Blood recent issues

Why some cells are "more equal" than others?

Ratajczak, M. Z., Shin, D.-M. — Thu, 03 Dec 2009 09:02:28 PST

Fine-tuning targeted therapy of CML

Shah, N. P. — Thu, 03 Dec 2009 09:02:28 PST

Ready to analyze genetically modified human platelets

Gawaz, M. — Thu, 03 Dec 2009 09:02:28 PST

Blast crisis

Thu, 03 Dec 2009 09:02:28 PST

The role of B cells in the pathogenesis of graft-versus-host disease

Thu, 03 Dec 2009 09:02:28 PST

Allogeneic hematopoietic stem cell transplantation is an established treatment modality for malignant and nonmalignant hematologic diseases. Acute and chronic graft-versus-host diseases (GVHDs) are a major cause of morbidity and mortality after allogeneic stem cell transplantation. T cells have been identified as key players in the graft-versus-host reaction and, therefore, most established drugs used against GVHD target T cells. Despite our knowledge on the pathogenesis of the GVH reaction, success of established therapies for prevention and treatment of GHVD is unsatisfactory. Recently, animal and human studies demonstrated that B cells are involved in the immunopathophysiology of acute and chronic GVHD. Early phase clinical trials of B-cell depletion with rituximab have shown beneficial effects on both acute and chronic GVHD. This review summarizes the current experimental and clinical evidence for the involvement of B cells in the pathogenesis of acute and chronic GVHD and discusses the clinical implications for the management of patients undergoing allogeneic stem cell transplantation.

Evidence of serum immunoglobulin abnormalities up to 9.8 years before diagnosis of chronic lymphocytic leukemia: a prospective study

Thu, 03 Dec 2009 09:02:28 PST

Immune-related deficiencies are well-known complications of chronic lymphocytic leukemia (CLL). Although recent data indicate that almost all CLL patients are preceded by a monoclonal B-cell lymphocytosis precursor state, patterns of immune defects preceding CLL diagnosis are unclear. We identified 109 persons who developed CLL from the prospective and nationwide Prostate, Lung, Colorectal and Ovarian Cancer Screening Trial with 77 469 participants, with serially collected prediagnostic serum samples. We assayed monoclonal (M)–proteins, / free light chains (FLCs) in prediagnostic obtained up to 9.8 years before CLL diagnosis. The prevalence of an abnormal FLC ratio, M-protein, and hypogamma-globulinemia before CLL diagnosis was 38% (95% confidence interval, 29%-47%), 13% (7%-21%), and 3% (1%-8%), respectively. M-proteins and abnormal FLC ratios were detected up to 9.8 years before CLL diagnosis in a total of 48 persons (44%). Hypogammaglobulinemia was not present until 3 years before the diagnosis of CLL. Among 37 patients with information on tumor cell immunophenotype, an association between immunophenotype and involved FLC (P = .024, Fisher exact test) was observed. Among 61 persons with a normal FLC ratio and without an M-protein, 17 had elevated and/or FLC levels, indicating polyclonal B-cell activation in 17 of 109 (16%) patients. These findings support a role for chronic immune stimulation in CLL genesis.

Nilotinib for the frontline treatment of Ph+ chronic myeloid leukemia

Thu, 03 Dec 2009 09:02:28 PST

Nilotinib has a higher binding affinity and selectivity for BCR-ABL with respect to imatinib and is an effective treatment of chronic myeloid leukemia (CML) after imatinib failure. In a phase 2 study, 73 early chronic-phase, untreated, Ph⁺ CML patients, received nilotinib at a dose of 400 mg twice daily. The primary endpoint was the complete cytogenetic response (CCgR) rate at 1 year. With a median follow-up of 15 months, the CCgR rate at 1 year was 96%, and the major molecular response rate 85%. Responses were rapid, with 78% CCgR and 52% major molecular response at 3 months. During the first year, the treatment was interrupted at least once in 38 patients (52%). The mean daily dose ranged between 600 and 800 mg in 74% of patients, 400 and 599 mg in 18% of patients, and was less than 400 mg in 8% of patients. Dose interruptions were mainly due to nonhematologic and biochemical side effects. Myelosuppression was irrelevant. One patient progressed to blastic crisis after 6 months; one went off-treatment for lipase increase grade 4 (no pancreatitis). Nilotinib is safe and very active in early chronic-phase CML. These data support a role for nilotinib for the frontline treatment of CML. This study was registered at ClinicalTrials.gov as NCT00481052.

Chronic myeloid leukemia: a prospective comparison of interphase fluorescence in situ hybridization and chromosome banding analysis for the definition of complete cytogenetic response: a study of the GIMEMA CML WP

Thu, 03 Dec 2009 09:02:28 PST

In chronic myeloid leukemia, different methods are available to monitor the response to therapy: chromosome banding analysis (CBA), interphase fluorescence in situ hybridization (I-FISH), and real-time quantitative polymerase chain reaction (RT-Q-PCR). The GIMEMA CML WP (Gruppo Italiano Malattie Ematologiche Adulto Chronic Myeloid Leukemia Working Party) has performed a prospective study to compare CBA and I-FISH for the definition of complete cytogenetic response (CCgR). Samples (n = 664) were evaluated simultaneously by CBA and I-FISH. Of 537 cases in CCgR, the number of positive nuclei by I-FISH was less than 1% in 444 cases (82.7%). Of 451 cases with less than 1% positive nuclei by I-FISH, 444 (98.4%) were classified as CCgR by CBA. The major molecular response rate was significantly greater in cases with I-FISH less than 1% than in those with I-FISH 1% to 5% (66.8% vs 51.6%, P < .001) and in cases with CCgR and I-FISH less than 1% than in cases with CCgR and I-FISH 1% to 5% (66.1% vs 49.4%, P = .004). I-FISH is more sensitive than CBA and can be used to monitor CCgR. With appropriate probes, the cutoff value of I-FISH may be established at 1%. These trials are registered at http://www.clinicaltrials.gov as NCT00514488 and NCT00510926.

Dasatinib treatment of chronic-phase chronic myeloid leukemia: analysis of responses according to preexisting BCR-ABL mutations

Thu, 03 Dec 2009 09:02:28 PST

Dasatinib is a BCR-ABL inhibitor with 325-fold higher potency than imatinib against unmutated BCR-ABL in vitro. Imatinib failure is commonly caused by BCR-ABL mutations. Here, dasatinib efficacy was analyzed in patients recruited to phase 2/3 trials with chronic-phase chronic myeloid leukemia with or without BCR-ABL mutations after prior imatinib. Among 1043 patients, 39% had a preexisting BCR-ABL mutation, including 48% of 805 patients with imatinib resistance or suboptimal response. Sixty-threedifferent BCR-ABL mutations affecting 49 amino acids were detected at baseline, with G250, M351, M244, and F359 most frequently affected. After 2 years of follow-up, dasatinib treatment of imatinib-resistant patients with or without a mutation resulted in notable response rates (complete cytogenetic response: 43% vs 47%) and durable progression-free survival (70% vs 80%). High response rates were achieved with different mutations except T315I, including highly imatinib-resistant mutations in the P-loop region. Impaired responses were observed with some mutations with a dasatinib median inhibitory concentration (IC₅₀) greater than 3nM; among patients with mutations with lower or unknown IC₅₀, efficacy was comparable with those with no mutation. Overall, dasatinib has durable efficacy in patients with or without BCR-ABL mutations. All trials were registered at http://www.clinicaltrials.gov as NCT00123474, NCT00101660, and NCT00103844.

Abnormal serum free light chain ratio in patients with multiple myeloma in complete remission has strong association with the presence of oligoclonal bands: implications for stringent complete remission definition

Thu, 03 Dec 2009 09:02:28 PST

The prevalence of an abnormal serum free light chain (FLC) ratio in 34 patients with multiple myeloma in complete response (CR) after hematopoietic stem cell transplantation was studied. Fourteen of 34 patients (41.2%) showed an abnormal FLC ratio. The frequency of abnormal FLC ratio in patients with or without oligoclonal bands was 72.7% versus 26%, respectively (P = .023). The median value of FLC ratio was 2.55 (95% confidence interval, 1.89-3.20) in patients with oligoclonal bands versus 0.87 (95% confidence interval, 0.70-1.04) for those with no oligoclonal bands (P = .011). This is the first report showing that the presence of oligoclonal bands in patients with multiple myeloma in CR frequently results in an abnormal FLC ratio. Because an oligoclonal immune response is associated with a good outcome, our results question the current definition of stringent CR and support that the prognostic impact of oligoclonal bands should be also assessed on multivariate analysis.

Classification of amyloidosis by laser microdissection and mass spectrometry-based proteomic analysis in clinical biopsy specimens

Thu, 03 Dec 2009 09:02:29 PST

The clinical management of amyloidosis is based on the treatment of the underlying etiology, and accurate identification of the protein causing the amyloidosis is of paramount importance. Current methods used for typing of amyloidosis such as immunohistochemistry have low specificity and sensitivity. In this study, we report the development of a highly specific and sensitive novel test for the typing of amyloidosis in routine clinical biopsy specimens. Our approach combines specific sampling by laser microdissection (LMD) and analytical power of tandem mass spectrometry (MS)–based proteomic analysis. We studied 50 cases of amyloidosis that were well-characterized by gold standard clinicopathologic criteria (training set) and an independent validation set comprising 41 cases of cardiac amyloidosis. By use of LMD/MS, we identified the amyloid type with 100% specificity and sensitivity in the training set and with 98% in validation set. Use of the LMD/MS method will enhance our ability to type amyloidosis accurately in clinical biopsy specimens.

Induction of B-cell development in adult mice reveals the ability of bone marrow to produce B-1a cells

Thu, 03 Dec 2009 09:02:29 PST

To study B-cell development from bone marrow (BM), we generated recombination-activating gene 1 (Rag1)–targeted mice lacking mature lymphocytes. B-cell development can be induced in such mice by B cell–specific restoration of a functional Rag1 transcription unit. Follicular and marginal zone B cells populated the spleen when Rag1 expression was permitted. Notably, the peritoneal cavity was dominated by bona fide B-1a cells, as judged by surface markers and functional properties. These BM-derived B-1a cells exhibited a polyclonal VDJ repertoire with substantial N nucleotide insertions. Nevertheless, physiologic frequencies of phosphatidylcholine-specific B cells were detected. Importantly, the BM of young and 5-month-old mice was indistinguishable with regard to the potential to generate B-1a cells.

Undifferentiated hematopoietic cells are characterized by a genome-wide undermethylation dip around the transcription start site and a hierarchical epigenetic plasticity

Thu, 03 Dec 2009 09:02:29 PST

Evidence for the epigenetic regulation of hematopoietic stem cells (HSCs) is growing, but the genome-wide epigenetic signature of HSCs and its functional significance remain unclear. In this study, from a genome-wide comparison of CpG methylation in human CD34⁺ and CD34^– cells, we identified a characteristic undermethylation dip around the transcription start site of promoters and an overmethylation of flanking regions in undifferentiated CD34⁺ cells. This "bivalent-like" CpG methylation pattern around the transcription start site was more prominent in genes not associated with CpG islands (CGI^–) than CGI⁺ genes. Undifferentiated hematopoietic cells also exhibited dynamic chromatin associated with active transcription and a higher turnover of histone acetylation than terminally differentiated cells. Interestingly, inhibition of chromatin condensation by chemical treatment (5-azacytidine, trichostatin A) enhanced the self-renewal of "stimulated" HSCs in reconstituting bone marrows but not "steady-state" HSCs in stationary phase bone marrows. In contrast, similar treatments on more mature cells caused partial phenotypic dedifferentiation and apoptosis at levels correlated with their hematopoietic differentiation. Taken together, our study reveals that the undifferentiated state of hematopoietic cells is characterized by a unique epigenetic signature, which includes dynamic chromatin structures and an epigenetic plasticity that correlates to level of undifferentiation.

Cell-cell cooperation at the T helper cell/mast cell immunological synapse

Thu, 03 Dec 2009 09:02:29 PST

It has been suggested that mast cells might serve, under certain circumstances, as antigen-presenting cells (APCs) for T cells. However, whether cognate interactions between mast cells and class II–restricted CD4⁺ T cells actually occur is still an open question. We addressed this question by using peritoneal cell–derived mast cells (PCMCs) and freshly isolated peritoneal mast cells as APC models. Our results show that in vitro treatment of PCMCs with interferon- and interleukin-4 induced surface expression of mature major histocompatibility complex class II molecules and CD86. When interferon-/interleukin-4–primed PCMCs were used as APCs for CD4⁺ T cells, they induced activation of effector T cells but not of their naive counterparts as evidenced by CD69 up-regulation, proliferation, and cytokine production. Confocal laser scanning microscopy showed that CD4⁺ T cells formed immunological synapses and polarized their secretory machinery toward both antigen-loaded PCMCs and freshly isolated peritoneal mast cells. Finally, on cognate interaction with CD4⁺ T cells, mast cells lowered their threshold of activation via FcRI. Our results show that mast cells can establish cognate interactions with class II–restricted helper T cells, implying that they can actually serve as resident APCs in inflamed tissues.

B-cell follicle development remodels the conduit system and allows soluble antigen delivery to follicular dendritic cells

Bajenoff, M., Germain, R. N. — Thu, 03 Dec 2009 09:02:29 PST

Afferent lymph is transported throughout lymph nodes (LNs) by the conduit system. Whereas this conduit network is dense in the T-cell zone, it is sparse in B-cell follicles. In this study, we show that this differential organization emerges during lymph node development. Neonatal LNs lack B follicles, but have a developed T-cell zone and a dense conduit network. As new T and B cells enter the developing LN, the conduit network density is maintained in the T, but not the B zone, leading to a profound remodeling of the follicular network that nevertheless maintains its connectivity. In adults, the residual follicular conduits transport soluble antigen to deep regions, where follicular dendritic cells are abundant and appear to replace the fibroblastic reticular cells that enwrap conduits in the T zone. This strategic location correlates with the capacity of the follicular dendritic cells to capture antigen even in the absence of antigen-specific antibodies. Together, these results describe how the stromal organization of the T and B regions of LNs diverges during development, giving rise to distinct antigen transport and delivery modes in the 2 compartments.

Appearance of peripheral blood plasma cells and memory B cells in a primary and secondary immune response in humans

Thu, 03 Dec 2009 09:02:29 PST

In humans, the kinetics of the appearance of memory B cells and plasma cells during primary immunization are not well defined. In this study, we assessed the primary B-cell response of rabies-antigen naive volunteers during a 3-dose course of rabies vaccine compared with the B-cell response to a booster dose of rabies vaccine given to previously immunized volunteers. After a single dose of vaccine, in the naive group plasma and memory B cells appeared later (peak at day 10) than in the primed group (peak at day 7) and were at lower frequency. The most rapid responses (day 4) were detected after a third immunization in the naive group. This is the first study to document the detailed kinetics of the plasma cell and memory B-cell responses to immunization in adult humans and to demonstrate differences in the responses that relate to the preexisting immune status of the persons.

ATG-induced expression of FOXP3 in human CD4+ T cells in vitro is associated with T-cell activation and not the induction of FOXP3+ T regulatory cells

Broady, R., Yu, J., Levings, M. K. — Thu, 03 Dec 2009 09:02:29 PST

Several recent reports have suggested that in vitro exposure of CD4⁺ T cells to rabbit antithymocyte globulin (rATG), which is commonly used to prevent and treat graft-versus-host disease and allograft rejection, is an effective method to induce CD4⁺CD25⁺FOXP3⁺ T regulatory cells (Tregs). We and others, however, have shown that FOXP3 is also expressed in activated T cells. We therefore investigated whether the induction of FOXP3 expression by rATG resulted in a stable population of suppressive Tregs. We found that exposure of peripheral blood mononuclear cells (PBMCs) or conventional T cells to rATG resulted in induction of transient rather than stable expression of CD25 and FOXP3. Furthermore, rATG-treated T effector cells acquired neither an immunosuppressive profile of cytokine production nor suppressive capacity, even at the time of maximal FOXP3 expression. These findings indicate that the notion that rATG can be used to induce Tregs in vitro for cellular therapy in vivo should be re-evaluated.

Characterization of a rituximab variant with potent antitumor activity against rituximab-resistant B-cell lymphoma

Thu, 03 Dec 2009 09:02:29 PST

Despite widespread use of the anti-CD20 monoclonal antibody (mAb), rituximab, in treating B-cell lymphomas, its efficacy remains variable and often modest. A better understanding of rituximab-mediated killing mechanisms is essential to develop more effective therapeutic agents. In this study, we modulated the binding property of rituximab by introducing several point mutations in its complementarity-determining regions. The data showed that changing the binding avidity of rituximab in the range from 10^–8 to 10^–10 M could regulate its antibody-dependent cellular cytotoxicity but not affect its complement-dependent cytotoxicity and apoptosis-inducing activity in B-lymphoma cells. Contradictory to previous findings, we found that the complement-dependent cytotoxicity potency of CD20 mAb was independent of the off-rate. Despite still being a type I CD20 mAb, a rituximab triple mutant (H57DE/H102YK/L93NR), which had a similar binding avidity to a double mutant (H57DE/H102YK), was unexpectedly found to have extremely potent apoptosis-inducing activity. Moreover, this triple mutant, which was demonstrated to efficiently initiate both caspase-dependent and -independent apoptosis, exhibited potent in vivo therapeutic efficacy, even in the rituximab-resistant lymphoma model, suggesting that it might be a promising therapeutic agent for B-cell lymphomas.

TC-PTP is required for the maintenance of MYC-driven B-cell lymphomas

Young, R. M., Polsky, A., Refaeli, Y. — Thu, 03 Dec 2009 09:02:29 PST

We sought to determine the contributions of protein tyrosine phosphatases (PTPs) to the pathogenesis of B-cell lymphomas. We found that T-cell PTP (TC-PTP) was overexpressed in transformed B cells. We hypothesized that TC-PTP may be a tumor-promoting gene that is regulated by MYC overexpression in B cells. Knockdown of TC-PTP in murine tumors resulted in decreased cell viability in vitro because of an arrest in the G₁ phase of the cell cycle. Furthermore, cells with reduced TC-PTP expression were unable to either engraft or expand in vivo. Taken together, these data indicate that TC-PTP is required for B-cell tumor maintenance. Our data also suggested a correlation between TC-PTP expression and MYC overexpression. To investigate this further, we used malignant murine B cells that contain a doxycycline-repressible MYC transgene. We found that repression of MYC overexpression with doxycycline reduced TC-PTP expression. Moreover, enforced expression of TC-PTP showed partial rescue of the expansion of tumor cells after suppression of MYC overexpression. These results suggest that MYC overexpression induces TC-PTP overexpression, which in turn promotes tumor proliferation, implicating TC-PTP as an important effector of the MYC-driven proliferation program in B-cell lymphomas. Thus, TC-PTP may be a suitable molecular target for the treatment of B-cell lymphomas.

Cotreatment with panobinostat and JAK2 inhibitor TG101209 attenuates JAK2V617F levels and signaling and exerts synergistic cytotoxic effects against human myeloproliferative neoplastic cells

Thu, 03 Dec 2009 09:02:29 PST

The mutant JAK2V617F tyrosine kinase (TK) is present in the majority of patients with BCR-ABL–negative myeloproliferative neoplasms (MPNs). JAK2V617F activates downstream signaling through the signal transducers and activators of transcription (STAT), RAS/mitogen-activated protein kinase (MAPK), and phosphatidylinositol 3 (PI3)/AKT pathways, conferring proliferative and survival advantages in the MPN hematopoietic progenitor cells (HPCs). Treatment with the pan-histone deacetylase (HDAC) inhibitor panobinostat (PS) is known to inhibit the chaperone function of heat shock protein 90, as well as induce growth arrest and apoptosis of transformed HPCs. Here, we demonstrate that PS treatment depletes the autophosphorylation, expression, and downstream signaling of JAK2V617F. Treatment with PS also disrupted the chaperone association of JAK2V617F with hsp90, promoting proteasomal degradation of JAK2V617F. PS also induced apoptosis of the cultured JAK2V617F-expressing human erythroleukemia HEL92.1.7 and Ba/F3-JAK2V617F cells. Treatment with the JAK2 TK inhibitor TG101209 attenuated JAK2V617F autophosphorylation and induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with PS and TG101209 further depleted JAK/STAT signaling and synergistically induced apoptosis of HEL92.1.7 and Ba/F3-JAK2V617F cells. Cotreatment with TG101209 and PS exerted greater cytotoxicity against primary CD34⁺ MPN cells than normal CD34⁺ HPCs. These in vitro findings suggest combination therapy with HDAC and JAK2V617F inhibitors is of potential value for the treatment of JAK2V617F-positive MPN.

FLT3-ITD up-regulates MCL-1 to promote survival of stem cells in acute myeloid leukemia via FLT3-ITD-specific STAT5 activation

Thu, 03 Dec 2009 09:02:29 PST

Myeloid cell leukemia-1 (MCL-1) is an essential survival factor for hematopoiesis. In humans, hematopoietic stem cells (HSCs) express MCL-1 at the highest level in response to FMS-like tyrosine kinase-3 (FLT3) signaling. We here show that this FLT3-dependent stem cell maintenance system also plays a critical role in survival of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). The CD34⁺CD38^– LSC fraction expresses high levels of FLT3 as well as MCL-1, even compared with normal HSCs. Treatment with FLT3 ligand induced further MCL-1 up-regulation in LSCs in all AML cases tested. Interestingly, the group of samples expressing the highest levels of MCL-1 constituted AML with FLT3–internal tandem duplications (ITD). In FLT3-ITD AML cell lines, cells expressed a high level of MCL-1, and an inhibition of MCL-1 induced their apoptotic cell death. A tyrosine kinase inhibitor suppressed MCL-1 expression, and induced apoptosis that was reversed by the enforced MCL-1 expression. Finally, transduction of FLT3-ITD into HSCs strongly activated MCL-1 expression through its signal transducer and activator of transcription 5 (STAT5)–docking domains. This effect was completely abrogated when STAT5 activation was blocked. Thus, the acquisition of FLT3-ITD ensures LSC survival by up-regulating MCL-1 via constitutive STAT5 activation that is independent of wild-type FLT3 signaling.

Human platelets produced in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice upon transplantation of human cord blood CD34+ cells are functionally active in an ex vivo flow model of thrombosis

Thu, 03 Dec 2009 09:02:29 PST

Xenotransplantation systems have been used with increasing success to better understand human hematopoiesis and thrombopoiesis. In this study, we demonstrate that production of human platelets in nonobese diabetic/severe combined immunodeficient mice after transplantation of unexpanded cord-blood CD34⁺ cells was detected within 10 days after transplantation, with the number of circulating human platelets peaking at 2 weeks (up to 87 x 10³/µL). This rapid human platelet production was followed by a second wave of platelet formation 5 weeks after transplantation, with a population of 5% still detected after 8 weeks, attesting for long-term engraftment. Platelets issued from human hematopoietic stem cell progenitors are functional, as assessed by increased CD62P expression and PAC1 binding in response to collagen-related peptide and thrombin receptor-activating peptide activation and their ability to incorporate into thrombi formed on a collagen-coated surface in an ex vivo flow model of thrombosis. This interaction was abrogated by addition of inhibitory monoclonal antibodies against human glycoprotein Ib (GPIb) and GPIIb/IIIa. Thus, our mouse model with production of human platelets may be further explored to study the function of genetically modified platelets, but also to investigate the effect of stimulators or inhibitors of human thrombopoiesis in vivo.

Urokinase-type plasminogen activator increases hepatocyte growth factor activity required for skeletal muscle regeneration

Sisson, T. H., Nguyen, M.-H., Yu, B., Novak, M. L., Simon, R. H., Koh, T. J. — Thu, 03 Dec 2009 09:02:29 PST

The plasminogen system plays a crucial role in the repair of a variety of tissues, including skeletal muscle. We hypothesized that urokinase-type plasminogen activator (uPA) promotes muscle regeneration by activating hepatocyte growth factor (HGF), which, in turn, stimulates proliferation of myoblasts required for regeneration. In our studies, levels of active HGF and phosphorylation of the HGF receptor c-met were increased after muscle injury in wild-type mice. Compared with wild-type animals, mice deficient in uPA (uPA^–/–) had markedly reduced HGF levels and c-met activation after muscle damage. This reduced HGF activity in uPA^–/– animals was associated with decreased cell proliferation, myoblast accumulation, and new muscle fiber formation. On the other hand, HGF activity was enhanced at early time points in PAI-1^–/– mice compared with wild-type mice and the PAI-1^–/– animals exhibited accelerated muscle fiber regeneration. Furthermore, administration of exogenous uPA rescued HGF levels and muscle regeneration in uPA^–/– mice, and an HGF-blocking antibody reduced HGF activity and muscle regeneration in wild-type mice. We also found that uPA promotes myoblast proliferation in vitro through its proteolytic activity, and this process was inhibited by an HGF-blocking antibody. Together, our findings demonstrate that uPA promotes muscle regeneration through HGF activation and subsequent myoblast proliferation.

Inducing the tryptophan catabolic pathway, indoleamine 2,3-dioxygenase (IDO), for suppression of graft-versus-host disease (GVHD) lethality

Thu, 03 Dec 2009 09:02:29 PST

During graft-versus-host disease (GVHD), donor T cells become activated and migrate to tissue sites. Previously, we demonstrated a crucial role for the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) in GVHD regulation. Here, we show that upon arrival in the colon, activated donor T cells produced interferon- that up-regulated IDO, causing T-cell anergy and apoptosis. IDO induces GCN2 kinase, up-regulating a T-cell stress response implicated in IDO immunosuppression. Donor T cells did not require GCN2 kinase to respond to IDO, suggesting toxic IDO metabolites, and not tryptophan depletion, were responsible for suppression. When exogenous metabolites were administered, GVHD lethality was reduced. To determine whether IDO could be induced before transplantation for enhanced GVHD suppression, we first determined whether antigen-presenting cells (APCs) or epithelial cells were primarily responsible for IDO expression and subsequent GVHD suppression. Recipients with wild-type versus IDO^–/– APCs had increased survival, regardless of epithelial-cell expression of IDO, suggesting that APCs were suitable targets for inducing IDO. Administration of an agonist to toll-like receptor-7/8, a receptor expressed primarily on APCs, induced IDO and reduced injury in the colon and ameliorated lethality. We conclude that IDO up-regulation may have therapeutic potential for preventing GVHD in the clinic.

The transfer of adaptive immunity to CMV during hematopoietic stem cell transplantation is dependent on the specificity and phenotype of CMV-specific T cells in the donor

Thu, 03 Dec 2009 09:02:29 PST

The successful reconstitution of adaptive immunity to human cytomegalovirus (CMV) in hematopoietic stem cell transplantation (HSCT) recipients is central to the reduction of viral reactivation-related morbidity and mortality. Here, we characterized the magnitude, specificity, phenotype, function, and clonotypic composition of CMV-specific T-cell responses in 18 donor-recipient pairs both before and after HSCT. The principal findings were: (1) the specificity of CMV-specific T-cell responses in the recipient after HSCT mirrors that in the donor; (2) the maintenance of these targeting patterns reflects the transfer of epitope-specific T-cell clonotypes from donor to recipient; (3) less differentiated CD27⁺CD57^– CMV-specific memory T cells are more likely to persist in the recipient after HSCT compared with more terminally differentiated CD27^– CD57⁺ CMV-specific memory T cells; (4) the presence of greater numbers of less differentiated CD8⁺ CMV-specific T cells in the donor appears to confer protection against viral reactivation in the recipient after HSCT; and (5) CMV-specific T cells acquire a more differentiated phenotype and a restricted functional profile after HSCT. Overall, these findings define the immunologic factors that influence the successful adoptive transfer of antigen-specific T-cell immunity during HSCT, which enables the identification of recipients at particular risk of CMV reactivation after HSCT.

An impaired transendothelial migration potential of chronic lymphocytic leukemia (CLL) cells can be linked to ephrin-A4 expression

Thu, 03 Dec 2009 09:02:29 PST

Chronic lymphocytic leukemia (CLL) cell migration into lymphoid tissues is an important aspect of the pathobiology of this disease. Here, we investigated the role of ephrin-A4 (EFNA4) in the transendothelial migration (TEM) capacity of CLL and normal B cells through interacting with endothelial EphA2 (erythropoietin-producing hepatocellular carcinoma). CLL cells showed a remarkable impairment in the adhesion to and transmigration through human umbilical vein endothelial cell (HUVEC) monolayers, correlating with their higher EFNA4 expression. In vitro, TEM was mediated by EFNA4 binding to endothelial EphA2 receptor, which is highly expressed in tumor necrosis factor-–activated HUVECs as well as in the CD31⁺ endothelial cells of human lymph nodes. The pretreatment of CLL cells with EphA2 homodimers further impaired their adhesion to and transmigration through HUVEC monolayers, whereas pretreatment of HUVECs with EFNA4 homodimers improved those phenomena in both CLL and normal B cells, suggesting that EFNA4 signaling negatively contributed to TEM. In fact, EFNA4 signaling into CLL cells significantly reduced their adhesion to intercellular adhesion molecule 1, vascular cell adhesion molecule 1, and several extracellular matrix molecules and impaired CCL-19–mediated TEM and chemotaxis. Our results suggest that EFNA4-EphA2 interactions are involved in CLL cell trafficking between blood and the tissues and therefore may become a therapeutic target in the future.

Pericyte recruitment during vasculogenic tube assembly stimulates endothelial basement membrane matrix formation

Stratman, A. N., Malotte, K. M., Mahan, R. D., Davis, M. J., Davis, G. E. — Thu, 03 Dec 2009 09:02:29 PST

We show that endothelial cell (EC)–generated vascular guidance tunnels (ie, matrix spaces created during tube formation) serve as conduits for the recruitment and motility of pericytes along EC ablumenal surfaces to facilitate vessel maturation events, including vascular basement membrane matrix assembly and restriction of EC tube diameter. During quail development, pericyte recruitment along microvascular tubes directly correlates with vascular basement membrane matrix deposition. Pericyte recruitment to EC tubes leads to specific induction of fibronectin and nidogen-1 (ie, matrix-bridging proteins that link together basement membrane components) as well as perlecan and laminin isoforms. Coincident with these events, up-regulation of integrins, ₅β₁, ₃β₁, ₆β₁, and ₁β₁, which bind fibronectin, nidogens, laminin isoforms, and collagen type IV, occurs in EC-pericyte cocultures, but not EC-only cultures. Integrin-blocking antibodies to these receptors, disruption of fibronectin matrix assembly, and small interfering RNA suppression of pericyte tissue inhibitor of metalloproteinase (TIMP)-3 (a known regulator of vascular tube stabilization) all lead to decreased EC basement membrane, resulting in increased vessel lumen diameter, a key indicator of dysfunctional EC-pericyte interactions. Thus, pericyte recruitment to EC-lined tubes during vasculogenesis is a stimulatory event controlling vascular basement membrane matrix assembly, a fundamental maturation step regulating the transition from vascular morphogenesis to stabilization.

Endothelial cell ADAMTS-13 and VWF: production, release, and VWF string cleavage

Turner, N. A., Nolasco, L., Ruggeri, Z. M., Moake, J. L. — Thu, 03 Dec 2009 09:02:29 PST

Human umbilical vein endothelial cell (HUVEC)–released ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin repeats) and HUVEC-secreted von Willebrand factor (VWF) strings were investigated under static conditions that allow the accumulation and analysis of ADAMTS-13. The latter was released constitutively from HUVECs and cleaved the secreted and cell-anchored VWF strings progressively during 15 minutes in Ca²⁺/Zn²⁺-containing buffer. HUVEC ADAMTS13 mRNA expression was approximately 1:100 of VWF monomeric subunit expression. In contrast to multimeric VWF stored within Weibel-Palade bodies and secreted rapidly in response to cell stimulation, ADAMTS-13 was released directly from the Golgi to the cell exterior without an organelle storage site. The constitutive release of ADAMTS-13 continued at the same slow rate regardless of the presence or absence of histamine stimulation of HUVECs. Consequently, the percentage of VWF strings cleaved by ADAMTS-13 at VWF Y¹⁶⁰⁵-M¹⁶⁰⁶ decreased as the rate of VWF string secretion was increased by cell stimulation. Blockade of HUVEC ADAMTS-13 activity by antibodies to different ADAMTS-13 domains made it possible to detect the attachment of ADAMTS-13 all along the lengths of HUVEC-secreted VWF strings. Constitutive ADAMTS-13 released from endothelial cells may contribute to the maintenance of cell surfaces free of hyperadhesive VWF multimeric strings.

The proof is in the crystal

Lopez, J. A., Munday, A. — Thu, 26 Nov 2009 09:01:59 PST

Childhood ITP: knowing when to worry?

Lambert, M. P. — Thu, 26 Nov 2009 09:01:59 PST

Arming CTLs against immunosuppressors

Comoli, P. — Thu, 26 Nov 2009 09:01:59 PST

Chronic lymphocytic leukemia: interplay between noncoding RNAs and protein-coding genes

Calin, G. A., Croce, C. M. — Thu, 26 Nov 2009 09:01:59 PST

One of the most unexpected and fascinating discoveries in oncology over the past few years is the interplay between abnormalities in protein-coding genes and noncoding RNAs (ncRNAs) that is causally involved in cancer initiation, progression, and dissemination. MicroRNAs (miRNAs), small regulatory ncRNAs, are involved in the pathogenesis of all types of human cancers, including leukemias, mainly via dysregulation of expression of cancer genes. Increasing evidence shows that miRNAs can work as tumor suppressors (inhibiting malignant potential) or oncogenes (activating malignant potential). Researchers first identified this new paradigm of molecular oncology in patients with chronic lymphocytic leukemia (CLL). Understanding the roles of miRNAs and other ncRNAs in leukemic cells is not only uncovering a new layer of gene regulation but also providing new markers for improved diagnosis and prognosis, as well as novel therapeutic options for CLL patients. Herein we focus on the roles of miRNAs and ultraconserved ncRNA genes in CLL, highlighting what is already known about their function, proposing a novel model of CLL predisposition and progression, and describing the challenges for the near future.

Primary radiotherapy showed favorable outcome in treating extranodal nasal-type NK/T-cell lymphoma in children and adolescents

Thu, 26 Nov 2009 09:01:59 PST

Extranodal nasal-type natural killer (NK)/T-cell lymphoma is rarely observed in children and adolescents. We aim to investigate the clinical features, prognosis, and treatment outcomes in these patients. Thirty-seven patients were reviewed. There were 19, 14, 2, and 2 patients with stage I, stage II, stage III, and stage IV diseases, respectively. Among the patients with stage I and II disease, 19 patients received initial radiotherapy with or without chemotherapy, and 14 patients received chemotherapy followed by radiotherapy. The 4 patients with stage III and IV disease received primary chemotherapy and radiation of the primary tumor. Children and adolescents with extranodal nasal-type NK/T-cell lymphoma usually presented with early-stage disease, high frequency of B symptoms, good performance, low-risk age-adjusted international prognostic index, and chemoresistance. The complete response rate after initial radiotherapy was 73.7%, which was significantly higher than the response rate after initial chemotherapy (16.7%; P = .002). The 5-year overall survival (OS) and progression-free survival (PFS) rates for all the patients were 77.0% and 68.5%, respectively. The corresponding OS and PFS rates for patients with stage I and II disease were 77.6% and 72.3%, respectively. Children and adolescents with early-stage extranodal nasal-type NK/T-cell lymphoma treated with primary radiotherapy had a favorable prognosis.

Intracranial hemorrhage (ICH) in children with immune thrombocytopenia (ITP): study of 40 cases

Psaila, B., Petrovic, A., Page, L. K., Menell, J., Schonholz, M., Bussel, J. B. — Thu, 26 Nov 2009 09:01:59 PST

Intracranial hemorrhage (ICH) is a rare but devastating complication of childhood immune thrombocytopenia purpura (ITP). A survey of ICH from 1987 to 2000 identified cases of ICH in childhood ITP in the United States. Forty patients with ICH and 80 matched ITP control subjects were accrued. The estimated incidence of ICH was 0.19% to 0.78%. Platelet counts were less than 20 x 10⁹/L in 90% and less than 10 x 10⁹/L in 75% of children with ICH. Eighteen (45%) children developed ICH within 7 days of diagnosis of ITP; for 10 of these, ICH was the presenting feature of ITP. Twelve (30%) children had chronic ITP. Head trauma and hematuria were the most prominent features associated with ICH, identified in 33% and 22.5% of the patients with ICH and 1 and none of the controls (both P < .001). Bleeding beyond petechiae and ecchymoses was also linked to ICH. Mortality was 25%; a further 25% had neurologic sequelae. Strategies by which high-risk children could be identified were considered, and the costs of preventive combination treatment were estimated. Children with severe thrombocytopenia plus head trauma and/or hematuria appeared to be at particularly high risk of ICH. Aggressive treatment of these children may be appropriate.

Generation of Epstein-Barr virus-specific cytotoxic T lymphocytes resistant to the immunosuppressive drug tacrolimus (FK506)

Thu, 26 Nov 2009 09:02:00 PST

Adoptive transfer of autologous Epstein-Barr virus–specific cytotoxic T lymphocytes (EBV-CTLs) to solid organ transplant (SOT) recipients has been shown safe and effective for the treatment of EBV-associated posttransplantation lymphoproliferative disorders (PTLDs). SOT recipients, however, require the continuous administration of immunosuppressive drugs to prevent graft rejection, and these agents may significantly limit the long-term persistence of transferred EBV-CTLs, precluding their use as prophylaxis. Tacrolimus (FK506) is one of the most widely used immunosuppressive agents in SOT recipients, and its immunosuppressive effects are largely dependent on its interaction with the 12-kDa FK506-binding protein (FKBP12). We have knocked down the expression of FKBP12 in EBV-CTLs using a specific small interfering RNA (siRNA) stably expressed from a retroviral vector and found that FKBP12-silenced EBV-CTLs are FK506 resistant. These cells continue to expand in the presence of the drug without measurable impairment of their antigen specificity or cytotoxic activity. We confirmed their FK506 resistance and anti-PTLD activity in vivo using a xenogenic mouse model, suggesting that the proposed strategy may be of value to enhance EBV-specific immune surveillance in patients at high risk of PTLD after transplantation.

Generation of EBV-specific cytotoxic T cells that are resistant to calcineurin inhibitors for the treatment of posttransplantation lymphoproliferative disease

Thu, 26 Nov 2009 09:02:00 PST

Epstein-Barr virus (EBV)–driven posttransplantation lymphoproliferative disease (PTLD) is a serious complication of immunosuppression after either stem cell transplantation (SCT) or solid organ transplantation (SOT). Adoptive transfer of EBV-specific cytotoxic T lymphocytes (EBV-CTLs) is an effective prophylaxis and treatment for PTLD after SCT, but not for PTLD after SOT when pharmacologic immunosuppression cannot be discontinued. We report the generation of calcineurin (CN) mutants that render EBV-CTL resistant to the immunosuppressants tacrolimus (FK506) and cyclosporin A (CsA): mutant CNa12 confers resistance to CsA but not FK506, and mutant CNa22 confers resistance to FK506 but not CsA, whereas mutant CNb30 renders CTLs resistant to both calcineurin inhibitors. Untransduced EBV-CTLs do not proliferate in the presence of FK506/CsA. However, EBV-CTLs transduced with a retroviral vector coding for these mutants retain the ability to both proliferate and secrete normal levels of interferon- in the presence therapeutic levels of FK506 (CNa12), CsA (CNa22), or both (CNb30). The cytotoxicity and phenotype of EBV-CTL lines were unaffected by expression of these mutant CNs. This approach should allow effective immunotherapy with EBV-CTLs in the SOT setting without risking the graft by reduction in immunosuppression, and represents a generic approach to improving immunotherapy in the face of immunosuppression.

Systematic in vivo structure-function analysis of p300 in hematopoiesis

Kimbrel, E. A., Lemieux, M. E., Xia, X., Davis, T. N., Rebel, V. I., Kung, A. L. — Thu, 26 Nov 2009 09:02:00 PST

Cyclic adenosine monophosphate response element binding (CREB)–binding protein (CBP) and p300 are multidomain transcriptional coactivators that help assemble large regulatory complexes at sites of active transcription. Nullizygosity of CBP or p300 results in pervasive defects in hematopoiesis. To systematically assess the structural domains of p300 required for normal hematopoiesis, we used recombinase-mediated cassette exchange to create an allelic series of coisogenic embryonic stem cells, each expressing a different mutant of p300 from the endogenous locus. We found that deletion of either the KIX or CH1 domain caused profound and pervasive defects in hematopoiesis, whereas the loss of most other domains had only lineage-restricted effects. When expressed from the p300 locus, an extra copy of CBP largely compensated for a lack of p300. Surprisingly, mutation of the p300 histone acetyltransferase (HAT) domain had minimal effects on hematopoiesis, and actually increased progenitor and stem cell numbers and proliferative potential. Our results suggest that, in distinct contrast to other organ systems, HAT activity does not provide a critical function for hematopoietic development and emphasizes the importance of enzyme-independent functions of p300.

Sox7-sustained expression alters the balance between proliferation and differentiation of hematopoietic progenitors at the onset of blood specification

Thu, 26 Nov 2009 09:02:00 PST

The molecular mechanisms that regulate the balance between proliferation and differentiation of precursors at the onset of hematopoiesis specification are poorly understood. By using a global gene expression profiling approach during the course of embryonic stem cell differentiation, we identified Sox7 as a potential candidate gene involved in the regulation of blood lineage formation from the mesoderm germ layer. In the present study, we show that Sox7 is transiently expressed in mesodermal precursors as they undergo specification to the hematopoietic program. Sox7 knockdown in vitro significantly decreases the formation of both primitive erythroid and definitive hematopoietic progenitors as well as endothelial progenitors. In contrast, Sox7-sustained expression in the earliest committed hematopoietic precursors promotes the maintenance of their multipotent and self-renewing status. Removal of this differentiation block driven by Sox7-enforced expression leads to the efficient differentiation of hematopoietic progenitors to all erythroid and myeloid lineages. This study identifies Sox7 as a novel and important player in the molecular regulation of the first committed blood precursors. Furthermore, our data demonstrate that the mere sustained expression of Sox7 is sufficient to completely alter the balance between proliferation and differentiation at the onset of hematopoiesis.

Functionally distinct subsets of human NK cells and monocyte/DC-like cells identified by coexpression of CD56, CD7, and CD4

Thu, 26 Nov 2009 09:02:00 PST

The lack of natural killer (NK) cell–specific markers, as well as the overlap among several common surface antigens and functional properties, has obscured the delineation between NK cells and dendritic cells. Here, novel subsets of peripheral blood CD3/14/19^neg NK cells and monocyte/dendritic cell (DC)–like cells were identified on the basis of CD7 and CD4 expression. Coexpression of CD7 and CD56 differentiates NK cells from CD56⁺ monocyte/DC-like cells, which lack CD7. In contrast to CD7⁺CD56⁺ NK cells, CD7^negCD56⁺ cells lack expression of NK cell–associated markers, but share commonalities in their expression of various monocyte/DC-associated markers. Using CD7, we observed approximately 60% of CD4⁺CD56⁺ cells were CD7^neg cells, indicating the actual frequency of activated CD4⁺ NK cells is much lower in the blood than previously recognized. Functionally, only CD7⁺ NK cells secrete gamma interferon (IFN) and degranulate after interleukin-12 (IL-12) plus IL-18 or K562 target cell stimulation. Furthermore, using CD7 to separate CD56⁺ NK cells and CD56⁺ myeloid cells, we demonstrate that unlike resting CD7⁺CD56⁺ NK cells, the CD7^negCD56⁺ myeloid cells stimulate a potent allogeneic response. Our data indicate that CD7 and CD56 coexpression discriminates NK cells from CD7^negCD56⁺ monocyte/DC-like cells, thereby improving our ability to study the intricacies of NK-cell subset phenotypes and functions in vivo.

In vivo intraclonal and interclonal kinetic heterogeneity in B-cell chronic lymphocytic leukemia

Thu, 26 Nov 2009 09:02:00 PST

Clonal evolution and outgrowth of cellular variants with additional chromosomal abnormalities are major causes of disease progression in chronic lymphocytic leukemia (CLL). Because new DNA lesions occur during S phase, proliferating cells are at the core of this problem. In this study, we used in vivo deuterium (²H) labeling of CLL cells to better understand the phenotype of proliferating cells in 13 leukemic clones. In each case, there was heterogeneity in cellular proliferation, with a higher fraction of newly produced CD38⁺ cells compared with CD38^– counterparts. On average, there were 2-fold higher percentages of newly born cells in the CD38⁺ fraction than in CD38^– cells; when analyzed on an individual patient basis, CD38⁺ ²H-labeled cells ranged from 6.6% to 73%. Based on distinct kinetic patterns, interclonal heterogeneity was also observed. Specifically, 4 patients exhibited a delayed appearance of newly produced CD38⁺ cells in the blood, higher leukemic cell CXC chemokine receptor 4 (CXCR4) levels, and increased risk for lymphoid organ infiltration and poor outcome. Our data refine the proliferative compartment in CLL based on CD38 expression and suggest a relationship between in vivo kinetics, expression of a protein involved in CLL cell retention and trafficking to solid tissues, and clinical outcome.

Genetic variation in CXCR4 and risk of chronic lymphocytic leukemia

Thu, 26 Nov 2009 09:02:00 PST

A genome-wide linkage scan has provided evidence for a chronic lymphocytic leukemia (CLL) susceptibility locus at 2q21 to which the chemokine receptor CXCR4 gene maps. Recent data provide some evidence for common variation in CXCR4 according to the polymorphic variant rs2228014 defining CLL risk. To examine the role of genetic variation in CXCR4 on CLL risk, we screened 188 familial CLL cases and 213 controls for germline mutations in the coding regions of CXCR4 and genotyped rs2228014 in 1058 CLL cases and 1807 controls. No association between rs2228014 and risk of CLL was seen (P = .83). One truncating (W195X) and 2 missense mutations with possible functional consequences (V139I and G335S) were identified among 186 familial cases and 0 in 213 controls sequenced. Our analysis provides no evidence that common variation in CXCR4 defined by rs228014 influences the risk of CLL, but that functional coding mutations in CXCR4 may contribute to familial CLL.

Relationship of differential gene expression profiles in CD34+ myelodysplastic syndrome marrow cells to disease subtype and progression

Sridhar, K., Ross, D. T., Tibshirani, R., Butte, A. J., Greenberg, P. L. — Thu, 26 Nov 2009 09:02:00 PST

Microarray analysis with 40 000 cDNA gene chip arrays determined differential gene expression profiles (GEPs) in CD34⁺ marrow cells from myelodysplastic syndrome (MDS) patients compared with healthy persons. Using focused bioinformatics analyses, we found 1175 genes significantly differentially expressed by MDS versus normal, requiring a minimum of 39 genes to separately classify these patients. Major GEP differences were demonstrated between healthy and MDS patients and between several MDS subgroups: (1) those whose disease remained stable and those who subsequently transformed (tMDS) to acute myeloid leukemia; (2) between del(5q) and other MDS patients. A 6-gene "poor risk" signature was defined, which was associated with acute myeloid leukemia transformation and provided additive prognostic information for International Prognostic Scoring System Intermediate-1 patients. Overexpression of genes generating ribosomal proteins and for other signaling pathways was demonstrated in the tMDS patients. Comparison of del(5q) with the remaining MDS patients showed 1924 differentially expressed genes, with underexpression of 1014 genes, 11 of which were within the 5q31-32 commonly deleted region. These data demonstrated (1) GEPs distinguishing MDS patients from healthy and between those with differing clinical outcomes (tMDS vs those whose disease remained stable) and cytogenetics [eg, del(5q)]; and (2) molecular criteria refining prognostic categorization and associated biologic processes in MDS.

Expression profiling of a hemopoietic cell survival transcriptome implicates osteopontin as a functional prognostic factor in AML

Thu, 26 Nov 2009 09:02:00 PST

Deregulated cell survival programs are a classic hallmark of cancer. We have previously identified a serine residue (Ser585) in the βc subunit of the granulocyte-macrophage colony-stimulating factor receptor that selectively and independently promotes cell survival. We now show that Ser585 phosphorylation is constitutive in 20 (87%) of 23 acute myeloid leukemia (AML) patient samples, indicating that this survival-only pathway is frequently deregulated in leukemia. We performed a global expression screen to identify gene targets of this survival pathway and report a 138-gene βc Ser585-regulated transcriptome. Pathway analysis defines a gene network enriched for PI3-kinase target genes and a cluster of genes involved in cancer and cell survival. We show that one such gene, osteopontin (OPN), is a functionally relevant target of the Ser585-survival pathway as shown by siRNA-mediated knockdown of OPN expression that induces cell death in both AML blasts and CD34⁺CD38^–CD123⁺ leukemic progenitors. Increased expression of OPN at diagnosis is associated with poor prognosis with multivariate analysis indicating that it is an independent predictor of overall patient survival in normal karyotype AML (n = 60; HR = 2.2; P = .01). These results delineate a novel cytokine-regulated Ser585/PI3-kinase signaling network that is deregulated in AML and identify OPN as a potential prognostic and therapeutic target.

Neutrophil-specific deletion of Syk kinase results in reduced host defense to bacterial infection

Van Ziffle, J. A., Lowell, C. A. — Thu, 26 Nov 2009 09:02:00 PST

Leukocyte-specific CD18 integrins are critical in mediating cell recruitment and activation during host defense responses to bacterial infection. The signaling pathways downstream of CD18 integrins are dependent on the spleen tyrosine kinase, Syk. To investigate the role integrin signaling plays in host defense, we examined the responses of Syk-deficient neutrophils to bacterial challenge with serum-opsonized Staphylococcus aureus and Escherichia coli. Syk-conditional knockout mice lacking this kinase specifically in myeloid cells or just neutrophils were also used to investigate host responses in vivo. Syk-deficient neutrophils manifested impaired exocytosis of secondary and tertiary granules, reduced cytokine release, and very poor activation of the NADPH oxidase in response to serum-opsonized S aureus and E coli. These functional defects correlated with impaired activation of c-Cbl, Pyk2, Erk1/2, and p38 kinases. Bacterial phagocytosis, neutrophil extracellular trap formation, and killing were also reduced in Syk-deficient cells, with a more profound effect after S aureus challenge. In vivo, loss of Syk in myeloid cells or specifically in neutrophils resulted in reduced clearance of S aureus after subcutaneous or intraperitoneal infection, despite normal recruitment of inflammatory cells. These results indicate that loss of Syk kinase-mediated integrin signaling impairs leukocyte activation, leading to reduced host defense responses.

Glycoprotein Ib{alpha} inhibitor complex structure reveals a combined steric and allosteric mechanism of von Willebrand factor antagonism

McEwan, P. A., Andrews, R. K., Emsley, J. — Thu, 26 Nov 2009 09:02:00 PST

Platelet glycoprotein Ib (GpIb) interactions with von Willebrand factor (VWF) are a critical early event in platelet adhesion, which contributes to hemostasis and thrombosis. Here we report the structure of a complex between GpIb and a potent peptide inhibitor. The cyclic peptide (CTERMALHNLC) was isolated from a cysteine-constrained phage display library, and in the complex this forms one and a half turns of an amphipathic -helix, the curvature of which facilitates contacts with the curved concave face of the GpIb leucine-rich repeats. The peptide has only limited overlap with the VWF binding site. It effectively inhibits by stabilizing an alternative -helical conformation of a regulatory loop that forms an extended β-hairpin upon VWF binding. The structure defines a previously unrecognized binding site within GpIb and represents a clear strategy for developing antiplatelet agents targeting the GpIb-VWF interaction allosterically.

Contributions of extravascular and intravascular cells to fibrin network formation, structure, and stability

Thu, 26 Nov 2009 09:02:00 PST

Fibrin is essential for hemostasis; however, abnormal fibrin formation is hypothesized to increase thrombotic risk. We previously showed that in situ thrombin generation on a cell's surface modulates the 3-dimensional structure and stability of the fibrin network. Currently, we compared the abilities of extravascular and intravascular cells to support fibrin formation, structure, and stability. Extravascular cells (fibroblasts, smooth muscle) supported formation of dense fibrin networks that resisted fibrinolysis, whereas unstimulated intravascular (endothelial) cells produced coarse networks that were susceptible to fibrinolysis. All 3 cell types produced a fibrin structural gradient, with a denser network near, versus distal to, the cell surface. Although fibrin structure depended on cellular procoagulant activity, it did not reflect interactions between integrins and fibrin. These findings contrasted with those on platelets, which influenced fibrin structure via interactions between β3 integrins and fibrin. Inflammatory cytokines that induced prothrombotic activity on endothelial cells caused the production of abnormally dense fibrin networks that resisted fibrinolysis. Blocking tissue factor activity significantly reduced the density and stability of fibrin networks produced by cytokine-stimulated endothelial cells. Together, these findings indicate fibrin structure and stability reflect the procoagulant phenotype of the endogenous cells, and suggest abnormal fibrin structure is a novel link between inflammation and thrombosis.

Role of tyrosine phosphatase SHP-1 in the mechanism of endorepellin angiostatic activity

Thu, 26 Nov 2009 09:02:00 PST

Endorepellin, the C-terminal domain of perlecan, is a powerful angiogenesis inhibitor. To dissect the mechanism of endorepellin-mediated endothelial silencing, we used an antibody array against multiple tyrosine kinase receptors. Endorepellin caused a widespread reduction in phosphorylation of key receptors involved in angiogenesis and a concurrent increase in phosphatase activity in endothelial cells and tumor xenografts. These effects were efficiently hampered by function-blocking antibodies against integrin 2β1, the functional endorepellin receptor. The Src homology-2 protein phosphatase-1 (SHP-1) coprecipitated with integrin 2 and was phosphorylated in a dynamic fashion after endorepellin stimulation. Genetic evidence was provided by lack of an endorepellin-evoked phosphatase response in microvascular endothelial cells derived from integrin 2β1^–/– mice and by response to endorepellin in cells genetically engineered to express the 2β1 integrin, but not in cells either lacking this receptor or expressing a chimera harboring the integrin 2 ectodomain fused to the 1 intracellular domain. siRNA-mediated knockdown of integrin 2 caused a dose-dependent reduction of SHP-1. Finally, the levels of SHP-1 and its enzymatic activity were substantially reduced in multiple organs from 2β1^–/– mice. Our results show that SHP-1 is an essential mediator of endorepellin activity and discover a novel functional interaction between the integrin 2 subunit and SHP-1.

Insufficient evidence to suggest less stringent therapy in hemophilia B?

den Uijl, I. E. M., Roosendaal, G., Fischer, K. — Thu, 26 Nov 2009 09:02:00 PST

Response: Comparing joint arthroplasties in severe hemophilia A with severe hemophilia B

Tagariello, G., Iorio, A., Mannucci, P. M. — Thu, 26 Nov 2009 09:02:00 PST

Delayed but functional neutrophil extracellular trap formation in neonates

Thu, 26 Nov 2009 09:02:00 PST

Response: Gestational age as a factor in neutrophil extracellular trap formation

Yost, C. C., Zimmerman, G. A. — Thu, 26 Nov 2009 09:02:00 PST

Macrophage fusion cuisine

Sica, A., Mantovani, A. — Thu, 19 Nov 2009 09:02:19 PST

IG genes and hairy cell leukemia

Caligaris-Cappio, F. — Thu, 19 Nov 2009 09:02:19 PST

Cracking the platelet WIP

Poole, A. W. — Thu, 19 Nov 2009 09:02:19 PST

Mechanisms underlying neutrophil-mediated monocyte recruitment

Soehnlein, O., Lindbom, L., Weber, C. — Thu, 19 Nov 2009 09:02:19 PST

Extravasation of polymorphonuclear leukocytes (PMNs) to the site of inflammation precedes a second wave of emigrating monocytes. That these events are causally connected has been established a long time ago. However, we are now just beginning to understand the molecular mechanisms underlying this cellular switch, which has become even more complex considering the emergence of monocyte subsets, which are affected differently by signals generated from PMNs. PMN granule proteins induce adhesion as well as emigration of inflammatory monocytes to the site of inflammation involving β₂-integrins and formyl-peptide receptors. Furthermore, modification of the chemokine network by PMNs and their granule proteins creates a milieu favoring extravasation of inflammatory monocytes. Finally, emigrated PMNs rapidly undergo apoptosis, leading to the discharge of lysophosphatidylcholine, which attracts monocytes via G2A receptors. The net effect of these mechanisms is the accumulation of inflammatory monocytes, thus promoting proinflammatory events, such as release of inflammation-sustaining cytokines and reactive oxygen species. As targeting PMNs without causing serious side effects seems futile, it may be more promising to aim at interfering with subsequent PMN-driven proinflammatory events.

How I treat postthrombotic syndrome

Kahn, S. R. — Thu, 19 Nov 2009 09:02:19 PST

Postthrombotic syndrome (PTS) is a chronic complication of deep venous thrombosis (DVT) that reduces quality of life and has important socioeconomic consequences. More than one-third of patients with DVT will develop PTS, and 5% to 10% of patients will develop severe PTS, which may manifest as venous ulceration. The principal risk factors for PTS are persistent leg symptoms 1 month after the acute episode of DVT, extensive DVT, recurrent ipsilateral DVT, obesity, and older age. Daily use of elastic compression stockings (ECSs) for 2 years after proximal DVT appears to reduce the risk of PTS; however, there is uncertainty about optimal duration of use and compression strength of ECSs and the magnitude of their effect. The cornerstone of managing PTS is compression therapy, primarily using ECSs. Venoactive medications such as aescin and rutoside may provide short-term relief of PTS symptoms. The likelihood of developing PTS after DVT should be discussed with patients, and symptoms and signs of PTS should be monitored during clinical follow-up. Further studies to elucidate the pathophysiology of PTS, to identify clinical and biologic risk factors, and to test new preventive and therapeutic approaches to PTS are needed to ultimately improve the long-term prognosis of patients with DVT.

Serum ferritin level changes in children with sickle cell disease on chronic blood transfusion are nonlinear and are associated with iron load and liver injury

Thu, 19 Nov 2009 09:02:19 PST

Chronic blood transfusion is increasingly indicated in patients with sickle cell disease. Measuring resulting iron overload remains a challenge. Children without viral hepatitis enrolled in 2 trials for stroke prevention were examined for iron overload (STOP and STOP2; n = 271). Most received desferrioxamine chelation. Serum ferritin (SF) changes appeared nonlinear compared with prechelation estimated transfusion iron load (TIL) or with liver iron concentrations (LICs). Averaged correlation coefficient between SF and TIL (patients/observations, 26 of 164) was r = 0.70; between SF and LIC (patients/observations, 33 of 47) was r = 0.55. In mixed models, SF was associated with LIC (P = .006), alanine transaminase (P = .025), and weight (P = .026). Most patients with SF between 750 and 1500 ng/mL had a TIL between 25 and 100 mg/kg (72.8% ± 5.9%; patients/observations, 24 of 50) or an LIC between 2.5 and 10 mg/g dry liver weight (75% ± 0%; patients/observations, 8 of 9). Most patients with SF of 3000 ng/mL or greater had a TIL of 100 mg/kg or greater (95.3% ± 6.7%; patients/observations, 7 of 16) or an LIC of 10 mg/g dry liver weight or greater (87.7% ± 4.3%; patients/observations, 11 of 18). Although SF changes are nonlinear, levels less than 1500 ng/mL indicated mostly acceptable iron overload; levels of 3000 ng/mL or greater were specific for significant iron overload and were associated with liver injury. However, to determine accurately iron overload in patients with intermediately elevated SF levels, other methods are required. These trials are registered at www.clinicaltrials.gov as #NCT00000592 and #NCT00006182.

Relationship of erythropoietin, fetal hemoglobin, and hydroxyurea treatment to tricuspid regurgitation velocity in children with sickle cell disease

Thu, 19 Nov 2009 09:02:19 PST

Hydroxyurea and higher hemoglobin F improve the clinical course and survival in sickle cell disease, but their roles in protecting from pulmonary hypertension are not clear. We studied 399 children and adolescents with sickle cell disease at steady state; 38% were being treated with hydroxyurea. Patients on hydroxyurea had higher hemoglobin concentration and lower values for a hemolytic component derived from 4 markers of hemolysis (P ≤ .002) but no difference in tricuspid regurgitation velocity compared with those not receiving hydroxyurea; they also had higher hemoglobin F (P < .001) and erythropoietin (P = .012) levels. Hemoglobin F correlated positively with erythropoietin even after adjustment for hemoglobin concentration (P < .001). Greater hemoglobin F and erythropoietin each independently predicted higher regurgitation velocity in addition to the hemolytic component (P ≤ .023). In conclusion, increase in hemoglobin F in sickle cell disease may be associated with relatively lower tissue oxygen delivery as reflected in higher erythropoietin concentration. Greater levels of erythropoietin or hemoglobin F were independently associated with higher tricuspid regurgitation velocity after adjustment for degree of hemolysis, suggesting an independent relationship of hypoxia with higher systolic pulmonary artery pressure. The hemolysis-lowering and hemoglobin F–augmenting effects of hydroxyurea may exert countervailing influences on pulmonary blood pressure in sickle cell disease.

Identification of novel regulators of hematopoietic stem cell development through refinement of stem cell localization and expression profiling

Mascarenhas, M. I., Parker, A., Dzierzak, E., Ottersbach, K. — Thu, 19 Nov 2009 09:02:19 PST

The first adult-repopulating hematopoietic stem cells (HSCs) are detected starting at day 10.5 of gestation in the aorta-gonads-mesonephros (AGM) region of the mouse embryo. Despite the importance of the AGM in initiating HSC production, very little is currently known about the regulators that control HSC emergence in this region. We have therefore further defined the location of HSCs in the AGM and incorporated this information into a spatial and temporal comparative gene expression analysis of the AGM. The comparisons included gene expression profiling (1) in the newly identified HSC-containing region compared with the region devoid of HSCs, (2) before and after HSC emergence in the AGM microenvironment, and (3) on populations enriched for HSCs and their putative precursors. Two genes found to be up-regulated at the time and place where HSCs are first detected, the cyclin-dependent kinase inhibitor p57Kip2/Cdkn1c and the insulin-like growth factor 2, were chosen for further analysis. We demonstrate here that they play a novel role in AGM hematopoiesis. Interestingly, many genes involved in the development of the tissues surrounding the dorsal aorta are also up-regulated during HSC emergence, suggesting that the regulation of HSC generation occurs in coordination with the development of other organs.

The role and regulation of friend of GATA-1 (FOG-1) during blood development in the zebrafish

Thu, 19 Nov 2009 09:02:19 PST

The nuclear protein FOG-1 binds transcription factor GATA-1 to facilitate erythroid and megakaryocytic maturation. However, little is known about the function of FOG-1 during myeloid and lymphoid development or how FOG-1 expression is regulated in any tissue. We used in situ hybridization, gain- and loss-of-function studies in zebrafish to address these problems. Zebrafish FOG-1 is expressed in early hematopoietic cells, as well as heart, viscera, and paraspinal neurons, suggesting that it has multifaceted functions in organogenesis. We found that FOG-1 is dispensable for endoderm specification but is required for endoderm patterning affecting the expression of late-stage T-cell markers, independent of GATA-1. The suppression of FOG-1, in the presence of normal GATA-1 levels, induces severe anemia and thrombocytopenia and expands myeloid-progenitor cells, indicating that FOG-1 is required during erythroid/myeloid commitment. To functionally interrogate whether GATA-1 regulates FOG-1 in vivo, we used bioinformatics combined with transgenic assays. Thus, we identified 2 cis-regulatory elements that control the tissue-specific gene expression of FOG-1. One of these enhancers contains functional GATA-binding sites, indicating the potential for a regulatory loop in which GATA factors control the expression of their partner protein FOG-1.

Alternatively activated macrophages engage in homotypic and heterotypic interactions through IL-4 and polyamine-induced E-cadherin/catenin complexes

Thu, 19 Nov 2009 09:02:19 PST

Alternatively activated macrophages (AAMs), triggered by interleukin-4 (IL-4) and IL-13, play a modulating role during Th2 cytokine-driven pathologies, but their molecular armament remains poorly characterized. Here, we established E-cadherin (Cdh1) as a selective marker for IL-4/IL-13–exposed mouse and human macrophages, which is STAT6-dependently induced during polarized Th2 responses associated with Taenia crassiceps helminth infections or allergic airway inflammation. The IL-4–dependent, arginase-1/ornithine decarboxylase–mediated production of polyamines is important for maximal Cdh1 induction, unveiling a novel mechanism for IL-4–dependent gene transcription. At the macrophage surface, E-cadherin forms a functional complex with the catenins that accumulates at sites of cell contact. Macrophage-specific deletion of the Cdh1 gene illustrates the implication of E-cadherin in IL-4–driven macrophage fusion and heterotypic interactions with CD103⁺ and KLRG1⁺ T cells. This study identifies the E-cadherin/catenin complex as a discriminative, partly polyamine-regulated feature of IL-4/IL-13–exposed alternatively activated macrophages that contributes to homotypic and heterotypic cellular interactions.

High TCL1 levels are a marker of B-cell receptor pathway responsiveness and adverse outcome in chronic lymphocytic leukemia

Thu, 19 Nov 2009 09:02:20 PST

Although activation of the B-cell receptor (BCR) signaling pathway is implicated in the pathogenesis of chronic lymphocytic leukemia (CLL), its clinical impact and the molecular correlates of such response are not clearly defined. T-cell leukemia 1 (TCL1), the AKT modulator and proto-oncogene, is differentially expressed in CLL and linked to its pathogenesis based on CD5⁺ B-cell expansions arising in TCL1-transgenic mice. We studied here the association of TCL1 levels and its intracellular dynamics with the in vitro responses to BCR stimulation in 70 CLL cases. The growth kinetics after BCR engagement correlated strongly with the degree and timing of induced AKT phospho-activation. This signaling intensity was best predicted by TCL1 levels and the kinetics of TCL1-AKT corecruitment to BCR membrane activation complexes, which further included the kinases LYN, SYK, ZAP70, and PKC. High TCL1 levels were also strongly associated with aggressive disease features, such as advanced clinical stage, higher white blood cell counts, and shorter lymphocyte doubling time. Higher TCL1 levels independently predicted an inferior clinical outcome (ie, shorter progression-free survival, P < .001), regardless of therapy regimen, especially for ZAP70⁺ tumors. We propose TCL1 as a marker of the BCR-responsive CLL subset identifying poor prognostic cases where targeting BCR-associated kinases may be therapeutically useful.

VH4-34+ hairy cell leukemia, a new variant with poor prognosis despite standard therapy

Arons, E., Suntum, T., Stetler-Stevenson, M., Kreitman, R. J. — Thu, 19 Nov 2009 09:02:20 PST

Hairy cell leukemia variant (HCLv) presents with high disease burden, lack of typical antigens like CD25, and poor response to standard treatments like cladribine. Occasionally, patients with classic HCL respond poorly. Clinical and molecular features of HCL and HCLv has not been compared. Rearrangements expressing immunoglobulin VH chain were sequenced, including 22 from 20 patients with HCLv and 63 from 62 patients with classic HCL. Most patients were seeking relapsed/refractory trials, representing a poor-prognosis population. VH4-34, a gene commonly used in autoimmune disorders, was observed in 8 (40%) HCLv and 6 (10%) classic (P = .004) HCL patients. Compared with 71 VH4-34^– rearrangements, 14 VH4-34⁺ rearrangements were more frequently (P < .001) unmutated, defined as greater than 98% homologous to germline sequence. VH4-34⁺ patients had greater white blood cell counts at diagnosis (P = .002), lower response rate (P < .001) and progression-free survival (P = .007) after initial cladribine, and shorter overall survival from diagnosis (P < .001). Response and survival were more closely related to VH4-34 status than to whether or not patients had HCLv. VH4-34⁺ HCL is an important disorder that only partly overlaps with the previously described HCLv. Response to initial single-agent cladribine therapy is suboptimal; these patients should be considered for alternative approaches, including antibody-related therapy.

Hairy cell leukemias with unmutated IGHV genes define the minor subset refractory to single-agent cladribine and with more aggressive behavior

Thu, 19 Nov 2009 09:02:20 PST

Hairy cell leukemia (HCL) is generally responsive to single-agent cladribine, and only a minority of patients are refractory and with poor prognosis. HCLs generally express mutated (M) and, in a minority, unmutated (UM) IGHV. In a multicenter clinical trial in newly diagnosed HCL, we prospectively investigated clinical and molecular parameters predicting response and event-free survival after single-agent cladribine. Of 58 HCLs, 6 expressed UM-IGHV (UM-HCL) and 52 M-IGHV (M-HCL). Beneficial responses were obtained in 53 of 58 patients (91%), whereas treatment failures were observed in 5 of 58 patients (9%). Failures were associated significantly with UM-IGHV (5 of 5 failures vs 1 of 53 beneficial responses had UM-IGHV, P < .001), leukocytosis (3 of 5 vs 3 of 53, P = .006), and bulky spleen (4 of 5 vs 4 of 53, P < .001). The UM-HCL not benefiting from cladribine characteristically had bulky spleen (4 of 5, 80%), leukocytosis (3 of 5, 60%), and TP53 defects (2 of 5, 40%), and progressed rapidly after first treatment (median event-free survival, 7.5 months). Our data suggest that UM-HCLs identify the minor subgroup failing cladribine treatment and with more aggressive disease. High incidence of TP53 dysfunction indicates a potential mechanism of resistance to cladribine in the UM-HCL group. Overall, our data provide new molecular elements relevant for treatment concerns in HCL.

TAPP2 links phosphoinositide 3-kinase signaling to B-cell adhesion through interaction with the cytoskeletal protein utrophin: expression of a novel cell adhesion-promoting complex in B-cell leukemia

Thu, 19 Nov 2009 09:02:20 PST

Tandem pleckstrin homology domain proteins (TAPPs) are recruited to the plasma membrane via binding to phosphoinositides produced by phosphoinositide 3-kinases (PI3Ks). Whereas PI3Ks are critical for B-cell activation, the functions of TAPP proteins in B cells are unknown. We have identified 40 potential interaction partners of TAPP2 in B cells, including proteins involved in cytoskeletal rearrangement, signal transduction and endocytic trafficking. The association of TAPP2 with the cytoskeletal proteins utrophin and syntrophin was confirmed by Western blotting. We found that TAPP2, syntrophin, and utrophin are coexpressed in normal human B cells and B-chronic lymphocytic leukemia (B-CLL) cells. TAPP2 and syntrophin expression in B-CLL was variable from patient to patient, with significantly higher expression in the more aggressive disease subset identified by zeta-chain–associated protein kinase of 70 kDa (ZAP70) expression and unmutated immunoglobulin heavy chain (IgH) genes. We examined whether TAPP can regulate cell adhesion, a known function of utrophin/syntrophin in other cell types. Expression of membrane-targeted TAPP2 enhanced B-cell adhesion to fibronectin and laminin, whereas PH domain–mutant TAPP2 inhibited adhesion. siRNA knockdown of TAPP2 or utrophin, or treatment with PI3K inhibitors, significantly inhibited adhesion. These findings identify TAPP2 as a novel link between PI3K signaling and the cytoskeleton with potential relevance for leukemia progression.

Follicular lymphoma cells induce T-cell immunologic synapse dysfunction that can be repaired with lenalidomide: implications for the tumor microenvironment and immunotherapy

Thu, 19 Nov 2009 09:02:20 PST

An important hallmark of cancer progression is the ability of tumor cells to evade immune recognition. Understanding the relationship between neoplastic cells and the immune microenvironment should facilitate the design of improved immunotherapies. Here we identify impaired T-cell immunologic synapse formation as an active immunosuppressive mechanism in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). We found a significant reduction in formation of the F-actin immune synapse in tumor-infiltrating T cells (P < .01) from lymphoma patients compared with age-matched healthy donor cells. Peripheral blood T cells exhibited this defect only in patients with leukemic-phase disease. Moreover, we demonstrate that this T-cell defect is induced after short-term tumor cell contact. After 24-hour coculture with FL cells, previously healthy T cells showed suppressed recruitment of critical signaling proteins to the synapse. We further demonstrate repair of this defect after treatment of both FL cells and T cells with the immunomodulatory drug lenalidomide. Tissue microarray analysis identified reduced expression of the T-cell synapse signature proteins, including the cytolytic effector molecule Rab27A associated with poor prognosis, in addition to reduced T-cell numbers and activity with disease transformation. Our results highlight the importance of identifying biomarkers and immunotherapeutic treatments for repairing T-cell responses in lymphoma.

The transcription factors STAT5A/B regulate GM-CSF-mediated granulopoiesis

Thu, 19 Nov 2009 09:02:20 PST

Neutrophils play a vital role in the immune defense, which is evident by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. Both the GM-CSF–permitted survival of mature neutrophils and the generation of granulocytes from granulocyte-macrophage progenitors (GMPs) were markedly reduced in Stat5a/b mutants. GMPs showed impaired colony-formation ability with reduced number and size of colonies on GM-CSF stimulation. Moreover, continuous cell fate analyses by time-lapse microscopy and single cell tracking revealed that Stat5a/b-null GMPs showed both delayed cell-cycle progression and increased cell death. Finally, transcriptome analysis indicated that STAT5A/B directs GM-CSF signaling through the regulation of proliferation and survival genes.

Platelet-associated IgAs and impaired GPVI responses in platelets lacking WIP

Thu, 19 Nov 2009 09:02:20 PST

The role of the Wiskott-Aldrich syndrome protein (WASp) in platelet function is unclear because platelets that lack WASp function normally. WASp constitutively associates with WASp-interacting protein (WIP) in resting and activated platelets. The role of WIP in platelet function was investigated using mice that lack WIP or WASp. WIP knockout (KO) platelets lack WASp and thus are double deficient. WIP KO mice have a thrombocytopenia, similar to WASp KO mice, resulting in part from enhanced platelet clearance. Most WIP KO, but not WASp KO, mice evolved platelet-associated immunoglobulins (Ig) of the IgA class, which normalize their platelet survival but diminish their glycoprotein VI (GPVI) responses. Protein tyrosine phosphorylation, including that of phospholipase C-2, and calcium mobilization are impaired in IgA-presenting WIP KO platelets stimulated through GPVI, resulting in defects in -granule secretion, integrin IIbβ3 activation, and actin assembly. The anti-GPVI antibody JAQ1 induces the irreversible loss of GPVI from circulating platelets in wild-type mice, but not in WIP KO mice that bear high levels of platelet-associated IgAs. Together, the data indicate that platelet-associated IgAs negatively modulate GPVI signaling and function in WIP KO mice.

Platelet protein disulfide isomerase is localized in the dense tubular system and does not become surface expressed after activation

van Nispen tot Pannerden, H. E., van Dijk, S. M., Du, V., Heijnen, H. F. G. — Thu, 19 Nov 2009 09:02:20 PST

Evidence is accumulating that circulating tissue factor (TF) contributes to the initiation of coagulation and the formation of fibrin. The majority of circulating TF is cryptic, and it has been suggested that close vicinity with anionic phospholipids on the cell surface increases the active conformation of TF. Two recent papers have shown that encryption of TF and initiation of coagulation are facilitated by the enzyme protein disulfide isomerase (PDI), possibly on the surface of activated platelets or endothelial cells. In this brief report, we demonstrate that the majority of PDI in platelets is intracellular where it is exclusively located in the dense tubular system. On activation, PDI remains confined to the intracellular stores of the dense tubular system and is neither released nor targeted to the cell surface. Similar results were obtained in endothelium where PDI remains exclusively localized in the endoplasmic reticulum, both at steady state and after thrombin stimulation.

A role for thrombin in the initiation of the immune response to therapeutic factor VIII

Skupsky, J., Zhang, A.-H., Su, Y., Scott, D. W. — Thu, 19 Nov 2009 09:02:20 PST

Administration of human factor VIII (FVIII) to FVIII knockout hemophilia mice is a useful small animal model to study the physiologic response in patients iatrogenically immunized to this therapeutic protein. These mice manifest a robust, T cell–dependent, antibody response to exogenous FVIII treatment, even when encountered through traditionally tolerogenic routes. Thus, FVIII given via these routes elicits both T- and B-cell responses, whereas a control, foreign protein, such as ovalbumin (OVA), is poorly immunogenic. When FVIII is heat inactivated, it loses function and much of its immunogenicity. This suggests that FVIII's immunogenicity is principally tied to its function and not its structure. If mice are treated with the anticoagulant warfarin, which depletes other coagulation factors including thrombin, there is a reduced immune response to FVIII. Furthermore, when mice are treated with the direct thrombin inhibitor, hirudin, the T-cell responses and the serum anti-FVIII antibody concentrations are again significantly reduced. Notably, when FVIII is mixed with OVA, it acts to increase the immune response to OVA. Finally, administration of thrombin with OVA is sufficient to induce immune responses to OVA. Overall, these data support the hypothesis that formation of thrombin through the procoagulant activity of FVIII is necessary to induce costimulation for the immune response to FVIII treatment.

Mutation of the H-bond acceptor S119 in the ADAMTS13 metalloprotease domain reduces secretion and substrate turnover in a patient with congenital thrombotic thrombocytopenic purpura

Thu, 19 Nov 2009 09:02:20 PST

Hereditary thrombotic thrombocytopenic purpura is caused by mutations in a disintegrin and metalloprotease with thrombospondin motifs (ADAMTS13) resulting in defective processing of von Willebrand factor (VWF) that causes intravascular platelet aggregation culminating in thrombocytopenia with shistocytic anemia. In this study the functional and structural role of a recently identified ADAMTS13 metalloprotease domain mutation S119F was investigated. Secretion from heterologous cells was hampered but not completely eliminated. Secreted S119F was active toward multimeric VWF and FRETS-VWF73 but with abnormal kinetics, having a significantly reduced overall catalytic rate (k_cat; 0.88 ± 0.04 s^–1 vs 2.78 ± 0.11 s^–1) and slightly smaller Michaelis constant (K_M; 1.4 ± 0.2µM vs 2.3 ± 0.3µM). A computational model of the metalloprotease domain demonstrates both steric and polar interaction effects caused by S119F. Interestingly, mutant S119A has properties similar to S119F (k_cat = 0.82 ± 0.03 s^–1 and K_M = 1.1 ± 0.1µM), allowing to assign distorted kinetics to the loss of the H-bond with conserved residue W262. We conclude that the S119-W262 H-bond is crucial for maximal turnover.

Limited role of MHC class I chain-related gene A (MICA) typing in assessing graft-versus-host disease risk after fully human leukocyte antigen-matched unrelated donor transplantation

Thu, 19 Nov 2009 09:02:20 PST

Response: MHC class I chain-related gene A (MICA) in unrelated donor transplantation

Fernandez-Vina, M., Parmar, S., Champlin, R., de Lima, M. — Thu, 19 Nov 2009 09:02:20 PST

Platelet CFH: in search of the source

Akkerman, J. W. N. — Thu, 12 Nov 2009 09:38:04 PST

Hunting for the Achilles' heel of CLL

Byrd, J. C. — Thu, 12 Nov 2009 09:38:04 PST

Mining ferritin iron: 2 pathways

Theil, E. C. — Thu, 12 Nov 2009 09:38:04 PST

Acute graft-versus-host disease: from the bench to the bedside

Socie, G., Blazar, B. R. — Thu, 12 Nov 2009 09:38:04 PST

During the past decade, progress in basic immunology has been impressive. In parallel, whereas our understanding of the pathophysiology of acute graft-versus-host disease (GVHD) has greatly improved, so has our knowledge of the complexities of the immune system. Much of the immunobiology of acute GVHD has been gleaned from preclinical models and far less from correlations with clinical observations or therapeutic interventions. In this review, we summarize some of the major advances in GVHD pathophysiology, including the translation of these from the bench to the bedside, and discuss preclinical approaches that warrant further exploration in the clinic.

How I treat mycosis fungoides and Sezary syndrome

Prince, H. M., Whittaker, S., Hoppe, R. T. — Thu, 12 Nov 2009 09:38:04 PST

The most common subtypes of primary cutaneous T-cell lymphomas are mycosis fungoides (MF) and Sézary syndrome (SS). The majority of patients have indolent disease; and given the incurable nature of MF/SS, management should focus on improving symptoms and cosmesis while limiting toxicity. Management of MF/SS should use a "stage-based" approach; treatment of early-stage disease (IA-IIA) typically involves skin directed therapies that include topical corticosteroids, phototherapy (psoralen plus ultraviolet A radiation or ultraviolet B radiation), topical chemotherapy, topical or systemic bexarotene, and radiotherapy. Systemic approaches are used for recalcitrant early-stage disease, advanced-stage disease (IIB-IV), and transformed disease and include retinoids, such as bexarotene, interferon-, histone deacetylase inhibitors, the fusion toxin denileukin diftitox, systemic chemotherapy including transplantation, and extracorporeal photopheresis. Examples of drugs under active investigation include new histone deacetylase inhibitors, forodesine, monoclonal antibodies, proteasome inhibitors, and immunomodulatory agents, such as lenalidomide. It is appropriate to consider patients for novel agents within clinical trials if they have failed front-line therapy and before chemotherapy is used.

Evaluation of pentostatin in corticosteroid-refractory chronic graft-versus-host disease in children: a Pediatric Blood and Marrow Transplant Consortium study

Thu, 12 Nov 2009 09:38:05 PST

There is no standard therapy for steroid-refractory chronic graft-versus-host disease (GVHD). This problem is particularly daunting in children with chronic GVHD, whereby the effects of the disease and its treatment may impair normal growth and development. Children are also particularly vulnerable to failure and/or toxicity of therapy; for example, joint contractures or joint damage may result in life-long disability. The Pediatric Blood and Marrow Transplant Consortium performed a phase 2 trial of pentostatin for steroid-refractory chronic GVHD in 51 children (median age, 9.8 years) from 24 institutions. Overall response was 53% (95% confidence interval, 40%-64%), with a response of 59% (95% confidence interval, 42%-75%) in sclerosis. Thirteen subjects (25%) had toxicity requiring them to stop pentostatin. The drug had a significant steroid-sparing effect in those that responded. A trend was also observed toward increased survival at 3 years in responders versus nonresponders (69% vs 50%; P = .06). The intravenous administration of the drug ensures compliance in a patient group in which oral therapy is difficult to monitor. Pentostatin has activity in refractory chronic GVHD in children, and future studies, including treatment of children newly diagnosed with high-risk chronic GVHD, are warranted. The trial was registered at www.Clinicaltrials.gov as #NCT00144430.

The use of nilotinib or dasatinib after failure to 2 prior tyrosine kinase inhibitors: long-term follow-up

Thu, 12 Nov 2009 09:38:05 PST

Responses can be achieved with dasatinib or nilotinib after failure of 2 prior tyrosine kinase inhibitors (TKIs). We report on 48 chronic myeloid leukemia patients sequentially treated with 3 TKIs: 34 with dasatinib after imatinib/nilotinib failure and 14 with nilotinib after imatinib/dasatinib failure. Before the third TKI, 25 patients were in chronic phase (CP), 10 in accelerated phase (AP), and 13 in blast phase (BP). Best response to third TKI in CP was 5 major molecular responses (MMR), 3 complete cytogenetic (CCyR), 2 partial cytogenetic (PCyR), 3 minor cytogenetic (mCyR), 6 complete hematologic responses (CHR), and 6 with no response (NR). In AP, 1 patient achieved MMR, 1 CCyR, 2 PCyR, 1 mCyR, 4 CHR, and 1 NR. In BP, 1 achieved MMR, 2 CCyR, 1 PCyR, 1 mCyR, 2 returned to CP, and 6 NR. Median CCyR duration was 16.3 months; 3 CP patients achieving CCyR had a response more than 12 months. Median failure-free survival was 20 months for patients in CP, 5 months in AP, and 3 months in BP. Use of second-generation TKI after failure to 2 TKIs may induce responses, but these are usually not durable except in some CP patients. New treatment options are needed.

The persistence of immunophenotypically normal residual bone marrow plasma cells at diagnosis identifies a good prognostic subgroup of symptomatic multiple myeloma patients

Thu, 12 Nov 2009 09:38:05 PST

Multiparameter flow cytometry immunophenotyping allows discrimination between normal (N-) and myelomatous (MM-) plasma cells (PCs) within the bone marrow plasma cell compartment (BMPCs). Here we report on the prognostic relevance of detecting more than 5% residual normal plasma cells from all bone marrow plasma cells (N-PCs/BMPCs) by multiparameter flow cytometry in a series of 594 newly diagnosed symptomatic MM patients, uniformly treated according to the Grupo Español de MM 2000 (GEM2000) protocol. Our results show that symptomatic MM patients with more than 5% N-PCs/BMPCs (n = 80 of 594; 14%) have a favorable baseline clinical prospect, together with a significantly lower frequency of high-risk cytogenetic abnormalities and higher response rates. Moreover, this group of patients had a significantly longer progression-free survival (median, 54 vs 42 months, P = .001) and overall survival (median, not reached vs 89 months, P = .04) than patients with less than or equal to 5% N-PCs/BMPCs. Our findings support the clinical value of detecting residual normal PCs in MM patients at diagnosis because this reveals a good prognostic category that could benefit from specific therapeutic approaches. This trial was registered at www.clinicaltrials.gov as NCT00560053.

Anti-CD3 antibodies modulate anti-factor VIII immune responses in hemophilia A mice after factor VIII plasmid-mediated gene therapy

Peng, B., Ye, P., Rawlings, D. J., Ochs, H. D., Miao, C. H. — Thu, 12 Nov 2009 09:38:05 PST

One major obstacle in gene therapy is the generation of immune responses directed against transgene product. Five consecutive anti-CD3 treatments concomitant with factor VIII (FVIII) plasmid injection prevented the formation of inhibitory antibodies against FVIII and achieved persistent, therapeutic levels of FVIII gene expression in treated hemophilia A mice. Repeated plasmid gene transfer is applicable in tolerized mice without eliciting immune responses. Anti-CD3 treatment significantly depleted both CD4⁺ and CD8⁺ T cells, whereas increased transforming growth factor-β levels in plasma and the frequency of both CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25^–Foxp3⁺ regulatory T cells in the initial few weeks after treatment. Although prior depletion of CD4⁺CD25⁺ cells did not abrogate tolerance induction, adoptive transfer of CD4⁺ cells from tolerized mice at 6 weeks after treatment protected recipient mice from anti-FVIII immune responses. Anti-CD3–treated mice mounted immune responses against both T-dependent and T-independent neo-antigens, indicating that anti-CD3 did not hamper the immune systems in the long term. Concomitant FVIII plasmid + anti-CD3 treatment induced long-term tolerance specific to FVIII via a mechanism involving the increase in transforming growth factor-β levels and the generation of adaptive FVIII-specific CD4⁺Foxp3⁺ regulatory T cells at the periphery. Furthermore, anti-CD3 can reduce the titers of preexisting anti-FVIII inhibitory antibodies in hemophilia A mice.

Necdin restricts proliferation of hematopoietic stem cells during hematopoietic regeneration

Kubota, Y., Osawa, M., Jakt, L. M., Yoshikawa, K., Nishikawa, S.-I. — Thu, 12 Nov 2009 09:38:05 PST

Hematopoietic stem cell (HSC) proliferation is tightly regulated by a poorly understood complex of positive and negative cell-cycle regulatory mechanisms. Necdin (Ndn) is an evolutionally conserved multifunctional protein that has been implicated in cell-cycle regulation of neuronal cells. Here, we provide evidence that necdin plays an important role in restricting excessive HSC proliferation during hematopoietic regeneration. We identify Ndn as being preferentially expressed in the HSC population on the basis of gene expression profiling and demonstrate that mice deficient in Ndn show accelerated recovery of the hematopoietic system after myelosuppressive injury, whereas no overt abnormality is seen in steady-state hematopoiesis. In parallel, after myelosuppression, Ndn-deficient mice exhibit an enhanced number of proliferating HSCs. Based on these findings, we propose that necdin functions in a negative feedback loop that prevents excessive proliferation of HSCs during hematopoietic regeneration. These data suggest that the inhibition of necdin after clinical myelosuppressive treatment (eg, chemotherapy, HSC transplantation) may provide therapeutic benefits by accelerating hematologic recovery.

BMP4 regulates the hematopoietic stem cell niche

Thu, 12 Nov 2009 09:38:05 PST

Bone morphogenetic protein 4 (BMP4) is required for mesoderm commitment to the hematopoietic lineage during early embryogenesis. However, deletion of BMP4 is early embryonically lethal and its functional role in definitive hematopoiesis is unknown. Consequently, we used a BMP4 hypomorph to investigate the role of BMP4 in regulating hematopoietic stem cell (HSC) function and maintaining steady-state hematopoiesis in the adult. Reporter gene expression shows that Bmp4 is expressed in cells associated with the hematopoietic microenvironment including osteoblasts, endothelial cells, and megakaryocytes. Although resting hematopoiesis is normal in a BMP4-deficient background, the number of c-Kit⁺, Sca-1⁺, Lineage^– cells is significantly reduced. Serial transplantation studies reveal that BMP4-deficient recipients have a microenvironmental defect that reduces the repopulating activity of wild-type HSCs. This defect is even more pronounced in a parabiosis model that demonstrates a profound reduction in wild-type hematopoietic cells within the bone marrow of BMP4-deficient recipients. Furthermore, wild-type HSCs that successfully engraft into the BMP4-deficient bone marrow show a marked decrease in functional stem cell activity when tested in a competitive repopulation assay. Taken together, these findings indicate BMP4 is a critical component of the hematopoietic microenvironment that regulates both HSC number and function.

Defects in osteoblast function but no changes in long-term repopulating potential of hematopoietic stem cells in a mouse chronic inflammatory arthritis model

Thu, 12 Nov 2009 09:38:05 PST

Recent studies support the notion that there is an intricate relationship between hematopoiesis and bone homeostasis in normal steady states. Using mice undergoing chronic inflammatory arthritis, we investigated the relationship between hematopoiesis and bone homeostasis in pathologic conditions. We demonstrate that mice undergoing chronic inflammatory arthritis displayed osteoporosis resulting from a severe defect in osteoblast function. Despite the defective osteoblast function, however, the hematopoietic stem cells from these mice exhibited normal properties in either long-term repopulation or cell cycling. Therefore, the bone-forming capacity of osteoblasts is distinct from their ability to maintain hematopoietic stem cells in chronic inflammatory conditions.

Homeostasis of dendritic cells in lymphoid organs is controlled by regulation of their precursors via a feedback loop

Thu, 12 Nov 2009 09:38:05 PST

Dendritic cells (DCs) are key coordinators of the immune response, governing the choice between tolerance and immunity. Despite their importance, the mechanisms controlling the size of the DC compartment are largely unknown. Using a mouse model allowing continuous DC depletion, we show that maintenance of DC numbers in spleen is an active process mediated by Flt3-L–dependent regulation of precursor differentiation into DCs, rather than by changes in proliferation of the differentiated DCs. In particular, the frequency and differentiation potential of intrasplenic DC precursors increased in response to reduced DC numbers. Levels of Flt3-L, a cytokine required for DC differentiation, increased in the blood after DC depletion and returned to normal levels once the DC compartment filled up again. Our data suggest a feedback regulation of DC homeostasis whereby reduction of the DC pool size promotes differentiation of their precursors, via increased Flt3-L availability. This mechanism is different to those known for other immune cell types, such as the B- and T-cell compartments, whereby lymphopenia induces proliferation of already differentiated lymphocytes.

CD56+ human blood dendritic cells effectively promote TH1-type {gamma}{delta} T-cell responses

Gruenbacher, G., Gander, H., Rahm, A., Nussbaumer, W., Romani, N., Thurnher, M. — Thu, 12 Nov 2009 09:38:05 PST

CD56⁺ human dendritic cells (DCs) have recently been shown to differentiate from monocytes in response to GM-CSF and type 1 interferon in vitro. We show here that CD56⁺ cells freshly isolated from human peripheral blood contain a substantial subset of CD14⁺CD86⁺HLA-DR⁺ cells, which have the appearance of intermediate-sized lymphocytes but spontaneously differentiate into enlarged DC-like cells with substantially increased HLA-DR and CD86 expression or into fully mature CD83⁺ DCs in response to appropriate cytokines. Stimulation of CD56⁺ cells containing both DCs and abundant T cells with zoledronate and interleukin-2 (IL-2) resulted in the rapid expansion of T cells as well as in IFN-, TNF-, and IL-1β but not in IL-4, IL-10, or IL-17 production. IFN-, TNF-, and IL-1β production were almost completely abolished by depleting CD14⁺ cells from the CD56⁺ subset before stimulation. Likewise, depletion of CD14⁺ cells dramatically impaired T-cell expansion. IFN- production could also be blocked by neutralizing the effects of endogenous IL-1β and TNF-. Conversely, addition of recombinant IL-1β, TNF-, or both further enhanced IFN- production and strongly up-regulated IL-6 production. Our data indicate that CD56⁺ DCs from human blood are capable of stimulating CD56⁺ T cells, which may be harnessed for immunotherapy.

The thymus-independent immunity conferred by a pneumococcal polysaccharide is mediated by long-lived plasma cells

Thu, 12 Nov 2009 09:38:05 PST

It was recently shown that bacterial thymus-independent (TI) antigens confer long-lasting immunity and generate memory B lymphocytes. However, reactivation of TI memory B cells is repressed in immunocompetent mice, thus raising the issue of the mechanism whereby TI vaccines confer immune protection. Here, we propose an explanation to this apparent paradox by showing that a Streptococcus pneumoniae capsular polysaccharide (PS) generates long-lived bone marrow (BM) plasma cells which frequency can be increased by CpG oligodeoxynucleotides (ODNs). The adjuvant effect of CpG ODNs on the PS3 Ab response is directly targeted to B cells and does not involve B-1a cells. We also demonstrated that BM plasma cells generated in response to the thymus-dependent (TD) form of the PS vaccine have a higher secretion capacity than those produced after immunization with the CpG-adjuvanted PS vaccine. Finally, we show that the PS-specific BM plasma cell compartment is sufficient to confer full protection of vaccinated mice against S pneumoniae infection. Altogether, our results show that TI antigens like their TD counterparts can generate both the lymphoid and the plasma cell component of B-cell memory. They also provide a framework for the improvement and widespread usage of TI vaccines.

Diverse marrow stromal cells protect CLL cells from spontaneous and drug-induced apoptosis: development of a reliable and reproducible system to assess stromal cell adhesion-mediated drug resistance

Thu, 12 Nov 2009 09:38:05 PST

Marrow stromal cells (MSCs) provide important survival and drug resistance signals to chronic lymphocytic leukemia (CLL) cells, but current models to analyze CLL–MSC interactions are heterogeneous. Therefore, we tested different human and murine MSC lines and primary human MSCs for their ability to protect CLL cells from spontaneous and drug-induced apoptosis. Our results show that both human and murine MSCs are equally effective in protecting CLL cells from fludarabine-induced apoptosis. This protective effect was sustained over a wide range of CLL–MSC ratios (5:1 to 100:1), and the levels of protection were reproducible in 4 different laboratories. Human and murine MSCs also protected CLL cells from dexamethasone- and cyclophosphamide-induced apoptosis. This protection required cell–cell contact and was virtually absent when CLL cells were separated from the MSCs by micropore filters. Furthermore, MSCs maintained Mcl-1 and protected CLL cells from spontaneous and fludarabine-induced Mcl-1 and PARP cleavage. Collectively, these studies define common denominators for CLL cocultures with MSCs. They also provide a reliable, validated tool for future investigations into the mechanism of MSC–CLL cross talk and for drug testing in a more relevant fashion than the commonly used suspension cultures.

Stage-specific Arf tumor suppression in Notch1-induced T-cell acute lymphoblastic leukemia

Volanakis, E. J., Williams, R. T., Sherr, C. J. — Thu, 12 Nov 2009 09:38:05 PST

Frequent hallmarks of T-cell acute lymphoblastic leukemia (T-ALL) include aberrant NOTCH signaling and deletion of the CDKN2A locus, which contains 2 closely linked tumor suppressor genes (INK4A and ARF). When bone marrow cells or thymocytes transduced with a vector encoding the constitutively activated intracellular domain of Notch1 (ICN1) are expanded ex vivo under conditions that support T-cell development, cultured progenitors rapidly induce CD4⁺/CD8⁺ T-ALLs after infusion into healthy syngeneic mice. Under these conditions, enforced ICN1 expression also drives formation of T-ALLs in unconditioned CD-1 nude mice, bypassing any requirements for thymic maturation. Retention of Arf had relatively modest activity in suppressing the formation of T-ALLs arising from bone marrow–derived ICN1⁺ progenitors in which the locus is epigenetically silenced, and all resulting Arf ^+/+ tumors failed to express the p19^Arf protein. In striking contrast, retention of Arf in thymocyte-derived ICN1⁺ donor cells significantly delayed disease onset and suppressed the penetrance of T-ALL. Use of cultured thymocyte-derived donor cells expressing a functionally null Arf-GFP knock-in allele confirmed that ICN1 signaling can induce Arf expression in vivo. Arf activation by ICN1 in T cells thereby provides stage-specific tumor suppression but also a strong selective pressure for deletion of the locus in T-ALL.

Extensive intraclonal diversification in a subgroup of chronic lymphocytic leukemia patients with stereotyped IGHV4-34 receptors: implications for ongoing interactions with antigen

Thu, 12 Nov 2009 09:38:05 PST

Several studies indicate that the development of chronic lymphocytic leukemia (CLL) may be influenced by antigen recognition through the clonotypic B-cell receptors (BCRs). However, it is still unclear whether antigen involvement is restricted to the malignant transformation phase or whether the putative antigen(s) may continuously trigger the CLL clone and affect not only the progenitor cell but also the leukemic cells themselves. To address this issue, we conducted a large-scale subcloning study of rearranged immunoglobulin heavy variable (IGHV) genes of diverse mutational status from 71 CLL cases (total, 1496 subcloned sequences), belonging to both the common IgM/IgD variant and the rare IgG-positive variant. Although most cases showed no or low levels of intraclonal diversification (ID), we report intense ID in the IGHV genes of selected cases, especially a subgroup of 13 IgG-switched cases expressing stereotyped, mutated IGHV4-34 rearrangements (subset 4). We demonstrate that the ID evident in subset 4 cases cannot be attributed to IGHV4-34 usage, IGHV gene-mutated status, class-switch recombination, or BCR stereotypy in general; rather, it represents a unique phenomenon strongly correlated with the distinctive BCR of subset 4. In such cases, the observed ID patterns may imply a stereotyped response to an active, ongoing interaction with antigen(s).

Chronic lymphocytic leukemia of E{micro}-TCL1 transgenic mice undergoes rapid cell turnover that can be offset by extrinsic CD257 to accelerate disease progression

Thu, 12 Nov 2009 09:38:05 PST

Results of heavy-water labeling studies have challenged the notion that chronic lymphocytic leukemia (CLL) represents an accumulation of noncycling B cells. We examined leukemia cell turnover in Eµ-TCL1 transgenic (TCL1-Tg) mice, which develop a CLL-like disease at 8 to 12 months of age. We found that leukemia cells in these mice not only had higher proportions of proliferating cells but also apoptotic cells than did nonleukemic lymphocytes. We crossed TCL1-Tg with BAFF-Tg mice, which express high levels of CD257. TCL1xBAFF-Tg mice developed CLL-like disease at a significantly younger age and had more rapid disease progression and shorter survival than TCL1-Tg mice. Leukemia cells of TCL1xBAFF-Tg mice had similar proportions of proliferating cells, but fewer proportions of dying cells, than did the CLL cells of TCL1-Tg mice. Moreover, leukemia cells from either TCL1xBAFF-Tg or TCL1-Tg mice produced more aggressive disease when transferred into BAFF-Tg mice than into wild-type (WT) mice. Neutralization of CD257 resulted in rapid reduction in circulating leukemia cells. These results indicate that the leukemia cells of TCL1-Tg mice undergo high levels of spontaneous apoptosis that is offset by relatively high rates of leukemia cell proliferation, which might allow for acquisition of mutations that contribute to disease evolution.

Generation of CD8+ T cell-mediated immunity against idiotype-negative lymphoma escapees

Thu, 12 Nov 2009 09:38:05 PST

We investigated the ability of CpG-oligodeoxynucleotide to generate an anti-tumor CD8⁺ T-cell immune response and to synergize with passive antibody therapy. For these studies, we generated an antibody against the idiotype on the A20 B-cell lymphoma line. This antibody caused the regression of established tumors, but ultimately the tumors relapsed. The escaping surface IgG-negative tumor cells were resistant to both antibody-dependent cellular cytotoxicity and signaling-induced cell death. Addition of intratumoral CpG to antibody therapy cured large established tumors and prevented the occurrence of tumor escapees. The failure of the combination therapy in mice deficient for CD8⁺ T cells demonstrates the critical role of CD8⁺ T cells in tumor eradication. When mice were inoculated with 2 tumors and treated systemically with antibody followed by intratumoral CpG in just one tumor, both tumors regressed, indicating that a systemic immune response was generated. Although antibody therapy can eliminate tumor cells bearing the target antigen, it frequently selects for antigen loss variants. However, when a poly-specific T-cell response was generated against the tumor by intratumoral CpG, even large established tumors were cured. Such an immune response can prevent the emergence of antibody selected tumor escapees and provide long-lasting tumor protection.

Gene expression-based classification and regulatory networks of pediatric acute lymphoblastic leukemia

Thu, 12 Nov 2009 09:38:05 PST

Pediatric acute lymphoblastic leukemia (ALL) contains cytogenetically distinct subtypes that respond differently to cytotoxic drugs. Subtype classification can be also achieved through gene expression profiling. However, how to apply such classifiers to a single patient and correctly diagnose the disease subtype in an independent patient group has not been addressed. Furthermore, the underlying regulatory mechanisms responsible for the subtype-specific gene expression patterns are still largely unknown. Here, by combining 3 published microarray datasets on 535 mostly white children's samples and generating a new dataset on 100 Chinese children's ALL samples, we were able to (1) identify a 62-gene classifier with 97.6% accuracy from the white children's samples and validated it on the completely independent set of 100 Chinese samples, and (2) uncover potential regulatory networks of ALL subtypes. The classifier we identified was, thus far, the only one that could be applied directly to a single sample and that sustained validation in a large independent patient group. Our results also suggest that the etiology of ALL is largely the same among different ethnic groups, and that the transcription factor hubs in the predicted regulatory network might play important roles in regulating gene expression and development of ALL.

A human monoclonal antibody drug and target discovery platform for B-cell chronic lymphocytic leukemia based on allogeneic hematopoietic stem cell transplantation and phage display

Thu, 12 Nov 2009 09:38:05 PST

Allogeneic hematopoietic stem cell transplantation (alloHSCT) is the only potentially curative treatment available for patients with B-cell chronic lymphocytic leukemia (B-CLL). Here, we show that post-alloHSCT antibody repertoires can be mined for the discovery of fully human monoclonal antibodies to B-CLL cell-surface antigens. Sera collected from B-CLL patients at defined times after alloHSCT showed selective binding to primary B-CLL cells. Pre-alloHSCT sera, donor sera, and control sera were negative. To identify post-alloHSCT serum antibodies and subsequently B-CLL cell-surface antigens they recognize, we generated a human antibody-binding fragment (Fab) library from post-alloHSCT peripheral blood mononuclear cells and selected it on primary B-CLL cells by phage display. A panel of Fab with B-CLL cell-surface reactivity was strongly enriched. Selection was dominated by highly homologous Fab predicted to bind the same antigen. One Fab was converted to immunoglobulin G1 and analyzed for reactivity with peripheral blood mononuclear cells from B-CLL patients and healthy volunteers. Cell-surface antigen expression was restricted to primary B cells and up-regulated in primary B-CLL cells. Mining post-alloHSCT antibody repertoires offers a novel route to discover fully human monoclonal antibodies and identify antigens of potential therapeutic relevance to B-CLL and possibly other cancers. Trials described herein were registered at www.clinicaltrials.gov as nos. NCT00055744 and NCT00003838.

Inactivating SOCS1 mutations are caused by aberrant somatic hypermutation and restricted to a subset of B-cell lymphoma entities

Thu, 12 Nov 2009 09:38:05 PST

STATs are constitutively activated in several malignancies. In primary mediastinal large B-cell lymphoma and Hodgkin lymphoma (HL), inactivating mutations in SOCS1, an inhibitor of JAK/STAT signaling, contribute to deregulated STAT activity. Based on indications that the SOCS1 mutations are caused by the B cell–specific somatic hypermutation (SHM) process, we analyzed B-cell non-HL and normal B cells for mutations in SOCS1. One-fourth of diffuse large B-cell lymphoma and follicular lymphomas carried SOCS1 mutations, which were preferentially targeted to SHM hotspot motifs and frequently obviously inactivating. Rare mutations were observed in Burkitt lymphoma, plasmacytoma, and mantle cell lymphoma but not in tumors of a non–B-cell origin. Mutations in single-sorted germinal center B cells were infrequent relative to other genes mutated as byproducts of normal SHM, indicating that SOCS1 inactivation in primary mediastinal large B-cell lymphoma, HL, diffuse large B-cell lymphoma, and follicular lymphoma is frequently the result of aberrant SHM.

The BH3-only protein Bim plays a critical role in leukemia cell death triggered by concomitant inhibition of the PI3K/Akt and MEK/ERK1/2 pathways

Thu, 12 Nov 2009 09:38:05 PST

Mechanisms underlying apoptosis induced by concomitant interruption of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase 1/2 (MEK/ERK1/2) and phosphatidylinositol 3-kinase (PI3K)/Akt pathways were investigated in human leukemia cells. Inhibition of these pathways using the MEK inhibitor PD184352 or U0126 and the PI3K/Akt inhibitor perifosine strikingly induced apoptosis in multiple malignant human hematopoietic cells, and substantially reduced the colony-forming capacity of primary acute myeloblastic leukemia, but not normal CD34⁺ cells. These events were associated with pronounced Bim up-regulation, Mcl-1 down-regulation, marked Bak/Bax conformational change accompanied by Bax membrane translocation, and a pronounced increase in Bax/Bak association. Molecular studies using tet-inducible Akt, constitutively active MEK1, dominant-negative Akt, and MEK1 small interfering RNA revealed that inhibition of both MEK/ERK1/2 and Akt pathways plays a critical functional role in perifosine/PD184352-mediated lethality. Ectopic Mcl-1 expression potently inhibited perifosine/PD184352-induced apoptosis, as did Bak or Bax knockdown. Notably, knockdown of Bim, but not Bad, blocked Bak and Bax conformational change, inhibited Bax membrane translocation, diminished Bax/Bak binding, and sharply attenuated perifosine/PD184352-induced apoptosis. Finally, enforced expression of Bim significantly enhanced apoptosis induced by PI3K/Akt inhibitors, analogous to the effects of MEK1/2 inhibitors. Collectively, these findings suggest that Bim, and Mcl-1, but not Bad, integrate death signaling triggered by concomitant disruption of the PI3K/Akt and MEK1/2/ERK1/2 pathways in human leukemia cells.

GM-CSF and IL-4 induce dendritic cell differentiation and disrupt osteoclastogenesis through M-CSF receptor shedding by up-regulation of TNF-{alpha} converting enzyme (TACE)

Thu, 12 Nov 2009 09:38:05 PST

Monocytes give rise to macrophages, osteoclasts (OCs), and dendritic cells (DCs). Macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappaB (RANK) ligand induce OC differentiation from monocytes, whereas granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) trigger monocytic differentiation into DCs. However, regulatory mechanisms for the polarization of monocytic differentiation are still unclear. The present study was undertaken to clarify the mechanism of triggering the deflection of OC and DC differentiation from monocytes. GM-CSF and IL-4 abolished monocytic differentiation into OCs while inducing DC differentiation even in the presence of M-CSF and RANK ligand. GM-CSF and IL-4 in combination potently up-regulate tumor necrosis factor- (TNF-) converting enzyme (TACE) and activity in monocytes, causing ectodomain shedding of M-CSF receptor, resulting in the disruption of its phosphorylation by M-CSF as well as the induction of osteoclastogenesis from monocytes by M-CSF and RANK ligand. Interestingly, TACE inhibition robustly causes the resumption of the surface expression of M-CSF receptor on monocytes, facilitating M-CSF–mediated phosphorylation of M-CSF receptor and macrophage/OC differentiation while impairing GM-CSF– and IL-4–mediated DC differentiation from monocytes. These results reveal a novel proteolytic regulation of M-CSF receptor expression in monocytes to control M-CSF signaling and monocytic differentiation into macrophage/OC-lineage cells or DCs.

The small Rho GTPase Cdc42 regulates neutrophil polarity via CD11b integrin signaling

Szczur, K., Zheng, Y., Filippi, M.-D. — Thu, 12 Nov 2009 09:38:05 PST

Neutrophil migration to sites of infection is the first line of cellular defense. A key event of migration is the maintenance of a polarized morphology, which is characterized by a single leading edge of filamentous actin and a contractile uropod devoid of filamentous actin protrusions. Using a mouse model of high Cdc42 activity, we previously demonstrated the importance of Cdc42 activity in neutrophil migration. However, the specific functions of Cdc42 in this process remain to be understood. Using neutrophils genetically deficient in Cdc42, we show that Cdc42 regulates directed migration by maintaining neutrophil polarity. Although it is known to be activated at the front, Cdc42 suppresses protrusions at the uropod. Interestingly, Cdc42 makes use of the integrin CD11b during this process. Cdc42 determines the redistribution of CD11b at the uropod. In turn, using CD11b-null cells and CD11b crosslinking experiments, we show that CD11b modulates myosin light chain phosphorylation to suppress lateral protrusions. Our results uncover a new mechanism in which Cdc42 regulates the uropod through CD11b signaling to maintain polarity in migrating neutrophils. It also reveals new functions for CD11b in neutrophil polarity.

Platelet-associated complement factor H in healthy persons and patients with atypical HUS

Thu, 12 Nov 2009 09:38:06 PST

Atypical hemolytic uremic syndrome (aHUS) is associated with complement system dysregulation, and more than 25% of pediatric aHUS cases are linked to mutations in complement factor H (CFH) or CFH autoantibodies. The observation of thrombocytopenia and platelet-rich thrombi in the glomerular microvasculature indicates that platelets are intimately involved in aHUS pathogenesis. It has been reported that a releasable pool of platelet CFH originates from -granules. We observed that platelet CFH can arise from endogenous synthesis in megakaryocytes and that platelets constitutively lacking -granules contain CFH. Electron and high-resolution laser fluorescence confocal microscopy revealed that CFH was present throughout the cytoplasm and on the surface of normal resting platelets with no evident concentration in -granules, lysosomes, or dense granules. Therapeutic plasma transfusion in a CFH-null aHUS patient revealed that circulating platelets take up CFH with similar persistence of CFH in platelets and plasma in vivo. Washed normal platelets were also observed to take up labeled CFH in vitro. Exposure of washed normal platelets to plasma of an aHUS patient with CFH autoantibodies produced partial platelet aggregation or agglutination, which was prevented by preincubation of platelets with purified CFH. This CFH-dependent response did not involve P-selectin mobilization, indicating a complement-induced platelet response distinct from -granule secretion.

Specific iron chelators determine the route of ferritin degradation

De Domenico, I., Ward, D. M., Kaplan, J. — Thu, 12 Nov 2009 09:38:06 PST

Deferoxamine (DFO) is a high-affinity Fe (III) chelator produced by Streptomyces pilosus. DFO is used clinically to remove iron from patients with iron overload disorders. Orally administered DFO cannot be absorbed, and therefore it must be injected. Here we show that DFO induces ferritin degradation in lysosomes through induction of autophagy. DFO-treated cells show cytosolic accumulation of LC3B, a critical protein involved in autophagosomal-lysosomal degradation. Treatment of cells with the oral iron chelators deferriprone and desferasirox did not show accumulation of LC3B, and degradation of ferritin occurred through the proteasome. Incubation of DFO-treated cells with 3-methyladenine, an autophagy inhibitor, resulted in degradation of ferritin by the proteasome. These results indicate that ferritin degradation occurs by 2 routes: a DFO-induced entry of ferritin into lysosomes and a cytosolic route in which iron is extracted from ferritin before degradation by the proteasome.

The therapeutic effect of bone marrow-derived liver cells in the phenotypic correction of murine hemophilia A

Thu, 12 Nov 2009 09:38:06 PST

The transdifferentiation of bone marrow cells (BMCs) into hepatocytes has created enormous interest in applying this process to the development of cellular medicine for degenerative and genetic diseases. Because the liver is the primary site of factor VIII (FVIII) synthesis, we hypothesized that the partial replacement of mutated liver cells by healthy cells in hemophilia A mice could manage the severity of the bleeding disorder. We perturbed the host liver with acetaminophen to facilitate the engraftment and hepatic differentiation of lineage-depleted enhanced green fluorescent protein-expressing BMCs. Immunohistochemistry experiments with the liver tissue showed that the donor-derived cells expressed the markers of both hepatocytes (albumin and cytokeratin-18) and endothelial cells (von Willebrand factor). The results of fluorescent in situ hybridization and immunocytochemistry experiments suggested that differentiation was direct in this model. The BMC-recipient mice expressed FVIII protein and survived in a tail clip challenge experiment. Furthermore, a coagulation assay confirmed that the plasma FVIII activity was maintained at 20.4% (± 3.6%) of normal pooled plasma activity for more than a year without forming its inhibitor. Overall, this report demonstrated that BMCs rescued the bleeding phenotype in hemophilia A mice, suggesting a potential therapy for this and other related disorders.

Recombinant canine B-domain-deleted FVIII exhibits high specific activity and is safe in the canine hemophilia A model

Thu, 12 Nov 2009 09:38:06 PST

Production of recombinant B-domain–deleted canine factor VIII (cFVIII-BDD) unexpectedly revealed superior protein yields with 3-fold increased specific activity relative to human FVIII-BDD (hFVIII-BDD). We also determined that activated cFVIII-BDD is more stable than activated hFVIII-BDD. Furthermore, cFVIII-BDD is efficient at inducing hemostasis in human plasma containing FVIII inhibitors. Infusion of cFVIII-BDD in hemophilia A dogs resulted in correction of the disease phenotype with a pharmacokinetic profile similar to clinical experience with hFVIII-BDD. Notably, immune tolerance challenges with cFVIII-BDD in young and adult hemophilia A dogs did not induce the formation of neutralizing or nonneutralizing antibodies to cFVIII. These data establish the framework to quantitatively investigate the efficacy and safety in preclinical studies of novel therapies for hemophilia A.

Bone marrow as an alternative site for islet transplantation

Thu, 12 Nov 2009 09:38:06 PST

The liver is the current site for pancreatic islet transplantation, but has many drawbacks due to immunologic and nonimmunologic factors. We asked whether pancreatic islets could be engrafted in the bone marrow (BM), an easily accessible and widely distributed transplant site that may lack the limitations seen in the liver. Syngeneic islets engrafted efficiently in the BM of C57BL/6 mice rendered diabetic by streptozocin treatment. For more than 1 year after transplantation, these animals showed parameters of glucose metabolism that were similar to those of nondiabetic mice. Islets in BM had a higher probability to reach euglycemia than islets in liver (2.4-fold increase, P = .02), showed a compact morphology with a conserved ratio between and β cells, and affected bone structure only very marginally. Islets in BM did not compromise hematopoietic activity, even when it was strongly induced in response to a BM aplasia-inducing infection with lymphocytic choriomeningitis virus. In conclusion, BM is an attractive and safe alternative site for pancreatic islet transplantation. The results of our study open a research line with potentially significant clinical impact, not only for the treatment of diabetes, but also for other diseases amenable to treatment with cellular transplantation.

Superagonistic CD28 stimulation of allogeneic T cells protects from acute graft-versus-host disease

Beyersdorf, N., Ding, X., Hunig, T., Kerkau, T. — Thu, 12 Nov 2009 09:38:06 PST

Acute graft-versus-host disease (aGVHD) often precludes successful immunotherapy of hematologic malignancies with allogeneic T cells. Therefore, we investigated the effect of immunomodulatory superagonistic anti-CD28 monoclonal antibodies (CD28-SA) on the capacity of allogeneic T cells to mediate both aGVHD and the protective graft-versus-tumor (GVT) response. In vivo pretreatment of donor C57BL/6 mice or short-term in vitro culture of donor lymph node cells with a CD28-SA efficiently protected BALB/c recipient mice from aGVHD. This protection strongly relied on the presence of CD28-SA–activated CD4⁺ CD25⁺ Foxp3⁺ regulatory T cells in the donor T-cell inoculum. With respect to the GVT response, CD28-SA–prestimulated T cells were still as potent in clearing lymphoma cells as were T cells without CD28-SA preactivation. Taken together, our data suggest that CD28-SA stimulation of bulk leukocyte cultures in vitro markedly increases the therapeutic window for adoptive immunotherapy with allogeneic T cells in vivo.

Selectin-mediated activation of endothelial cells induces expression of CCL5 and promotes metastasis through recruitment of monocytes

Laubli, H., Spanaus, K.-S., Borsig, L. — Thu, 12 Nov 2009 09:38:06 PST

Hematogenous metastasis is promoted by interactions of tumor cells with leukocytes, platelets, and the endothelium in the local intravascular microenvironment. Here we show that the activation of the microvascular endothelium results in recruitment of monocytes to metastatic tumor cells and promotes the establishment of the metastatic microenvironment. This inflammatory-like endothelial response was observed in microvascular endothelial cells only. Microarray analysis of microvascular endothelial cells cocultured with tumor cells in the presence of leukocytes and platelets revealed a specific gene expression profile. Selectin-mediated interactions of tumor cells with platelets and leukocytes activated endothelial cells and induced production of C-C chemokine ligand 5 (CCL5). Inhibition of CCL5-dependent monocyte recruitment during the early phase of metastasis by a CCL5 receptor antagonist strongly reduced tumor cell survival and attenuated metastasis. Collectively, these findings demonstrate that the endothelial expression of CCL5 contributes to the formation of a permissive metastatic microenvironment.

A novel interplay between Epac/Rap1 and mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) regulates thrombospondin to control angiogenesis

Thu, 12 Nov 2009 09:38:06 PST

Tumors depend upon angiogenesis for growth and metastasis. It is therefore critical to understand the inhibitory signaling mechanisms in endothelial cells that control angiogenesis. Epac is a cyclic adenosine 5'-monophosphate–activated guanine nucleotide exchange factor for Rap1. In this study, we show that activation of Epac or Rap1 leads to potent inhibition of angiogenesis in vivo. Epac/Rap1 activation down-regulates inhibitor of differentiation 1 (Id1), which negatively regulates thrombospondin-1 (TSP1), an inhibitor of angiogenesis. Consistent with this mechanism, activation of Epac/Rap 1 induces expression of TSP1; conversely, depletion of Epac reduces TSP1 levels in endothelial cells. Blockade of TSP1 binding to its receptor, CD36, rescues inhibition of chemotaxis or angiogenesis by activated Epac/Rap1. Mitogen-activated protein kinase kinase 5, a downstream mediator of vascular endothelial growth factor, antagonizes the effects of Epac/Rap1 by inducing Id1 and suppressing TSP1 expression. Finally, TSP1 is also secreted by fibroblasts in response to Epac/Rap1 activation. These results identify Epac and Rap1 as inhibitory regulators of the angiogenic process, implicate Id1 and TSP1 as downstream mediators of Epac/Rap1, and highlight a novel interplay between pro- and antiangiogenic signaling cascades involving multiple cell types within the angiogenic microenvironment.

The role of cytogenetic abnormalities in acute myeloid leukemia with NPM1 mutations and no FLT3 internal tandem duplication

Thu, 12 Nov 2009 09:38:06 PST

Response: NPM1-mutated AML is an entity irrespective of whether or not chromosomal aberrations are present

Haferlach, C., Vignetti, M., Haferlach, T., Falini, B. — Thu, 12 Nov 2009 09:38:06 PST

Elevated fibrinogen {gamma}' ratio is associated with cardiovascular diseases and acute phase reaction but not with clinical outcome

Thu, 12 Nov 2009 09:38:06 PST

Response: Elevated fibrinogen {gamma}' ratios and clinical outcomes

Nowak-Gottl, U., Weiler, H., Stoll, M. — Thu, 12 Nov 2009 09:38:06 PST

Allogeneic transplantation for children and adolescents with Hodgkin lymphoma

Thu, 12 Nov 2009 09:38:06 PST

van de Vosse et al. Antisense-mediated exon skipping to correct IL-12R{beta}1 deficiency in T cells. Blood. 2009;113(19):4548-4555.

Thu, 12 Nov 2009 09:38:06 PST