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Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans. In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways. Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells. Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions.

Marijuana abuse is very prominent among pregnant women. Although marijuana cannabinoids have been shown to exert immunosuppression in adults, virtually nothing is known about the effects of marijuana use during pregnancy on the developing immune system of the fetus and during postnatal life. We noted that murine fetal thymus expressed high levels of the cannabinoid receptors CB1 and CB2. Moreover, perinatal exposure to Δ(9)-tetrahydrocannabinol (THC) had a profound effect on the fetus as evidenced by a decrease in thymic cellularity on gestational days 16, 17, and 18 and postgestational day 1 and marked alterations in T cell subpopulations. These outcomes were reversed by CB1/CB2 antagonists, suggesting that THC-mediated these effects through cannabinoid receptors. Thymic atrophy induced in the fetus correlated with caspase-dependent apoptosis in thymocytes. Thymic atrophy was the result of direct action of THC and not based on maternal factors inasmuch as THC was able to induce T cell apoptosis in vitro in fetal thymic organ cultures. It is noteworthy that perinatal exposure to THC also had a profound effect on the immune response during postnatal life. Peripheral T cells from such mice showed decreased proliferative response to T cell mitogen as well as both T cell and antibody response to HIV-1 p17/p24/gp120 antigens. Together, our data demonstrate for the first time that perinatal exposure to THC triggers profound T cell dysfunction, thereby suggesting that the offspring of marijuana abusers who have been exposed to THC in utero may be at a higher risk of exhibiting immune dysfunction and contracting infectious diseases including HIV.

Allogeneic hematopoietic cell transplantation (HCT) is widely used to treat patients with life-threatening malignant and nonmalignant hematological diseases. However, allogeneic HCT often is accompanied by severe and lethal complications from graft-versus-host disease (GVHD), in which activated donor T cells recognize histocompatibility antigenic mismatches and cause significant toxicity in the recipient. In the current study, we tested the hypothesis that activation of cannabinoid receptors on donor-derived T cells may prevent GVHD. We tested the effect of Δ(9)-tetrahydrocannabinol (THC) in an acute model of GVHD that was induced by transferring parental C57BL/6 (B6) spleen cells into (C57BL/6 × DBA/2) F(1)(BDF1) mice. Transfer of B6 cells into BDF1 mice produced severe acute GVHD in the recipient, characterized by lymphoid hyperplasia, weight loss, T helper l cytokine production and mortality. THC administration led to early recovery from body weight loss, reduced tissue injury in the liver and intestine, as well as complete survival. THC treatment reduced the expansion of donor-derived effector T cells and blocked the killing of host-derived immune cells while promoting Foxp3(+) regulatory T cells. Impaired hematopoiesis seen during GVHD was rescued by treatment with THC. The ability of THC to reduce the clinical GVHD was reversed, at least in part, by administration of cannabinoid receptor (CB) 1 and CB2 antagonists, thereby demonstrating that THC-mediated amelioration of GVHD was cannabinoid receptor-dependent. Our results demonstrate for the first time that targeting cannabinoid receptors may constitute a novel treatment modality against acute GVHD.

ABSTRACTThere are two important mechanisms of activation of invariant natural killer T cells (iNKT cells) by microbes: direct activation of the invariant T-cell receptor (TCR) by microbial glycolipids presented by CD1d and indirect activation, mediated by the responses of antigen-presenting cells to microbes. In this study, we provide evidence for a novel CD1d-independent direct activation of iNKT cells involving a microbial protein superantigen presented in the context of major histocompatibility complex class II (MHC-II), which plays a critical role in pathogenesis, thereby redefining the role of iNKT cells. Intranasal exposure to staphylococcal enterotoxin B (SEB) in C57BL/6 wild-type mice caused acute lung injury (ALI) characterized by vascular leak, cytokine storm, and infiltration of mononuclear cells in the lungs. In contrast, the vascular leak and inflammation were decreased by ∼50% in NKT cell-deficient Jα18−/−and CD1d−/−mice following SEB exposure, which was reversed following adoptive transfer of iNKT cells into CD1d−/−mice. In vitro, SEB could directly stimulate iNKT cells in a CD1d-independent manner via MHC-II/TCR interaction, specifically involving Vβ8. These studies not only demonstrate that iNKT cells can be activated directly by a bacterial protein superantigen independent of CD1d but also indicate that in addition to the conventional T cells, iNKT cells play a critical role in SEB-mediated ALI.

Inflammatory bowel disease is a chronic, relapsing, and tissue-destructive disease. Resveratrol (3,4,5-trihydroxy-trans-stilbene), a naturally occurring polyphenol that exhibits beneficial pleiotropic health effects, is recognized as one of the most promising natural molecules in the prevention and treatment of chronic inflammatory disease and autoimmune disorders. In the present study, we investigated the effect of resveratrol on dextran sodium sulfate (DSS)-induced colitis in mice and found that it effectively attenuated overall clinical scores as well as various pathological markers of colitis. Resveratrol reversed the colitis-associated decrease in body weight and increased levels of serum amyloid A, tumor necrosis factor-alpha, interleukin (IL-6), and IL-1beta. After resveratrol treatment, the percentage of CD4(+) T cells in mesenteric lymph nodes (MLN) of colitis mice was restored to normal levels, and there was a decrease in these cells in the colon lamina propria (LP). Likewise, the percentages of macrophages in MLN and the LP of mice with colitis were decreased after resveratrol treatment. Resveratrol also suppressed cyclooxygenase-2 (COX-2) expression induced in DSS-exposed mice. Colitis was associated with a decrease in silent mating type information regulation-1 (SIRT1) gene expression and an increase in p-inhibitory kappaB expression and nuclear transcription factor-kappaB (NF-kappaB) activation. Resveratrol treatment of mice with colitis significantly reversed these changes. This study demonstrates for the first time that SIRT1 is involved in colitis, functioning as an inverse regulator of NF-kappaB activation and inflammation. Furthermore, our results indicate that resveratrol may protect against colitis through up-regulation of SIRT1 in immune cells in the colon.

Cannabinoids are a group of compounds that mediate their effects through cannabinoid receptors. The discovery of 9-tetrahydrocannabinol (THC) as the major psychoactive principle in marijuana, as well as the identification of cannabinoid receptors and their endogenous ligands, has led to a significant growth in research aimed at understanding the physiological functions of cannabinoids. Cannabinoid receptors include CB1, which is predominantly expressed in the brain, and CB2, which is primarily found on the cells of the immune system. The fact that both CB1 and CB2 receptors have been found on immune cells suggests that cannabinoids play an important role in the regulation of the immune system. Recent studies demonstrated that administration of THC into mice triggered marked apoptosis in T cells and dendritic cells, resulting in immunosuppression. In addition, several studies showed that cannabinoids downregulate cytokine and chemokine production and, in some models, upregulate T-regulatory cells (Tregs) as a mechanism to suppress inflammatory responses. The endocannabinoid system is also involved in immunoregulation. For example, administration of endocannabinoids or use of inhibitors of enzymes that break down the endocannabinoids, led to immunosuppression and recovery from immune-mediated injury to organs such as the liver. Manipulation of endocannabinoids and/or use of exogenous cannabinoids in vivocan constitute a potent treatment modality against inflammatory disorders. This review will focus on the potential use of cannabinoids as a new class of anti-inflammatory agents against a number of inflammatory and autoimmune diseases that are primarily triggered by activated T cells or other cellular immune components.

CD44 is involved in several biological processes owing to its dual role as a cell adhesion and signaling molecule. In an allogeneic dendritic cell (DC)–T cell interaction model, we show here that CD44 gets clustered at the contact between T cells with mature but not immature DCs. Also, CD44 colocalized with lipid rafts at the immunological synapse (IS). Using DCs or T cells derived from CD44‐deficient mice, we observed that the presence of CD44 on DCs and T cells is important for the formation of DC–T cell tight conjugates. However, deficiency of CD44 on DCs but not T cells affected the functional IS, as indicated by decreased phosphotyrosine and protein kinase C‐θ enrichment at the synapse. Also, CD44‐deficient DCs induced significantly decreased proliferation as well as IL‐2 and IFN‐γ production from allogeneic T cells. The polarization of CD44 at the synapse was also noted in an antigen (OVA)‐specific, syngeneic DC–T cell interaction using OVA‐specific T cells derived from OT‐II mice. It was believed that large molecules such as CD44 were excluded from the IS. Results presented here show for the first time that CD44 is recruited to the IS during allogeneic DC and T cell interactions and plays an important role in subsequent T cell activation.

Immune-mediated liver diseases including autoimmune and viral hepatitis are a major health problem worldwide. Natural cannabinoids such as Δ9-tetrahydrocannabinol (THC) effectively modulate immune cell function, and they have shown therapeutic potential in treating inflammatory diseases. We investigated the effects of THC in a murine model of concanavalin A (ConA)-induced hepatitis. Intraperitoneal administration of THC after ConA challenge inhibited hepatitis as shown by significant decrease in liver enzymes and reduced liver tissue injury. Furthermore, THC treatment resulted in significant suppression of crucial inflammatory cytokines in ConA-induced hepatitis. It is noteworthy that THC treatment in ConA-injected mice led to significant increase in absolute number of Forkhead helix transcription factor p3+T regulatory cells in liver. We were surprised to find that select cannabinoid receptor (CB1 or CB2) agonists were not able to block hepatitis either independently or in combination. However, CB1/CB2 mixed agonists were able to efficiently attenuate hepatitis similar to THC. The modulatory effect of THC in ConA-induced hepatitis was reversed by both CB1 and CB2 antagonists. We also observed that endogenous cannabinoid anandamide was able to reduce hepatitis by suppressing cytokine levels. In addition, deficiency or inhibition of endocannabinoid hydrolyzing enzyme fatty acid amide hydrolase (FAAH), which leads to increased levels of endogenous cannabinoids, resulted in decreased liver injury upon ConA challenge. Our data demonstrate that targeting cannabinoid receptors using exogenous or endogenous cannabinoids and use of FAAH inhibitors may constitute novel therapeutic modalities to treat immune-mediated liver inflammation.

Although the toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on T cells in vivo have been well characterized, attempts to reproduce these findings in vitro have not been successful. In the current study, we examined whether activation or the presence of dendritic cells (DCs) would make primary naive T cells from C57BL/6 mice susceptible to TCDD-induced apoptosis in vitro. Although nonactivated primary T cells cultured with 10 to 1000 nM TCDD were relatively resistant to apoptosis, they became sensitive to apoptosis upon activation with concanavalin A (ConA). Moreover, ConA-activated T cells cultured in the presence of DCs showed highest levels of TCDD-induced apoptosis. Likewise, primary T cells from OT.II.2a mice cultured with specific ovalbumin peptide and syngeneic DCs showed higher levels of apoptosis compared with similar nonactivated T cells. T-cell activation led to up-regulation of aryl hydrocarbon receptor (AhR), Fas, and Fas-ligand (FasL) expression. In addition, DC maturation and culture with TCDD caused significant induction of FasL. TCDD-mediated apoptosis in activated peripheral T cells was AhR-dependent. Analysis of why nonactivated T cells are more resistant, whereas activated T cells are sensitive to TCDD-induced apoptosis revealed that TCDD treatment of activated but not nonactivated T cells led to down-regulation of cellular FLICE inhibitory protein (c-FLIP), an inhibitor of apoptosis. Moreover, down-regulation of c-FLIP using small interfering RNA in nonactivated T cells made them sensitive to TCDD-induced apoptosis. The current study demonstrates for the first time that TCDD can induce apoptosis in vitro in peripheral T cells upon activation and in the presence of DCs and that this may be mediated by down-regulation of c-FLIP.

Resveratrol (trans-3,5,4'-trihydroxystilbene), a polyphenolic compound found in plant products, including red grapes, exhibits anticancer, antioxidant, and anti-inflammatory properties. Using an animal model of multiple sclerosis (MS), we investigated the use of resveratrol for the treatment of autoimmune diseases. We observed that resveratrol treatment decreased the clinical symptoms and inflammatory responses in experimental allergic encephalomyelitis (EAE)-induced mice. Furthermore, we observed significant apoptosis in inflammatory cells in spinal cord of EAE-induced mice treated with resveratrol compared with the control mice. Resveratrol administration also led to significant down-regulation of certain cytokines and chemokines in EAE-induced mice including tumor necrosis factor-α, interferon-γ, interleukin (IL)-2, IL-9, IL-12, IL-17, macrophage inflammatory protein-1α (MIP-1α), monocyte chemoattractant protein-1 (MCP-1), regulated on activation normal T-cell expressed and secreted (RANTES), and Eotaxin. In vitro studies on the mechanism of action revealed that resveratrol triggered high levels of apoptosis in activated T cells and to a lesser extent in unactivated T cells. Moreover, resveratrol-induced apoptosis was mediated through activation of aryl hydrocarbon receptor (AhR) and estrogen receptor (ER) and correlated with up-regulation of AhR, Fas, and FasL expression. In addition, resveratrol-induced apoptosis in primary T cells correlated with cleavage of caspase-8, caspase-9, caspase-3, poly(ADP-ribose) polymerase, and release of cytochrome c. Data from the present study demonstrate, for the first time, the ability of resveratrol to trigger apoptosis in activated T cells and its potential use in the treatment of inflammatory and autoimmune diseases including, MS.

We have demonstrated previously that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) up-regulates Fas and FasL in immune cells, although the molecular mechanisms remain unknown. We investigated the regulation of Fas or FasL promoter by TCDD in EL4 T cells using luciferase reporter constructs. We observed 20 +/- 5- and 14 +/- 4-fold induction of promoter activity for Fas and FasL, respectively, after TCDD exposure. The induction of luciferase was significantly reduced (2 +/- 1-fold) in the presence of alpha-naphthoflavone, an aryl hydrocarbon receptor (AhR) antagonist. We noted the presence of a dioxin response element (DRE) and five nuclear factor-kappaB (NF-kappaB) motifs on Fas promoter, and no DRE but two NF-kappaB motifs on FasL promoter. When we investigated the role of DRE and NF-kappaB, we observed varying levels of luciferase induction (9 +/- 2-fold for DRE and 8 +/- 2-fold for NF-kappaBs of Fas promoter and 6 +/- 3-fold for NF-kappaBs of FasL promoter). Mutations in DRE of Fas promoter or NF-kappaBs of FasL promoter led to decreased luciferase induction, further supporting our results. Probes for DRE or NF-kappaB motifs of Fas and/or FasL promoters demonstrated mobility shift in the presence of nuclear extract from TCDD-treated EL4 cells. Furthermore, we observed supershift in mobility when DRE and NF-kappaB probes were incubated in the presence of anti-mouse AhR, and anti-NF-kappaB (RelA/p65 and p50) antibodies, respectively. Administration of TCDD into mice caused significant increase in Fas and FasL transcripts in thymus and liver. These data demonstrate that TCDD regulates Fas and FasL promoters through DRE and/or NF-kappaB motifs via AhR.

T regulatory cells (CD4+CD25+) play an important role in the regulation of the immune response. However, little is known about the ability of T regulatory cells to regulate endothelial cell (EC) damage following activation of lymphocytes with IL-2. Therefore, in the current study, we examined the role of T regulatory cells and the subsequent Th1/Th2 bias in IL-2-mediated EC injury using the well-characterized C57BL/6 (Th1-biased) and BALB/c (Th2-biased) models. Following IL-2 treatment, BALB/c mice were less susceptible to IL-2-induced vascular leak syndrome (VLS) compared with C57BL/6 mice. Splenocytes from BALB/c mice displayed less cytotoxicity against ECs compared with those from C57BL/6 mice. Interestingly, BALB/c mice had significantly higher numbers of CD4+CD25+ T regulatory cells, which proliferated more profoundly following IL-2 treatment, compared with CD4+CD25+ T regulatory cells from C57BL/6 mice. In addition, T regulatory cells from naive BALB/c mice were more potent suppressors of anti-CD3 mAb-stimulated proliferation of T cells than similar cells from C57BL/6 mice. Depletion of T regulatory cells in both BALB/c and C57BL/6 mice led to a significant increase in IL-2-induced VLS. Together, the results from this study suggest that CD4+CD25+ T regulatory cells play an important role in the regulation of IL-2-induced EC injury.

In the current study, we examined the effects of the nonpsychoactive cannabinoid, cannabidiol, on the induction of apoptosis in leukemia cells. Exposure of leukemia cells to cannabidiol led to cannabinoid receptor 2 (CB2)-mediated reduction in cell viability and induction in apoptosis. Furthermore, cannabidiol treatment led to a significant decrease in tumor burden and an increase in apoptotic tumors in vivo. From a mechanistic standpoint, cannabidiol exposure resulted in activation of caspase-8, caspase-9, and caspase-3, cleavage of poly(ADP-ribose) polymerase, and a decrease in full-length Bid, suggesting possible cross-talk between the intrinsic and extrinsic apoptotic pathways. The role of the mitochondria was further suggested as exposure to cannabidiol led to loss of mitochondrial membrane potential and release of cytochrome c. It is noteworthy that cannabidiol exposure led to an increase in reactive oxygen species (ROS) production as well as an increase in the expression of the NAD(P)H oxidases Nox4 and p22(phox). Furthermore, cannabidiol-induced apoptosis and reactive oxygen species (ROS) levels could be blocked by treatment with the ROS scavengers or the NAD(P)H oxidase inhibitors. Finally, cannabidiol exposure led to a decrease in the levels of p-p38 mitogen-activated protein kinase, which could be blocked by treatment with a CB2-selective antagonist or ROS scavenger. Together, the results from this study reveal that cannabidiol, acting through CB2 and regulation of Nox4 and p22(phox) expression, may be a novel and highly selective treatment for leukemia.

Exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), an environmental pollutant, has been shown to cause thymic atrophy and apoptosis. However, whether TCDD alters the process of T-cell selection in the thymus is not clear. To this end, we investigated the effects of TCDD in the context of the HY-T-cell receptor (TCR) transgenic (Tg) mouse model. We noted that negatively selecting male HY-TCR Tg mice were significantly more sensitive to the thymotoxic effects of TCDD relative to positively selecting female HY-TCR Tg mice, including increased reduction in cellularity and increased induction of apoptosis. TCDD exposure also altered the thymocyte subset composition in HY-TCR Tg male but not female mice. In addition, TCDD treatment resulted in increased extracellularly regulated kinase phosphorylation and lymphocyte-specific protein tyrosine kinase expression in thymocytes of HY-TCR Tg male but not female mice. The increase in proportion of CD8+ mature thymocytes noted in HY-TCR Tg male mice was reflected in the periphery, with TCDD-exposed HY-TCR Tg male mice having increased numbers of CD8+ T cells. Finally, we noted that the proliferative response of HY-TCR Tg male T cells to HY(self)-Ag was enhanced after exposure to TCDD, whereas that of HY-TCR Tg female mice was decreased. Taken together, these data suggest that TCDD alters the process of thymic selection, possibly by enhancing negative thymocyte selection, whereas at the same time allowing autoreactive T cells to escape deletion in the thymus and immigrate to the periphery.

ABSTRACTExposure to bacterial superantigens such as staphylococcal enterotoxin B (SEB) leads to the induction of toxic shock syndrome which results in multiorgan failure, including liver damage. In the present study, we investigated the role of CD44 in SEB-induced liver injury. Injection of SEB into d-galactosamine-sensitized CD44 wild-type (WT) mice led to a significant increase in CD44 expression on liver T cells, NK cells, and NKT cells. Administration of SEB to CD44 knockout (KO) mice caused significantly enhanced liver damage which correlated with elevated numbers of T cells, NK cells, NKT cells, and macrophages in the liver and increased production of tumor necrosis factor alpha and gamma interferon compared to CD44 WT mice. Furthermore, liver mononuclear cells from CD44 KO mice were resistant to SEB-induced apoptosis, and cDNA microarray analysis revealed that SEB activation of such cells led to the induction of several antiapoptotic genes and repression of proapoptotic genes. Examination of CD44 isoforms revealed that SEB exposure altered CD44 variant 7 (v7) isoform expression. Interestingly, mice bearing a specific deletion of the CD44v7 exon exhibited increased susceptibility to SEB-induced hepatitis. Finally, treatment of CD44 WT mice with anti-CD44 monoclonal antibodies reduced expression of CD44 in liver mononuclear cells and caused increased susceptibility to SEB-induced liver injury. Together, these data demonstrate that the expression of CD44 and/or CD44v7 on SEB-activated liver mononuclear cells facilitates their rapid apoptosis, thereby preventing severe liver injury in wild-type mice, and suggest that CD44 plays an important role in the regulation and elimination of immune cells in the liver.

Patients with mutations in Fas develop autoimmune lymphoproliferative disease (ALPS), while their family members with similar mutations are often normal, thereby suggesting that additional factors may play a role in the development of ALPS. In the current study, we tested the role of CD44 in the development of lymphoproliferative disease by generating CD44(-/-)/Fas(-/-) mice, which failed to express CD44 and Fas, and compared them to CD44(+/+)/Fas(-/-) mice that expressed CD44, but not Fas. The results showed that CD44(-/-)/Fas(-/-) mice developed a more severe lymphoproliferative and autoimmune disease when compared to CD44(+/+)/Fas(-/-) mice. This was indicated by increased numbers of cells in their lymph nodes, and a greater proportion of B220(+)CD4(-)CD8(-) (double-negative) T cells as well as antibodies against single-stranded DNA and chromatin. The heightened severity of lymphoproliferative disease seen in CD44(-/-)/Fas(-/-) mice correlated with increased resistance of T cells, but not B cells, to undergo activation-induced cell death (AICD). The current study suggests that deficiency in CD44 in combination with a defect in one of the molecules involved in the death receptor family such as Fas can further down-regulate AICD, and exacerbate the lymphoproliferative and autoimmune disease.

T cells upon activation are known to up‐regulate CD44 expression. However, the precise function of CD44 on activated T cells is not clear. In this report, we demonstrate that signaling through CD44 plays an important role in activation‐induced cell death (AICD). CD44 knockout (KO) mice had an elevated in vivo primary and in vitro secondary response to challenge with conalbumin, anti‐CD3 mAb and staphylococcal enterotoxin A (SEA), which correlated with reduced AICD when compared to CD44 wild‐type mice. In addition, CD44 KO mice exhibited increased delayed‐type hypersensitivity response to dinitrofluorobenzene. In a model examining in vitro AICD, splenocytes from CD44 KO mice showed resistance to TCR‐mediated apoptosis when compared to splenocytes from CD44 wild‐type mice. In addition, signaling through CD44 led to increased apoptosis in TCR‐activated but not resting T cells from CD44 wild‐type mice without affecting Fas expression. Injection of SEA into mice deficient in CD44 and Fas (CD44 KO/lpr) led to an increased primary response when compared to mice that expressed CD44 but not Fas (CD44 WT/lpr), suggesting that the enhanced response to SEA was dependent on CD44 but not Fas expression. Administration of anti‐CD44 mAb into CD44 wild‐type mice caused a significant decrease in antigen‐specific T cell response. Together, these data implicate CD44 as an important regulator of AICD in T cells. Furthermore, targeting CD44 in vivo may constitute a novel approach to induce apoptosis in activated T cells, and therefore to treat autoimmune diseases, allograft rejection and graft versus host disease.

Delta(9)-tetrahydrocannabinol (THC), the main psychoactive component of marijuana has been shown to suppress the immune response. However, the exact mechanism of THC-induced immunosuppression remains unclear. In the current study, we tested the hypothesis that exposure to THC leads to the induction of apoptosis in lymphocyte populations. Splenocytes of C57BL/6 mice cultured in the presence of 10 microM or greater concentrations of THC showed significantly reduced proliferative response to mitogens, including anti-CD3 monoclonal antibodies (mAbs), concanavalin A (Con A), and lipopolysaccharide (LPS) in vitro. Thymocytes and naive and activated splenocytes exposed to 10 microM or 20 microM THC showed significantly increased levels of apoptosis. Treatment with CB2 antagonist inhibited THC-induced apoptosis in thymocytes and activated splenocytes. Administration of 10 mg/kg body weight of THC into C57BL/6 mice led to thymic and splenic atrophy as early as 6 h after treatment. This effect could be partially inhibited by treatment with a caspase inhibitor in vivo. THC exposure led to reductions in the numbers of all subpopulations of splenocytes and thymocytes examined. Functional studies revealed that splenocytes from THC-treated mice had significantly reduced proliferative response to anti-CD3 mAbs, Con A, and LPS in vitro. Finally, thymocytes and splenocytes exposed to THC in vivo exhibited apoptosis upon in vitro culture. Together, these results suggest that in vivo exposure to THC can lead to significant suppression of the immune response by induction of apoptosis.

In the current study, we examined whether ligation of CB2 receptors would lead to induction of apoptosis in tumors of immune origin and whether CB2 agonist could be used to treat such cancers. Exposure of murine tumors EL-4, LSA, and P815 to delta-9-tetrahydrocannabinol (THC) in vitro led to a significant reduction in cell viability and an increase in apoptosis. Exposure of EL-4 tumor cells to the synthetic cannabinoid HU-210 and the endogenous cannabinoid anandamide led to significant induction of apoptosis, whereas exposure to WIN55212 was not effective. Treatment of EL-4 tumor-bearing mice with THC in vivo led to a significant reduction in tumor load, increase in tumor-cell apoptosis, and increase in survival of tumor-bearing mice. Examination of a number of human leukemia and lymphoma cell lines, including Jurkat, Molt-4, and Sup-T1, revealed that they expressed CB2 receptors but not CB1. These human tumor cells were also susceptible to apoptosis induced by THC, HU-210, anandamide, and the CB2-selective agonist JWH-015. This effect was mediated at least in part through the CB2 receptors because pretreatment with the CB2 antagonist SR144528 partially reversed the THC-induced apoptosis. Culture of primary acute lymphoblastic leukemia cells with THC in vitro reduced cell viability and induced apoptosis. Together, the current data demonstrate that CB2 cannabinoid receptors expressed on malignancies of the immune system may serve as potential targets for the induction of apoptosis. Also, because CB2 agonists lack psychotropic effects, they may serve as novel anticancer agents to selectively target and kill tumors of immune origin.

In the current study, we investigated whether the growth of FasL-bearing tumor cells would induce apoptosis and toxicity in organs that express high level of Fas. Sera from C57BL/6 +/+(wild-type) mice injected with syngeneic FasL+ tumors, LSA, or EL-4, showed significantly higher levels of soluble FasL than that from the nontumor-bearing mice. Furthermore, the soluble FasL was functional inasmuch as the sera from tumor-bearing mice were able to induce apoptosis in Fas+ but not Fas−targets. Histopathologic studies and in situ TUNEL assay to detect apoptosis were carried out in C57BL/6 +/+(Fas+) or C57BL/6 lpr/lpr (Fas−) mice injected with syngeneic LSA and EL-4 tumor cells. The morphology of the liver and thymus from tumor bearing C57BL/6 +/+ mice showed marked damage and tissue destruction. In contrast, the liver and thymus from tumor-bearing C57BL/6 lpr/lpr mice showed minimal damage. Furthermore, the tumor-bearing C57BL/6 +/+, but not the C57BL/6 lpr/lpr, mice exhibited significant apoptosis in the liver and thymus. The FasL responsible for toxicity was tumor derived rather than host derived; tumor-bearing C57BL/6 gld/gld(FasL-defective) mice also exhibited significant apoptosis in the liver and thymus. Together, these data suggested that the in vivo growth of FasL-bearing tumor cells can induce significant apoptosis and toxicity in Fas+ tissues of the host. Such toxicity may be mediated by the soluble FasL produced by tumor cells.

More than two third share of electricity come from coal fired power plants in India. Coal fired power plants are the largest source of anthropogenic CO2 emissions per unit of electricity generation among all fossil fuel based power plants. There has been climate change and global warming globally due to increasing anthropogenic emission of greenhouse gas (GHG) into the atmosphere. This paper examines life cycle GHG emission such as CH4, CO2 and N2O of a National Thermal Power Corporation (NTPC) Limited power plant using life cycle approach. The various stages involved in the assessment of life cycle GHG emissions in the present study include coal mining, transportation of coal to the power plant and coal combustion for electricity generation. The results show that direct CO2 emission from coal combustion is about 890 g CO2-e/kWh, whereas life cycle GHG emissions amount to 929.1 g CO2-e/kWh. Indirect GHG emissions add up to 4.2% of total emissions. Coal mine methane leakage into atmosphere in India is low since more than 90% of the coal mining is surface mining.

In the current study, we investigated whether the naive, poly I:C or interleukin-2 (IL-2)–induced natural killer (NK)/lymphokine-activated killer (LAK) cells use perforin and/or Fas ligand (FasL) to mediated cytotoxicity. We correlated these findings with the ability of mice to reject syngeneic Fas+ and Fas- tumor cells either spontaneously or after IL-2 treatment. The spontaneous NK-cell–mediated cytotoxicity was primarily perforin based, whereas the poly I:C and IL-2–induced NK/LAK activity was both FasL and perforin dependent. L1210 Fas+ tumor targets were more sensitive than L1210 Fas- targets to poly I:C and IL-2–induced cytotoxicity in wild-type, gld/gld, and perforin knockout mice. When L1210 Fas+ and Fas– tumor cells were injected subcutaneously (sc) or intraperitoneally into syngeneic mice, Fas- tumor cells caused mortality earlier than Fas+ tumor cells. Also, approximately 20% of the mice injected sc with L1210 Fas+ tumor cells survived the challenge(>60 days), whereas all mice injected similarly with L1210 Fas- tumor cells died. When immunotherapy using IL-2 (10,000 U, three times/d for a week, followed by once/d for an additional week) was attempted in mice injected sc with tumor cells, IL-2 treatment was very effective against mice bearing L1210 Fas+ (40% survival) but not L1210 Fas- (0% survival) tumors. These data correlated with the finding that the LAK cells from IL-2–injected mice caused increased cytotoxicity against L1210 Fas+ when compared with L1210 Fas- targets. Also, L1210 Fas+tumor-bearing mice showed increased tumor-specific cytotoxic T lymphocyte (CTL) activity when compared with those bearing L1210 Fas- tumor cells. Together our studies show for the first time that expression of Fas on tumor targets makes them more immunogenic as well as susceptible to CTL- and IL-2–induced LAK activity. The Fas+ tumor cells are also more responsive to immunotherapy with IL-2.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental pollutant well known for its toxicity to the thymus. Recent studies from our laboratory demonstrated that TCDD induces apoptosis in thymocytes. In the current study, we investigated the mechanism of TCDD-induced apoptosis. Administration of a single dose of TCDD at 0.1, 1, 5, and 50 μg/kg body wt intraperitoneally, into C57BL/6 +/+ (wild-type) mice caused a dose-dependent decrease in thymic cellularity. In contrast, a similar treatment with TCDD, in Fas-deficient C57BL/6 lpr/lpr(lpr) or Fas-ligand defective C57BL/6 gld/gld(gld), mice failed to induce thymic atrophy at 0.1–5 μg/kg body wt of TCDD. In lprand gldmice, significant thymic atrophy was seen only at 50 μg/kg body wt of TCDD. Injection of TCDD caused apoptosis only in wild-type but not in lpror gldmice. The sera from TCDD-treated wild-type mice exhibited increased levels of soluble Fas ligand, inasmuch as incubation of Fas+, but not Fas−cells with the sera, triggered apoptosis. Also, TCDD-induced apoptosis in thymocytes was inhibited both in vitroand in vivoby caspase inhibitors. TCDD treatment caused significant up-regulation in the expression of FasL but not Fas mRNA in the thymocytes of wild-type mice. Also, such thymocytes exhibited marked alterations in the surface markers, characteristic of cells undergoing apoptosis. In contrast, TCDD treatment caused minimal phenotypic changes in thymocytes from lprand gldmice. Together, the current study demonstrates that Fas–Fas ligand interactions play an important role in TCDD-mediated induction of apoptosis and immunotoxicity.

3'-Azido-2',3'-dideoxythymidine (AZT)3 is extensively used in the treatment of human immunodeficiency virus (HIV) infection. Currently, it is debated whether AZT should be offered to symptomless HIV-infected individuals in the hope of delaying or even preventing progression to acquired immunodeficiency syndrome (AIDS). However, before the chronic use of AZT, it is essential to establish whether this drug alters the differentiation and functions of T cells particularly because HIV infects CD4+ T cells, as well as investigate whether AZT would alter the T cell response to HIV-encoded antigens. In the current study, therefore, we investigated the effect of administration of AZT into mice for 7-14 days on T cell differentiation in the thymus and the ability of T cells to respond to HIV antigens in the periphery. Our data demonstrated that AZT, when administered orally at 500 mg/kg body wt or higher concentrations for 14 days, caused marked thymic involution with significant decrease in the percentage of CD4+CD8+ T cells and increase in the percentage of CD4−CD8−, CD4+ and CD8+ T cells. AZT treatment did not affect the cellularity of the spleen or the ratios of T cells in the periphery. Also, splenic T cells from AZT-treated mice did not demonstrate marked decrease in their ability to respond to mitogens in vitro. However, when AZT-treated mice were immunized with foreign antigens, such as gp 120 of HIV or conalbumin, the T cells demonstrated significant decrease in their ability to respond to these antigens. When AZT was added to cultures, it was found to inhibit the proliferation of T cells to mitogens as well as the differentiation of cytotoxic T lymphocytes (CTL). Interestingly, addition of exogenous IL-2 to CTL cultures reconstituted the decreased CTL response. Furthermore, administration of IL-2 into AZT-treated mice could also reconstitute the decrease in thymic cellularity induced by AZT. These data indicate that AZT, when present at the time of T cell differentiation or responsiveness to antigens in vivo, can mediate significant inhibition of such functions thereby suggesting that AZT may affect the immune response to HIV antigens.Copyright 1995, 1999 Academic Press, Inc.

There is growing evidence to suggest that tumorigenic transformation of cells may result from aberrant regulation of autocrine growth factor production. In the current study we describe the spontaneous in vitro transformation of T-lymphocyte cell lines during routine cell culture as evidenced by autonomous growth without any requirement for stimulation or exogenous interleukin 2 (IL-2). These cells constitutively expressed the IL-2 gene and were inhibited from proliferating by addition of antibodies against IL-2, the IL-2 receptor, or IL-2 antisense oligonucleotides, thereby suggesting that the cell transformation resulted from IL-2-mediated autocrine growth. The transformed cells when injected into nude but not normal mice induced tumors that were inhibited by antibodies against IL-2 and the IL-2 receptor as well as by immunosuppressive drugs such as cyclosporin A. These studies demonstrate that aberrant regulation of IL-2 production can lead to spontaneous transformation of T cells in vitro, capable of inducing tumors in vivo. Our studies not only provide evidence for the important role played by autocrine growth factors in tumorigenicity but also stress the need to use caution while performing immunotherapy using in vitro-cultured T cells against cancer and viral infections, particularly in an immunodeficient host, to exclude any possible transfer of transformed mutant cells.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is well known for its immunotoxic effects particularly on the thymus. The exact mechanism by which TCDD induces thymic atrophy is not clear. In the current study, we investigated whether TCDD triggers apoptosis in thymocytes, when administeredin vivo,by using the TdT-mediated FITC–dUTP nick end labeling method and analyzing the cell flow cytometrically. Significant apoptosis was detected at 8–12 hr after the TCDD injection but not at 24 hr or beyond, up to 120 hr of study. Furthermore, the induction of apoptosis was confirmed using the JAM test in which thymocytes from TCDD-treated mice, labeled with [3H]thymidine, exhibited increased DNA fragmentation when compared to the controls. Similar to TCDD treatment, administration of dexamethasone (5 or 100 mg/kg) into C57BL/6 mice triggered apoptosis that was only detected at 12 hr after administration of the drug and not thereafter. When thymocytes from TCDD- or dexamethasone-treated mice were culturedin vitrofor 24 hr, they exhibited marked increase in apoptosis when compared to the vehicle-treated controls. However, TCDD, when added toin vitrocultures of thymocytes, failed to trigger apoptosis. Together, our studies demonstrate that TCDD can induce apoptosis in thymocytesin vivo.This can be detected only at an early stage following TCDD administration, possibly because of rapid clearance of apoptotic cells by the phagocytic cellsin vivo.

In the present communication, we describe the unique observation that Lyt-1+2-, L3T4+ T cells but not Lyt-2+ T cells isolated from the spleens of normal, unimmunized H-2d mice proliferate strongly and directly to an irradiated, syngeneic, Lyt-1+2-, L3T4+, Ia- autoreactive T cell line/clone. In contrast, Lyt-1+2- T cells failed to proliferate when stimulated by long-term, antigen-specific, H-2d-restricted T cell lines. Supernatants from the cultures of autoreactive T cells or recombinant interleukin 2 (IL-2) alone, failed to induce proliferation of the Lyt-1+2- T cells, suggesting that cell-cell interaction is essential for growth. In addition, the proliferative response of Lyt-1+2- T cells was independent of Ia+ antigen-presenting cells and was not blocked by either anti-Iad or anti-H-2d antibodies, but was inhibited by anti-L3T4 antibodies. All these observations suggested that the responding Lyt-1+2- T cells were recognizing the anti-self-Ia receptor expressed on autoreactive T cells and that such T cells might therefore express the internal image of self-Ia determinants. We suggest that the T-T interactions observed may represent interactions between two helper T cell subpopulations at the idiotope level and that autoreactive T cells may function as an important feature of the regulatory network and/or lead to the expansion of the T cell repertoire.

In the current study, we investigated the repercussions of the interaction between tumor cells (LSA) and the tumor-specific cytotoxic T lymphocyte (CTL) (PE-9) when both expressed Fas and Fas ligand (FasL). The CTL clone, PE-9, expressed high levels of Fas and FasL upon activation through the T-cell receptor (TCR). Furthermore, the activated PE-9 cells used both perforin- and FasL-based pathways to kill Fas-positive (Fas+) LSA tumor cells. Interestingly, LSA tumor cells also constitutively expressed FasL but not perforin, and killed Fas+ PE-9 CTLs and Fas+ but not Fas-negative (Fas−) activated T cells and thymocytes, as detected using the JAM test. PE-9 CTLs, cultured for 24 hours in the presence of cell lysates of FasL-bearing LSA cells but not FasL-deficient P815 cells, exhibited significant apoptosis as detected using the TUNEL method. Moreover, another FasL+ T-cell lymphoma line, EL-4, induced apoptosis in Fas+ but not in Fas− T cells in a similar fashion. The current study demonstrates for the first time that not only can the tumor-specific CTL mediate Fas-based killing of tumor cells, but FasL+ tumor cells can kill the Fas+ tumor-specific CTL. Thus, the survival of the tumor or the host may depend on which cell can accomplish this task more efficiently. The current study also suggests that FasL-based killing of CTLs by specific tumor cells may constitute a major limiting factor in successful immunotherapy.

CD44 is a widely distributed cell surface glycoprotein whose principal ligand has been identified as hyaluronic acid (HA), a major component of the extracellular matrix (ECM). Recent studies have demonstrated that activation through CD44 leads to induction of effector function in T cells and macrophages. In the current study, we investigated whether HA or monoclonal antibodies (MoAbs) against CD44 would induce a proliferative response in mouse lymphocytes. Spleen cells from normal and nude, but not severe combined immunodeficient mice, exhibited strong proliferative responsiveness to stimulation with soluble HA or anti-CD44 MoAbs. Furthermore, purified B cells, but not T cells, were found to respond to HA. HA was unable to stimulate T cells even in the presence of antigen presenting cells (APC) and was unable to act as a costimulus in the presence of mitogenic or submitogenic concentrations of anti-CD3 MoAbs. In contrast, stimulation of B cells with HA in vitro, led to B-cell differentiation as measured by production of IgM antibodies in addition to increased expression of CD44 and decreased levels of CD45R. The fact that the B cells were responding directly to HA through its binding to CD44 and not to any contaminants or endotoxins was demonstrated by the fact that F(ab)2 fragments of anti-CD44 MoAbs or soluble CD44 fusion proteins could significantly inhibit the HA-induced proliferation of B cells. Also, HA-induced proliferation of B cells was not affected by the addition of polymixin B, and B cells from lipopolysaccharide (LPS)-unresponsive C3H/HeJ strain responded strongly to stimulation with HA. Furthermore, HA, but not chondroitin-sulfate, another major component of the ECM, induced B-cell activation. It was also noted that injection of HA intraperitoneally, triggered splenic B cell proliferation in vivo. Together, the current study demonstrates that interaction between HA and CD44 can regulate murine B-cell effector functions and that such interactions may play a critical role during normal or autoimmune responsiveness of B cells.

In the current study, we investigated the role of interleukin-2 (IL-2) and IL-4 as autocrine growth factors responsible for autonomous growth of four murine tumor cell lines: LSA, a radiation leukemia virus-induced T-cell lymphoma; EL-4, a chemically triggered T-cell lymphoma; PE-3T, a T-cell line that underwent spontaneous transformation ex vivo; and P815, a mastocytoma. All tumor cell lines screened constitutively expressed IL-2 receptor (IL-2R) and IL-4R genes. However, only LSA and PE-3T cells expressed IL-2 and IL-4 genes constitutively, whereas EL-4 and P815 tumor cells expressed only IL-4 but not IL-2. Monoclonal antibodies (MoAbs) against IL-2, IL-4, or a combination of these, as well as MoAbs against IL-2R significantly inhibited the proliferation of LSA but not that of other tumor cell lines ex vivo. To exclude the possibility that, in other tumor cell lines, the autocrine growth factor may interact with its receptor within the cell, the ability of antisense phosphorothioate oligonucleotides to inhibit the growth of the tumor cells was tested. The antisense phosphorothioate oligonucleotides specific for IL-2, IL-4, IL-2Rβ, or IL-2Rγ chains, added in culture, could markedly inhibit the growth of LSA but not that of the other tumor cell lines screened. Inasmuch as IL-2Rβ and IL-2Rγ subunits also serve as a component of the receptors for IL-4, IL-7, IL-9, and IL-15, the above data suggested that such cytokine redundancy was not responsible for autonomous growth of the other tumor cell lines. Addition of exogenous IL-2 or IL-4 to the tumor cell cultures caused significant enhancement in the proliferation of PE-3T cells, whereas other cell lines were either not significantly affected or slightly inhibited from growing. Interestingly, the LSA tumor growth in nude mice was significantly inhibited after treatment of these mice with a combination of MoAbs against IL-2 and IL-4. Together, our studies show for the first time that IL-2 and IL-4 may serve as autocrine growth factors in the autonomous proliferation of tumor cells, particularly those that are retrovirally induced. Second, some tumor cell lines, despite expressing certain cytokines and their receptors constitutively, may not depend exclusively on such factors for autocrine growth.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a highly toxic environmental pollutant and is well known for inducing thymic atrophy in mice, although the exact mechanism of its action remains unclear. Recent studies from our laboratory demonstrated that TCDD induces apoptosis in thymocytes and that Fas−mice (lpr/lpr) were more resistant to TCDD-induced immunotoxicity when compared to the Fas+wild-type mice. Inasmuch as induction of apoptosis is associated with alterations in adhesion molecule expression, in the current study we analyzed the expression of a variety of surface molecules on thymocytes treated with TCDDin vivo.Interestingly, in thymocytes from mice treated with a single dose of 50 μg/kg body wt of TCDD, there was a significant increase in the density of expression of CD3, αβTCR, CD44, and IL-2R, and a decrease in the expression of J11d, CD4, and CD8 molecules when compared to the control thymocytes. These alterations were first visible 3 days after TCDD treatment and increased on Days 5 and 10 posttreatment. Furthermore, most of the alterations in the density of expression of various markers were dose dependent with minimal but significant changes at 0.1 μg and maximum alterations at 50 μg/kg body wt of TCDD. At most lower concentrations (0.1–5 μg/kg), TCDD caused alterations in the density of cell surface markers but not in the percentage of cells expressing a specific molecule. It is striking that the phenotypic alterations were similar to those seen in normal thymocytes undergoing spontaneous apoptosisin vitroas previously reported. Together, the current study suggests that TCDD treatment induces phenotypic changes in thymocytes that are similar to those seen in normal thymocytes undergoing apoptosis. Also, because detection of apoptosisin vivois difficult, phenotypic alterations in the density of thymocyte surface molecules may serve as a useful biomarker for toxicity involving apoptosis.

We have recently demonstrated that nitrosoureas such as 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) and chlorozotocin (CLZ) can cure almost 100% of mice bearing LSA tumor, syngeneic to C57BL/6 mice. In contrast, similar or higher doses of streptozotocin (STZ) completely failed to cure LSA-bearing mice. Further studies revealed that the efficacy of nitrosoureas may depend on their immunomodulating properties. In the current study, therefore, we investigated the effect of these nitrosoureas on the immune system of normal mice. Treatment of C57BL/6 mice with 5 intraperitoneal injections of 20 mg/kg body weight of BCNU or CLZ caused an increase in the percentage of CD4−CD8−T cells and a decrease in the percentage of CD4+CD8+T cells in the thymus. In addition, such treatment also caused an increase in the percentage of CD4+T cells without significantly affecting the CD8+T cells in the thymus. However, when total cellularity of the thymus was studied, BCNU and CLZ were found to decrease the total number of CD4+CD8+T cells without significantly affecting the other subsets. In contrast, similar or higher (100 mg/kg body weight) doses of STZ had no significant effect on the total number and percentages of T cell subsets in the thymus. Also BCNU and CLZ but not STZ treatment caused a 50% decrease in the total number of CD4+and CD8+T cells in the spleen. When T cells in the spleens of nitrosourea-treated mice were functionally analysed, it was observed that BCNU and CLZ caused a dramatic decrease in the T cell responsiveness to Con A, anti-CD3 and phorbol myristate acetate plus calcium ionophore stimulation. In contrast, STZ treatment failed to significantly inhibit the T cell responsiveness to these activation signals. Using the accessory cell-dependent and -independent assays, BCNU and CLZ were found to suppress the functions of both T cells and macrophages. Also, addition of growth factors such as IL-1, IL-2, IL-4 and IL-6 failed to reconstitute the defective responsiveness of BCNU- and CLZ-treated T cells and macrophages. Together our data suggest that nitrosoureas have varying immunomodulating properties and this may in turn determine their efficacy in the treatment of cancer.

IMPORTANCE: Operating room (OR) turnaround times (TATs) and on-time first-case starts (FCSs) are commonly used measures of OR efficiency. Prolonged TATs and late FCSs occur frequently at academic medical centers. OBJECTIVE: To test the hypothesis that establishing a financial incentive program (FIP) for OR teams would improve efficiency, leading to decreased TATs and improved on-time FCSs. DESIGN, SETTING, AND PARTICIPANTS: Prospective study to evaluate the effect of an FIP on OR efficiency between March 1, 2013, and December 31, 2013, at a freestanding academic trauma hospital. Participants were all OR team members and included anesthesiologists, certified registered nurse anesthetists, nurses, and technicians. INTERVENTIONS: Operating room efficiency awareness education was conducted before FIP implementation beginning in February 2013. Each eligible OR team member achieving a TAT of 60 minutes or less or an on-time FCS was awarded 1 point. Reports listing individual performances were posted. Pay bonuses were awarded for achieving 1 of 3 progressive point totals in any month. MAIN OUTCOMES AND MEASURES: Outcomes were TAT, which was defined as “wheels out” to “wheels in,” and on-time FCS, which was defined as “wheels in” within 6 minutes of the scheduled start time. RESULTS: Before FIP implementation, the mean TAT varied between 77 and 83 minutes, with only 18% to 26% of TATs being 60 minutes or less; on-time FCSs averaged 29% to 34%. After FIP implementation, on-time FCSs improved from 31% to 64% (P < .001), and TATs of 60 minutes or less increased from 24% to 52% (P < .001). The cost of a 2-month FIP was $8340. We saved 13 minutes per TAT, for an estimated savings of $177 000. We estimate an additional savings of $33 000 for on-time FCSs, for a total hospital savings of $210 000. CONCLUSIONS AND RELEVANCE: A novel FIP improved OR efficiency. Given the small amount of money involved, it seems unlikely that financial incentives were solely responsible. Effectively communicating the importance of TATs and on-time FCSs and publishing individual results more likely increased staff awareness. Teamwork created by linking assignment of points to a team result likely contributed to success.