Combined use of topical cyclosporin A and silver sulphadiazine on allografts infected with Pseudomonas in burned rats

In this study, we investigated the effects of a combination of topical Cyclosporin A (CyA) and silver sulphadiazine on infected and normal skin allografts in burned and unburned rats. Buffalo (Buf) allografts on unburned Lewis (Lew) recipients survived 16.8 +/- 0.9 days after a 10-day course of topical CyA and silver sulphadiazine, as compared to 7.4 +/- 1.1 days for untreated allografts and 18.6 +/- 0.9 days for those receiving CyA only for 10 days. Allografts infected with Pseudomonas aeruginosa and placed on burned recipients survived significantly longer (13.7 +/- 1.1 days) when treated with silver sulphadiazine and topical CyA for 7 days compared to a similar group of animals treated with silver sulphadiazine alone (8.4 +/- 1.0 days). The mortality rate for burned, infected and allografted animals receiving no medication or topical CyA was 50 per cent compared with zero in animals treated with silver sulphadiazine only or combined silver sulphadiazine and topical CyA.     

1alpha,25-dihydroxyvitamin D3 shows strong and additive immunomodulatory effects with cyclosporine A in rat renal allotransplants.

BACKGROUND: Vitamin D3 and its metabolites have long been found to exert immunosuppressive effects both in vivo and in vitro. The present study investigated the effect of 1alpha,25-dihydroxycholecalciferol (1,25DHC) on vascularized renal allografts in rats. METHODS: Three days prior to transplantation, two groups of animals were subjected to 1,25DHC (1 microg/kg/day IP) and a low calcium diet, which was continued until the end of the experiments. Recipient organs were removed and single allografts were transplanted in a high responder strain combination (ACI --> Lewis). Following transplantation, low-dose cyclosporine A (3.2 mg/kg/day CsA) administration was started in two experimental groups of recipients (one group receiving 1,25 DHC additionally) whereas the control allograft recipients received no immunosuppression (control III). Graft survival and renal function was monitored until death or the end of experiments and allograft rejection was assessed histologically using the Banff classification. RESULTS: 1,25DHC significantly prolonged allograft survival in comparison to control III (9.6 +/- 1 vs. 5.7 +/- 0.2 days; P=0.009). In addition, a combination of 1,25DHC and low-dose CsA increased allograft survival compared to CsA administration alone (24 +/- 0.9 vs. 13 +/- 0.3 days; P=0.008). 1,25DHC preserved renal creatinine clearance and decreased proteinuria in comparison to control III, and the combination of 1,25DHC and low-dose CsA again showed an additive effect on preservation of renal function. 1,25DHC and low-dose CsA both decreased interleukin (IL)-2 and IL-12 expression levels in serum and allografts, and a combination treatment produced the strongest attenuation of IL-2 and IL-12 expression. In addition, 1,25DHC increased IL-4 and IL-10 expression levels in allografts, whereas CsA alone did not alter IL-4 and IL-10 expression. In contrast, combination of 1,25DHC and low-dose CsA showed a significant increase in IL-10 expression levels whereas IL-4 expression was not elevated. CONCLUSION: Monotherapy with 1,25DHC significantly prolongs survival of renal allografts and preserves graft function in rats. A combination of 1,25DHC and CsA caused an additive effect on graft survival with differential regulation of pro- and anti-inflammatory cytokines, as compared to 1,25DHC administration alone.     

Low-dose immunosuppression in a rat hind-limb transplantation model

Composite tissue allografts (CTAs) offer an alternative to conventional reconstructive methods. However, the toxicity of the drugs that are required to prevent rejection has prevented its widespread clinical application. The purpose of this study was to determine whether a low-dose, corticosteroid-free combination regimen of tacrolimus and mycophenolate mofetil (MMF) would prevent rejection in a rat hind-limb model, with minimal toxic side effects. Three groups were used in this study. In group I, Wistar Furth (WF) rats received a syngeneic WF hind-limb. In groups II and III, WF rats received an ACI hind-limb. The latter were treated with tacrolimus–MMF. Assessment for rejection, flow cytometry, and mixed lymphocyte reactions was performed. Biopsies were taken regularly and at the time of killing. Combination therapy with low-dose tacrolimus–MMF effectively prolonged CTA survival indefinitely, with minimal side effects. Toxicity associated with immunosuppressive drugs can be avoided in a low-dose combination corticosteroid-free regimen.     

Low-dose immunosuppression in a rat hind-limb transplantation model

Composite tissue allografts (CTAs) offer an alternative to conventional reconstructive methods. However, the toxicity of the drugs that are required to prevent rejection has prevented its widespread clinical application. The purpose of this study was to determine whether a low-dose, corticosteroid-free combination regimen of tacrolimus and mycophenolate mofetil (MMF) would prevent rejection in a rat hind-limb model, with minimal toxic side effects. Three groups were used in this study. In group I, Wistar Furth (WF) rats received a syngeneic WF hind-limb. In groups II and III, WF rats received an ACI hind-limb. The latter were treated with tacrolimus-MMF. Assessment for rejection, flow cytometry, and mixed lymphocyte reactions was performed. Biopsies were taken regularly and at the time of killing. Combination therapy with low-dose tacrolimus-MMF effectively prolonged CTA survival indefinitely, with minimal side effects. Toxicity associated with immunosuppressive drugs can be avoided in a low-dose combination corticosteroid-free regimen.     

Delayed rejection of MHC class II-disparate skin allografts in mice treated with farnesyltransferase inhibitors

Farnesyltransferase inhibitors (FTIs), developed as anti-cancer drugs, have the potential to modulate immune responses without causing nonspecific immune suppression. We have investigated the possibility that FTIs, by affecting T cell cytokine secretion, can attenuate alloreactive immune responses. The effects of FTIs on murine alloreactive T cells were determined both in vitro, by measuring cytokine secretion or cell proliferation in mixed lymphocyte cultures, and in vivo, by performing skin allografts from H-2(bm12) mice to MHC class II-disparate B6 mice. We found that two different FTIs, ABT-100 and L-744,832, blocked secretion of IFN-gamma, IL-2, IL-4, and TNF-alpha from na?ve T cells in vitro. ABT-100 and L-744,832 blocked cytokine production from both CD4(+) and CD8(+) na?ve T cells stimulated with CD3 and CD28 antibodies, but only if the cells were pretreated with the FTIs for 48 h. Proliferation of alloreactive T cells in mixed lymphocyte cultures was blocked by either FTI. We also found that the proliferation of enriched T cells stimulated with IL-2 was blocked by ABT-100 treatment. In mice with an MHC class II-disparate skin graft, rejection of primary allografts was significantly delayed by treatment with either ABT-100 or L-744,832. Secondary rejection in mice previously primed to the alloantigen was found to be unaffected by L-744,832 treatment. We have shown that FTIs can block T cell cytokine secretion and attenuate alloreactive immune responses. The ability of FTIs to block secretion of cytokines, including IFN-gamma and IL-4, from na?ve T cells provides a likely biological mechanism for the specific suppression of class II MHC-mediated allorejection. These results demonstrate that FTIs may have useful immunomodulatory activity due to their ability to delay priming to alloantigens.     

The modulatory effect of diltiazem on human in vitro alloreactivity when used alone or in combination with cyclosporin A and/or methylprednisolone

The calcium channel blocker diltiazem is often included in post-transplant regimens in combination with other immunosuppressive drugs such as cyclosporin A (CyA). It is primarily used because of its antinephrotoxic and antihypertensive effects, so that undesirable side effects induced by the immunosuppressive therapy can be reduced. Its alleged ability to induce direct immunosuppression may explain the encouraging results from its clinical use and would appear to encourage a much wider use of this drug. The present study shows the effect of diltiazem on the human in vitro alloresponse when used alone or in combination with cyclosporin A (CyA) and methylprednisolone (MP). The results show that, when administered alone, diltiazem exerts a suppressive effect, but only at high, non-therapeutic doses. Interestingly, in combination with CyA or MP, diltiazem enhances the suppressive effect of these two drugs on in vitro alloresponses at lower doses. This additional effect of diltiazem may contribute to better graft survival in clinical transplantation. Received: 26 February 1997 Received after revision: 9 June 1997 Accepted: 3 July 1997     

Immune responsiveness in renal transplant recipients: mycophenolic acid severely depresses humoral immunity in vivo

BACKGROUND: Current immunosuppressive drug treatments for renal transplant recipients result in high one-year graft survival rates. Despite adequate suppression of the immune response directed to the allograft, the immune system remains able to cope with many infectious agents. METHODS: To define the influence of distinct immunosuppressive treatment protocols, primary and secondary cellular and humoral immune responses in groups of renal transplant recipients were studied: patient treated with prednisolone and cyclosporine A (P/CsA); with IgA CD3 monoclonal antibody as a rejection treatment superimposed on prednisolone and cyclosporine A (IgA CD3 mAb+P/CsA); and with prednisolone, cyclosporine A and mycophenolate mofetil (P/CsA/MMF). RESULTS: Primary in vitro proliferative responses to the protein antigen keyhole limpet hemocyanin (KLH) were not significantly disturbed in P/CsA treated patients, or in IgA CD3 mAb+P/CsA and P/CsA/MMF treated patients. In vitro proliferative responses to the recall antigen tetanus toxoid (TT) were similarly unaffected. Antigen-specific antibody responses to immunization with KLH and TT were not affected by treatment with P/CsA, or by IgA CD3 mAb+P/CsA, but were severely disturbed in patients treated with P/CsA/MMF. All patients displayed a profound inhibition of the delayed-type hypersensitivity skin reactivity to KLH and recall antigens. Nevertheless, in most patients with P/CsA treatment, T cell infiltrates were observed in skin biopsies from the site of KLH challenge, while expression of intercellular cell adhesion molecule-1 (ICAM-1) expression in challenged skin was significantly decreased in these patients. The balance between T helper 1 and T helper 2 cells was unaffected by immunosuppressive treatments during one year of follow-up. CONCLUSIONS: Immunosuppressive drug treatment with P/CsA inhibits delayed-type hypersensitivity skin reactions to both primary and frequently encountered antigens. Histological studies indicate an effect on ICAM-1 expression, leaving the influx of CD3pos T cells unaffected. Administration of a 10-day course of IgA CD3 mAb does not add profound immunosuppressive effects on the measured parameters. In contrast, addition of treatment with MMF profoundly decreases both primary and secondary humoral immune responsiveness in vivo. Finally, no effect of the studied immunosuppressive drugs on Th1/Th2 balance in vivo was measured.     

The survival and nature of the immune response to soft-tissue and composite-tissue allografts in rats treated with low-dose cyclosporine

We studied a variety of soft-tissue and composite-tissue allografts (CTA) in a histoincompatible rat model to determine the outcome and the nature of the immunologic responses to these tissues using continuous low-dose cyclosporine (CsA) therapy. Brown-Norway (RT1n) rats served as donors of soft tissue and CTA to Lewis (RT1l) rat recipients given low-dose CsA immunosuppressive therapy by gavage. Nine groups were studied. Three control groups were not treated with CsA: group 1, skin grafts alone; group 2, skin flaps alone; and group 3, skin grafts and delayed vessel allotransplants. Six groups were treated with CsA: group 4, skin grafts alone; group 5, skin flaps alone; group 6, skin grafts and delayed vessel allotransplants; group 7, aortas alone; group 8, muscle flaps alone; and group 9, bone grafts alone. Isografts were performed in all groups as technical controls. The appearance posttransplant of donor-directed cytotoxic antibodies was determined in recipient serum using a complement-mediated cytotoxicity assay and was compared to control and pretransplant sera. In the absence of CsA therapy, recipients in groups 1, 2, and 3 rejected their allografts early (8.5-9.4 days) and developed profound antidonor cytotoxic antibody activity posttransplant by day 7. Groups 4, 5, 6, 7, and 9 had prolonged graft survival in the presence of low-dose CsA, despite the presence of antidonor antibody activity. By contrast, group 8 (muscle flaps) were all uniformly rejected in the presence of profound recipient cytotoxic antidonor antibody activity. These results suggest that long-term soft-tissue and CTA survival can be achieved in histoincompatible rat recipients using continuous low-dose CsA immunosuppressive therapy despite the presence of cytotoxic antidonor antibodies.     

Differential effects of immunosuppressive drugs on chemokine receptor CCR7 in human monocyte-derived dendritic cells: selective upregulation by rapamycin

BACKGROUND: Appropriate recruitment of dendritic cells (DC) at sites of inflammation and migration to secondary lymphoid organs is of critical importance for the initiation of Ag-specific immune responses. The proper localization of DC in selected tissues is guided primarily by the coordinated expression of chemokine receptors (CKR). Here we show that immunosuppressive drugs have divergent effects on the modulation of CKR in maturing DC. METHODS AND RESULTS: Dexamethazone (DEX) and IL-10 inhibited human DC migration to CCL19 in vitro and mouse DC migration to lymph nodes (LN) in vivo, by impairing CCR7 expression. The calcineurin inhibitors cyclosporine A (CsA) and tacrolimus (FK506) were characterized by the inability to modulate CKR expression and migratory activity. Rapamycin (RAPA) increased DC migration to CCL19 in vitro and to LN in vivo by enhancing CCR7 expression. This effect could be mediated, in LPS-maturing DC, by the inhibition of autocrine IL-10 production. The in vivo data obtained with ex vivo RAPA treated DC were confirmed in a model of in vivo drug administration in mice, suggesting a potential clinical relevance. CONCLUSIONS: These findings demonstrate that immunosuppressive agents differently modulate the CKR switch associated with maturing DC; in particular, RAPA selectively up-regulates CCR7 and enhances the migration of differentiated DC to regional LN. This study contributes to a better understanding of the role of immunosuppressive therapy on DC migration, a potentially relevant check point of immunosuppressive treatment.     

Effects of immunosuppressive drugs on functions of activated T lymphocytes. Cyclosporine inhibition of gamma interferon production in the presence of interleukin

Expression of HLA DR by tubular cells of renal allografts of patients treated with cyclosporine (CsA) is less than that from patients treated with a combination of methylprednisolone (MPRED) and azathioprine (AZA). To examine the reason for this difference, the effects of immunosuppressive drugs on functions of alloactivated mononuclear cells, which had been primed in culture without added immunosuppressive drugs, was compared. CsA, 0.1 microgram/ml, inhibited gamma interferon production by 79 +/- 7% and in the presence of interleukin 2 (IL-2) by 82 +/- 10%. CsA, 0.1 microgram/ml, inhibited cytotoxic effector function by 11 +/- 12% and proliferation of cells that had been washed to remove lymphokines by 61 +/- 17% but only 17 +/- 8% in the presence of IL-2. MPRED, 20 micrograms/ml, inhibited gamma interferon production by washed alloactivated cells by 79 +/- 12% and 59 +/- 7% with IL-2. MPRED, 20 micrograms/ml, inhibited proliferation of washed cells by 36 +/- 4 and 86 +/- 3% with or without IL-2, respectively, and it inhibited cytotoxic effector function by 71 +/- 16%. AZA and its metabolites 6-mercaptopurine and 6-thioinosinic acid had little inhibitory effect on any tested function of activated lymphocytes at a concentration of 0.2 microgram/ml. These results indicate that CsA has a greater inhibitory effect on gamma interferon production by activated lymphocytes in the presence of IL-2 than MPRED or AZA in vitro, which may explain their differential effects on renal tubular cell HLA DR expression in vivo. Gamma interferon production was the only activated lymphocyte function tested that was inhibited by CsA in the presence of IL-2. MPRED was able to inhibit all functions, albeit to a lesser degree, in the presence of IL-2 or of IL-2-containing culture supernatants.     

Mechanism of the immunosuppressive effect in vivo of novel immunosuppressive drug beta-SQAG9, which inhibits the response of the CD62L+ T-cell subset

INTRODUCTION: We synthesized sulfo-glycolipid, beta-SQAG9 (designate square beta-SQAG9 liposome, because it efficiently forms a liposome structure) that possessed immunosuppressive effects such as inhibition of T-cell responses in human allogeneic MLR and skin allograft survival in rats, and bound to CD62L (L-selectin) in vitro. In this study, we further investigated the immunosuppressive mechanism in vivo by beta-SQAG9 liposome in a skin-allografted rat model. METHODS: ACI rats (RT1(a)) were grafted skin of LEW rats (RT1(1)) treated with PBS or beta-SQAG9 liposome IV once a day for 7 days. Subsequently, we investigated the population of T cells and CD62L(+) T-cell subset in the spleen, axillary lymph nodes (ALNs), and peripheral blood of skin-allografted rats by two-color flow cytometry. RESULTS: Five of 11 (45.5%) rats that were treated with 50 mg/kg beta-SQAG9 liposome showed graft survival and another showed moderate rejection in graft. The CD62L(+) T-cell subset population in ALNs of beta-SQAG9 liposome-treated rats decreased in a dose-dependent manner. No significant difference in the T-cell population was observed between the beta-SQAG9 and control groups. These data suggest that beta-SQAG9 could bind to the CD62L(+) T-cell subset in vivo as well as in vitro and affect T-cell migration, which might lead to T-cell tolerance in vivo.     

Low-dose cyclosporine mediates donor hyporesponsiveness in a fully mismatched rat kidney transplant model

Tolerance induction protocols have been successfully tested in animal models, yet their compatibility with immunosuppressive drugs remains to be fully elucidated. Our own previous data have indicated that cyclosporine A (CsA) affects the balance of effector and regulatory mechanisms with low-dose CsA doses promoting hyporesponsiveness. Here, we present a fully mismatched rat kidney model in which low-dose CsA treatment induces donor hyporesponsiveness to secondary renal allografts. Lewis recipients of DA kidney grafts received low, medium or high doses of CsA × 10 days. By 30 days, the primary transplant was removed and a second transplant of donor origin was engrafted. Following low-dose CsA, but not high-dose CsA treatment of the primary recipient, secondary transplants were accepted long-term in the absence of immunosuppression. Regulatory T-cell function was unimpaired and independent of the CyA dosage. Of note, low-dose CsA significantly reduced alloantibody titers in primary recipients. Adoptive transfer of graft infiltrating cells or splenocytes from hyporesponsive recipients supported long-term acceptance of donor kidney allografts. These results demonstrate a dose-dependent and transferable "pro-tolerogenic" effect of low-dose CsA treatment. This model is of clinical relevance to test the interference of CsA with tolerance induction in the absence of additional immunosuppression.     

Immunosuppressive activities of rat antisera raised against supernates from skin-graft-primed cells

Rat antisera raised against supernates derived from draining lymph node cells of skin-graft-primed mice exhibit a number of immunosuppressive effects in vitro and in vivo. The skin graft-induced, cell-free-supernates had been demonstrated to contain a number of helper activities that led to an antigen-specific induction of cytolytic T lymphocytes and/or to the induction of interleukin-2 synthesis. The rat antisera administered to skin graft recipients resulted in prolongation of major-histocompatibility-complex-incompatible skin graft survival. The rat antisera appear to have a specificity for the inhibition of T cell responses in vitro, although binding to B and T cells was apparent. The responses of unprimed cells to T cell mitogens and alloantigens are blocked, whereas B cell responses to the lipopolysaccharide mitogen are not blocked by the antisera. The generation of cytolytic T lymphocytes and the cytolytic functions of such cells are both blocked by the rat antisera. The inhibition of the differentiation pathway in cells cultured continuously with the antisera was overcome only through the addition of conditioned medium obtained from stimulated concanavalin A rat spleen cells, as opposed to mouse cell conditioned media. The rat antisera do not appear to block T cell responses via the IL-2 receptor, and were found to be substantially less effective against activated and proliferating T cells. These rat antisera have allowed us to further examine the pathways involved in T cell responses.     

Attenuation of airway obliteration by ciprofloxacin in experimental posttransplant bronchiolitis obliterans

BACKGROUND: Ciprofloxacin is widely used to treat respiratory tract infections. Like other fluoroquinolones, ciprofloxacin has immunomodulatory effects; however, it is unknown whether these effects are beneficial in the setting of lung transplantation. We investigated potential immunomodulatory effects of ciprofloxacin in a model of posttransplant bronchiolitis obliterans. METHODS: The heterotopic tracheal transplantation model in rats was used. Three groups received ciprofloxacin and underwent different immunosuppressive regimens of cyclosporine A, that is, no immunosuppression, insufficient immunosuppression, or low-dose immunosuppression. Three groups underwent the same immunosuppressive regimen but had no ciprofloxacin treatment. Tracheas were harvested after 21 days and examined with respect to histology and expression of selected cytokines. RESULTS: The allografts of animals treated with ciprofloxacin showed less airway obliteration compared with allografts of untreated animals. When combined with low-dose immunosuppression ciprofloxacin showed beneficial effects in preventing airway obliteration and rejection of the respiratory epithelium. Cytokine gene expression of the allografts treated with ciprofloxacin was higher with respect to transforming growth factor-beta and equal with respect to tumor necrosis factor-alpha and interferon-gamma compared with controls. When applied in combination with cyclosporine A, ciprofloxacin lowered the expression of transforming growth factor-beta and tumor necrosis factor-alpha and increased interferon-gamma expression. CONCLUSION: Ciprofloxacin attenuates airway rejection after tracheal transplantation. Genetic expression of mediators that are known to play an important role in mediating rejection in this model supports an immunomodulatory and antifibrotic role of ciprofloxacin. These findings suggest that further clinical studies are needed to investigate whether ciprofloxacin in addition to its bactericidal effect might be beneficial in the treatment of human posttransplant bronchiolitis obliterans.     

Allo-specific T-Cells Encoding for Viral IL-10 Exert Strong Immunomodulatory Effects in vitro but Fail to Prevent Graft Rejection

Recently, we demonstrated the capacity of allo-specific gene-engineered T lymphocytes as transport vehicle for therapeutic transgenes into allografts. In this study, the influence of viral IL-10 as therapeutic transgene was addressed. Lewis rat T-cell lines specific for DA rat alloantigens were engineered to express vIL-10 by using a retroviral gene expression system. Like T regulatory 1 cells, vIL-10 transgenic T lymphocytes express the phenotype CD4(+)25(+) and secrete, in addition to vIL-10, rat IL-10 and IFN-gamma but no IL-4. First, the capacity of vIL-10 transgenic T-cell lines to modulate alloantigen-specific immune responses was evaluated in vitro. In comparison to control MLR with no transgenic cells or equal numbers of control T(EGFP)-lymphocytes, the proliferation as well as production of IFN-gamma by naive responder cells were significantly diminished. Despite this regulatory capacity in vitro, T(vIL-10)-lymphocytes were not able, either alone or in combination with suboptimal doses of Cyclosporine A, to prolong the survival of either DA rat cardiac or renal allografts in Lewis rat recipients. These data demonstrate that intra-graft IL-10 over-expression is not sufficient to prolong allograft survival in a high-responder strain combination and that the regulatory capacity of T cells in vitro does not predict their in vivo efficiency.     

Enhancement of skin allograft survival by soluble alloantigen is serum-mediated.

H-2d mice treated before skin grafting with either soluble alloantigen prepared from H-2b cells or with serum obtained from similarly sensitized mice showed a significantly prolonged survival of H-2b skin allografts. This in vivo enhancement was paralleled by a depression of cell-mediated cytotoxicity to [51Cr]H-2b target cells in vitro. Such in vitro and in vivo effects were abrogated when mice were repeatedly injected with soluble alloantigen before and after skin grafting, demonstrating that enhancing antibody present could be neutralized by this mode of antigen administration. The data suggest that serum factors present in mice pretreated with soluble alloantigen lead to specific blocking at the induction phase of the cell-mediated immune response and result in skin graft enhancement.     

Renal allograft immunosuppression

The long-term effects of different immunosuppressive drugs and regimens on renal allograft histology are virtually unknown. Therefore, in order to investigate the long-term effects of triple drug treatment versus different combinations of two immunosuppressive drugs on allograft histology, a prospective, randomized trial was performed. One group received triple therapy consisting of low-dose cyclosporin (CyA), azathioprine (Aza), and methylprednisolone (MP), and three groups received combinations of two drugs, i.e., Aza plus CyA, Aza plus MP, and CyA plus MP. At 2 years, there were no significant differences with regard to graft (80%) or patient (87%) survival, or to graft function between the four groups. After 2 years, a protocol core biopsy was taken of all 102 patients having a functioning graft. Of these patients, 61 (60%) were still following the original, randomized treatment protocol; in the remaining cases, changes had occurred in the original protocol and so these cases were considered drop-outs in this study. Histological specimens were examined blindly by two independent observers. Most of the 34 histological variables examined showed no changes. Diffuse fibrosis was most frequent in the CyA plus MP group (70%) and significantly more severe than in the triple therapy group. Mesangial matrix increase in glomeruli was significantly less common in the triple therapy group (8%) than in any one of the double drug combination groups (47%). Two other changes in glomeruli--Bowman capsular thickening and global glomerular sclerosis--were also less frequent in the triple therapy group.(ABSTRACT TRUNCATED AT 250 WORDS)