Sirolimus: a therapeutic advance for dermatologic disease.

Sirolimus, also known as rapamycin (SRL, Rapamune®), was approved in 1999 by the US Food and Drug Administration to prevent graft rejection in renal transplantation. As a member of the mammalian target of rapamycin (mTOR) inhibitor class, its potent immunosuppressant, anti-angiogenic and anti-proliferative properties are well recognized. When compared to other immunosuppressants, SRL has a lower risk of renal, neurologic and lymphoproliferative complications. It has become a promising treatment modality for angiofibromas, Kaposi's sarcoma and other inflammatory and malignant disorders of the skin. With the recent discovery that mTOR inhibitors extend the lifespan of mice, sirolimus and other rapamycin analogs (rapalogs) are emerging as therapeutic targets for the treatment and prevention of age-related diseases.

Blockade of K(ATP) channels reduces endothelial hyperpolarization and leukocyte recruitment upon reperfusion after hypoxia.

Ischemia/reperfusion injury in renal transplantation leads to slow or initial nonfunction, and predisposes to acute and chronic rejection. In fact, severe ischemia reperfusion injury can significantly reduce graft survival, even with modern immunosuppressive agents. One of the mechanisms by which ischemia/reperfusion causes injury is activation of endothelial cells resulting in inflammation. Although several therapies can be used to prevent leukocyte recruitment to ischemic vessels (e.g. antiadhesion molecule antibodies), there have been no clinical treatments reported that can prevent initial immediate neutrophil recruitment upon reperfusion. Using intravital microscopy, we describe abrogation of immediate neutrophil recruitment to ischemic microvessels by the K(ATP) antagonist glibenclamide (Glyburide). Further, we show that glibenclamide can reduce leukocyte recruitment in vitro under physiologic flow conditions. ATP-regulated potassium channels (K(ATP)) are important in the control of cell membrane polarization. Here we describe profound hyperpolarization of endothelial cells during hypoxia, and the reduction of this hyperpolarization using glibenclamide. These findings suggest that control of endothelial membrane potential during ischemia may be an important therapeutic tool in avoiding ischemia/reperfusion injury, and therefore, enhancing transplant long-term function.

Reducing the risk of viral infection in renal transplantation.

Viruses are under constant surveillance by the immune system. With the introduction of more potent immunosuppressive regimens in transplantation, the increased risk of infectious diseases accompanies the decreased risk of acute rejection. Is the overall burden of immunosuppression the prime consideration or do the various immunosuppressive agents contribute individual risks? Do some immunosuppressive agents actually protect against viral disease? Cytomegalovirus (CMV) was initially a significant complication of transplantation, but the incidence of severe CMV disease has decreased with the identification of high-risk groups and the introduction of screening and prophylactic strategies to control reactivation and de novo infection. Antiviral agents with specificity against CMV have been developed; however, CMV has developed resistance to antiviral agents, causing concern. BK polyomavirus is an emerging threat to renal transplantation, as BK nephropathy can cause significant graft loss, often within the first few years of the transplant. Return to dialysis carries increased morbidity and mortality and reduced quality of life. This brief overview examines the clinical literature regarding these viruses and discusses the potential for manipulating intracellular signaling pathways using specific immunosuppressive agents to inhibit viral reactivation and replication.

Autoantibodies in lupus nephritis patients requiring renal transplantation.

The goal of this nested case-control study was to compare autoantibody profiles in systemic lupus erythematosus (SLE) patients with lupus nephritis (LN), lupus nephritis patients requiring renal transplantation (LNTP) and a SLE control group without nephritis (CON). Sera were assayed for a variety of autoantibodies by addressable laser bead immunoassay (ALBIA) and enzyme-linked immunoassay (ELISA) and to dsDNA by Crithidia luciliae assay. The frequency of nucleosome autoantibodies was significantly greater in the LNTP group (79%) compared to the LN (18%) and CON (9%) groups (P < 0.0005). The frequency of other autoantibodies, including anti-dsDNA, did not differ significantly between groups. Among patients with LN, the odds of progressing to renal transplantation was 16-fold higher (OR 16.5 [95% CI 2.5, 125.7], P = 0.0005) in patients testing positive for anti-nucleosome antibodies compared to those who tested negative. Furthermore, the level of anti-nucleosome antibodies was significantly ( P < 0.00005) higher in the LNTP group (3.69 +/- 2.79) than the LN (0.51 +/- 0.51) and CON (0.34 +/- 0.44) groups. Review of 48 renal biopsies from 29 patients indicated that there was no difference in renal histological classification among patients with anti-nucleosome antibodies compared to those who tested negative. Our observations suggest that nucleosome autoantibodies are a biomarker for more severe SLE renal disease requiring transplantation.

Osmotic stimulation of the Na+/H+ exchanger NHE1: relationship to the activation of three MAPK pathways.

The Na+/H+ exchanger (NHE) becomes activated by hyperosmolar stress, thereby contributing to cell volume regulation. The signaling pathway(s) responsible for the shrinkage-induced activation of NHE, however, remain unknown. A family of mitogen-activated protein kinases (MAPK), encompassing p42/p44 Erk, p38 MAPK and SAPK, has been implicated in a variety of cellular responses to changes in osmolarity. We therefore investigated whether these kinases similarly signal the hyperosmotic activation of NHE. The time course and osmolyte concentration dependence of hypertonic activation of NHE and of the three sub-families of MAPK were compared in U937 cells. The temporal course and dependence on osmolarity of Erk and p38 MAPK activation were found to be similar to that of NHE stimulation. However, while pretreatment of U937 cells with the kinase inhibitors PD98059 and SB203580 abrogated the osmotic activation of Erk and p38 MAPK, respectively, it did not prevent the associated stimulation of NHE. Thus, Erk1/2 and/or p38 MAPK are unlikely to mediate the osmotic regulation of NHE. The kinetics of NHE activation by hyperosmolarity appeared to precede SAPK activation. In addition, hyperosmotic activation of NHE persisted in mouse embryonic fibroblasts lacking SEK1/MKK4, an upstream activator of SAPK. Moreover, shrinkage-induced activation of NHE still occurred in COS-7 cells that were transiently transfected with a dominant-negative form of SEK1/MKK4 (SEK1/MKK4-A/L) that is expected to inhibit other isoforms of SEK as well. Together, these results demonstrate that the stimulation of NHE and the activation of Erk, p38 MAPK and SAPK are parallel but independent events.

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway.

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.

The stress-activated protein kinase pathways.

Part of the cellular response to toxins, physical stresses and inflammatory cytokines occurs by signalling via the stress-activated protein kinase (SAPK) and p38 reactivating kinase pathways. This results in modification of cellular gene expression. These stress-responsive kinase pathways are structurally similar, but functionally distinct, from the archetypal mitogen-activated protein kinases (MAPKs or ERKs). The ERK pathway is a hierarchical cascade originating at the cell membrane with receptors for mitogens or growth factors, which recruit, via adapter proteins and exchange factors, the small guanosine triphosphatase (GTPase) Ras (see fig. 1). Ras activates raf, a serine threonine kinase, which activates MEK (MAPK/ERK kinase). MEK, in turn, phosphorylates and activates ERK1 and ERK2, which translocate to the nucleus and transactivate transcription factors, changing gene expression to promote growth, differentiation or mitosis. By transducing signals through a cascade of kinases, several options for control are introduced for amplifying and/or modifying the output signal. The SAPK and p38 pathways are also hierarchically arranged, but less is known about the upstream components and the downstream effects of stimulation of these pathways. Among the processes modulated by stress-responsive pathways are apoptosis, transformation, development, immune activation, inflammation and adaptation to environmental changes. This review outlines the upstream componentry of these pathways that interact with a variety of agonists to modify the activity of SAPK and p38, and explores the downstream functions of this activation.

HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway.

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR-based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.

MLK-3 activates the SAPK/JNK and p38/RK pathways via SEK1 and MKK3/6.

Mixed lineage kinase-3 (MLK-3) is a 97 kDa serine/threonine kinase with multiple interaction domains, including a Cdc42 binding motif, but unknown function. Cdc42 and the related small GTP binding protein Rac1 can activate the SAPK/JNK and p38/RK stress-responsive kinase cascades, suggesting that MLK-3 may have a role in upstream regulation of these pathways. In support of this role, we demonstrate that MLK-3 can specifically activate the SAPK/JNK and p38/RK pathways, but has no effect on the activation of ERKs. Immunoprecipitated MLK-3 catalyzed the phosphorylation of SEK1 in vitro, and co-transfected MLK-3 induced phosphorylation of SEK1 and MKK3 at sites required for activation, suggesting direct regulation of these protein kinases. Furthermore, interactions between MLK-3 and SEK and MLK-3 and MKK6 were observed in co-precipitation experiments. Finally, kinase-dead mutants of MLK-3 blocked activation of the SAPK pathway by a newly identified mammalian analog of Ste20, germinal center kinase, but not by MEKK, suggesting that MLK-3 functions to activate the SAPK/JNK and p38/RK cascades in response to stimuli transduced by Ste20-like kinases.

The stress-activated protein kinase pathway mediates cell death following injury induced by cis-platinum, UV irradiation or heat.

BACKGROUND: Stimuli that stress cells, including inflammatory cytokines, ultra-violet irradiation, DNA-damaging chemotherapeutic drugs and heat shock, stimulate a recently identified cytoplasmic signaling system that is structurally related to the mitogen-activated protein kinase pathway. This pathway consists of a cascade of protein kinases including stress-activated protein kinase (SAPK), also termed Jun N-terminal kinase (JNK), and two kinases that activate it, MEKK and SEK/MKK4. Despite rapid progress in delineating the components of this pathway, the cellular consequence of its activation remains to be defined. RESULTS: We have screened cells for defects in SAPK signaling and identified a cell line, previously characterized for its thermotolerance properties, as being more refractive to SAPK activation induced by heat stress than its thermosensitive parental line. Stable expression of dominant inhibiting SEK mutants in thermosensitive parental cells specifically and effectively blocked SAPK activation after heat shock. These lines also became markedly resistant to the cytocidal effects of thermal stress, confirming the phenotype of the thermotolerant line. These cell lines defective in SAPK activation were also resistant to the lethal effects of the DNA-damaging drug cis-platinum. CONCLUSIONS: Experimentally induced stable blockade of SAPK activation in cells with normal thermosensitivity is sufficient to confer resistance to cell death induced by diverse stimuli including heat and the chemotherapeutic agent cis-platinum. These results suggest that activation of the SAPK pathway by diverse cell stressors plays a critical part in mediating the toxicity of these treatments and inducing cell death. SAPK activation in this context could broadly influence cellular response to stress, modulate apoptosis during development or determine the clinical response of tumor cells to cytotoxic therapies.

Anti-B cell antibodies for the treatment of monoclonal Epstein-Barr virus-induced lymphoproliferative syndrome after multivisceral transplantation.

Epstein-Barr virus-induced lymphoproliferative syndrome (EBV-LPS) is associated with OKT3 therapy in transplant patients. Response to chemotherapy or radiation is generally poor, while polyclonal EBV-LPS has had favorable responses to therapy with CD21 and CD24 monoclonal antibodies. Oligoclonal disease has not been previously reported to respond to therapy with CD21 and CD24. We report a 27-y old woman who developed a monoclonal EBV-LPS (confirmed by southern analysis of tumour for EBV DNA) after 180 mg of OKT3 for a multivisceral transplant. The patient achieved clinical remission for more than 2 months, but later died from cytomegalovirus pneumonia. Levels of CD21 and CD24 were > 2000 ng/ml during therapy and no human anti-mouse antibodies were formed. Peripheral blood B cells were cleared during therapy. We conclude that CD21 and CD24 monoclonal antibodies may be of value in the therapy of oigoclonal EBV-LPS, and merit further study.