Suppression Of Graft Rejection Research Articles

Actions of progesterone on uterine immunosuppression and endometrial gland development in the uterine gland knockout (UGKO) ewe

In ewes, the uterine gland knockout (UGKO) phenotype is caused by neonatal exposure to norgestomet to arrest uterine gland development and produce an adult which has a uterus characterized by the lack of endometrial glands. Since endometrial glands in the sheep produce the lymphocyte-inhibitory protein, ovine uterine serpin (OvUS), an experiment was conducted with ewes of the UGKO phenotype to evaluate whether the inhibitory actions of progesterone on tissue rejection responses in utero are dependent upon the presence of endometrial glands. Control and UGKO ewes were ovariectomized and subsequently treated with either 100 mg/day progesterone or corn oil vehicle for 30 days. An autograft and allograft of skin were then placed in each uterine lumen and treatments were continued for an additional 30 days before grafts were examined for survival. All autografts survived and had a healthy appearance after histological analysis. Allografts were generally rejected in ewes treated with vehicle but were present for hormone-treated ewes, regardless of uterine phenotype. Analysis of the histoarchitecture and protein synthetic capacity of the uterus revealed that progesterone induced differentiation of endometrial glands and synthesis and secretion of OvUS in UGKO ewes. The UGKO ewes had reduced density of CD45R+ lymphocytes in the endometrial epithelium and there was a tendency for progesterone to reduce this effect in luminal epithelium. Taken together, results confirm the actions of progesterone to inhibit graft rejection response in utero. Responses of UGKO ewes to progesterone indicate that the hormone can induce de novo development and differentiation of endometrial glands, at least when skin grafts are in the uterus.

Adenovirus-Mediated XIAP Gene Transfer Reverses the Negative Effects of Immunosuppressive Drugs on Insulin Secretion and Cell Viability of Isolated Human Islets

Immunosuppressive drugs are routinely used to provide tolerance after whole pancreas and islet cell transplantations. While they are essential in inhibiting graft rejection, little is known about their effect on islet function and beta-cell viability. In this study, we report that tacrolimus, sirolimus, and mycophenolic acid, when added to cultures of freshly isolated human islets, induce a downregulation of the synthesis and secretion of insulin. These functional changes are associated with decreased islet cell viability. All three agents induce a decrease of intracellular levels of Bcl-2 and Bcl-xL, with an increased level of Smac, indicating that they are capable of promoting a downregulation of anti-apoptotic factors and an accumulation of pro-apoptotic mediators. Transduction of islet cells with the anti-apoptotic gene XIAP prevents the negative effects of these drugs on the function and viability of islets. XIAP-infected cells show a higher expression of phospho-CREB (cAMP-responsive element binding protein) and a reduced level of Smac, resulting in a significant reduction of apoptotic cells and a preservation of the glucose-dependent secretion of insulin. In conclusion, the present study demonstrates that genetically modified human islets expressing XIAP are resistant to the negative effects of immunosuppressive drugs on insulin secretion and cell viability.

Effect of ciclosporin A on the organogenesis and function of embryonic metanephroi allografted into adult rats

To evaluate the effect of ciclosporin A (CsA) on the organogenesis and function of embryonic metanephroi allografted into adult rats. The whole metanephroi from the 15, 16 and 17 embryonic day-old (E15, E16, E17) embryos of Sprague-Dawley (SD) rat were allografted into the omenta of SD adult rats with their left kidneys resected, which were divided into 2 categories: 3 CsA-treated groups of 10 rats (E15CsASD, E16CsASD, and E17CsASD) and 3 non-CsA-treated groups of 10 rats (E15SD, E16SD, and E17SD). Thirty SD rats without kidney resection were divided into 6 equal groups and underwent allografting embryonic metanephroi in the same manner as mentioned above. The E15 metanephroi of Lewis rats were allografted into the omenta of Thirty adult Brown Norway (BN) rats with one kidney resected. Were divided into 6 equal groups, received allografting of embryonic metanephroi, and injected with CsA of normal saline in the manner as mentioned above (E15CsABN and E15BN). Two to 4 weeks after implantation, the metanephroi allografted in host rats were removed for histopathological examination or anastomosed for renal function measurement 4 weeks later. (1) Four weeks after implantation, the E17SD and E16SD metanephroi showed signs of acute rejection as hypercellular glomeruli and mononuclear cell infiltration in the interstitium. The E16CsASD and E17CsASD metanephroi formed mature nephrons and collecting ducts with few lymphocytic infiltrates. After CsA was discontinued, the E16CsASD and E17CsASD metanephroi were rejected fully within 21 days. (2) 4 weeks after implantation, the E15SD metanephroi were enlarged, became vascularized, and developed mature tubules and glomeruli; however, they were rejected by 100 days after implantation. The E15 Lewis metanephroi were fully rejected within two weeks in the BN adult rats. With CsA administrated, the E15 Lewis metanephroi developed normal mature nephrons and collecting ducts within the adult BN rats. If CsA was discontinued, the E15CsABN metanephroi were rejected. (3) The E15CsASD metanephroi had significantly lower values of wet weight (P = 0.006) and higher values of creatinine clearances (P = 0.007) than the E15SD metanephros transplants, but were identical to those of the E16CsASD metanephroi (P = 0.948, P = 0.840). (4) The metanephroi did not grow or differentiate in the rats without host kidney resection. (1) Cyclosporin A may suppress graft rejection, thus normalizing the growth and function of fetal metanephroi in the omenta of host rats. (2) A variety of factors affect the growth and development of allografted metanephroi, whereas rejection remains the major one.

Cardiovascular risk profile in patients treated with sirolimus after renal transplantation

Renal transplant patients are inherently predisposed to cardiovascular disease (CVD) as a result of prolonged exposure to multiple cardiovascular risk factors. Approximately one half of all late graft losses are due to death with a functioning graft, and CVD is the most frequent cause of death with a functioning graft among these patients. Immunosuppressive therapies associated with a reduced burden of risk for CVD would therefore greatly decrease post-transplantation morbidity and mortality. The nephrotoxic effects observed with the use of calcineurin inhibitors (CNIs), such as cyclosporine (CsA), run counter to the goal of renal transplant therapy. Sirolimus, a more recent immunosuppressive agent with a unique mechanism of action, offers an alternative to CsA. Recent data from a 4-year study investigating early CsA withdrawal from a sirolimus-CsA-steroid (SRL-CsA-ST) combination demonstrated significantly better renal function, lower blood pressure, and improved graft survival after CsA withdrawal. During that trial, the increase in serum lipids induced by sirolimus was generally manageable with lipid-lowering therapy. Further investigation is warranted to evaluate the value of CNI-free therapy compared with CNI-based regimens in reducing cardiovascular risk factors and improving patient and graft survival.

High-Affinity Small Molecule Inhibitors of T Cell Costimulation: Compounds for Immunotherapy

Costimulatory molecules are important regulators of T cell activation and thus favored targets for therapeutic manipulation of immune responses. One of the key costimulatory receptors is CD80, which binds the T cell ligands, CD28, and CTLA-4. We describe a set of small compounds that bind with high specificity and low nanomolar affinity to CD80. The compounds have relatively slow off-rates and block both CD28 and CTLA-4 binding, implying that they occlude the shared ligand binding site. The compounds inhibit proinflammatory cytokine release in T cell assays with submicromolar potency, and as such, they represent promising leads for the development of novel therapeutics for immune-mediated inflammatory disease. Our results also suggest that other predominantly beta proteins, such as those that dominate the cell surface, may also be accessible as potentially therapeutic targets.

Rapamycin-treated, alloantigen-pulsed host dendritic cells induce ag-specific T cell regulation and prolong graft survival.

Tolerogenic properties of dendritic cells (DC), particularly those in the immature state, and their therapeutic potential are increasingly being recognized. Among several distinct approaches to generate stably immature DC, pharmacologic manipulation stands out as a promising and clinically applicable option. We have shown recently that the immunophilin ligand rapamycin (Rapa) can inhibit DC maturation and their effector functions. Here, we examined the impact of Rapa exposure on subsequent alloantigen (Ag) presentation by myeloid DC via the indirect pathway. Rapa-treated, allogeneic lysate-pulsed host DC (Rapa-DC) were inferior stimulators of syngeneic T cells, compared to lysate-pulsed control DC. Rapa exposure did not block alloAg uptake by DC nor impair their in vivo homing to splenic T cell areas after adoptive transfer. T cells primed by Rapa-treated, alloAg-pulsed DC showed decreased capacity to produce IL-2 and IFNgamma, and were hyporesponsive to subsequent challenge via both the direct and indirect pathways, in an Ag-specific manner. When infused 1 week before transplantation, these Rapa-DC significantly prolonged alloAg-specific heart graft survival. This effect was reversed by systemic IL-2 administration but enhanced by either repeated infusion of the cells or a short post-transplant course of FK506. These therapeutic effects, achieved by targeting both major pathways of allorecognition, provide the basis for a clinically applicable strategy to suppress graft rejection.

Combined treatment of glatiramer acetate and low doses of immunosuppressive drugs is effective in the prevention of graft rejection

The immunomodulator glatiramer acetate (GA, copolymer 1, Copaxone, GLAT), currently used for the treatment of multiple sclerosis, is a well-tolerated drug with a high safety profile. We have previously demonstrated that GA suppresses the immune rejection manifested in graft versus host disease, as well as in graft rejection. In an attempt to reduce the dosage and toxicity of the current immunosuppressive regimens, we have now tested the ability of GA, combined with low doses of cyclosporin (CyA) or tacrolimus (FK506), to suppress the rejection of mismatched allografts across major histocompatibility barriers. We report herewith that such combination therapy was effective in several animal models: (1) it led to a significant delay of the vigorous process of skin rejection in mice, manifested by evidential prolongation in skin graft survival (higher than that obtained with at least double dose of the immunosuppressive drug alone). (2) The combined treatment led to efficient inhibition of the functional deterioration of thyroid grafts in mice, manifested by 2.2- to 20.1-fold increase in iodine absorbance of the transplanted thyroids, as compared to each drug alone. (3) Combination therapy inhibited significantly the rejection of vascularized heart transplants in rats. Thus, cardiac allograft survival following the combined treatment with GA and low dose of CyA was longer than the survival obtained by fourfold higher dose of CyA alone. In all transplantation systems, combination therapy of GA with either CyA or FK506 significantly suppressed graft rejection and was more effective than treatment with either GA or the immunosuppressive drug alone, suggesting that such treatment may be beneficial for human transplantation.

Clinical Snippets

There are about 90 human tyrosine kinases and about half of those are receptors. Yu and colleagues analyzed cultured rat cells and found that follicular papilla cells and dermal fibroblasts expressed different tyrosine kinases. Consistent with their data, c-met (HGF receptor) kinase preferentially stimulated growth in rat follicular papilla cells and PDGF receptor preferentially stimulated rat fibroblasts. The recognition that kinases are somewhat hair follicle-specific may lead to drugs that inhibit their expression and therefore, can serve in hair removal. J Invest Dermatol 123:283–290, 2004. Patients with advanced stages of mycosis fungoides and Sézary syndrome often receive poor prognoses, and novel therapies are needed. Zhang and colleagues examined the capacity for CDDO and bexarotene to treat MF/SS cells. CDDO, a multifunctional synthetic oleanane triterpenoid, inhibits proliferation of many human tumor cell lines, and bexarotene, a retinoid RXR ligand, reduces cutaneous manifestations of MF/SS in both early-stage and late-stage disease. Since CDDO is so powerful in inducing apoptosis, and many chemotherapeutic agents work similarly, the clinical potential of CDDO, either alone or in combination with bexarotene, is a promising area for study. J Invest Dermatol 123:380–387, 2004. Langerhans cells (LC) are considered the immune system's “sentinels”, presenting antigens that stimulate T cell-mediated defenses against viral infections and neoplasias of the skin or mucosa. This information is important for those pursuing vaccine design and immunotherapy, and provides insights into the pathogenetic mechanisms for cutaneous diseases. Here, von Bubnoff and co-workers show that specialized LC can produce indoleamine 2,3-dioxygenase (IDO), which inhibits T cell response. This new view of LC as serving an immunoregulatory function in promoting T cell tolerance will help investigations with allogeneic fetuses, the inhibition of graft rejection, or, possibly, in some individuals, the prevention of allergic disease. J Invest Dermatol 123:298–304, 2004. Alopecia areata, an autoimmune disease that targets actively growing hair follicles, affect nearly 2% of the general population. Sundberg and colleagues, using mouse cross-strains, identified expanded intervals for AA genetic susceptibility on two mouse chromosomes where a variety of immunoregulatory genes are located. This provides data for analyzing the genetic candidates responsible for AA as well as insights into other conditions, such as thyroid disorders, which have been associated with AA subsets. Random genome-wide linkage screening in mice and humans can lead to greater understandings of AA and other complex polygenic diseases. J Invest Dermatol 123:294–297, 2004. Human papillomavirus (HPV) genomes are often retrieved from tumor biopsies, but cutaneous HPV are common on the top of skin lesions and in healthy skin. Forslund and colleagues, comparing samples from skin lesions, same-tumor biopsies “stripped” of superficial cells, and healthy skin, observed that HPV DNA prevalence was greater in lesions than in biopsies, but viral rates were equivalent in healthy sites and tumor tops. Apparently, the presence of virus is not specifically associated with the tumor below. These findings will inform studies on HPV in non-melanoma skin cancers—a risk for renal and other post-transplant patients. J Invest Dermatol 123:388–394, 2004. In humans, nevi are both indicators for developing melanoma and short-term markers of adverse reactions to melanoma-inducing sun exposure. To link light-wavelength ranges to pathogenesis, Menzies and colleagues exposed guinea pigs to ultraviolet (UV)B to near UVA2 light, UVA, visible light, a full solar spectrum, or no irradiation. As sun-exposure increased, so did nevi, and the sun-exposure patterns similarly induced both nevi and melanoma. The most powerful findings for nevi induction was in the UVB-treated group compared with all groups. Surprisingly, wavelengths beyond UVB may have a protective role. J Invest Dermatol 123:354–360, 2004.

A highly selective inhibitor of IkappaB kinase, BMS-345541, augments graft survival mediated by suboptimal immunosuppression in a murine model of cardiac graft rejection.

We previously demonstrated in vitro and in vivo that an IkappaB kinase (IKK) inhibitor blocks cytokine production and suppresses immune responses. These results indicate that a potent IKK inhibitor may have the potential of being a novel therapeutic agent for the prevention of graft rejection. The IKK inhibitor BMS-345541 was tested in mice for its ability to inhibit anti-CD3-induced interleukin (IL)-2 and tumor necrosis factor (TNF)-alpha production and T-cell proliferation in an in vivo mixed lymphocyte reaction. BMS-345541 was further tested for its ability to suppress graft rejection in a murine nonvascularized heterotopic cardiac allograft model. BMS-345541 was tested as a single agent and in combination with other immunomodulators for inhibition of T-cell proliferation and graft rejection in vivo. BMS-345541 suppressed, in a dose-dependent manner, the production of both IL-2 and TNF-alpha in mice stimulated with an injection of anti-CD3 antibody. Approximately 70% inhibition of both IL-2 and TNF were observed at a dose of 100 mg/kg. When BMS-345541 was administered at 100 mg/kg as a single agent, in vivo T-cell proliferation was not inhibited. However, when combined with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin (200 microg), a synergistic antiproliferative effect was observed, resulting in 77% inhibition of CD4+ T-cell proliferation. In the murine heterotopic heart transplant model, BMS-345541 did not prolong graft survival when administered at 50 mg/kg as a single agent. However, when administered with a suboptimal dose of cytotoxic T-lymphocyte antigen-4 immunoglobulin or cyclosporine A (15 mg/kg), graft survival was significantly increased compared with either agent alone. These results indicate that inhibition of IKK may serve as novel adjunctive therapy for the prevention of graft rejection.

Antibody response and allogeneic mixed lymphocyte reaction in mu-, delta-, and kappa-opioid receptor knockout mice

The implication of opioid receptors in immune response has been studied using mu-, delta- and kappa-opioid receptor knockout mice. The mutant animals were compared to their wild-type (WT) counterparts for antibody (Ab) response to the prototype Ag keyhole limpet hemocyanin (KLH). Kappa-receptor deficient mice displayed higher Ab titers for either total Ig, IgM, IgG1 or IgG2a isotypes, whereas mu and delta animals behaved as wild-type mice. Therefore, endogenous kappa-receptor activation would tonically inhibit Ab response. Opioid receptor deficient mice were also used to investigate the immunosuppressive action of naltrindole, a delta-opioid receptor antagonist, shown earlier to inhibit graft rejection and the allogeneic mixed lymphocyte reaction (MLR) in vitro. Naltrindole and two related compounds inhibited MLR performed with lymphocytes from wild-type and delta-opioid receptor knockout mice. These compounds also suppressed MLR assayed with cells from triple mu/delta/kappa-opioid receptor mutants. We therefore demonstrate that naltrindole immunosuppressive activity is not mediated by any of the three mu-, delta- or kappa-opioid receptors, but by a target which remains to be discovered.

Conversion from Cyclosporin A to Tacrolimus as a Non‐Surgical Alternative to Reduce Gingival Enlargement: A Preliminary Case Series

Gingival enlargement (GE) is a frequent side effect that occurs in organ transplant recipients (OTR) after the administration of cyclosporin A (CsA). The availability of new drugs used to suppress graft rejection in OTR offers an opportunity to manage GE non-surgically. This preliminary case series aimed to analyze the effect of CsA withdrawal and its substitution by another immunosuppressant in OTR with severe GE. Four organ transplant recipients who had received a liver or renal allograft were recruited for this study. All OTR had developed clinically severe CsA-induced GE. GE scores were assessed for each patient at baseline and at weeks 2, 4, 8, 12, 16, and 54 following conversion to tacrolimus. Scaling and root planing were initially performed and repeated monthly during the first 6 months. Careful polishing of the teeth was carried out once every 2 weeks until month 6 and then monthly until month 12. Hygiene instructions and reinforcement to optimize oral hygiene were maintained throughout the study. The four patients showed a rapid decrease in their gingival symptoms and in the size of the gingivae. This change was clinically evident 8 weeks after conversion to tacrolimus. One year later, all the patients experienced GE regression, although some anatomic irregularities persisted in the interdental papillae of one of the patients. No adverse effects from tacrolimus were observed during the study except in one patient who presented headaches. It seems that CsA withdrawal and its conversion to tacrolimus in organ transplant recipients who develop severe gingival enlargement, together with an extensive plaque control program, provide an effective means to control/eliminate gingival hyperplasia, with minimal risk of graft dysfunction.

The mosaic of immunosuppressive drugs

Graft rejections as well as tolerance are true representation of the specificity, sophistication and redundancy of an elegantly and meticulously designed immune system. Tolerance is in a way similar to the process of self-recognition where lymphoid clones, during development, baring self-reactive receptor are eliminated or rendered in active by “clonal deletion” leading to a state of accommodation and acceptance (anergic). On the other hand, both acute and chronic rejections are manifestation of the purpose of existence of the immune system, which is to defend the host against foreign invaders. Thus, in order to treat (control) graft rejection it is necessary to determine and understand the steps leading to recognition, stimulation, activation, and amplification of the immune system. The first step leading to the initiation of the immune system cascade is recognition. Which can either be direct where donor antigens of the major histocompatibility complex (MHC) expressed on the donor cells (passenger leukocytes) or tissues are recognised by the host immune system. The direct recognition pathway initiates acute graft rejection. Alternatively processed donor MHC peptides presented by the recipient antigen presenting cells (APC) initiate the indirect pathway of immune response, which is as important as the direct recognition especially in chronic rejection. Recognition is followed by the ligation of a series of adhesion molecules starting with an antigen to its specific T-cell receptor (TCR)/cluster of differentiation (CD) complex, expressed on the surface of the T cell. In order for the activation to precede additional costimulatory signals, such as ligation of the CD28/B7, CD4/HLA class II and CD/HLA class I antigens are required. The activation process is accompanied by an increase of cytokines production such as interleukin (IL)-2, IL-12, interferon (INF) and tumour necrosis factor (TNF) by the primed T cell. The complexity and the polymorphic nature of the immune system have necessitated designing agents that inhibit the immune system at different levels. Cyclosporine and Tacrolimus, collectively known as calcineurin inhibitors, seems to act on the IL-2 by inhibiting its production thus leading to a decrease in the proliferation of the activated lymphocyte. Rapamycin, which is similar to Tacrolimus, inhibits graft rejection by blocking IL-2 activation and phosphorylation of 70 S6 kinase thus inhibiting the progression of T-cell from G to S phase. While Cellcept (MMF) reduce the proliferation of T cell by inhibiting purine synthesis and by its action on ionosine monophosphate dehydrogenase. Anti-lymphocyte antibodies (ATG) deplete circulating lymphocytes while selective monoclonal antibodies are directed against IL-2 receptor thus reducing the rate of proliferation of activated T cells. Recently, antibodies to the CD40/CD40 ligand have been shown to induce long-term graft survival with the inhibition of the Th1 cytokines (INF), IL-2 and IL-12 and upregulating the Th2 cytokines IL-4 and IL-10. Lastly graft rejection can be reduced by blockade of the B7/CD28 costimulation pathway with the fusion protein CTLA-4Ig. With the availability of such potent and diverse agents it is now possible to develop multi drug regiments that can depress the immune system at the different steps of the activation cascade, with minimal side effects, thus improving graft and patient survival rates.

Immunotherapeutic Effects of CTLA4Ig Fusion Protein on Murine EAE and GVHD

Background: CTLA4 (CD152), which is expressed on the surface of T cells following activation, has a much higher affinity for B7 molecules comparing to CD28, and is a negative regulator of T cell activation. In contrast to stimulating and agonistic capabilities of monoclonal antibodies specific to CTLA-4, CTLA4Ig fusion protein appears to act as CD28 antagonist and inhibits in vitro and in vivo T cell priming in variety of immunological conditions. We've set out to confirm whether inhibition of the CD28-B7 costimulatory response using a soluble form of human CTLA4Ig fusion protein would lead to persistent inhibition of alloreactive T cell activation. Methods: We have used CHO-dhfr cell-line to produce CTLA4Ig fusion protein. After serum free culture of transfected cell line we purified this recombinant molecule by using protein A column. To confirm characterization of fusion protein, we carried out a series of Western blot, SDS-PAGE and silver staining analyses. We have also investigated the efficacy of CTLA4Ig in vitro such as mixed lymphocyte reaction (MLR) & cytotoxic T lymphocyte (CTL) response and in vivo such as experimental autoimmune encephalomyelitis (EAE), graft versus host disease (GVHD) and skin-graft whether this fusion protein could inhibit alloreactive T cell activation and lead to immunosuppression of activated T cell. Results: In vitro assay, CTLA4Ig fusion protein inhibited immune response in T cell-specific manner: 1) Human CTLA4Ig inhibited allogeneic stimulation in murine MLR; 2) CTLA4Ig prevented the specific killing activity of CTL. In vivo assay, human CTLA4Ig revealed the capacities to induce alloantigen-specific hyporesponsiveness in mouse model: 1) GVHD was efficiently blocked by dose-dependent manner; 2) Clinical score of EAE was significantly decreased compared to nomal control; 3) The time of skin-graft rejection was not different between CTLA4Ig treated and control group. Conclusion: Human CTLA4Ig suppress the T cell-mediated immune response and efficiently inhibit the EAE, GVHD in mouse model. The mechanism of T cell suppression by human CTLA4Ig fusion protein may be originated from the suppression of activity of cytotoxic T cell. Human CTLA4Ig could not suppress the rejection in mouse skin-graft, this finding suggests that other mechanism except the suppression of cytotoxic T cell may exist on the suppression of graft rejection. (Immune Network 2003;3(4):302-309)

Clinical Study on Surgical Outcome of Penetrating Keratoplasty for Herpetic Leukoma

To evaluate prognostic factors, rejection, and recurrence of herpetic keratitis, in keratoplasty for herpetic leukoma. We compared surgical outcome between patients who underwent keratoplasty for herpetic leukoma and those who received it for non-herpetic disorders. We compared the eyes of 24 patients in the herpetic group with 17 eyes of 20 patients with non-herpetic corneal disorders, i.e., the non-herpetic group. The rejection rate was 45.8% for the herpetic group and 5.00% for the non-herpetic group, showing significant difference(p < 0.05). Transparent grafts were obtained for 75.0% of the herpetic group and 85.0% of the non-herpetic group, giving no significant difference. In the herpetic group, graft transparency in eyes with recurrences were 94.1%(p < 0.05). Graft rejection occurred mostly within 1 year postoperatively. RecuRrent herpetic keratitis tended to occur within 3 years and it was later than graft rejection. In patients who underwent keratoplasty for herpetic leukoma, steroid therapy is important for 1 postoperative year to suppress graft rejection, but after that, steroid treatment should be carefully considered to minimize the risk of recurrences of herpetic keratitis.

Intraocular dexamethasone delivery system for corneal transplantation in an animal model.

To assess the efficacy of a new intraocular biodegradable polymer dexamethasone drug delivery system (DEX DDS) in a high-risk corneal transplantation model. Lewis rats that received orthotopic corneal transplants (Balb/c mice donors) were divided into three groups (six rats in each); group 1 received no treatment and served as controls, group 2 was treated with 0.1% betamethasone eyedrops three times daily for 6 weeks, and group 3 received DEX DDS in the anterior chamber at the time of transplantation. All grafts in the untreated control group were rejected within 8 days. In the betamethasone eyedrop group, five eyes (83%) were rejected during the 8-week study period. None of the grafts in the DEX DDS group was rejected. The administration of DEX DDS significantly prolonged the survival rate of the corneal grafts (p < 0.001, log-rank test). DEX DDS is effective in suppressing graft rejection in high-risk corneal transplantation.

Heme oxygenase-1, a protective gene that prevents the rejection of transplanted organs.

Endothelial cells (EC) play a pivotal role in regulating inflammatory reactions such as those involved in the rejection of transplanted organs. This occurs through the expression of a series of pro- and anti-inflammatory genes that are associated with the activation of these cells. Presumably, the expression of pro-inflammatory genes promotes events that lead to graft rejection, while expression of anti-inflammatory (protective) genes suppresses those events and thus contributes in sustaining graft survival. Understanding how the expression of these genes is regulated and their mechanism of action are important issues for the development of new therapeutic strategies to suppress graft rejection. We have studied this phenomenon using experimental models of transplantation in rats. We discuss here data that supports the concept that grafts can express anti-inflammatory (protective) genes that mitigate inflammatory reactions leading to graft rejection. The data reviewed focus on the role of one of such genes, the stress responsive gene heme oxygenase-1, and of its byproduct carbon monoxide, which can suppress graft rejection and lead to long-term graft survival.

Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants.

Mouse-to-rat cardiac transplants survive long term after transient complement depletion by cobra venom factor and T cell immunosuppression by cyclosporin A. Expression of heme oxygenase-1 (HO-1) by the graft vasculature is critical to achieve graft survival. In the present study, we asked whether this protective effect was attributable to the generation of one of the catabolic products of HO-1, carbon monoxide (CO). Our present data suggests that this is the case. Under the same immunosuppressive regimen that allows mouse-to-rat cardiac transplants to survive long term (i.e., cobra venom factor plus cyclosporin A), inhibition of HO-1 activity by tin protoporphyrin, caused graft rejection in 3--7 days. Rejection was associated with widespread platelet sequestration, thrombosis of coronary arterioles, myocardial infarction, and apoptosis of endothelial cells as well as cardiac myocytes. Under inhibition of HO-1 activity by tin protoporphyrin, exogenous CO suppressed graft rejection and restored long-term graft survival. This effect of CO was associated with inhibition of platelet aggregation, thrombosis, myocardial infarction, and apoptosis. We also found that expression of HO-1 by endothelial cells in vitro inhibits platelet aggregation and protects endothelial cells from apoptosis. Both these actions of HO-1 are mediated through the generation of CO. These data suggests that HO-1 suppresses the rejection of mouse-to-rat cardiac transplants through a mechanism that involves the generation of CO. Presumably CO suppresses graft rejection by inhibiting platelet aggregation that facilitates vascular thrombosis and myocardial infarction. Additional mechanisms by which CO overcomes graft rejection may involve its ability to suppress endothelial cell apoptosis.

STEM-CELL TRANSPLANTATION FOR INHERITED IMMUNODEFICIENCY DISORDERS

For patients with well-characterized, rapidly fatal, nonmalignant immunodeficiency disorders, such as SCID, the decision to proceed with allogeneic SCT is clear-cut. For patients with many other disorders, this decision can be extremely difficult. Disorders such as LAD or CGD have a variable natural history. Each patient must be considered individually, with the risk for SCT-related morbidity and mortality carefully weighed against that of the underlying disease. Significant advances during the past 10 years have made SCT a much safer procedure. Use of nonmyeloablative conditioning regimens as a means of reducing toxicity of high-dose chemotherapy and irradiation hold great promise. Highly immunosuppressive, nonchemotherapeutic agents that inhibit graft rejection or GVHD by blocking the critical costimulatory component of the T-cell receptor-antigen interaction are beginning to emerge and may be ideal for SCT of nonmalignant diseases. Therefore, the risk-benefit equation must be reassessed each year as the severity of patients' disorders is better defined and techniques of SCT improve.

Dominant tolerance and linked suppression induced by therapeutic antibodies do not depend on Fas-FasL interactions.

Nonlytic anti-CD4 monoclonal antibody therapy can be used to induce transplantation tolerance in rodent models. Such tolerance is often associated with dominant regulation, mediated by CD4+ cells, and characterized by infectious tolerance and linked suppression. Understanding the mechanisms by which CD4+ regulatory cells function may improve the manner in which current immunosuppressants are applied and may lead to the development of new tolerance-inducing therapeutics. Fas-mediated apoptosis has been characterized as an important mechanism of peripheral self-tolerance and we here examine whether it has any role in anti-CD4 monoclonal antibody-induced dominant tolerance. Tolerance to transplanted skin and bone marrow, mismatched for multiple minor histocompatibility antigens, was induced in Fas mutant and control mice using anti-CD4 and anti-CD8 monoclonal antibodies. To test for linked suppression, animals were transplanted with a second graft-bearing tolerated and third party antigens. The ability of splenocytes from tolerant animals to suppress graft rejection was assessed by transfer into partially immunocompromised recipients. Monoclonal antibody therapy rendered Fas mutant mice tolerant of minor disparate skin and bone marrow. Splenocytes from these and control tolerant animals when transferred into partially immunocompromised Fas mutant or control recipients, induced antigen-specific suppression of graft rejection. Additionally, tolerant Fas mutant mice accepted grafts bearing tolerated and third party antigens. Signal transduction through the Fas receptor plays no essential role in the induction of tolerance using anti-CD4 and anti-CD8 monoclonal antibodies or its maintenance by active regulation.

Low-density lipoprotein-independent effects of statins.

Statins have pleiotropic properties that complement their cholesterol-lowering effects. These properties may partly account for their established benefit in the prevention of coronary artery disease beyond the reduction of LDL-cholesterol levels. The most widely recognized properties are reviewed here. They include: (i) nitric oxide-mediated improvement of endothelial dysfunction and upregulation of endothelin-1 expression; (ii) antioxidant effects; (iii) anti-inflammatory properties; (iv) inhibition of cell proliferation with anticarcinogenic actions in animals; (v) stabilization of atherosclerotic plaques; (vi) anticoagulant effects; and (vii) inhibition of graft rejection after heart and kidney transplantation. As advances are made in our knowledge, new properties are steadily being uncovered. Pleiotropic effects are currently being given consideration when instituting combination therapy for patients at high cardiovascular risk. Some pleiotropic effects are negative, and may account for occasional untoward drug interactions. For many of these new properties, the clinical relevance has not been established. The challenge for the future will be to design and carry out appropriate clinical trials to establish their relative importance in the prevention of coronary artery disease.