Dendritic Cells Inflamed by Coagulation
By Robert Flaumenhaft, MD, PhD
Dr. Flaumenhaft indicated no relevant conflicts of interest.
Niessen F, Schaffner F, Furlan-Freguia C, et al. Dendritic cell PAR1–S1P3 signaling couples coagulation and inflammation. Nature. 2008;452:654-8.
That crosstalk occurs between coagulation and inflammation during sepsis has been appreciated for decades. This link has been spotlighted with the use of activated protein C in sepsis therapy. Yet, a more detailed understanding of the coupling of coagulation and inflammation is required to identify superior strategies for treating decompensated inflammatory responses. Recent work by Niessen, et al. demonstrates that coupling of protease-activated receptor 1 (PAR1) signaling to the sphingosine-1-phosphate 3 (S1P3) receptor on dendritic cells provides a critical connection between coagulation and inflammation.
PAR1 is a thrombin receptor that becomes activated following tissue factor-initiated production of thrombin. The investigators found that delayed inflammatory responses and mortality from a 90 percent lethal dose of lipopolysaccharide (LPS) were significantly decreased in PAR1-/- mice, by use of a direct thrombin inhibitor, or by a PAR1 antagonist. Based on previous studies linking PAR1 stimulation to sphingosine phosphate signaling,1 the researchers evaluated the role of sphingosine kinase 1 (SphK1) and S1P3 in PAR1-mediated delayed inflammatory responses. Delayed inflammatory responses were diminished in SphK1-/- and S1P3-/- mice and could be restored in PAR1
-/-
mice via stimulation of S1P3. These observations suggested that SphK1 and S1P3 acted as downstream effectors of PAR1.
The investigators' original hypothesis was that vascular cells mediated the effects of PAR1 stimulation in inflammation. Unexpectedly, reconstitution of PAR1-/- mice with wild-type bone marrow restored normal, delayed inflammation. Multiplex cytokine profiles suggested that dendritic cells mediated the inflammatory response. Adaptive transfer of wild-type dendritic cells into PAR1-/-, SphK1-/-, or S1P3-/- mice restored delayed inflammatory response and reversed the survival advantage to LPS challenge observed in these phenotypes. The observation that only wild-type dendritic cells (not PAR1-/- or S1P3-/- dendritic cells) were able to restore systemic inflammation demonstrated that PAR1 and S1P3 are coupled in an autocrine manner on dendritic cells.
The authors next analyzed local inflammation in mesenteric lymph nodes following intraperitoneal LPS administration. They found that loss of PAR1–S1P3 signaling resulted in increased dendritic cell sequestration in lymph nodes, consistent with previous data that S1P3 signaling enhances dendritic cell motility.2,3 When PAR1–S1P3 signaling was disrupted, IL-1β levels increased in the lymph nodes but were reduced in blood and lung. Dendritic cells express tissue factor. Impairment of PAR1–S1P3 crosstalk resulted in increased fibrin deposition in lymph nodes and decreased peripheral thrombin-antithrombin following LPS challenge. These results indicated that PAR1–S1P3 signaling on dendritic cells mediates dissemination of inflammation and coagulation from draining lymph nodes to the lungs and periphery.
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Schematic overview of the proposed pathway for lymphatic dissemination of coagulation and inflammation in deregulated immune responses.
Reprinted with permission from Macmillan Publishers Ltd: Niessen F, Schaffner F, Furlan-Freguia C, et al. Dendritic cell PAR1–S1P3 signaling couples coagulation and inflammation. Nature. 2008;452:654-8. |
Severe sepsis affects more than 750,000 patients a year and is associated with a mortality of approximately 30 percent. Systemic inflammation and disseminated intravascular coagulation resulting from excessive activation of the innate immune system are hallmarks of sepsis. Yet, how the coagulation and immune systems interact to contribute to dissemination of the inflammatory response is poorly understood.
Niessen and colleagues uncovered an important component of this response. They found that stimulation of dendritic cells through PAR1 results in stimulation of SphK and S1P3 leading to systemic dissemination of inflammation and coagulation. Interruption of PAR1–S1P3 crosstalk prevents delayed systemic inflammation and confines the inflammatory response to the lymph nodes. Pharmacological blockade of PAR1 confers a substantial survival advantage following LPS challenge or caecal ligation and puncture. These studies identify a potential strategy to control systemic inflammation without compromising host defense.
References
- Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1–phosphate receptor-1 crossactivation. Blood. 2005;105:3178-84.
- Czeloth N, Bernhardt G, Hofmann F, et al. Sphingosine-1-phosphate mediates migration of mature dendritic cells. J Immunol. 2005;175:2960-7.
- Maeda Y, Matsuyuki H, Shimano K, et al. Migration of CD4 T cells and dendritic cells toward sphingosine 1-phosphate (S1P) is mediated by different receptor subtypes: S1P regulates the functions of murine mature dendritic cells via S1P receptor type 3. J Immunol. 2007;178:3437-46.
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Increased Risk of VTE Associated With Impaired Clot Lysis: A New Dimension to Virchow's Triad
By Michael Linenberger, MD
Dr. Linenberger indicated no relevant conflicts of interest.
Meltzer ME, Lisman T, Doggen CJ, et al. Synergistic effects of hypofibrinolysis and genetic and acquired risk factors on the risk of a first venous thrombosis. PLoS Med. 2008;5:e97.
Acquired and familial hypercoagulable states include a number of well-characterized disorders that affect procoagulant proteins or cofactors, natural anticoagulants, and other mediators of hemostatic interactions with vascular endothelium. Although affected individuals are inherently prothrombotic, symptomatic deep vein thrombosis (DVT), pulmonary embolism (PE), and other types of venous thromboembolism (VTE) often occur only in the setting of additional "provocative" risk factors such as pregnancy, surgery, immobilization, obesity, advanced age, and/or exogenous female sex hormones. Since unprovoked VTE occurs in healthy young people with no identifiable predisposing hypercoagulable condition, other mechanisms must exist, and many studies have searched for prothrombotic defects related to impaired fibrinolysis. Aside from rare familial thrombotic dysfibrinogenemias and plasminogen deficiencies and a mild association with elevated levels of thrombin-activatable fibrinolysis inhibitor, no clear causal linkage has yet been established between VTE risk and specific abnormalities of tissue plasminogen activator (tPA), plasminogen activator inhibitor type 1, or α2-antiplasmin.
To further address this question, Meltzer, et al. performed a case-control study to determine whether hypofibrinolysis, as measured by a plasma clot lysis time (CLT) assay, might be an independent or cooperative risk factor for VTE. This investigation follows preliminary observations that patients in the Leiden Thrombophilia Study (LETS) with a plasma CLT at or above the 90th percentile had a two-fold increased risk of DVT.1 The CLT assay, performed by adding exogenous tissue factor and tPA to citrated plasma and measuring the time from half-maximal clot formation to half-maximal clot dissolution, reflects global plasma fibrinolytic potential and the interplay between the coagulation and fibrinolytic systems.1 In the current study, CLTs on 2,090 patients with a first idiopathic or provoked DVT and/or PE enrolled in the Multiple Environmental and Genetic Assessment (MEGA) were compared with 2,564 controls. Plasma samples were taken three months after discontinuation of anticoagulant therapy or one year after the VTE event. Hypofibrinolysis, defined as a CLT within the highest quartile of control values (i.e., the longest CLTs), was associated with an odds ratio for VTE (adjusted for age and sex; ORadj) of 2.4 in the absence of other prothrombotic risks. When hypofibrinolysis was combined with oral contraceptive pill (OCP) usage, immobilization, or factor V Leiden heterozygosity, a synergistic effect on VTE risk was observed with ORadj of 21.8, 10.3, and 8.1 compared to ORadj without hypofibrinolysis of 2.6, 4.3, and 3.5, respectively. In the control group, longer CLTs were seen with increasing age, male sex, increasing body mass index, diabetes, and the prothrombin 20210A mutation.
This large population-based study observed a 2.4-fold relative risk of a first VTE among individuals with hypofibrinolysis, which, by definition, was present in 25 percent of healthy controls. This is equivalent to the individual risks associated with exogenous female hormones, hyperhomocystinemia, and heterozygous prothrombin 20210A mutation. Concurrent immobilization, OCP usage, or factor V Leiden heterozygosity significantly augmented the VTE risk with hypofibrinolysis, reminiscent of the synergizing effects of additional clinical and genetic risks with other hypercoagulable states and consistent with the concept that VTE often results from overlapping predisposing conditions (see Figure). Additional data suggest that hypofibrinolysis may play a role in the pathogenesis of VTE with increasing age, obesity, diabetes, and the prothrombin 20210A mutation. Further studies are required, however, to define the genetic and functional mechanisms responsible for the impaired fibrinolytic potential as determined by the CLT assay. Prospective validation studies are also warranted to confirm that a prior VTE event does not affect CLT results.
From a clinical perspective, important next questions include:
- Is hypofibrinolysis associated with the risk of recurrent VTE, and can it be used to guide duration of anticoagulation?
- Is hypofibrinolysis related to VTE risks with other prothrombotic conditions (e.g., cancer, myeloproliferative disorders, antiphospholipid syndrome), and might it provide new perspectives on prevention and treatment in these settings?
- Can the CLT assay be validated for widespread clinical applicability, and what clinical and pharmacologic variables might confound its interpretation?
References
- Lisman T, de Groot PG, Meijers JC, et al. Reduced plasma fibrinolytic potential is a risk factor for venous thrombosis. Blood. 2005;105:1102-5.
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The "Eyes"(and Kidneys) Have It: A promoter polymorphism of the erythropoietin gene associated with proliferative diabetic retinopathy and end-stage renal disease
By Charles Parker, MD
Dr. Parker indicated no relevant conflicts of interest.
Tong Z, Yang Z, Patel S, et al. Promoter polymorphism of the erythropoietin gene in severe diabetic eye and kidney complications. Proc Natl Acad Sci USA. 2008;105:6998-7003.
Halcyon was the mythical bird that was said to breed in a nest floating at sea at the winter solstice, charming the wind and waves into calm. As an adjective, halcyon denotes a period of time in the past that was idyllically happy and peaceful. The halcyon days of erythropoietin supplementation lasted for about 15 years beginning in June 1989 when the U.S. Food and Drug Administration approved the recombinant human protein for treatment of anemia of chronic renal failure. The beginning of the end of those tranquil times occurred about five years ago with publication of a randomized study suggesting an adverse outcome for patients with head and neck cancer undergoing radiation therapy who received recombinant human erythropoietin for treatment of anemia.1 A subsequent study in mainly non-anemic patients with breast cancer receiving first-line chemotherapy also demonstrated inferior progression-free survival for those receiving the recombinant hormone.2 And although subsequent meta-analysis has suggested an overall survival benefit for cancer patients receiving supplemental erythropoietin,3 the calm could not be restored, and our once naïve view of erythropoietin as the simple, innocent regulator of erythropoiesis has been replaced by the current world-weary (sinister) concept of erythropoietin as a pleiotropic cytokine that effects a variety of physiologic and pathophysiologic processes. In addition to its primary function of regulating erythropoiesis, erythropoietin stimulates angiogenesis (both physiological and pathophysiological), upregulates tissue renin, enhances production of endothelin, and stimulates endothelial and vascular smooth muscle cell proliferation. Now compelling data from Tong and colleagues suggest a role for endogenous erythropoietin in neovascular complications of diabetes.
A remarkably high concordance rate (80 percent to 90 percent) exists between proliferative diabetic retinopathy (PDR) and end-stage renal disease (ESRD), and these complications show strong familial aggregation. Together, these observations suggest a genetic influence (susceptibility or resistance). Linkage studies have identified several potential loci, including a modifier of nephropathy on chromosome 7q21. To avoid the potentially confounding effects of phenotypic heterogeneity (especially as it applies to diabetic nephropathy), Tang, et al. used a case-controlled design that focused on patients with both PDR and ESRD. Given the important role of neovascularization in the pathophysiology of both PDR and ESRD, the investigators hypothesized that genes involved in angiogenesis effected susceptibility to these complications of diabetes. To test this hypothesis, 19 single nucleotide polymorphisms (SNPs) from 11 candidate genes were analyzed for allelic association. The initial genotyping involved 374 patients with both PDR and ESRD and 239 age- and ethnicity-matched controls from a Utah European-American type 2 diabetes cohort. Except for SNP rs1617640, located on 7q21, 1,125 bp upstream of the erythropoietin gene transcription start site (see Figure), no association was found. The linkage of SNP rs1617640 with diabetic microvascular complications was supported by analysis of two other large, independent type 1 diabetes cohorts. Compared to the GG genotype, erythropoietin concentration in the vitreous body was 7.5 times higher in samples from individuals with the TT risk genotype, and the T allele enhanced expression of a luciferase reporter construct by 25-fold compared to the G allele. The TT genotype appears to modulate erythropoietin expression by creating a binding site for transcription factors EVI1/MEL1 or AP1. Analysis of gene expression in murine models provided additional support for a role for erythropoietin in both diabetic retinopathy and nephropathy.
 |
Relationship between SNP rs1617640 and the erythropoietin gene. The SNP is located 1,125 bp upstream of the EPO transcription site. The findings of Tong, et al. suggest an additive allele-dose model with the heterozygous risk allele being GT and the homozygous risk allele being TT. The T allele appears to enhance EPO expression by creating a matrix match with EVI1/MEL1 or AP1 transcription enhancer binding sites.
To simplify the Figure, the region of EPO containing the coding sequence is illustrated by the uninterrupted yellow rectangle and is not drawn to scale. The blue rectangle represents noncoding sequence upstream of the EPO start site and is not drawn to scale. |
Recombinant erythropoietin is used extensively to treat patients with the anemia of renal failure (many of whom have diabetic nephropathy), and patients whose hemoglobin concentration is maintained above 13.5 gm/dl have a higher rate of cardiovascular complications than those maintained at a hemoglobin concentration below 11.5 gm/dl.4 The studies of Tong and colleagues provide a plausible explanation for the observed concordance and genetic predisposition to PDR and ESRD and send yet another cautionary note to physicians who prescribe the recombinant protein.
References
- Henke M, Laszig R, Rübe C, et al. Erythropoietin to treat head and neck cancer patients with anaemia undergoing radiotherapy: randomised, double-blind, placebo-controlled trial. Lancet. 2003;362:1255-60.
- Leyland-Jones B, Semiglazov V, Pawlicki M, et al. Maintaining normal hemoglobin levels with epoetin alfa in mainly nonanemic patients with metastatic breast cancer receiving first-line chemotherapy: a survival study. J Clin Oncol. 2005;23:5960-72.
- Bohlius J, Langensiepen S, Schwarzer G, et al. Recombinant human erythropoietin and overall survival in cancer patients: results of a comprehensive meta-analysis. J Natl Cancer Inst. 2005;97:489-98.
- Singh AK, Szczech L, Tang KL, et al. Correction of anemia with epoetin alfa in chronic kidney disease. N Engl J Med. 2006;355:2085-98.
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Gene Addiction in Myeloma
By Kenneth Anderson, MD
Dr. Anderson indicated no relevant conflicts of interest.
Shaffer AL, Emre NC, Lamy L, et al. IRF4 addiction in multiple myeloma. Nature. 2008;454:226-31.
Progress in the treatment of myeloma has directly translated from an improved understanding of the mechanisms of myeloma cell growth, survival, and drug resistance within the bone marrow microenvironment.1 Specifically, microarray profiling can identify the gene signature of myeloma cells before and after binding of tumor cells to bone marrow stromal cells and show induced changes in tumor as well as stromal cells due to cell-cell contact as well as cytokines. Importantly, targets and targeted therapies within tumor cells and the microenvironment can be validated preclinically in this model (for example, proteasome activity is upregulated in myeloma cells within the bone marrow milieu, and proteasome inhibitors can induce cytotoxicity against myeloma cells by overcoming cell adhesion-mediated drug resistance to conventional therapies).2 Excitingly, such targeted therapies can then rapidly translate from the bench to the bedside (for example, proteasome inhibitors have progressed rapidly to FDA approval for treatment of relapsed refractory to relapsed and only recently to newly diagnosed myeloma).3,4,5 Moreover, combination therapy informed by preclinical studies can also quickly move from the laboratory to the clinic. For example, the demonstration that proteasome inhibitors block DNA damage repair6 provided the basis for preclinical and clinical studies showing that proteasome inhibitors can sensitize or overcome resistance to DNA-damaging agents, ultimately culminating in the FDA approval of pegylated liposomal doxorubicin and bortezomib7 for treatment of relapsed myeloma.
Genetic knock-down and overexpression studies in myeloma cells now allow for stringent validation of a target as critical for myeloma cell growth.8 Shaffer and colleagues have recently carried out elegant small hairpin RNA (shRNA) screening studies, which show that interferon regulatory factor 4 (IRF4) is required for tumor cell viability, and confirmed by the ability of IRF4 overexpression to rescue myeloma cells from lethality induced by IRF4 shRNA. Importantly, there are no intrinsic genetic abnormalities of IRF4 within myeloma cell lines representing the spectrum of known genetic abnormalities in myeloma. Having shown the survival function of IRF4, these investigators utilized gene profiling and genome-wide chromatin analysis to demonstrate IRF4 target genes, such as MYC. Most importantly, IRF4 was also a target of MYC activation, both suggesting that genetic abnormalities of MYC in myeloma can upregulate IRF4 and confirming a MYC-IRF4 autoregulatory growth mechanism in myeloma cells.
These studies both identify hallmark genetic mechanisms underlying myeloma growth and suggest a novel therapeutic target. They demonstrate the power of genetic technologies for functionally validating a target gene and pathway underlying myeloma cell development. Indeed, a novel mechanism of autoregulatory myeloma cell growth has been identified. These studies also provide the basis for genetic mouse models, which may more closely mimic human myeloma. Finally, they identify a novel target and circuit for novel targeted therapies. It would be of interest to see whether any currently available myeloma therapies modulate this pathway. Importantly, it is likely that IRF4 and related circuits are modulated in the bone marrow milieu, and analogous studies in models of myeloma within the tumor microenvironment would further validate both the importance of these findings in myeloma pathogenesis and their potential therapeutic application.
References
- Hideshima T, Mitsiades C, Tonon G, et al. Understanding multiple myeloma pathogenesis in the bone marrow to identify new therapeutic targets. Nat Rev Cancer. 2007;7:585-98.
- Hideshima T, Richardson P, Chauhan D, et al. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 2001;61:3071-6.
- Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med. 2003;348:2609-17.
- Richardson PG, Sonneveld P, Schuster MW, et al. Bortezomib or high-dose dexamethasone for relapsed multiple myeloma. N Engl J Med. 2005;352:2487-98.
- San Miguel J, Schlag R, Khuageva N, et al. Bortezomib plus melphalan-prednisone versus melphalan-prednisone in untreated multiple myeloma patients ineligible for stem cell transplantation. N Engl J Med. 2008. [In press]
- Mitsiades N, Mitsiades CS, Poulaki V, et al. Molecular sequelae of proteasome inhibition in human multiple myeloma cells. Proc Natl Acad Sci USA. 2002;99:14374-9.
- Orlowski RZ, Nagler A, Sonneveld P, et al. Randomized phase III study of pegylated liposomal doxorubicin plus bortezomib compared with bortezomib alone in relapsed or refractory multiple myeloma: combination therapy improves time to progression. J Clin Oncol. 2007;25:3892-901.
- Carrasco DR, Sukhdeo K, Protopopova M, et al. The differentiation and stress response factor XBP-1 drives multiple myeloma pathogenesis. Cancer Cell. 2007;11:349-60.
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MDM2 SNP May Be Important in Predicting Outcome of CLL Patients
By John C. Byrd, MD
Dr. Byrd indicated no relevant conflicts of interest.
Gryshchenko I, Hofbauer S, Stoecher M, et al. MDM2 SNP309 is associated with poor outcome in B-cell chronic lymphocytic leukemia. J Clin Oncol. 2008;26:2252-7.
The study of single-nucleotide polymorphism (SNP) function for important genes related to drug metabolism, genetic predisposition, and cancer has become a major research focus across multiple disciplines. With respect to CLL, several interesting polymorphisms in key genes such as Mcl-1, Bax, and Bcl-2 have been reported but not always validated by subsequent independent work. Reasons for this are many, including technique, study population, statistical interpretation, and blind chance of multiple exploratory analyses that identify an interesting SNP without subsequent verification. Many of these genes have demonstrable function, and the lack of clinical correlation does not imply lack of impact, but rather emphasizes the importance of multiple genes in producing a discernable phenotype.
One pathway that has been shown to be very important in CLL is the p53 pathway. Disruption of the p53 pathway has been implicated in resistance to alkylator-based therapy, nucleoside analog therapy, and rituximab therapy. p53 function can be inactivated through a variety of direct (mutation and/or deletion of p53) or indirect pathways such as overexpression of the MDM2 protein or mutation of genes such as ATM. Gryshchenko and colleagues reported that a G-versus-T polymorphism at nucleotide 309 of MDM2 intron 1 impacts CLL outcome. The original paper describing this MDM2 SNP3091 validated the mechanistic relevance and a follow-up study provided further relevance of its function.2 Gryshchenko's results support data from other tumor types in that the occurrence of the G/G form of SNP309 does not predispose to CLL. However, as in other tumor types, the G/G SNP309 does correlate with shortened treatment-free and overall survival in CLL patients.
The investigation of what we know about MDM2 SNP309 and the function of the p53 pathway provides clear rationale for concurrent evaluation of p53 defects with SNP assessment. This report is exemplary in that the investigators report that such defects (as assessed by both interphase cytogenetics and SSCP mutational studies of p53 mutations) do have a different impact on treatment-free survival when considered together with SNP309 status. The authors also perform one of the most important tasks in analyzing SNPs by using a second, independent cohort of patients to confirm their results. Even when a SNP has demonstrated function, confirming the relevance of such a biomarker on two independent patient data sets provides additional assurance that the finding is correct. This is especially true with smaller studies where confirmation in an independent population should be considered to avoid bias. What remains to be asked is the impact of the SNP309 with respect to IgVH mutational status and potentially other prognostic features in CLL. Additionally, the impact of the SNP309 on treatment outcome should also be explored.
So, the question remains relative to what these results mean to patients with CLL and the physicians who care for them. Grysh-chenko's results echo the data from other tumor types in that the occurrence of the G/G form of SNP309 does not predispose to CLL, but does impact time to first treatment and overall survival of CLL patients with this. Given the complexity of the pathway and the variance within populations of patients who exhibit one genotype or another, examination of this SNP to predict an individual patient's outcome will likely be ineffective outside of consideration of a wide range of other prognostic features relevant to this pathway and many others that are disordered in CLL. The SNP309 findings presented in this work add another layer of detail to the evaluation of p53 pathway function that will require physicians to consider implications as therapeutic efforts move forward toward individualized treatment decisions based upon SNP and acquired genetic findings present in the tumor cell.
References
- Bond GL, Hu W, Bond EE, et al. A single nucleotide polymorphism in the MDM2 promoter attenuates the p53 tumor suppressor pathway and accelerates tumor formation in humans. Cell. 2004;119:591-602.
- Arva NC, Gopen TR, Talbott KE, et al. A chromatin-associated and transcriptionally inactive p53-Mdm2 complex occurs in mdm2 SNP309 homozygous cells. J Biol Chem. 2005;280:26776-87.
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Home Sweet Home
By Nelson Chao, MD
Dr. Chao indicated no relevant conflicts of interest.
Sackstein R, Merzaban JS, Cain DW, et al. Ex vivo glycan engineering of CD44 programs human multipotent mesenchymal stromal cell trafficking to bone. Nat Med. 2008;14:181-7.
Hematopoietic stem cell transplantation is possible because progenitors and stem cells have the ability to home to their niches to re-establish normal hematopoiesis. This is a highly orchestrated process that requires specific homing receptor-ligand interactions. Imagine that one of these stems cells is plucked out in a bone marrow harvest and then re-infused into a central vein. That cell will circulate in the vasculature until it finds the appropriate receptors. Cellular recruitment to bone occurs within specialized marrow vessels that constitutively express vascular E-selectin, a lectin that recognizes specific sugars (sialofucosylated determinants) on its various ligands. Then that cell has to transmigrate from the vascular endothelium into the interstitial space and again into the bone marrow cavity and find the proper niche. There it will be able to self-renew and also differentiate into committed progenitors. If that cell is not able to find the appropriate niche, it is likely to die or possibly remain quiescent, unable to provide the necessary contribution to hematopoietic reconstitution. Similarly, other adoptive cellular therapies (i.e., antigen-specific T cells) rely on the potential that the infused cells will home to the appropriate sites for their effector function. But how do they find the right home?
The publication by Sackstein, et al. demonstrates the importance and potential clinical application of ensuring the proper address. Multipotent mesenchymal stromal cells (MSCs, also termed mesenchymal stem cells) have the potential for a variety of therapeutic uses. However, clinically their use is constrained by the poor osteotropism of infused MSCs. Human MSCs do not express E-selectin ligands, but express a CD44 glycoform bearing α-2,3-sialyl modifications. Using an α-1,3-fucosyltransferase preparation and enzymatic conditions specifically designed for treating live cells, they converted the native CD44 glycoform on MSCs into hematopoietic cell E-selectin/L-selectin ligand (HCELL), which conferred potent E-selectin binding without effects on cell viability or multipotency. Real-time intravital microscopy in immunocompromised (NOD/SCID) mice showed that intravenously infused HCELL+ MSCs infiltrated marrow within hours of infusion, with ensuing rare foci of endosteally localized cells and human osteoid generation. Following extravasation, there was reversion to the native CD44 glycoform.
The ability to modify the decorated sugars on the cell surface without changes in multipotency or survival is an exciting finding and raises the prospect for clinical application. Despite the lack of the necessary effectors for homing to bone, osteotropism was conferred by ex vivo modification of the sugars of a single glycoprotein (CD44) creating a potent E-selectin ligand HCELL. Engineering HCELL expression was achieved by rational design to specifically drive surface α-1,3-fucosylation. Therefore, this technology could significantly improve the use of MSCs for hematopoietic reconstitution. Moreover, E-selectin expression is upregulated in areas of inflammation and ischemia; therefore, programmed HCELL expression should provide the proper address for targeted migration and infiltration of these cells for therapeutic purposes. In a broader sense, increasing E-selectin ligand activity could be expanded to other populations of cells. A variety of physiologic and pathologic processes, including immune diseases, infectious diseases, and cancer, are accompanied by upregulated E-selectin expression in affected endothelial beds. Regulatory T cells or cytotoxic T cells could be programmed for specific cellular trafficking by chemical modification of their cell surface sugars on a distinct membrane glycoprotein.
Who says you can't go home again?
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Pit Stop in the Liver for Primitive Erythrocytes to Enucleate
By Diane Krause, MD, PhD, and Stephanie Halene, MD
Drs. Krause and Halene indicated no relevant conflicts of interest.
Isern J, Fraser ST, He Z, et al. The fetal liver is a niche for maturation of primitive erythroid cells. Proc Natl Acad Sci USA. 2008;105:6662-7.
Blood formation in development is characterized by two distinct stages. Primitive hematopoiesis, which is initiated by a population of primitive hematopoietic stem cells, is progressively replaced by definitive hematopoiesis, which is derived from definitive hematopoietic stem cells that maintain hematopoiesis throughout adulthood. During murine embryogenesis, primitive erythroid cells (EryP) develop in the yolk sac blood islands on embryonic day 7.5 (E7.5), enter the circulation by E9.5, and remain nucleated until E12.5. In contrast, definitive erythroid cells (EryD), which appear later in fetal development, are enucleated prior to entering the blood stream.
In order to better understand how enucleation of EryP is regulated, the investigators designed an elegant system to track EryP. In this system, the promoter elements of the ε-globin gene drive expression of green fluorescent protein (GFP), such that GFP expression is restricted to EryP. This allows tracking of EryP throughout development even during E12 through E16 when progressively greater numbers of EryD appear. The investigators followed maturation and expansion of EryP as well as surface markers on the cells before, during, and after enucleation. They discovered a transient upregulation of particular integrins from E12.5 to E14.5, with little expression of these integrins on EryP before or after this time frame. The investigators hypothesized that upregulation of these integrins promotes adhesion of EryP to macrophages in the developing fetal liver forming so-called erythroid blood islands in which developing erythroid cells form rosettes around central macrophages, which then phagocytose the nuclei as the erythroid cells enucleate. To test this hypothesis, the investigators engineered erythroid blood islands in vitro using isolated fetal liver-derived macrophages from one specific stage of fetal liver development and EryP from multiple different stages of development. They found that the EryP from days E12.5 through E14.5 adhered to the macrophages, while those from earlier and later stages did not. The two-day window during which EryP adhere corresponds to the time during embryogenesis when EryP upregulate integrins and enucleate. By E15.5, enucleation of EryP is complete.
As an erythrocyte enucleates, the nucleus is not just expelled from the cell, but remains surrounded by a thin rim of plasma and plasma membrane and is pinched off from the remaining erythroblast by formation of an actin ring. This results in two cells. One is the young enucleated RBC, which can re-enter the circulation, and the other contains the membrane-bound nucleus. A key feature of this process is the asymmetric distribution of plasma membrane components between the resulting RBC versus the expelled nucleus. To test the hypothesis that integrins are enriched on the plasma membrane surrounding the expelled nucleus may be responsible for the retention of the nuclei near the macrophages, while allowing the RBC to re-enter the circulation, the investigators took their model a step further. They targeted the GFP protein expressed under the control of the ε-globin gene to enter the nucleus, which allowed them to specifically track the "pinched-off" nucleus. As predicted, expression of the integrins was much higher on the nuclei than on the enucleated RBCs, and the nuclei were retained in the fetal liver.
The findings presented in this work not only represent an astute observation of the distinct development of primitive versus definitive erythropoiesis, but also employ an elegant model to elucidate the mechanisms underlying these differences. In an era when exploration into stem-cell therapies employing more and more primitive cell types is turning into a realistic hope, a deeper understanding of developmental mechanisms will certainly contribute to the field.
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Myelodysplastic Syndromes: Will WHO classification and WPSS become tools for hematopoietic stem cell
By Gérard Socié, MD, PhD
Dr. Socié indicated no relevant conflicts of interest.
Alessandrino EP, Della Porta MG, Bacigalupo A, et al. WHO classification and WPSS predict post-transplant outcome in patients with myelodysplastic syndrome: a study from the GITMO. Blood. 2008. [Epub ahead of print]
Currently, allogeneic stem cell transplantation (SCT) is considered to be the only potentially curative therapy for myelodysplastic syndromes (MDS). However, since most patients with MDS are older than 60, few are candidates for myeloablative transplantation. Approximately 25 percent of patients with MDS are younger than 60 and may be considered for transplantation. Trials of SCT demonstrate long-term survival rates between 25 percent and 70 percent. The Inter-national Prognostic Scoring System (IPSS) is reported to be a useful predictor of transplantation outcome.1 The optimal timing of bone marrow transplantation from HLA-identical siblings or unrelated donors for MDS is unknown. Many patients enjoy a long period after diagnosis without obvious disease progression. For these patients, the risks of immediate morbidity and mortality associated with transplantation are unacceptably high. Eventually, however, most patients with MDS develop symptomatic cytopenias, or their disease evolves to a more aggressive phenotype or transforms into AML, at which time SCT is less likely to be successful. Prospective comparisons of different transplantation timing strategies are not available and, unfortunately, are unlikely to be performed.
In a decision analysis, Cutler, et al. reported analyses using individual patient risk-assessment data from transplantation and non-transplantation registries performed for all four IPSS risk groups with adjustments for quality of life (QoL).1 For low and intermediate-1 IPSS groups, they reported that delayed transplantation maximized overall survival. Transplantation prior to leukemic transformation was associated with a greater number of years of life than transplantation at the time of leukemic progression. In a cohort of patients under the age of 40, an even more marked survival advantage for delayed transplantation was noted. For intermediate-2 and high IPSS groups, transplantation at diagnosis maximized overall survival. No changes in the optimal transplantation strategies were noted when QoL adjustments were incorporated. Thus, the authors claimed that for low- and intermediate-1-risk MDS, delayed BMT is associated with maximal life expectancy, whereas immediate transplantation for intermediate-2- and high-risk disease is associated with maximal life expectancy.1
In 2002, the WHO formulated a new proposal for the classification of MDS. The distinction of multi-lineage dysplasia and the recognition of two categories of RAEB represented an improvement in the ability to predict survival and leukemic evolution. Data have also emerged on the ability of the WHO classification to guide clinical decision-making regarding therapeutic choice. A WHO classification-based prognostic scoring system (WPSS) has been recently defined and validated in untreated patients. The WPSS is based on WHO categories, karyotype abnormalities, and transfusion requirement, and is able to identify five risk groups of MDS patients with difference in survival and risks of leukemic progression. The impact of WHO classification and WPSS on the outcome of MDS patients receiving allogeneic SCT remains, however, to be clarified.
In their study, Alessandrino, et al., for the Italian group for transplantation (GITMO), studied the impact of WHO classification and WPSS on the outcome of patients with MDS receiving allogeneic SCT. They studied 365 patients reported to the GITMO between 1990 and 2006. Five-year overall survival (OS) was 80 percent in refractory anemia, 57 percent in refractory cytopenias with multilineage dysplasia, 51 percent in RAEB-1, 28 percent in RAEB-2, and 25 percent in acute leukemia from MDS (p=0.001). Five-year probability of relapse was 9 percent, 22 percent, 24 percent, 56 percent, and 53 percent, respectively (p<0.001), while five-year transplant-related mortality (TRM) was 14 percent, 39 percent, 38 percent, 34 percent, and 44 percent, respectively. In multivariate analysis, WHO classification showed a significant effect on OS and probability of relapse. Transfusion-dependency was associated with a reduced OS and increased TRM. In multivariate analysis, WPSS showed a prognostic significance on both OS and probability of relapse. In patients without excess blasts, multilineage dysplasia and transfusion-dependency significantly affected OS and were associated with an increased TRM. In patients without excess blasts, WPSS identified two groups of patients (low vs. intermediate risk) with a significant difference in OS and TRM, while in the same group of patients, IPSS failed to stratify the prognosis.
Interestingly, both WHO and WPSS maintained their prognostic effect on post-transplantation outcome also in specific subsets of patients, such as subjects older than 50 as well as patients receiving reduced-intensity conditioning (RIC) regimen. This is clearly relevant in light of the increased number of RIC regimens performed in MDS in most recent years, after the demonstration of their efficacy in allowing engraftment and in decreasing TRM in patients ineligible for standard conditioning allogeneic SCT. Whether there is any advantage in administering chemotherapy to achieve remission before transplantation for MDS is the subject of debate. The GITMO data suggest that in patients with high-risk MDS according to WHO criteria (i.e., RAEB-1, RAEB-2), achieving a complete remission before a standard allogeneic SCT seems not to be associated with a better post-transplant outcome. In patients receiving RIC, complete remission was associated with a trend to a reduced relapse rate. As expected, a significant impact of disease status at transplant was present in patients affected with AML from MDS (formerly classified as RAEB-t according to FAB criteria).
Nowadays, however, the real question is what the impact of new chemotherapeutic agents in MDS before SCT, such as 5-azacytidine, will be. These new drugs seem to be able to increase the survival of patients with MDS; their role, if any, before SCT is still under investigation.
References
- Cutler CS, Lee SJ, Greenberg P, et al. A decision analysis of allogeneic bone marrow transplantation for the myelodysplastic syndromes: delayed transplantation for low-risk myelodysplasia is associated with improved outcome. Blood. 2004;104:579-85.
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