The role of gut microbiota in shaping the relapse-remitting and chronic-progressive forms of multiple sclerosis in mouse models.

Using a mouse model of multiple sclerosis (MS), experimental autoimmune encephalitis (EAE), we evaluated the role of gut microbiota in modulating chronic-progressive (CP) versus relapse-remitting (RR) forms of the disease. We hypothesized that clinical courses of EAE may be shaped by differential gut microbiota. Metagenomic sequencing of prokaryotic 16S rRNA present in feces from naïve mice and those exhibiting CP-EAE or RR-EAE revealed significantly diverse microbial populations. Microbiota composition was considerably different between naïve strains of mice, suggesting microbial components present in homeostatic conditions may prime mice for divergent courses of disease. Additionally, there were differentially abundant bacteria in CP and RR forms of EAE, indicating a potential role for gut microbiota in shaping tolerant or remittance-favoring, and pathogenic or pro-inflammatory-promoting conditions. Furthermore, immunization to induce EAE led to significant alterations in gut microbiota, some were shared between disease courses and others were course-specific, supporting a role for gut microbial composition in EAE pathogenesis. Moreover, using Linear Discriminant Analysis (LDA) coupled with effect size measurement (LEfSe) to analyze microbial content, biomarkers of each naïve and disease states were identified. Our findings demonstrate for the first time that gut microbiota may determine the susceptibility to CP or RR forms of EAE.

Resveratrol (3, 5, 4'-Trihydroxy-trans-Stilbene) Attenuates a Mouse Model of Multiple Sclerosis by Altering the miR-124/Sphingosine Kinase 1 Axis in Encephalitogenic T Cells in the Brain.

Resveratrol (3,5,4'-trihydroxy-trans-stilbene) (RES) is a naturally-derived phytoestrogen found in the skins of red grapes and berries and has potential as a novel and effective therapeutic agent. In the current study, we investigated the role of microRNA (miRNA) in RES-mediated attenuation of experimental autoimmune encephalomyelitis (EAE), a murine model of multiple sclerosis. Administration of RES effectively decreased disease severity, including inflammation and central nervous system immune cell infiltration. miRNA microarray analysis revealed an altered miRNA profile in encephalitogenic CD4+ T cells from EAE mice exposed to RES treatment. Additionally, bioinformatics and in silico pathway analysis suggested the involvement of RES-induced miRNA in pathways and processes that regulated cellular proliferation. Additional studies confirmed that RES affected cell cycle progression and apoptosis in activated T cells, specifically in the brain. RES treatment significantly upregulated miR-124 during EAE, while suppressing associated target gene, sphingosine kinase 1 (SK1), and this too was specific to mononuclear cells in the brains of treated mice, as peripheral immune cells remained unaltered upon RES treatment. Collectively, these studies demonstrate that RES treatment leads to amelioration of EAE development through mechanism(s) potentially involving suppression of neuroinflammation via alteration of the miR-124/SK1 axis, thereby halting cell-cycle progression and promoting apoptosis in activated encephalitogenic T cells. Graphical Abstract Resveratrol alters the miR-124/sphingosine kinase 1 (SK1) axis in encephalitogenic T cells, promotes cell-cycle arrest and apoptosis, and decreases neuroinflammation in experiemental autoimmune encephalomyelitis (EAE).

Novel sphingosine kinase-1 inhibitor, LCL351, reduces immune responses in murine DSS-induced colitis.

Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid metabolite which has been implicated in many diseases including cancer and inflammatory diseases. Recently, sphingosine kinase 1 (SK1), one of the isozymes which generates S1P, has been implicated in the development and progression of inflammatory bowel disease (IBD). Based on our previous work, we set out to determine the efficacy of a novel SK1 selective inhibitor, LCL351, in a murine model of IBD. LCL351 selectively inhibits SK1 both in vitro and in cells. LCL351, which accumulates in relevant tissues such as colon, did not have any adverse side effects in vivo. In mice challenged with dextran sodium sulfate (DSS), a murine model for IBD, LCL351 treatment protected from blood loss and splenomegaly. Additionally, LCL351 treatment reduced the expression of pro-inflammatory markers, and reduced neutrophil infiltration in colon tissue. Our results suggest inflammation associated with IBD can be targeted pharmacologically through the inhibition and degradation of SK1. Furthermore, our data also identifies desirable properties of SK1 inhibitors.

Sphingosine kinase 1 regulates tumor necrosis factor-mediated RANTES induction through p38 mitogen-activated protein kinase but independently of nuclear factor κB activation.

Sphingosine kinase 1 (SK1) produces the pro-survival sphingolipid sphingosine 1-phosphate and has been implicated in inflammation, proliferation, and angiogenesis. Recent studies identified TRAF2 as a sphingosine 1-phosphate target, implicating SK1 in activation of the NF-κB pathway, but the functional consequences of this connection on gene expression are unknown. Here, we find that loss of SK1 potentiates induction of the chemokine RANTES (regulated on activation, normal T cell expressed and secreted; also known as CCL5) in HeLa cells stimulated with TNF-α despite RANTES induction being highly dependent on the NF-κB pathway. Additionally, we find that SK1 is not required for TNF-induced IKK phosphorylation, IκB degradation, nuclear translocation of NF-κB subunits, and transcriptional NF-κB activity. In contrast, loss of SK1 prevented TNF-induced phosphorylation of p38 MAPK, and inhibition of p38 MAPK, like SK1 knockdown, also potentiates RANTES induction. Finally, in addition to RANTES, loss of SK1 also potentiated the induction of multiple chemokines and cytokines in the TNF response. Taken together, these data identify a potential and novel anti-inflammatory function of SK1 in which chemokine levels are suppressed through SK1-mediated activation of p38 MAPK. Furthermore, in this system, activation of NF-κB is dissociated from SK1, suggesting that the interaction between these pathways may be more complex than currently thought.

Epidermal growth factor-induced cellular invasion requires sphingosine-1-phosphate/sphingosine-1-phosphate 2 receptor-mediated ezrin activation.

Ezrin, radixin, and moesin (ERM) proteins link cortical actin to the plasma membrane and coordinate cellular events that require cytoskeletal rearrangement, including cell division, migration, and invasion. While ERM proteins are involved in many important cellular events, the mechanisms regulating their function are not completely understood. Our laboratory previously identified reciprocal roles for the sphingolipids ceramide and sphingosine-1-phosphate (S1P) in the regulation of ERM proteins. We recently showed that ceramide-induced activation of PP1α leads to dephosphorylation and inactivation of ERM proteins, while S1P results in phosphorylation and activation of ERM proteins. Following these findings, we aimed to examine known inducers of the SK/S1P pathway and evaluate their ability to regulate ERM proteins. We examined EGF, a known inducer of the SK/S1P pathway, for its ability to regulate the ERM family of proteins. We found that EGF induces ERM c-terminal threonine phosphorylation via activation of the SK/S1P pathway, as this was prevented by siRNA knockdown or pharmacological inhibition of SK. Using pharmacological, as well as genetic, knockdown approaches, we determined that EGF induces ERM phosphorylation via activation of S1PR2. In addition, EGF led to cell polarization in the form of lamellipodia, and this occurred through a mechanism involving S1PR2-mediated phosphorylation of ezrin T567. EGF-induced cellular invasion was also found to be dependent on S1PR2-induced T567 ezrin phosphorylation, such that S1PR2 antagonist, JTE-013, and expression of a dominant-negative ezrin mutant prevented cellular invasion toward EGF. In this work, a novel mechanism of EGF-stimulated invasion is unveiled, whereby S1P-mediated activation of S1PR2 and phosphorylation of ezrin T567 is required.

Regulation of the sphingosine kinase/sphingosine 1-phosphate pathway.

Sphingolipids have emerged as pleiotropic signaling molecules with roles in numerous cellular and biological functions. Defining the regulatory mechanisms governing sphingolipid metabolism is crucial in order to develop a complete understanding of the biological functions of sphingolipid metabolites. The sphingosine kinase/ sphingosine 1-phosphate pathway was originally thought to function in the irreversible breakdown of sphingoid bases; however, in the last few decades it has materialized as an extremely important signaling pathway involved in a plethora of cellular events contributing to both normal and pathophysiological events. Recognition of the SK/S1P pathway as a second messaging system has aided in the identification of many mechanisms of its regulation; however, a cohesive, global understanding of the regulatory mechanisms controlling the SK/S1P pathway is lacking. In this chapter, the role of the SK/S1P pathway as a second messenger is discussed, and its role in mediating TNF-α- and EGF-induced biologies is examined. This work provides a comprehensive look into the roles and regulation of the sphingosine kinase/ sphingosine 1-phosphate pathway and highlights the potential of the pathway as a therapeutic target.

Targeting the sphingosine kinase/sphingosine 1-phosphate pathway in disease: review of sphingosine kinase inhibitors.

Sphingosine 1-phosphate (S1P) is an important bioactive sphingolipid metabolite that has been implicated in numerous physiological and cellular processes. Not only does S1P play a structural role in cells by defining the components of the plasma membrane, but in the last 20 years it has been implicated in various significant cell signaling pathways and physiological processes: for example, cell migration, survival and proliferation, cellular architecture, cell-cell contacts and adhesions, vascular development, atherosclerosis, acute pulmonary injury and respiratory distress, inflammation and immunity, and tumorogenesis and metastasis [1,2]. Given the wide variety of cellular and physiological processes in which S1P is involved, it is immediately obvious why the mechanisms governing S1P synthesis and degradation, and the manner in which these processes are regulated, are necessary to understand. In gaining more knowledge about regulation of the sphingosine kinase (SK)/S1P pathway, many potential therapeutic targets may be revealed. This review explores the roles of the SK/S1P pathway in disease, summarizes available SK enzyme inhibitors and examines their potential as therapeutic agents. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Sphingosine 1-phosphate induces filopodia formation through S1PR2 activation of ERM proteins.

Previously we demonstrated that the sphingolipids ceramide and S1P (sphingosine 1-phosphate) regulate phosphorylation of the ERM (ezrin/radixin/moesin) family of cytoskeletal proteins [Canals, Jenkins, Roddy, Hernande-Corbacho, Obeid and Hannun (2010) J. Biol. Chem. 285, 32476-3285]. In the present article, we show that exogenously applied or endogenously generated S1P (in a sphingosine kinase-dependent manner) results in significant increases in phosphorylation of ERM proteins as well as filopodia formation. Using phosphomimetic and non-phosphorylatable ezrin mutants, we show that the S1P-induced cytoskeletal protrusions are dependent on ERM phosphorylation. Employing various pharmacological S1PR (S1P receptor) agonists and antagonists, along with siRNA (small interfering RNA) techniques and genetic knockout approaches, we identify the S1PR2 as the specific and necessary receptor to induce phosphorylation of ERM proteins and subsequent filopodia formation. Taken together, the results demonstrate a novel mechanism by which S1P regulates cellular architecture that requires S1PR2 and subsequent phosphorylation of ERM proteins.

Sphingosine kinase: Role in regulation of bioactive sphingolipid mediators in inflammation.

Sphingolipids and their synthetic enzymes are emerging as important mediators in inflammatory responses and as regulators of immune cell functions. In particular, sphingosine kinase (SK) and its product sphingosine-1-phosphate (S1P) have been extensively implicated in these processes. SK catalyzes the phosphorylation of sphingosine to S1P and exists as two isoforms, SK1 and SK2. SK1 has been shown to be activated by cytokines including tumor necrosis factor-alpha (TNF-alpha) and interleukin1-beta (IL1-beta). The activation of SK1 in this pathway has been shown to be, at least in part, required for mediating TNF-alpha and IL1-beta inflammatory responses in cells, including induction of cyclo-oxygenase 2 (COX2). In addition to their role in inflammatory signaling, SK and S1P have also been implicated in various immune cell functions including, mast cell degranulation, migration of neutrophils, and migration and maturation of lymphocytes. The involvement of sphingolipids and sphingolipid metabolizing enzymes in inflammatory signaling and immune cell functions has implicated these mediators in numerous inflammatory disease states as well. The contribution of these mediators, specifically SK1 and S1P, to inflammation and disease are discussed in this review.

Tolerance induction for solid organ grafts with donor-derived hematopoietic reconstitution.

Tolerance of transplanted tissue has been a focus of immunologists for decades. Indeed, to some the birth of immunology and the search for tolerance of the non-self are synonymous. One of the most powerful and reproducible methods of tolerance induction to allogeneic tissue has involved infusion of donor-specific hematopoietic cells. Under certain conditions, such infusion can result in hematopoietic reconstitution that can be experimentally accomplished at a variety of different time-points in the life of an organism from the in utero period through adulthood, reconstitution at each time-point involving consideration of a different set of immunological and physiological parameters. When high levels of donor-derived hematopoietic reconstitution are achieved, tolerance induction to donor-specific antigens is reproducible and long-lasting. Unfortunately, however, clinical efforts to achieve such high levels of hematopoietic reconstitution have historically been unsuccessful or fraught with complications. Transplantation efforts have been plagued by failure of engraftment, graft-vs-host disease (GVHD), or severe immunoincompetence of the recipient. Laboratory and clinical efforts during the last decade have resulted in a variety of developments that may overcome these barriers: (1) methods have been devised in which cells that cause GVHD can be depleted from the hematopoietic graft while hematopoietic reconstitution potential is preserved, (2) methods of harvesting large numbers of cells with multilineage reconstitution potential have been devised (an accomplishment that seems to allow the immunological barrier to be overwhelmed), and (3) capitalizing on the above two principles, minimally toxic preconditioning regimens have been designed that allow allogeneic engraftment. This review will focus on some of the experimental and clinical data of the past and the experimental and clinical issues that loom ahead.

CD8+TCR+ and CD8+TCR- cells in whole bone marrow facilitate the engraftment of hematopoietic stem cells across allogeneic barriers.

Although purified hematopoietic stem cells (HSC) are sufficient to engraft irradiated allogeneic recipients, bone marrow (BM) contains other cells that facilitate engraftment. Here, several candidate facilitators were tested by cotransplantation with HSC. Both TCR+ and TCR- CD8alpha+ BM subpopulations have facilitative potential. CD8+TCR+ cells are typical T lymphocytes. CD8+TCR- facilitators are CD3 , not CD3+, have a granular morphology, and are CD8beta- and CD11c+; they share phenotypic characteristics with CD8(alpha)alpha lymphoid dendritic cells and veto cells. We also demonstrate that lytic function is nqt necessary for facilitation and that the CD8alpha molecule is either important for facilitation or in the development of facilitators.

Systemic overexpression of BCL-2 in the hematopoietic system protects transgenic mice from the consequences of lethal irradiation.

A new transgenic mouse has been generated in which the proto-oncogene BCL-2 is ubiquitously overexpressed. H2K-BCL-2 transgenic mice overexpress BCL-2 in all cells of the hematolymphoid system and have been used to assess the role of BCL-2 in protecting cells of the hematolymphoid system from the consequences of ionizing radiation. We have expanded on previous studies that have demonstrated protection for specific (lymphoid) cell populations and show that systemic overexpression of BCL-2 can protect the hematopoietic system as a whole, including hematopoietic stem cells (HSC), thus increasing the radioresistance of the animal. The increase in radioresistance in H2K-BCL-2 transgenic mice has two components: an increase in the radioresistance of individual cells and, to a lesser extent, an increase in the size of certain critically important cell populations, such as HSC. Bone marrow transplantation experiments show that the increased radioresistance of the transgenic animals is provided by cells of the hematopoietic system. Protection against the consequences of irradiation is not limited to the increased expression levels of BCL-2 in transgenic mice; levels of endogenous BCL-2 are higher in lymphocyte populations that survive irradiation in wild-type mice. We show that ubiquitous overexpression of BCL-2 in the hematopoietic system can be used to increase the resistance of animals to lethal challenges such as irradiation.

Tolerance of allogeneic heart grafts in mice simultaneously reconstituted with purified allogeneic hematopoietic stem cells.

BACKGROUND: Animals reconstituted with allogeneic whole bone marrow (WBM) are often tolerant of donor-specific solid organ grafts. Clinical application of bone marrow transplantation in solid organ transplantation has been limited, however, principally by graft-versus-host disease. We previously demonstrated that hematopoietic stem cells (HSCs) reconstitute lethally irradiated allogeneic mice without producing graft-versus-host disease. The purpose of this study was to determine whether tolerance to solid organ grafts could be induced in mice reconstituted with HSCs. METHODS: BALB/c mice were lethally irradiated and reconstituted with allogeneic C57BL/Ka, Thy-1.1 WBM or HSCs. An isolated group was given a limited number of HSCs (250 cells) and a subpopulation of allogeneic cells known to facilitate HSC engraftment (facilitators). C57BL/Ka, Thy-1.1 neonatal heart grafts were placed in reconstituted animals either at the time of hematopoietic transplant or 35 days later. Third-party C3H grafts were placed over 2 months after hematopoietic reconstitution. Tolerance was defined as the persistence of cardiac contraction for the duration of evaluation (125-270 days). RESULTS: All surviving mice that were reconstituted with C57BL/Ka, Thy-1.1 HSCs, WBM, or HSCs and facilitators were tolerant of C57BL/Ka grafts long-term. Third-party C3H grafts placed in reconstituted animals were rejected by day 12, whereas those placed in unmanipulated mice were rejected by day 9. CONCLUSION: These data indicate that tolerance to concurrently or subsequently placed solid organ grafts can be reliably achieved with limited numbers of purified HSCs in a model where immunocompetence to third-party major histocompatibility complex antigens is delayed but intact.

Perinatal induction of immunotolerance to cardiac and pulmonary allografts.

OBJECTIVES: Tolerance appears to be more easily induced in the fetus before full immunocompetence is established, but elucidation of this process is needed. A model of perinatal tolerance induction to neonatal skin allografts followed by cardiac and pulmonary allografts is described. METHODS: Sixty Lewis (RT11) rat fetuses were inoculated intraperitoneally at 18 days gestation with 1 x 10(7) ACI (RT1a) rat fetal liver cells (group I); 20 Lewis fetuses were inoculated with 2 x 10(7) ACI fetal liver cells (Group II). control groups consisted of Lewis fetuses inoculated with saline solution (n = 25, group III) and fetuses that were not inoculated (n = 25, group IV). Twenty-five of the 50 surviving group I rats received ACI skin (< 24 hours old) and heart (8 to 10 weeks old) allografts (group IA); the remaining 25 rats received only ACI heart grafts (group IB). Groups II, III, and IV received ACI skin and cardiac allografts. Recipients tolerant to both skin and cardiac grafts received orthotopic ACI lung grafts and third-party skin grafts. Tolerance was indicated by graft survival for more than 100 days. Limiting dilution and flow cytometric analyses were performed. RESULTS: Abortion rates in groups I, II, III, and IV were 17% (10/60), 65% (13/20), 8% (2/25), and 4% (1/25), respectively. Specific tolerance to skin, cardiac, and lung allografts was observed in seven of 25 group IA recipients (28%) and seven of seven group II recipients (100%) compared with no tolerance in any group IB, III, or IV recipients (p = 0.03, chi 2 test). A 100-fold reduction of precursor cytotoxic T lymphocytes and significant splenocyte and bone marrow chimerism in tolerant versus nontolerant rats were noted (p = 0.0001, Student's t test). CONCLUSIONS: Using donor-strain fetal liver cells and neonatal skin grafts, we achieved higher frequencies of tolerance to solid organ grafts in adulthood with lower cell inocula and abortion rates than previously described. Chimerism and depressed precursor cytotoxic T lymphocyte frequencies in tolerant recipients suggest that hematopoietic stem cell engraftment and clonal deletion/anergy are involved in induction of perinatal tolerance.

From stem cells to lymphocytes: biology and transplantation.

We review the development of the hematopoietic system, focusing on the transition from hematopoietic stem cells (HSCs) to T cells. This includes the isolation of HSCs, and recent progress in understanding their ontogeny, homing properties, and differentiation. HSC transplantation is reviewed, including the kinetics of reconstitution, engraftment across histocompatibility barriers, the facilitation of allogeneic engraftment, and the mechanisms of graft rejection. We describe progress in understanding T-cell development in the bone marrow and thymus as well as the establishment of lymph nodes. Finally, the role of bcl-2 in regulating homeostasis in the hematopoietic system is discussed.

A rodent model of in utero chimeric tolerance induction.

BACKGROUND: In utero tolerance induction has potential application in pediatric heart transplantation. Immunotolerance appears to be more easily induced in the fetus before full immunocompetence is established; however, the mechanisms behind this phenomenon are still undefined. METHODS: One hundred thirty Lewis (RT1l) rat fetuses from 10 litters were inoculated intraperitoneally at 18 days gestation with 1 x 10(7) ACI (RT1a) rat fetal liver cells. Fifty of the 100 viable neonates successfully brought to term were grafted with neonatal ACI skin within 24 hours of birth and heterotopic ACI hearts at 8 to 10 weeks of age (group 1A); the remaining 50 neonates only received heterotopic ACI heart grafts at 8 to 10 weeks (group 1B). Control groups consisted of 50 Lewis fetuses (five litters) inoculated in utero with phosphate-buffered saline solution (group 2) and 50 Lewis fetuses (five litters) that received no inoculum (group 3); all of these surviving progeny received both neonatal ACI skin and adult ACI cardiac allografts. Skin and cardiac grafts were monitored by daily visual inspection and palpation, respectively. Limiting dilution analysis was performed among all groups to assess precursor cytotoxic lymphocyte frequencies. Likewise, peripheral blood lymphocyte and splenocyte populations were analyzed with flow cytometry to detect allogeneic chimerism. RESULTS: Abortion rates among groups 1, 2, and 3 were 23% (30/130 abortions), 10% (5/50 abortions), and 6% (3/50 abortions), respectively. Tolerance to both ACI skin and cardiac allografts was induced in 14 of the 50 group 1A Lewis recipients (28%). Tolerance was not achieved in any of the recipients in groups 1B, 2, or 3. Limiting dilution analysis among all groups revealed a marked reduction of precursor cytotoxic T-lymphocytes in tolerant allograft recipients compared with recipients in the other groups. Flow cytometry detected significant splenocyte chimerism among tolerant rats; significant peripheral blood chimerism was not noted. CONCLUSIONS: We describe allogeneic tolerance induction in utero to both rat skin and heart tissue by use of donor-strain fetal liver cells. Compared with previous studies with adult splenocytes as the tolerogen, we achieved a higher frequency of tolerance with a markedly lower cell inoculum and lower abortion rate. Allogeneic chimerism was noted in the tolerant recipients, suggesting hematopoietic stem cell engraftment. Cytotoxic T-lymphocyte precursor frequencies were markedly depressed in tolerant animals. Interestingly, both donor-strain fetal liver cells and neonatal skin grafts were required to induce tolerance. These data suggest a period of hematopoietic "education" during and shortly after hematopoietic stem cell engraftment.

Tolerance to cardiac allografts induced in utero with fetal liver cells.

BACKGROUND: Induction of immunological tolerance in utero has potential application in pediatric cardiac transplantation. We describe an inexpensive, reproducible, and well-characterized model of allogeneic tolerance induction in utero using donorstrain fetal liver cells. METHODS AND RESULTS: Each of 9 Lewis (LEW, RTl1) rat fetuses in one litter (group 1) and 10 LEW fetuses in another litter (group 2) were inoculated intraperitoneally at 17 to 18 days of gestation with 1 x 10(6) ACI (RTla) rat fetal liver cells. Ten LEW fetuses in a third litter inoculated with PBS (group 3) and 10 LEW noninoculated fetuses in a fourth litter (group 4) served as controls. The LEW rats were brought to term, and groups 1, 3, and 4 were grafted with neonatal ACI skin within 24 hours of birth and with heterotopic ACI hearts at 8 to 10 weeks of age; group 2 rats received only an ACI heart graft at 8 to 10 weeks. Skin and cardiac grafts were monitored by daily visual inspection and palpation, respectively. Peripheral blood lymphocyte (PBL) populations in all LEW recipients were analyzed with flow cytometry. All LEW fetuses survived to term and developed normally. The ACI skin and cardiac allografts on 3 of the 9 LEW rats in group 1 are viable to date (skin, > 170 days; cardiac, > 100 days). The remaining 6 recipients of this group and all animals in groups 2, 3, and 4 rejected their skin and cardiac grafts by postgrafting day 7. Significant PBL chimerism (1.57%) was observed in only 1 tolerant rat. CONCLUSIONS: We describe allogeneic tolerance induction in utero to both rat skin and cardiac tissue with donor-strain fetal liver cells. Compared with previous studies using adult splenocytes as the tolerogen, we achieved a higher frequency of tolerance with a markedly lower cell inoculum and no abortions. Interestingly, both donor-strain fetal liver cells and neonatal skin grafts were required to maintain tolerance into adulthood. Immunocompetence sufficient to reject allografts was noted in neonates, and PBL chimerism was not prominent in tolerant recipients.

Leflunomide prolongs pulmonary allograft and xenograft survival.

BACKGROUND: Leflunomide, an isoxazole derivative, has been shown to effectively prolong rodent allograft and cardiac xenograft survival. In vitro studies suggest that leflunomide inhibits the production of donor-specific antibodies and is capable of blocking both T- and B-cell proliferation. In light of the significant role that humoral immunity is believed to play in chronic pulmonary allograft rejection as well as hyperacute and accelerated acute xenograft rejection, we examined the efficacy of leflunomide in prolonging pulmonary allografts and xenografts and its effect on donor-specific antibody production. METHODS: Lungs from Brown Norway rats or Golden Syrian hamsters were orthotopically transplanted into Lewis rat recipients. Allograft recipients were treated daily for 14 days with vehicle, leflunomide (15 mg/kg/day orally), or cyclosporine (7.5 mg/kg/day orally) starting on the day of grafting (day 0). In xenograft recipients, leflunomide (20 mg/kg/day orally) or cyclosporine (7.5 mg/kg/day orally) treatment initiated on day 0 was continued until complete graft rejection; the leflunomide dosage was reduced to 10 mg/kg/day after day 14 because of weight loss and leukopenia. Graft viability was assessed with chest radiography in conjunction with open lung biopsies. Toxicity was monitored with body weight measurements, complete blood counts, and serum chemistries. Flow cytometric analysis of serum samples taken from graft recipients on day 7 was used to measure donor-specific immunoglobulin M and immunoglobulin G antibody titers. RESULTS: Allograft and xenograft control animals receiving vehicle yielded graft survival times of 6.0 +/- 0.0 and 5.4 +/- 0.6 days, respectively. Although xenograft recipients treated with cyclosporine (7.5 mg/kg/day orally) showed no significant graft prolongation, pulmonary allograft survival in recipients receiving cyclosporine alone was significantly prolonged to 28.2 +/- 0.7 days. Leflunomide-treated allograft (15 mg/kg/day orally) and xenograft (20 mg/kg/day orally) recipients displayed significant graft prolongation to 28.2 +/- 0.7 days and 15.8 +/- 3.3 days, respectively. Cyclosporine (7.5 mg/kg/day orally) enhanced the effect of leflunomide (20 mg/kg/day orally) in xenograft recipients with a mean graft survival time of 36.0 +/- 3.0 days achieved when both drugs were administered concomitantly. Cyclosporine significantly suppressed donor-specific immunoglobulin G antibody titers in both pulmonary allograft and xenograft recipients while not affecting immunoglobulin M levels. Leflunomide markedly suppressed both immunoglobulin G and immunoglobulin M donor-specific antibody titers in allograft and xenograft recipients. Except for mild leukopenia and anemia, both cyclosporine- and leflunomide-treated allograft recipients showed no evidence of toxic side effects after 14 days of therapy. However, leflunomide-treated xenograft recipients displayed significant weight loss, anemia, and leukopenia after 14 days of treatment with one death in each treatment group.

Aggressive blood conservation in coronary artery surgery: impact on patient care.

Data on 100 consecutive non-emergency coronary artery bypass (CABG) patients were analyzed retrospectively. Sixty-nine patients received no homologous blood (Group I). Thirty-one patients received a total of 118 units of blood products averaging 2.23 units of red cells (Group II). The average red cell transfusion rate for all patients was 0.7 units per patient. The median age for Group I was 61 and Group II was 68 years (p less than 0.05). The average number of grafts was the same for both (3 per patient) with 75% of Group I and 58% of Group II receiving internal mammary artery (IMA) grafts (p less than 0.05). Twelve of the Group II patients who received intraoperative transfusions on cardiopulmonary bypass to maintain adequate hemoglobin levels were older and had lower admission hematocrits: 36 +/- 0.8% compared to 41 +/- 0.5% for all other patients (p less than 0.05). Average postoperative blood loss was 889 +/- 38 ml for Group I and 1077 +/- 104 ml for Group II (p less than 0.05). Increased hemorrhage was correlated with bypass time and IMA use but not with preoperative heparin administration, pre-existing risk factors (diabetes, hypertension, etc.), bleeding time, post-bypass clotting time, age or number of grafts. Two patients in Group II and none in Group I required exploration for excessive postoperative hemorrhage. Mortality rate was 2% (both in Group II, neither transfusion related). Discharge hematocrits were the same for all at 29.4 +/- 0.4%. Among anemia-related postoperative symptoms, only sinus tachycardia was significantly higher in Group I (20%) compared to Group II (6.5%).(ABSTRACT TRUNCATED AT 250 WORDS)