C57BL/Ka bone marrow and spleen mixtures or low density fractions of C57BL/Ka mixtures. ...
The Journal of Immunology
The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mi... more The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta recep...
Transplantation proceedings, 1996
Transplantation, 2005
Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the ... more Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (␥c). Because mutations in genes encoding ␥c or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. Methods. Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (nϭ18) or its vehicle (controls, nϭ3) and were euthanized at day 90 or earlier if there was allograft rejection. Results. Mean survival time (Ϯ standard error of mean) in animals treated with CP-690,550 (53Ϯ7 days) was significantly longer than in control animals (7Ϯ1 days, Pϭ0.0003) and was positively correlated with exposure to the drug (rϭ0.79, PϽ0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46Ϯ7 days from transplantation vs. 7Ϯ1 days in controls, Pϭ0.0003). Persistent anemia, polyoma virus-like nephritis (nϭ2), and urinary calcium carbonate accretions (nϭ3) were seen in animals with high exposure. Natural killer cell and CD4 ϩ and CD8 ϩ T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. Conclusions. CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.
Transplantation, 1996
Anti-CD2 monoclonal antibody OX34 has been shown to suppress immunity in rodents in vitro and in ... more Anti-CD2 monoclonal antibody OX34 has been shown to suppress immunity in rodents in vitro and in vivo. To evaluate the effects of OX34 on vascularized allografts, Lewis (RT1(1)) hearts were transplanted heterotopically into Wistar Furth (RT1(u)) rats. A single 5 mg/kg intraperitoneal dose of OX34 administered at transplantation induced indefinite graft survival (mean survival time >140.3+/-12.3 vs. 12.7+/-0.7 control, P=0.001). The mixed lymphocyte response was partially inhibited at 60 days after transplant, returning to normal at 100 days. Donor-specific tolerance was confirmed by acceptance of second donor (>100 days, n=2) and rejection of third-party (mean survival time: 7.5+/-0.5 days, n=2) hearts. Immunohistochemical staining of allograft tissue from tolerant animals demonstrated abundant CD2+, CD4+, and CD8+ graft-infiltrating cells. To elucidate further the nature of these cells, we compared the expression of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma mRNA in allografted tissue from tolerant, acutely rejecting (AR), isografted, and naive animals using nonisotopic in situ hybridization. A significant increase in IL-2, IL-4, IL-10, and IFN-gamma mRNA was observed in graft-infiltrating cells of both tolerant and AR animals. IL-10 mRNA expression 4 days after transplant was significantly elevated in the OX34-treated compared to AR recipients. These data demonstrate that a single dose of OX34 at engraftment induces tolerance to vascularized allografts. Expression of both T helper 1 and T helper 2 cytokine mRNA profiles (IL-2/IFN-gamma and IL-4/ IL-10, respectively) are up-regulated locally in graft-infiltrating cells of AR and tolerant animal allografts.
Transplantation, 1996
Lewis rats were rendered tolerant to ACI heart allografts using a regimen of posttransplant total... more Lewis rats were rendered tolerant to ACI heart allografts using a regimen of posttransplant total lymphoid irradiation (TLI), rabbit antithymocyte or antilymphocyte globulin (RATG or RALG), and a single donor blood transfusion. All three treatment modalities were required to induce tolerance. The mechanism of the maintenance of tolerance was investigated by comparing the secretion of cytokines in the MLR, and the expression of cytokine mRNA in the allografts of tolerant and nontolerant Lewis rats. Although, the 3H-thymidine incorporation and secretion of IL-2 was frequently comparable in the MLR from tolerant and nontolerant rats, the secretion of IFN-gamma was markedly reduced in the tolerant rats. This was reflected in a markedly reduced frequency of cells expressing IFN-gamma mRNA in the allografts of tolerant as compared with nontolerant hosts. The frequency of cells expressing IL-2 and IL-10 mRNA was also reduced, but no significant difference was observed for cells with IL-4 mRNA. Spleen cells from nontolerant rats rapidly rejected ACI allografts in irradiated adoptive hosts, but spleen cells from tolerant rats did not. Evaluation of the cytokine mRNA expression at early and late time points in the allografts of adoptive hosts showed a pattern similar to that of the primary hosts. Thus, the tolerant state was associated with a maintenance or elevation of IL-4 expression and a marked reduction of IFN-gamma expression. Previous reports have shown that TLI alone induced this shift in the early recovery phase after irradiation.
Surgery, 1995
Immune regulation requires antigen recognition, signaling, activation, secretion of cytokines, an... more Immune regulation requires antigen recognition, signaling, activation, secretion of cytokines, and effector function by lymphocytes. Although there is redundancy in the activation and function of the immune response, some cytokines simultaneously promote and suppress different pathways of immunity. In the experiments reported here we compare cytokine gene expression within liver allografts from tolerant rats with normal and isografted liver tissue. We also compare the secretion of interferon-gamma (IFN-gamma) in the supernatant from mixed lymphocyte cultures by using peripheral blood lymphocytes stimulated against donor antigen. Orthotopic liver transplantations were performed using the cuff technique without hepatic artery revascularization. Nonisotopic in situ hybridization (ISH) was used to detect and localize messenger RNA to specific cells within tissue. Antisense DNA probes were generated to interleukin-2 (IL-2), IL-4, IL-10, and IFN-gamma. One-way mixed lymphocyte cultures were set up against irradiated donor splenocytes, and the supernatant was collected to measure IFN-gamma by enzyme-linked immunosorbent assay. Expression of IFN-gamma and IL-10 was up-regulated in tolerant animals versus normal or isografted liver (p = 0.0002 and 0.0001, IFN-gamma and IL-10, respectively). In situ hybridization localized the expression of messenger RNA predominantly to the cytoplasm of the hepatocytes. Levels of IFN-gamma were higher in the supernatant from proliferating peripheral lymphocytes against donor antigen from tolerant animals versus naive control animals. Expression of IFN-gamma and IL-10 is up-regulated in hepatocytes from allograft tissue after orthotopic liver transplantation. We believe that the up-regulation of IL-10 cross-regulates the effector function of IFN-gamma and supports cytokine-mediated immune dysregulation, which may be a mechanism of tolerance after orthotopic liver transplantation in rats.
Science, 2003
Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excel... more Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolongedsurvival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associatedwith hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation andin other clinical settings.
New England Journal of Medicine, 1989
... Samuel Strober, MD, Mohan Dhillon, MD, Mark Schubert, MD, Ph.D., Bari Holm, Edgar Engleman, M... more ... Samuel Strober, MD, Mohan Dhillon, MD, Mark Schubert, MD, Ph.D., Bari Holm, Edgar Engleman, MD, Claudia Benike, Richard Hoppe, MD, Richard Sibley, MD, J. Albertus Myburgh, MD, Geoffrey Collins, MD, and Barry Levin, MD. ... 13. Brent L, Opara SC. ...
Journal of Surgical Research, 2005
Background. Strategies to induce donor-specific allograft tolerance are best tested in preclinica... more Background. Strategies to induce donor-specific allograft tolerance are best tested in preclinical models developed in nonhuman primates (NHPs). Most protocols prepare the recipient by infusing hematopoietic cells from the donor. We report here a procedure to isolate and characterize large numbers of bone marrow cells (BMCs) from cynomolgus monkeys (cynos) that can then successfully be transplanted into conditioned recipients. Materials and methods. Vertebral columns of five cynos were excised en bloc and separated into individual vertebrae. The cancelous bone was extracted with a core puncher, fractionated, filtered, centrifuged, and resuspended in transplantation media before being analyzed by flow cytometry. In two instances, the collected BMCs were reinfused into allogeneic recipients preconditioned with a nonmyeloablative regimen. Chimerism was monitored using short-tandem repeat analysis. Results. The mean total BMCs yield was 25.5 ؋ 10 9 (range of 4.00 ؋ 10 9 to 59 ؋ 10 9) with mean cell viability of 93.4% (range: 90-96%). CD34 ؉ cells and CD3 ؉ cells averaged 0.34 and 3.91% of total BMCs, respectively. This resulted in absolute cell number yields of 1.02 ؋ 10 8 and 1.15 ؋ 10 9 for CD34 ؉ and CD3 ؉ cells, respectively. Graft-versus-host disease was absent in both bone marrow infused animals, and a maximum level of chimerism of 18% was detected at 3 weeks after BMCs infusion. Conclusion. We present here the first detailed report of a procedure to retrieve and characterize large numbers of BMCs from vertebral bodies of cynos and demonstrate that cells collected with this technique have the capability of engrafting in allogenic recipients.
Journal of Surgical Research, 1995
In some rodent haplotype combinations, spontaneous tolerance (ST) develops after orthotopic liver... more In some rodent haplotype combinations, spontaneous tolerance (ST) develops after orthotopic liver transplantation (OLT) without any immunosuppression [e.g., Lewis (Lew, RT1) into Wistar Furth (WF, RT1u)] whereas in other combinations vigorous, progressive rejection rapidly leads to the death of the recipients. We (and others) have induced tolerance (IT) in a rejecting strain combination [Dark Agouti (DA, RT1a) into Lew] by intrathymic inoculation of donor bone marrow cells and 1 cc of antilymphocyte serum (ALS) 7-14 days prior to OLT. We hypothesized that cellular immunity in the two groups of animals was similar. We first compared survival in each group of animals and found that there was no difference in the number of animals surviving > 100 days (8/11 vs 16/17, ST vs IT, respectively, P = 0.11). Liver function studies were similar in these animals at 2 and 4 weeks after OLT and comparable to syngeneic Lew into Lew OLT animals, but significantly lower than in the rejecting DA into Lew combination treated with only ALS. Animals that were unresponsive to their allografts demonstrated donor-specific tolerance by the acceptance of donor strain (n = 4, ST and IT) and rejection of third party (n = 1 and n = 2, ST and IT groups, respectively) heterotopic heart allografts. One-way mixed lymphocyte cultures (MLC) of peripheral blood lymphocytes against donor and third party antigen were suppressed to donor and third party stimulators versus the MLC of unmanipulated animals. Naive host strain responder lymph node cells and purified T cells demonstrated strong proliferative responses to donor strain antigen in both the ST and IT animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Surgical Research, 1996
immunosuppressive therapy. In a recent novel ap-We evaluated a combined posttransplant strategy p... more immunosuppressive therapy. In a recent novel ap-We evaluated a combined posttransplant strategy proach, donor-specific unresponsiveness was induced using antilymphocyte serum (ALS) at time of enin a rodent model by intrathymic (IT) inoculation of graftment followed by low dose total lymphoid irradiadonor islets along with a single dose of antilymphocyte tion (TLI) and donor bone marrow cell (BMC) inoculaserum (ALS), demonstrating the importance of the thytion administered either intrathymically (IT) or intramus as the site for educating maturing T cells in the venously (IV) in the vigorously rejecting strain development of donor-specific unresponsiveness [1]. Alcombination DA into Lew recipients. Allograft surthough this strategy is intriguing as a means of achievvival was significantly prolonged with administration ing unresponsiveness in clinical transplantation, the of ALS in combination with TLI and IT (105 { 28.6 applicability of this model in humans is still unclear. days) or IV (106.8 { 28.6 days) BMC compared to ad-This strategy has been confounded by the requirement ministration of ALS combined with either TLI (17.8 { of a low-responder rodent strain combination [2]. This 0.4 days) or BMC (9.0 { 0.0 days), or TLI combined approach also requires a pretransplant strategy which with BMC (11.5 { 0.5 days) (P õ 0.0001, experimental vs is not feasible in the setting of cadaveric transplantacontrol animals). There was no difference in survival tion. A further constraint is the requirement of IT inocbetween those animals who underwent IT or IV BMC ulation which may be inapplicable in adults where the inoculation. Third-party (WF) BMC inoculation did not thymus has involuted or may not be functional. significantly prolong allograft survival (10.0 { 1.0 To address these issues we evaluated a combined days). A mild to moderate cellular infiltrate was presstrategy in a high-responder strain combination using ent in allograft tissue after 100 days. To further characterize these cells, cytokine mRNA expression in allo-a posttransplant strategy. We were able to prolong allograft tissue (ú100 days posttransplant) was evaluated graft survival using donor antigen inoculated either IT using nonisotopic in situ hybridization. A similar cytoor IV. Graft infiltrating cells were identified ú100 days kine profile was demonstrated in allograft tissue composttransplant. To characterize further these cells, inpared to naive and isograft tissue, except for a slight tragraft cytokine mRNA expression was evaluated usincrease in IL-2 (P õ 0.02, control vs IV BMC; P Å NS, ing in situ hybridization. other groups). In summary, unresponsiveness was induced in a high-responder strain combination using MATERIALS AND METHODS a combined posttransplant strategy of ALS, TLI, and donor antigen either IT or IV. The cytokine profile of Animals. Lewis (Lew, RT1 1), Wistar Furth (WF, RT1 u), and blood the graft infiltrating cells was similar to that of normal group D Agouti (DA, RT1 a) rats were purchased from Harlan (Indiatissue. Unresponsiveness may be the result of funcnapolis, IN). Recipient animals weighed 200-250 gm. Animals were housed in the Research Animal Facility, a virus-free locked facility tional inactivation of these cells. ᭧ 1996 Academic Press, Inc. which conforms to NIH guidelines, with free access to water and rat chow. All procedures were carried out under sterile conditions using isoflurane inhalation anesthesia.
Journal of Immunotherapy, 1999
... & Wilkins, Inc., Philadelphia IL-13 Can Substitute for IL-4 in the Generation of Dendriti... more ... & Wilkins, Inc., Philadelphia IL-13 Can Substitute for IL-4 in the Generation of Dendritic Cells for the Induction of Cytotoxic T Lymphocytes and Gene Therapy Susan E. Alters, Jose R. Gadea, Bari Holm, Jane Lebkowski, and Ramila Philip RPR Gencell, Santa Clara, California ...
Human Immunology, 2004
Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased... more Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased infection susceptibility in newborns. We used multiparameter flow cytometry to characterize APC subsets in adult peripheral blood (APB) and cord blood (CB). APB had a higher proportion of CD11cϩ dendritic cells (DC), whereas CB mainly contained CD123ϩ DC. APB was enriched in CD16ϩCD11cϩ DC subset, whereas CD34ϩCD11c-CD123lo cells were prominent in CB. Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-␣ production was dampened in myeloid DC and monocytes from CB, whereas IL-1␣ production was not different. The reduction in TNF-␣ response did not appear to result from reduced surface detection of LPS, because CD14, toll-like receptor (TLR)-4 and TLR-2 levels were not reduced in CB APC compared with APB cells. Also, there was no correlation between TLR-2 or TLR-4 levels and TNF-␣ production in myeloid DC and monocytes. CB monocytes had lower surface HLA-DR immediately ex vivo. Both APB and CB monocytes upregulated HLA-DR after incubation, but an additional LPS-induced increase in HLA-DR was suggested only in APB monocytes. APB monocytes also showed a greater LPS-induced increase in CD40 expression. Together, our data show significant, selective differences in circulating APC between neonates and adults.
Transplant International, 1998
The availibility of a method to measure the effects of drugs on immune reactivity should be helpf... more The availibility of a method to measure the effects of drugs on immune reactivity should be helpful in optimizing treatment after organ transplantation. Since cyclosporine A (CSA) interferes with activation of T cells and cytokine synthesis, production of IL-2 and IFN-y might constitute a marker of this drug's effects. We measured the capacity for mitogen-stimulated production of these cytokines in whole blood by using immunostaining of intracellular and membrane antigens, followed by flow cytometry. The percentage of CD4' T cells producing IL-2 or IFN-y was strongly reduced in 20 transplant patients compared with 24 healthy controls. The capacity for IL-2 production of CD4+ and CD8' cells correlated inversely with CSA blood levels (P values 0.0087 and 0.0396, respectively). IFN-y production by CD4' T cells showed a negative correlation with the prednisolone dose (P = 0.0175) and, for the CD8' subset, with CSA trough levels (P = 0.0023). These data show that inhibition of T cell cytokine synthesis by CSA and prednisolone can be quantified.
The Journal of Immunology
Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by disc... more Fractionation of normal adult mouse spleen and bone marrow cells (C57BL/Ka) was performed by discontinuous Percoll density gradients. The fractionated low density (1.050-1.060 g/ml) C57BL/Ka spleen cells completely suppressed acute lethal graft vs host disease (GVHD) when coinjected with unfractionated C57BL/Ka spleen cells into sublethally irradiated (400 rad) BALB/c mice. In dose response experiments, as few as 0.5 x 10(6) low density cells from the spleen fractions suppressed acute GVHD induced by 2.5 x 10(6) unfractionated allogeneic spleen cells. Although the low density spleen fractions inhibited acute GVHD, the high density (1.075-1.090 g/ml) spleen fractions induced acute GVHD in sublethally irradiated BALB/c recipients. Fractionation of C57BL/Ka bone marrow cells showed that none of the high or low density fractions or unfractionated cells induced lethal GVHD. When these fractions were tested for their capacity to suppress GVHD by coinjection with C57BL/Ka unfractionated sp...
The Journal of Immunology
The survival of heterotopic heart allografts was determined in mongrel dogs treated with total ly... more The survival of heterotopic heart allografts was determined in mongrel dogs treated with total lymphoid irradiation (TLI) alone or in combination with other immunosuppressive agents. TLI alone (total dose, 1800 rad) minimally prolonged graft survival as compared with untreated controls. However, marked synergy was observed when TLI was combined with a 10-day post-transplant course of rabbit anti-dog thymocyte globulin (ATG). Approximately 40% of recipients given TLI and ATG showed specific unresponsiveness, as judged by the lack of rejection on serial biopsies for more than 1 yr and the prompt rejection of third party hearts. The addition of post-transplant azathioprine (90 to 180 days) to the TLI and ATG regimen increased the mortality of recipients and reduced the fraction of dogs showing specific unresponsiveness. Infusion of donor bone marrow cells at the time of heart transplantation failed to induce specific unresponsiveness in recipients given TLI alone or TLI in combination ...
The Journal of Immunology
C57BL/Ka bone marrow and spleen cells fractionated by density gradients were transplanted to leth... more C57BL/Ka bone marrow and spleen cells fractionated by density gradients were transplanted to lethally irradiated (800 rad) BALB/c recipients. Unfractionated bone marrow and spleen cell mixtures (1:1), or high density fractions of these cells induced acute lethal graft-vs-host disease (GVHD). In contrast, low and middle density fractions of bone marrow and spleen cell mixtures reconstituted the irradiated hosts, and the majority survived for at least 100 days. The latter cells contained sufficient hemopoietic cells for reconstitution, but were deficient in inducing GVHD. Examination of the T cell subsets in the low density fractions showed a reduction of typical alpha beta TCR+ CD4+ or CD8+ cells and little change in the proportion of alpha beta TCR+ CD4- CD8- cells. BALB/c mice injected with the BCL1 B cell leukemia/lymphoma were lethally irradiated and transplanted with unfractionated BALB/c or C57BL/Ka bone marrow and spleen mixtures or low density fractions of C57BL/Ka mixtures. ...
The Journal of Immunology
The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mi... more The predominant T cell subset in the bone marrow of specific pathogen-free C57BL/Ka and BALB/c mice expressed the alpha beta+ TCR CD4- CD8- surface phenotype. Purified C57BL/Ka alpha beta+ TCR CD4- CD8- marrow cells obtained by cell sorting suppressed the MLR of C57BL/Ka responder and BALB/c stimulator spleen cells. Although the percentage of typical T cells in the spleen was markedly reduced in adult nude mice or normal neonatal mice as compared to the normal adult, the percentage of alpha beta+ TCR CD4- CD8- cells in the spleen and marrow was not. The percentage of "self-reactive" V beta 5+ T cells in the BALB/c spleen was markedly reduced as compared to that in the C57BL/Ka spleen. However, the percentages in the bone marrow were similar. The results indicate that the predominant subset of marrow T cells in these pathogen-free mice differ with regard to surface marker phenotype, function, dependence on the adult thymus, and deletion of certain self-reactive V beta recep...
Transplantation proceedings, 1996
Transplantation, 2005
Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the ... more Background. Janus kinase 3 (JAK3) mediates signal transduction from cytokine receptors using the common chain (␥c). Because mutations in genes encoding ␥c or JAK3 result in immunodeficiency, we investigated the potential of a rationally designed inhibitor of JAK3, CP-690,550, to prevent renal allograft rejection in nonhuman primates. Methods. Life-supporting kidney transplantations were performed between mixed leukocyte reaction-mismatched, ABO blood group-matched cynomolgus monkeys. Animals were treated with CP-690,550 (nϭ18) or its vehicle (controls, nϭ3) and were euthanized at day 90 or earlier if there was allograft rejection. Results. Mean survival time (Ϯ standard error of mean) in animals treated with CP-690,550 (53Ϯ7 days) was significantly longer than in control animals (7Ϯ1 days, Pϭ0.0003) and was positively correlated with exposure to the drug (rϭ0.79, PϽ0.01). Four treated animals were euthanized at 90 days with a normal renal function and low-grade rejection at final pathology. Occurrence of rejection was significantly delayed in treated animals (46Ϯ7 days from transplantation vs. 7Ϯ1 days in controls, Pϭ0.0003). Persistent anemia, polyoma virus-like nephritis (nϭ2), and urinary calcium carbonate accretions (nϭ3) were seen in animals with high exposure. Natural killer cell and CD4 ϩ and CD8 ϩ T-cell numbers were significantly reduced in treated animals. Blood glucose, serum lipid levels, and arterial blood pressure were within normal range in treated animals, and no cancers were demonstrated. Conclusions. CP-690,550 is the first reported JAK3 inhibitor combining efficacy and good tolerability in a preclinical model of allotransplantation in nonhuman primates and thus has interesting potential for immunosuppression in humans.
Transplantation, 1996
Anti-CD2 monoclonal antibody OX34 has been shown to suppress immunity in rodents in vitro and in ... more Anti-CD2 monoclonal antibody OX34 has been shown to suppress immunity in rodents in vitro and in vivo. To evaluate the effects of OX34 on vascularized allografts, Lewis (RT1(1)) hearts were transplanted heterotopically into Wistar Furth (RT1(u)) rats. A single 5 mg/kg intraperitoneal dose of OX34 administered at transplantation induced indefinite graft survival (mean survival time >140.3+/-12.3 vs. 12.7+/-0.7 control, P=0.001). The mixed lymphocyte response was partially inhibited at 60 days after transplant, returning to normal at 100 days. Donor-specific tolerance was confirmed by acceptance of second donor (>100 days, n=2) and rejection of third-party (mean survival time: 7.5+/-0.5 days, n=2) hearts. Immunohistochemical staining of allograft tissue from tolerant animals demonstrated abundant CD2+, CD4+, and CD8+ graft-infiltrating cells. To elucidate further the nature of these cells, we compared the expression of interleukin (IL)-2, IL-4, IL-10, and interferon (IFN)-gamma mRNA in allografted tissue from tolerant, acutely rejecting (AR), isografted, and naive animals using nonisotopic in situ hybridization. A significant increase in IL-2, IL-4, IL-10, and IFN-gamma mRNA was observed in graft-infiltrating cells of both tolerant and AR animals. IL-10 mRNA expression 4 days after transplant was significantly elevated in the OX34-treated compared to AR recipients. These data demonstrate that a single dose of OX34 at engraftment induces tolerance to vascularized allografts. Expression of both T helper 1 and T helper 2 cytokine mRNA profiles (IL-2/IFN-gamma and IL-4/ IL-10, respectively) are up-regulated locally in graft-infiltrating cells of AR and tolerant animal allografts.
Transplantation, 1996
Lewis rats were rendered tolerant to ACI heart allografts using a regimen of posttransplant total... more Lewis rats were rendered tolerant to ACI heart allografts using a regimen of posttransplant total lymphoid irradiation (TLI), rabbit antithymocyte or antilymphocyte globulin (RATG or RALG), and a single donor blood transfusion. All three treatment modalities were required to induce tolerance. The mechanism of the maintenance of tolerance was investigated by comparing the secretion of cytokines in the MLR, and the expression of cytokine mRNA in the allografts of tolerant and nontolerant Lewis rats. Although, the 3H-thymidine incorporation and secretion of IL-2 was frequently comparable in the MLR from tolerant and nontolerant rats, the secretion of IFN-gamma was markedly reduced in the tolerant rats. This was reflected in a markedly reduced frequency of cells expressing IFN-gamma mRNA in the allografts of tolerant as compared with nontolerant hosts. The frequency of cells expressing IL-2 and IL-10 mRNA was also reduced, but no significant difference was observed for cells with IL-4 mRNA. Spleen cells from nontolerant rats rapidly rejected ACI allografts in irradiated adoptive hosts, but spleen cells from tolerant rats did not. Evaluation of the cytokine mRNA expression at early and late time points in the allografts of adoptive hosts showed a pattern similar to that of the primary hosts. Thus, the tolerant state was associated with a maintenance or elevation of IL-4 expression and a marked reduction of IFN-gamma expression. Previous reports have shown that TLI alone induced this shift in the early recovery phase after irradiation.
Surgery, 1995
Immune regulation requires antigen recognition, signaling, activation, secretion of cytokines, an... more Immune regulation requires antigen recognition, signaling, activation, secretion of cytokines, and effector function by lymphocytes. Although there is redundancy in the activation and function of the immune response, some cytokines simultaneously promote and suppress different pathways of immunity. In the experiments reported here we compare cytokine gene expression within liver allografts from tolerant rats with normal and isografted liver tissue. We also compare the secretion of interferon-gamma (IFN-gamma) in the supernatant from mixed lymphocyte cultures by using peripheral blood lymphocytes stimulated against donor antigen. Orthotopic liver transplantations were performed using the cuff technique without hepatic artery revascularization. Nonisotopic in situ hybridization (ISH) was used to detect and localize messenger RNA to specific cells within tissue. Antisense DNA probes were generated to interleukin-2 (IL-2), IL-4, IL-10, and IFN-gamma. One-way mixed lymphocyte cultures were set up against irradiated donor splenocytes, and the supernatant was collected to measure IFN-gamma by enzyme-linked immunosorbent assay. Expression of IFN-gamma and IL-10 was up-regulated in tolerant animals versus normal or isografted liver (p = 0.0002 and 0.0001, IFN-gamma and IL-10, respectively). In situ hybridization localized the expression of messenger RNA predominantly to the cytoplasm of the hepatocytes. Levels of IFN-gamma were higher in the supernatant from proliferating peripheral lymphocytes against donor antigen from tolerant animals versus naive control animals. Expression of IFN-gamma and IL-10 is up-regulated in hepatocytes from allograft tissue after orthotopic liver transplantation. We believe that the up-regulation of IL-10 cross-regulates the effector function of IFN-gamma and supports cytokine-mediated immune dysregulation, which may be a mechanism of tolerance after orthotopic liver transplantation in rats.
Science, 2003
Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excel... more Because of its requirement for signaling by multiple cytokines, Janus kinase 3 (JAK3) is an excellent target for clinical immunosuppression. We report the development of a specific, orally active inhibitor of JAK3, CP-690,550, that significantly prolongedsurvival in a murine model of heart transplantation and in cynomolgus monkeys receiving kidney transplants. CP-690,550 treatment was not associatedwith hypertension, hyperlipidemia, or lymphoproliferative disease. On the basis of these preclinical results, we believe JAK3 blockade by CP-690,550 has potential for therapeutically desirable immunosuppression in human organ transplantation andin other clinical settings.
New England Journal of Medicine, 1989
... Samuel Strober, MD, Mohan Dhillon, MD, Mark Schubert, MD, Ph.D., Bari Holm, Edgar Engleman, M... more ... Samuel Strober, MD, Mohan Dhillon, MD, Mark Schubert, MD, Ph.D., Bari Holm, Edgar Engleman, MD, Claudia Benike, Richard Hoppe, MD, Richard Sibley, MD, J. Albertus Myburgh, MD, Geoffrey Collins, MD, and Barry Levin, MD. ... 13. Brent L, Opara SC. ...
Journal of Surgical Research, 2005
Background. Strategies to induce donor-specific allograft tolerance are best tested in preclinica... more Background. Strategies to induce donor-specific allograft tolerance are best tested in preclinical models developed in nonhuman primates (NHPs). Most protocols prepare the recipient by infusing hematopoietic cells from the donor. We report here a procedure to isolate and characterize large numbers of bone marrow cells (BMCs) from cynomolgus monkeys (cynos) that can then successfully be transplanted into conditioned recipients. Materials and methods. Vertebral columns of five cynos were excised en bloc and separated into individual vertebrae. The cancelous bone was extracted with a core puncher, fractionated, filtered, centrifuged, and resuspended in transplantation media before being analyzed by flow cytometry. In two instances, the collected BMCs were reinfused into allogeneic recipients preconditioned with a nonmyeloablative regimen. Chimerism was monitored using short-tandem repeat analysis. Results. The mean total BMCs yield was 25.5 ؋ 10 9 (range of 4.00 ؋ 10 9 to 59 ؋ 10 9) with mean cell viability of 93.4% (range: 90-96%). CD34 ؉ cells and CD3 ؉ cells averaged 0.34 and 3.91% of total BMCs, respectively. This resulted in absolute cell number yields of 1.02 ؋ 10 8 and 1.15 ؋ 10 9 for CD34 ؉ and CD3 ؉ cells, respectively. Graft-versus-host disease was absent in both bone marrow infused animals, and a maximum level of chimerism of 18% was detected at 3 weeks after BMCs infusion. Conclusion. We present here the first detailed report of a procedure to retrieve and characterize large numbers of BMCs from vertebral bodies of cynos and demonstrate that cells collected with this technique have the capability of engrafting in allogenic recipients.
Journal of Surgical Research, 1995
In some rodent haplotype combinations, spontaneous tolerance (ST) develops after orthotopic liver... more In some rodent haplotype combinations, spontaneous tolerance (ST) develops after orthotopic liver transplantation (OLT) without any immunosuppression [e.g., Lewis (Lew, RT1) into Wistar Furth (WF, RT1u)] whereas in other combinations vigorous, progressive rejection rapidly leads to the death of the recipients. We (and others) have induced tolerance (IT) in a rejecting strain combination [Dark Agouti (DA, RT1a) into Lew] by intrathymic inoculation of donor bone marrow cells and 1 cc of antilymphocyte serum (ALS) 7-14 days prior to OLT. We hypothesized that cellular immunity in the two groups of animals was similar. We first compared survival in each group of animals and found that there was no difference in the number of animals surviving > 100 days (8/11 vs 16/17, ST vs IT, respectively, P = 0.11). Liver function studies were similar in these animals at 2 and 4 weeks after OLT and comparable to syngeneic Lew into Lew OLT animals, but significantly lower than in the rejecting DA into Lew combination treated with only ALS. Animals that were unresponsive to their allografts demonstrated donor-specific tolerance by the acceptance of donor strain (n = 4, ST and IT) and rejection of third party (n = 1 and n = 2, ST and IT groups, respectively) heterotopic heart allografts. One-way mixed lymphocyte cultures (MLC) of peripheral blood lymphocytes against donor and third party antigen were suppressed to donor and third party stimulators versus the MLC of unmanipulated animals. Naive host strain responder lymph node cells and purified T cells demonstrated strong proliferative responses to donor strain antigen in both the ST and IT animals.(ABSTRACT TRUNCATED AT 250 WORDS)
Journal of Surgical Research, 1996
immunosuppressive therapy. In a recent novel ap-We evaluated a combined posttransplant strategy p... more immunosuppressive therapy. In a recent novel ap-We evaluated a combined posttransplant strategy proach, donor-specific unresponsiveness was induced using antilymphocyte serum (ALS) at time of enin a rodent model by intrathymic (IT) inoculation of graftment followed by low dose total lymphoid irradiadonor islets along with a single dose of antilymphocyte tion (TLI) and donor bone marrow cell (BMC) inoculaserum (ALS), demonstrating the importance of the thytion administered either intrathymically (IT) or intramus as the site for educating maturing T cells in the venously (IV) in the vigorously rejecting strain development of donor-specific unresponsiveness [1]. Alcombination DA into Lew recipients. Allograft surthough this strategy is intriguing as a means of achievvival was significantly prolonged with administration ing unresponsiveness in clinical transplantation, the of ALS in combination with TLI and IT (105 { 28.6 applicability of this model in humans is still unclear. days) or IV (106.8 { 28.6 days) BMC compared to ad-This strategy has been confounded by the requirement ministration of ALS combined with either TLI (17.8 { of a low-responder rodent strain combination [2]. This 0.4 days) or BMC (9.0 { 0.0 days), or TLI combined approach also requires a pretransplant strategy which with BMC (11.5 { 0.5 days) (P õ 0.0001, experimental vs is not feasible in the setting of cadaveric transplantacontrol animals). There was no difference in survival tion. A further constraint is the requirement of IT inocbetween those animals who underwent IT or IV BMC ulation which may be inapplicable in adults where the inoculation. Third-party (WF) BMC inoculation did not thymus has involuted or may not be functional. significantly prolong allograft survival (10.0 { 1.0 To address these issues we evaluated a combined days). A mild to moderate cellular infiltrate was presstrategy in a high-responder strain combination using ent in allograft tissue after 100 days. To further characterize these cells, cytokine mRNA expression in allo-a posttransplant strategy. We were able to prolong allograft tissue (ú100 days posttransplant) was evaluated graft survival using donor antigen inoculated either IT using nonisotopic in situ hybridization. A similar cytoor IV. Graft infiltrating cells were identified ú100 days kine profile was demonstrated in allograft tissue composttransplant. To characterize further these cells, inpared to naive and isograft tissue, except for a slight tragraft cytokine mRNA expression was evaluated usincrease in IL-2 (P õ 0.02, control vs IV BMC; P Å NS, ing in situ hybridization. other groups). In summary, unresponsiveness was induced in a high-responder strain combination using MATERIALS AND METHODS a combined posttransplant strategy of ALS, TLI, and donor antigen either IT or IV. The cytokine profile of Animals. Lewis (Lew, RT1 1), Wistar Furth (WF, RT1 u), and blood the graft infiltrating cells was similar to that of normal group D Agouti (DA, RT1 a) rats were purchased from Harlan (Indiatissue. Unresponsiveness may be the result of funcnapolis, IN). Recipient animals weighed 200-250 gm. Animals were housed in the Research Animal Facility, a virus-free locked facility tional inactivation of these cells. ᭧ 1996 Academic Press, Inc. which conforms to NIH guidelines, with free access to water and rat chow. All procedures were carried out under sterile conditions using isoflurane inhalation anesthesia.
Journal of Immunotherapy, 1999
... & Wilkins, Inc., Philadelphia IL-13 Can Substitute for IL-4 in the Generation of Dendriti... more ... & Wilkins, Inc., Philadelphia IL-13 Can Substitute for IL-4 in the Generation of Dendritic Cells for the Induction of Cytotoxic T Lymphocytes and Gene Therapy Susan E. Alters, Jose R. Gadea, Bari Holm, Jane Lebkowski, and Ramila Philip RPR Gencell, Santa Clara, California ...
Human Immunology, 2004
Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased... more Defective antigen-presenting cell (APC) function has been hypothesized to contribute to increased infection susceptibility in newborns. We used multiparameter flow cytometry to characterize APC subsets in adult peripheral blood (APB) and cord blood (CB). APB had a higher proportion of CD11cϩ dendritic cells (DC), whereas CB mainly contained CD123ϩ DC. APB was enriched in CD16ϩCD11cϩ DC subset, whereas CD34ϩCD11c-CD123lo cells were prominent in CB. Lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-␣ production was dampened in myeloid DC and monocytes from CB, whereas IL-1␣ production was not different. The reduction in TNF-␣ response did not appear to result from reduced surface detection of LPS, because CD14, toll-like receptor (TLR)-4 and TLR-2 levels were not reduced in CB APC compared with APB cells. Also, there was no correlation between TLR-2 or TLR-4 levels and TNF-␣ production in myeloid DC and monocytes. CB monocytes had lower surface HLA-DR immediately ex vivo. Both APB and CB monocytes upregulated HLA-DR after incubation, but an additional LPS-induced increase in HLA-DR was suggested only in APB monocytes. APB monocytes also showed a greater LPS-induced increase in CD40 expression. Together, our data show significant, selective differences in circulating APC between neonates and adults.