Prior to 1990, many patients with inherited bleeding disorders were infected with hepatitis C virus (HCV). This study assessed the risk of end-stage liver disease (ESLD) in patients with hemophilia with chronic hepatitis C. Patients were infected between 1961 and 1990 and were followed up to August 2005. Of 847 anti-HCV(+) patients, 160 (19%) spontaneously cleared HCV and 687 (81%) developed chronic hepatitis C. Coinfection with HIV was present in 210 patients. After 35 years of infection the cumulative incidence of ESLD was 11.5% (95% CI, 8.2%-14.8%) in HIV(-) patients and 35.1% (95% CI, 29.2%-41.0%; P < .001) in patients coinfected with HIV. Independent risk factors of ESLD were HIV coinfection (hazard ratio 13.8; 95% CI, 7.5-25.3), older age at infection (hazard ratio 2.3 per 10 years; 95% CI, 2.0-2.8), alcohol abuse (hazard ratio 4.9; 95% CI, 2.5-9.6), and presence of HCV genotype 1 (hazard ratio 2.2; 95% CI, 1.1-4.2). With longer duration of HCV infection, the risk of developing ESLD is emerging in patients with inherited bleeding disorders. Risk factors for rapid progression to ESLD are alcohol abuse, coinfection with HIV, older age at infection, and presence of HCV genotype 1.

Thirty elderly (> 60 years) Philadelphia chromosome-positive (Ph(+)) patients with acute lymphoblastic leukemia (ALL) received imatinib, 800 mg daily, associated to steroids without further chemotherapy as frontline treatment. Median age was 69 years (range, 61-83 years). Twenty-nine patients were evaluable for response and all of them obtained a hematologic complete remission, with a median BCR-ABL reduction of 2.9 and 2.0 logs in p190(+) and p210(+) cases, respectively. Most of the induction treatment did not require admission of the patients. No major toxicities occurred and the treatment was well tolerated. Median survival from diagnosis was 20 months. This study shows that elderly Ph(+) patients with ALL-often considered eligible only for palliative treatment strategies-may benefit from an imatinib-steroids protocol, which does not require chemotherapy nor a long hospitalization, is feasible, highly active, and associated with a good quality of life.

Chronic myeloid leukemia (CML) stem and progenitor cells overexpress BcrAbl and are insensitive to imatinib mesylate (IM). We therefore investigated whether these cells were efficiently targeted by nilotinib. In K562, the inhibitory concentration (IC50) of nilotinib was 30 nM versus 600 nM for IM, consistent with its reported 20-fold-higher potency. However, in primary CD34(+) CML cells, nilotinib and IM were equipotent for inhibition of BcrAbl activity, producing equivalent but incomplete reduction in CrkL phosphorylation at 5 microM. CML CD34(+) cells were still able to expand over 72 hours with 5 microM of either drug, although there was a concentration-dependent restriction of amplification. As for IM, the most primitive cells (CFSE(max)) persisted and accumulated over 72 hours with nilotinib and remained caspase-3 negative. Furthermore, nilotinib with IM led to further accumulation of this population, suggesting at least additive antiproliferative effects. These results confirmed that, like IM, the predominant effect of nilotinib is antiproliferative rather than proapoptotic.

Plasma factor VIII coagulant activity (FVIII:C) level is a highly heritable quantitative trait that is strongly correlated with thrombosis risk. Polymorphisms within only 1 gene, the ABO blood-group locus, have been unequivocally demonstrated to contribute to the broad population variability observed for this trait. Because less than 2.5% of the structural FVIII gene (F8) has been examined previously, we resequenced all known functional regions in 222 potentially distinct alleles from 137 unrelated nonhemophilic individuals representing 7 racial groups. Eighteen of the 47 variants identified, including 17 single-nucleotide polymorphisms (SNPs), were previously unknown. As the degree of linkage disequilibrium across F8 was weak overall, we used measured-genotype association analysis to evaluate the influence of each polymorphism on the FVIII:C levels in 398 subjects from 21 pedigrees known as the Genetic Analysis of Idiopathic Thrombophilia project (GAIT). Our results suggested that 92714C>G, a nonsynonymous SNP encoding the B-domain substitution D1241E, was significantly associated with FVIII:C level. After accounting for important covariates, including age and ABO genotype, the association persisted with each C-allele additively increasing the FVIII:C level by 14.3 IU dL(-1) (P = .016). Nevertheless, because the alleles of 56010G>A, a SNP within the 3' splice junction of intron 7, are strongly associated with 92714C>G in GAIT, additional studies are required to determine whether D1241E is itself a functional variant.

Cladribine (2-chlorodeoxyadenosine, 2-CdA) treatment-associated infections may shorten potentially long-term survival in hairy cell leukemia (HCL). In search of the optimal mode of 2-CdA administration, 132 patients with untreated HCL were randomized to receive either standard 5-day 2-CdA protocol or a novel schedule of 6 weekly 2-CdA infusions suggested to be less toxic. Analysis of treatment response confirmed similar complete remission rates, overall response rates, progression-free survival, and overall survival in both 2-CdA protocols. However, we did not observe lower toxicity in the weekly schedule. Of special interest, no significant differences were found in the rate of grade 3/4 infections (18% for daily and 26% for weekly protocol, difference -8.2%; 95% confidence interval [CI] -23.2% to 6.9%; P = .28) and the rate of septic deaths (3% for daily and 2% for weekly protocol, difference 1.4%; 95% CI -4.3% to 7.0%; P = .64). In conclusion, HCL treatment with weekly 2-CdA infusions is equally effective but no safer than the standard 5-day 2-CdA protocol.

Acquired genomic aberrations have been shown to significantly impact survival in several hematologic malignancies. We analyzed the prognostic value of the most frequent chromosomal changes in a large series of patients with newly diagnosed symptomatic myeloma prospectively enrolled in homogeneous therapeutic trials. All the 1064 patients enrolled in the IFM99 trials conducted by the Intergroupe Francophone du Myélome benefited from an interphase fluorescence in situ hybridization analysis performed on purified bone marrow plasma cells. They were systematically screened for the following genomic aberrations: del(13), t(11;14), t(4;14), hyperdiploidy, MYC translocations, and del(17p). Chromosomal changes were observed in 90% of the patients. The del(13), t(11;14), t(4;14), hyperdiploidy, MYC translocations, and del(17p) were present in 48%, 21%, 14%, 39%, 13%, and 11% of the patients, respectively. After a median follow-up of 41 months, univariate statistical analyses revealed that del(13), t(4;14), nonhyperdiploidy, and del(17p) negatively impacted both the event-free survival and the overall survival, whereas t(11;14) and MYC translocations did not influence the prognosis. Multivariate analyses on 513 patients annotated for all the parameters showed that only t(4;14) and del(17p) retained prognostic value for both the event-free and overall survivals. When compared with the currently used International Staging System, this prognostic model compares favorably. In myeloma, the genomic aberrations t(4;14) and del(17p), together with beta2-microglobulin level, are important independent predictors of survival. These findings have implications for the design of risk-adapted treatment strategies.

HIV-1 strains use C-C-chemokine receptor 5, CCR5, as a coreceptor for host transmission. Human CCR5 chemokine ligands inhibit binding and infection, whereas CCR5 mutations also inhibit infection by preventing surface expression, resulting in delayed progression to AIDS. Here, we describe a human herpesvirus 6 (HHV-6A) chemokine, U83A, which binds CCR5 with higher affinity than human chemokines, displacing their binding and leading to inhibition of chemotaxis of human leukocytes. Similarly, U83A inhibits infection by HIV-1 strains which use CCR5, but not the CXCR4, coreceptor. Unlike human CCR5 chemokine ligands which induce rapid CCR5 internalization mediated via clathrin, treatment with U83A prevents internalization. A spliced truncated U83A isoform, U83A-N, also binds CCR5 albeit with lower affinity, and this correlates with lower HIV-1 infection inhibition, whereas further truncation abolishes binding and any inhibition. Confocal microscopy confirms CCR5 internalization inhibition by U83A treatment, whereas labeled transferrin uptake shows that endocytosis via clathrin is unaltered. Previous results show that, although U83A-N is an antagonist, U83A is an agonist for CCR1, CCR4, CCR6, and CCR8 present on immune effector and antigen-presenting cells and here also shown for CCR5. Thus, U83A could act as a novel inhibitor of HIV-1 infection while also stimulating local immunity to the virus.

Central nervous system (CNS) prophylaxis has led to a significant improvement in the outcome of patients with acute lymphocytic leukemia (ALL). Liposomal cytarabine (Enzon Pharmaceuticals, Piscataway, NJ; Skye Pharma, San Diego, CA), an intrathecal (IT) preparation of cytarabine with a prolonged half-life, has been shown to be safe and effective in the treatment of neoplastic meningitis. Liposomal cytarabine was given for CNS prophylaxis to 31 patients with newly diagnosed ALL. All patients were treated concurrently with hyper-CVAD chemotherapy (fractionated cyclophosphamide, vincristine, doxorubicin, and dexamethasone) including high-dose methotrexate (MTX) and cytarabine on alternating courses. Liposomal cytarabine 50 mg was given intrathecally on days 2 and 15 of hyper-CVAD and day 10 of high-dose MTX and cytarabine courses until completion of either 3, 6, or 10 IT treatments, depending on risk for CNS disease. Five patients (16%) experienced serious unexpected neurotoxicity, including seizures, papilledema, cauda equina syndrome (n = 2), and encephalitis after a median of 4 IT administrations of liposomal cytarabine. Toxicities usually manifested after the MTX and cytarabine courses. One patient died with progressive encephalitis. After a median follow-up of 7 months, no isolated CNS relapses have been observed. Liposomal cytarabine given via intrathecal route concomitantly with systemic chemotherapy that crosses the blood-brain barrier such as high-dose MTX and cytarabine can result in significant neurotoxicity.

Chemerin is a chemotactic agonist recently identified as the ligand of ChemR23, a serpentine receptor expressed by mononuclear phagocytes and dendritic cells (DCs). This study shows that blood CD56(low)CD16(+) natural killer (NK) cells selectively express functional ChemR23 and that this receptor is coexpressed with CXCR1, the CXCL8 receptor, and the KIR receptors. In vitro culturing of NK cells with IL-2 or IL-15 induced a delayed and time-dependent down-regulation of ChemR23 that was associated with the inhibition of NK cell migration to chemerin. Biopsies obtained from patients with oral lichen planus presented an infiltration of CD94(+)CD3(-)CD56(+) NK cells that coexpressed ChemR23. The same biopsies were infiltrated by myeloid, DC-SIGN(+) and plasmacytoid, CD123(+)BDCA2(+), ChemR23(+) dendritic cells that were occasionally associated with NK cells. In the same histologic sections, chemerin was expressed by inflamed dermal endothelium. These findings propose a role for the ChemR23/chemerin axis in the recruitment of blood NK cells and strongly implicate chemerin as a key factor for the colocalization of NK cells and DC subsets in pathologic peripheral tissues.

The National Marrow Donor Program maintains a registry of volunteer donors for patients in need of a hematopoietic stem cell transplantation. Strategies for selecting a partially HLA-mismatched donor vary when a full match cannot be identified. Some transplantation centers limit the selection of mismatched donors to those sharing mismatched antigens within HLA-A and HLA-B cross-reactive groups (CREGs). To assess whether an HLA mismatch within a CREG group ("minor") may result in better outcome than a mismatch outside CREG groups ("major"), we analyzed validated outcomes data from 2709 bone marrow and peripheral blood stem cell transplantations. Three-hundred and ninety-six pairs (15%) were HLA-DRB1 allele matched but had an antigen-level mismatch at HLA-A or HLA-B. Univariate and multivariate analyses of engraftment, graft-versus-host disease, and survival showed that outcome is not significantly different between minor and major mismatches (P = .47, from the log-rank test for Kaplan-Meier survival). However, HLA-A, HLA-B, and HLA-DRB1 allele-matched cases had significantly better outcome than mismatched cases (P < .001). For patients without an HLA match, the selection of a CREG-compatible donor as tested does not improve outcome.

CCR6, a homeostatic chemokine receptor, is shown here to characterize subsets of both central and effector memory T cells that secrete high levels of IL-2 and TNF-alpha in response to polyclonal and antigen-specific stimulation. CCR6(+) T lymphocytes disappeared dramatically from the peripheral blood of HIV-infected patients as HIV disease progressed. The capacity of CD4(+)CCR6(+) to secrete multiple cytokines remained intact among HIV-infected long-term nonprogressors but was partially lost from subjects with standard disease progression. CCR6(+) T lymphocytes, regardless of their CCR7 expression, accumulated in the spleen of HIV-infected patients, where they died by apoptosis. Assessment of CCR6 expression allowed us to describe novel memory T-cell subpopulations capable of high cytokine production and provided evidence of a pathologic CCR6-dependent pathway of memory T-cell homing that may participate in the loss of memory response against infections.

Repair of damage to DNA resulting from chemotherapy may influence drug toxicity and survival in response to treatment. We evaluated the role of polymorphisms in DNA repair genes APE1, XRCC1, ERCC1, XPD, and XRCC3 in predicting therapeutic outcomes of older adults with acute myeloid leukemia (AML) from 2 Southwest Oncology Group (SWOG) clinical trials. All patients received standard chemotherapy induction regimens. Using logistic and proportional hazards regression models, relationships between genotypes, haplotypes, and toxicities, response to induction therapy, and overall survival were evaluated. Patients with XPD Gln751C/Asp312G ('D') haplotype were more likely to have complete response (OR = 3.06; 95% CI, 1.44-6.70) and less likely to have resistant disease (OR = 0.32; 95%CI, 0.14-0.72) than patients with other haplotypes. ERCC1 polymorphisms were significantly associated with lung (P = .037) and metabolic (P = .041) toxicities, and patients with the XRCC3 241Met variant had reduced risk of liver toxicity (OR = 0.32; 95%CI, 0.11-0.95). Significant associations with other toxicities were also found for variant XPD genotypes/haplotypes. These data from clinical trials of older patients treated for AML indicate that variants in DNA repair pathways may have an impact on both outcomes of patients and toxicities associated with treatments. With validation of results in larger samples, these findings could lead to optimizing individual chemotherapy options.

Endorepellin, a C-terminal fragment of the vascular basement membrane proteoglycan perlecan, inhibits angiogenesis via the alpha2beta1-integrin receptor. Because this integrin is also implicated in platelet-collagen responses and because endorepellin or its fragments are generated in response to injury and inflammation, we hypothesized that endorepellin could also affect platelet biology. We discovered that endorepellin supported alpha2beta1-dependent platelet adhesion, without appreciably activating or aggregating platelets. Notably, endorepellin enhanced collagen-evoked responses in platelets, in a src kinase-dependent fashion, and enhanced the collagen-inhibitory effect of an alpha2beta1-integrin function-blocking antibody. Collectively, these results suggest that endorepellin/alpha2beta1-integrin interaction and effects are specific and dependent on cell type, differ from those emanated by exposure to collagen, and may be due to cellular differences in alpha2beta1-integrin activation/ligand affinity state. These studies also suggest a heretofore unrecognized role for angiostatic basement membrane fragments in platelet biology.

Human mesenchymal stem cells (hMSCs) represent promising tools in various clinical applications, including the regeneration of injured tissues by endogenous or transplanted hMSCs. The molecular mechanisms, however, that control hMSC mobilization and homing which require invasion through extracellular matrix (ECM) barriers are almost unknown. We have analyzed bone marrow-derivedhMSCs and detected strong expression and synthesis of matrix metalloproteinase 2 (MMP-2), membrane type 1 MMP (MT1-MMP), tissue inhibitor of metalloproteinase 1 (TIMP-1), and TIMP-2. The ability of hMSCs to traverse reconstituted human basement membranes was effectively blocked in the presence of synthetic MMP inhibitors. Detailed studies by RNA interference revealed that gene knock-down of MMP-2, MT1-MMP, or TIMP-2 substantially impaired hMSC invasion, whereas silencing of TIMP-1 enhanced cell migration, indicating opposing roles of both TIMPs in this process. Moreover, the inflammatory cytokines TGF-beta1, IL-1beta, and TNF-alpha up-regulated MMP-2, MT1-MMP, and/or MMP-9 production in these cells, resulting in a strong stimulation of chemotactic migration through ECM, whereas the chemokine SDF-1alpha exhibited minor effects on MMP/TIMP expression and cell invasion. Thus, induction of specific MMP activity in hMSCs by inflammatory cytokines promotes directed cell migration across reconstituted basement membranes in vitro providing a potential mechanism in hMSC recruitment and extravasation into injured tissues in vivo.

Using high-performance liquid chromatography-tandem mass spectrometry, we assessed trough imatinib plasma levels in 68 patients with chronic myeloid leukemia (CML) who responded or not to standard-dose imatinib, after at least 12 months' treatment. Mean trough imatinib plasma levels were significantly higher in the group with complete cytogenetic response (56 patients) than in the group without (12 patients; P = .03) and higher in the group with major molecular response (MMR) than in the group without (34 patients [1452 +/- 649 ng/mL] versus 34 patients [869 +/- 427 ng/mL]; P < .001). Regarding trough imatinib plasma levels and their discrimination potential for MMR, the area under receiver operating characteristic curve was 0.775, with best sensitivity (77%) and specificity (71%) at a plasma threshold of 1002 ng/mL. Therefore, monitoring of imatinib plasma levels could be very useful for the management of patients with CML or should at least be checked in the case of treatment failure or suboptimal response.

To identify previously unknown platelet receptors we compared the transcriptomes of in vitro differentiated megakaryocytes (MKs) and erythroblasts (EBs). RNA was obtained from purified, biologically paired MK and EB cultures and compared using cDNA microarrays. Bioinformatical analysis of MK-up-regulated genes identified 151 transcripts encoding transmembrane domain-containing proteins. Although many of these were known platelet genes, a number of previously unidentified or poorly characterized transcripts were also detected. Many of these transcripts, including G6b, G6f, LRRC32, LAT2, and the G protein-coupled receptor SUCNR1, encode proteins with structural features or functions that suggest they may be involved in the modulation of platelet function. Immunoblotting on platelets confirmed the presence of the encoded proteins, and flow cytometric analysis confirmed the expression of G6b, G6f, and LRRC32 on the surface of platelets. Through comparative analysis of expression in platelets and other blood cells we demonstrated that G6b, G6f, and LRRC32 are restricted to the platelet lineage, whereas LAT2 and SUCNR1 were also detected in other blood cells. The identification of the succinate receptor SUCNR1 in platelets is of particular interest, because physiologically relevant concentrations of succinate were shown to potentiate the effect of low doses of a variety of platelet agonists.

An urgent need exists to devise strategies to augment antiviral immune responses in patients with HIV who are virologically well controlled and immunologically stable on highly active antiretroviral therapy (HAART). The objective of this study was to compare the immunomodulatory effects of the cytokines interleukin (IL)-21 with IL-15 on CD8 T cells in patients with HIV RNA of less than 50 copies/mL and CD4 counts greater than 200 cells/mm.(3) Patient CD8 T cells displayed skewed maturation and decreased perforin expression compared with healthy controls. Culture of freshly isolated patient peripheral-blood mononuclear cells (PBMCs) for 5 hours to 5 days with IL-21 resulted in up-regulation of perforin in CD8 T cells, including memory and effector subsets and virus-specific T cells. IL-21 did not induce T-cell activation or proliferation, nor did it augment T-cell receptor (TCR)-induced degranulation. Treatment of patient PBMCs with IL-15 resulted in induction of perforin in association with lymphocyte proliferation and augmentation of TCR-induced degranulation. Patient CD8 T cells were more responsive to cytokine effects than the cells of healthy volunteers. We conclude that CD8 T cells of patients with HIV can be modulated by IL-21 to increase perforin expression without undergoing overt cellular activation. IL-21 could potentially be useful for its perforin-enhancing properties in anti-HIV immunotherapy.

A role for genetic susceptibility in non-Hodgkin lymphoma (NHL) is supported by the accumulating evidence of common genetic variations altering NHL risk. However, the pattern of NHL heritability remains poorly understood. We conducted a pooled analysis of 10 211 NHL cases and 11 905 controls from the International Lymphoma Epidemiology Consortium (InterLymph) to evaluate NHL risk among those with hematopoietic malignancies in first-degree relatives. Odds ratios (ORs) and 95% confidence intervals (CIs) of NHL and its subtypes were estimated from unconditional logistic regression models with adjustment for confounders. NHL risk was elevated for individuals who reported first-degree relatives with NHL (OR = 1.5; 95% CI = 1.2-1.9), Hodgkin lymphoma (OR = 1.6; 95% CI = 1.1-2.3), and leukemia (OR = 1.4; 95% CI = 1.2-2.7). Risk was highest among individuals who reported a brother with NHL (OR = 2.8; 95% CI = 1.6-4.8) and was consistent for all NHL subtypes evaluated. If a first-degree relative had Hodgkin lymphoma, NHL risk was highest if the relative was a parent (OR = 1.7; 95% CI = 1.0-2.9). If a first-degree relative had leukemia, NHL risk was highest among women who reported a sister with leukemia (OR = 3.0; 95% CI = 1.6-5.6). The pattern of NHL heritability appeared to be uniform across NHL subtypes, but risk patterns differed by specific hematopoietic malignancies and the sex of the relative, revealing critical clues to disease etiology.

The syndrome of leukocyte adhesion deficiency (LAD) combined with a severe Glanzmann-type bleeding disorder has been recognized as a separate disease entity. The variability in clinical and cell biological terms has remained largely unclear. We present data on 9 cases from 7 unrelated families, with 3 patients being actively followed for more than 12 years. The disease entity, designated LAD-1/variant syndrome, presents early in life and consists of nonpussing infections from bacterial and fungal origin, as well as a severe bleeding tendency. This is compatible with 2 major blood cell types contributing to the clinical symptoms (ie, granulocytes and platelets). In granulocytes of the patients, we found adhesion and chemotaxis defects, as well as a defect in NADPH oxidase activity triggered by unopsonized zymosan. This last test can be used as a screening test for the syndrome. Many proteins and genes involved in adhesion and signaling, including small GTPases such as Rap1 and Rap2 as well as the major Rap activity-regulating molecules, were normally present. Moreover, Rap1 activation was intact in patients' blood cells. Defining the primary defect awaits genetic linkage analysis, which may be greatly helped by a more precise understanding and awareness of the disease combined with the early identification of affected patients.

Human hematopoietic stem cells (HSCs) exposed to cytokines in vitro rapidly divide and lose their characteristic functional properties presumably due to the alteration of a genetic program that determines the properties of an HSC. We have attempted to reverse the silencing of this HSC genetic program by the sequential treatment of human cord blood CD34(+) cells with the chromatin-modifying agents, 5-aza-2'-deoxycytidine (5azaD) and trichostatin A (TSA). We determined that all CD34(+)CD90(+) cells treated with 5azaD/TSA and cytokines after 9 days of incubation divide, but to a lesser degree than cells exposed to only cytokines. When CD34(+)CD90(+) cells that have undergone extensive number of cell divisions (5-10) in the presence of cytokines alone were transplanted into immunodeficient mice, donor cell chimerism was not detectable. By contrast, 5azaD/TSA-treated cells that have undergone similar numbers of cell divisions retained their marrow repopulating potential. The expression of several genes and their products previously implicated in HSC self-renewal were up-regulated in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. These data indicate that HSC treated with chromatin-modifying agents are capable of undergoing repeated cell divisions in vitro while retaining their marrow-repopulating potential.