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Exposure to ionizing radiation increases the risk of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN), but such risks are not known in well-differentiated thyroid cancer (WDTC) patients treated with radioactive iodine (RAI). A total of 148 215 WDTC patients were identified from Surveillance, Epidemiology and End Results registries between 1973 and 2014, of whom 54% underwent definitive thyroidectomy and 46% received adjuvant RAI. With a median follow-up of 6.6 years, 77 and 66 WDTC patients developed MDS and MPN, respectively. Excess absolute risks for MDS and MPN from RAI treatment when compared to background rates in the US population were 6.6 and 8.1 cases per 100 000 person-years, respectively. Compared to background population rates, relative risks of developing MDS (3.85 (95% confidence interval, 1.7–7.6); P=0.0005) and MPN (3.13 (1.1–6.8); P=0.012) were significantly elevated in the second and third year following adjuvant RAI therapy, but not after thyroidectomy alone. The increased risk was significantly associated with WDTC size ⩾2 cm or regional disease. Development of MDS was associated with shorter median overall survival in WDTC survivors (10.3 vs 22.5 years; P<0.001). These data suggest that RAI treatment for WDTC is associated with increased risk of MDS with short latency and poor survival.

The biology, clinical phenotype and progression rate of chronic myelomonocytic leukemia (CMML) are highly variable due to diverse initiating and secondary clonal genetic events. To determine the effects of molecular features including clonal hierarchy in CMML, we studied whole-exome and targeted next-generation sequencing data from 150 patients with robust clinical and molecular annotation assessed cross-sectionally and at serial time points of disease evolution. To identify molecular lesions unique to CMML, we compared it to the related myeloid neoplasms (N=586), including juvenile myelomonocytic leukemia, myelodysplastic syndromes (MDS) and primary monocytic acute myeloid leukemia and discerned distinct molecular profiles despite similar pathomorphological features. Within CMML, mutations in certain pathways correlated with clinical classification, for example, proliferative vs dysplastic features. While most CMML patients (59%) had ancestral (dominant/co-dominant) mutations involving TET2, SRSF2or ASXL1genes, secondary subclonal hierarchy correlated with clinical phenotypes or outcomes. For example, progression was associated with acquisition of new expanding clones carrying biallelic TET2mutations or RASfamily, or spliceosomal gene mutations. In contrast, dysplastic features correlated with mutations usually encountered in MDS (for example, SF3B1and U2AF1). Classification of CMML based on hierarchies of ancestral and subclonal mutational events may correlate strongly with clinical features and prognosis.

The Revised International Prognostic Scoring System (IPSS-R) was developed for untreated myelodysplastic syndrome (MDS) patients based on clinical data. We created and validated a new model that incorporates mutational data to improve the predictive capacity of the IPSS-R in treated MDS patients. Clinical and mutational data from treated MDS patients diagnosed between January 2000 and January 2012 were used to develop the new prognostic system. A total of 508 patients were divided into training (n=333) and validation (n=175) cohorts. Independent significant prognostic factors for survival included age, IPSS-R, EZH2, SF3B1and TP53. Weighted coefficients for each factor were used to build the new linear predictive model, which produced four prognostic groups: low, intermediate-1, intermediate-2 and high with a median overall survival of 37.4, 23.2, 19.9 and 12.2 months, respectively, P<0.001. Significant improvement in the C-index of the new model (0.73) was observed compared with the IPSS-R (0.69). The new model predicted outcome both in a separate validation cohort and in another cohort of patients with paired samples at different time points during their disease course. The addition of mutational data to the IPSS-R makes it dynamic and enhances its predictive ability in treated MDS patients regardless of their initial or subsequent therapies.

Genetic and epigenetic aberrations contribute to the initiation and progression of acute myeloid leukemia (AML). GFI1, a zinc-finger transcriptional repressor, exerts its function by recruiting histone deacetylases to target genes. We present data that low expression of GFI1is associated with an inferior prognosis of AML patients. To elucidate the mechanism behind this, we generated a humanized mouse strain with reduced GFI1expression (GFI1-KD). Here we show that AML development induced by onco-fusion proteins such as MLL-AF9 or NUP98-HOXD13 is accelerated in mice with low human GFI1expression. Leukemic cells from animals that express low levels of GFI1show increased H3K9 acetylation compared to leukemic cells from mice with normal human GFI1expression, resulting in the upregulation of genes involved in leukemogenesis. We investigated a new epigenetic therapy approach for this subgroup of AML patients. We could show that AML blasts from GFI1-KDmice and from AML patients with low GFI1levels were more sensitive to treatment with histone acetyltransferase inhibitors than cells with normal GFI1expression levels. We suggest therefore that GFI1 has a dose-dependent role in AML progression and development. GFI1 levels are involved in epigenetic regulation, which could open new therapeutic approaches for AML patients.

Established prognostic tools in patients with myelodysplastic syndromes (MDS) were largely derived from untreated patient cohorts. Although azanucleosides are standard therapies for higher-risk (HR)-MDS, the relative prognostic performance of existing prognostic tools among patients with HR-MDS receiving azanucleoside therapy is unknown. In the MDS Clinical Research Consortium database, we compared the prognostic utility of the International Prognostic Scoring System (IPSS), revised IPSS (IPSS-R), MD Anderson Prognostic Scoring System (MDAPSS), World Health Organization-based Prognostic Scoring System (WPSS) and the French Prognostic Scoring System (FPSS) among 632 patients who presented with HR-MDS and were treated with azanucleosides as the first-line therapy. Median follow-up from diagnosis was 15.7 months. No prognostic tool predicted the probability of achieving an objective response. Nonetheless, all five tools were associated with overall survival (OS, P=0.025 for the IPSS, P=0.011 for WPSS and P<0.001 for the other three tools). The corrected Akaike Information Criteria, which were used to compare OS with the different prognostic scoring systems as covariates (lower is better) were 4138 (MDAPSS), 4156 (FPSS), 4196 (IPSS-R), 4186 (WPSS) and 4196 (IPSS). Patients in the highest-risk groups of the prognostic tools had a median OS from diagnosis of 11-16 months and should be considered for up-front transplantation or experimental approaches.

Risks of acute myeloid leukemia (AML) and/or myelodysplastic syndromes (MDS) are known to increase after cancer treatments. Their rise-and-fall dynamics and their associations with radiation have, however, not been fully characterized. To improve risk definition we developed SEERaBomb R software for Surveillance, Epidemiology and End Results second cancer analyses. Resulting high-resolution relative risk (RR) time courses were compared, where possible, to results of A-bomb survivor analyses. We found: (1) persons with prostate cancer receiving radiation therapy have increased RR of AML and MDS that peak in 1.5–2.5 years; (2) persons with non-Hodgkin lymphoma (NHL), lung and breast first cancers have the highest RR for AML and MDS over the next 1–12 years. These increased RR are radiation specific for lung and breast cancer but not for NHL; (3) AML latencies were brief compared to those of A-bomb survivors; and (4) there was a marked excess risk of acute promyelocytic leukemia in persons receiving radiation therapy. Knowing the type of first cancer, if it was treated with radiation, the interval from first cancer diagnosis to developing AML or MDS, and the type of AML, can improve estimates of whether AML or MDS cases developing in this setting are due to background versus other processes.

Abnormal activation of SETBP1through overexpression or missense mutations is highly recurrent in various myeloid malignancies; however, it is unclear whether such activation alone is able to induce leukemia development. Here we show that Setbp1overexpression in mouse bone marrow progenitors through retroviral transduction is capable of initiating leukemia development in irradiated recipient mice. Before leukemic transformation, Setbp1overexpression significantly enhances the self-renewal of hematopoietic stem cells (HSCs) and expands granulocyte macrophage progenitors (GMPs). Interestingly, Setbp1overexpression also causes transcriptional repression of critical hematopoiesis regulator gene Runx1and this effect is crucial for Setbp1-induced transformation. Runx1repression is induced by Setbp1-mediated recruitment of a nucleosome remodeling deacetylase (NuRD) complex to Runx1promoters and can be reversed by treatment with histone deacetylase (HDAC) inhibitors Entinostat and Vorinostat. Moreover, treatment with these inhibitors caused efficient differentiation of Setbp1activation-induced leukemia cells in vitro, and significantly extended the survival of mice transplanted with such leukemias, suggesting that HDAC inhibition could be an effective strategy for treating myeloid malignancies with SETBP1activation.

Cytomegalovirus (CMV) infections have a major impact on morbidity and mortality of transplant patients. Among the complex antiviral T‐cell response, CMV‐IE‐1 antigen‐specific CD8+ cells are crucial for preventing CMV disease but do not protect from recurring/lasting CMV reactivation. Recently, we confirmed that adoptive transfer of autologous IE‐1/pp65‐specific T‐cell lines was able to combat severe CMV disease; however, the control of CMV infection was only temporary. We hypothesized that CMV‐induced regulatory T cells (iTreg) might be related to recurring/lasting CMV infection.

Aim: To explore whether predisposition to bone marrow failure syndromes (BMF), such aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and myelosysplastic syndromes (MDS), is found in killer cell immunoglobulin-like receptor (KIR) and human leukocyte antigen (HLA) ligand (KIR-L) gene variations or cytokine polymorphisms. Patients: We studied a cohort of 77 patients with AA, 129 with MDS and 285 healthy controls for the frequencies of KIRL and KIR genotypes and 22 selected single nucleotide polymorphisms (SNPs) located within 10 cytokine (IL-1, IL-1, IL-2, IL-4, IL-6, IL-10, IL12, IFN-, TNF-, TGF-) and 3 cytokine receptor (IL-1R, IL-1RA, IL-4R) genes. Results: In AA we found a decreased frequency of inhibitory KIR-2DL3 genes. In MDS, no difference in the frequency of KIR genotype was identified; however, a decreased frequency of 2DL3 was found in hypocellular MDS. Analysis of the KIR genotype in correlation with the corresponding KIR-L profile, revealed a decreased frequency of stimulatory 2DS1/C2 mismatch both in AA and MDS. In AA and MDS cohorts, compared to controls, we found a higher frequency of TT codon 10 variant and of GG codon 25 variant of TGF- gene, consistent with a high secretory phenotype. This relationship was even more pronounced in PNH and hypocellular MDS. We confirm that the hypersecretory genotype T/T at position -874 of INF- gene was overrepresented only in AA and correlates with presence of a PNH clone. Instead in MDS patients, the frequency of G/A polymorphism at position -308 on the TNF- gene promoter, which correlates with higher TNF- production, was found significantly higher. Moreover, hypocellular MDS was characterized by a higher prevalence of IL-10 GCC/GCC haplotype, which is functionally associated with a low secretor phenotype. Conclusion: Our findings suggest that alterations in KIR/KIR-L matching, such as increased 3DL2 and decreased 2DS1 mismatch, and in the polymorphisms of TGF1, IFN-, TNF- and IL-10 may account for the propensity to immunemediated killing of hematopoietic stem cells and/or ineffective hematopoiesis characteristic of AA and MDS. Further studies are needed to elucidate whether these immunogenetic traits may be involved in increased risk of developing immune-mediated BMF.

This paper documents an instance of liquid metal induced embrittlement (LMIE) that occurred during the manufacture of automobile engine fuel lines. The fuel lines were constructed of low-carbon steel fittings press fit onto 304 stainless steel tube. The joints were subsequently furnace brazed with copper braze paste. The carbon steel fittings were occasionally observed lying separately as the part exited the brazing furnace. Failure analysis using metallographic techniques, scanning electron microscopy, and energy-dispersive spectroscopy indicated that the fractures were intergranular with no observable ductility. Copper from the braze filler metal was observed on the stainless steel tube fracture surfaces and within the tube wall grain boundaries. Copper and austenitic stainless steel are a well-known LMIE couple. Subsequent investigation indicated the stress required for rapid fracture was due to impacts of the parts against one another and the brazing furnace stationary components while the braze metal was liquid.

Abstract: Transmission electron microscopy (TEM) studies have been carried out on magnetron-sputtered coatings in the Al-rich region of the Al-Cu-Fe-Cr quasicrystalline phase field, focusing on the composition that exhibited the best tribological performance among a group of sputtered coatings. The size of the particles in the as-deposited coating is around 10 nm. The TEM experiments on annealed coatings (450 C for 6 hours+400 C for 7 hours) revealed that the material is composed of a decagonal quasicrystalline phase with a periodicity of 1.24 nm along the unique axis and a hexagonal crystalline phase, with a=2.48 nm and c=1.24 nm. These results show that a thermodynamically stable quasicrystalline phase exists in the Al-Cu-Fe-Cr alloy system, in addition to previously reported microcrystalline structures and orthorhombic and monoclinic approximants. The hexagonal phase is a crystalline approximant of the decagonal phase, structurally related to hexagonal crystalline phases previously reported in Al-Mn and Al-Cr-Ni quasicrystalline alloys.

Activation of Fas antigen, a cell surface receptor molecule, by its ligand results in transduction of a signal for cell death. The Fas system has been implicated in target cell recognition, clonal development of immune effector cells, and termination of the cellular immune response. Fas antigen expression on lymphocytes is regulated by interferon gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha), cytokines that also have inhibitory effects on hematopoiesis. We investigated Fas antigen expression on human marrow cells and the effects of Fas activation on hematopoiesis in vitro. Freshly isolated immature hematopoietic cells, as defined by the CD34 marker, did not express Fas antigen at levels detectable by fluorescent staining. CD34+ cells, which include progenitors and stem cells, showed low levels of Fas expression in culture, even in the presence of growth factors. Stimulation by TNF alpha and IFN gamma markedly increased Fas antigen expression on CD34+ cells. Anti-Fas antibody, which mimics the action of the putative ligand, enhanced IFN gamma- and TNF alpha-mediated suppression of colony formation by bone marrow (BM) in a dose-dependent manner. This effect did not require the presence of accessory cells. Colony formation from mature (CD34+ CD38+) and immature (CD34+ CD38-) progenitor cells and long-term culture initiating cells were susceptible to the inhibitory action of anti-Fas antibody in the presence of IFN gamma and TNF alpha. Apoptosis assays performed on total BM cells and CD34+ cells showed that anti-Fas antibody induced programmed cell death of CD34+ BM cells.(ABSTRACT TRUNCATED AT 250 WORDS)

THIS study was undertaken to evaluate the monocyte function of uraemic non-responders to hepatitis B vaccination. Therefore, some parameters concerning antigen processing by monocytes (Mo) as antigen presenting cells (APC) were analysed. It was found that in uraemic non-responders, (1) the internalization of HBsAg by monocytes was significantly decreasjed—HBsAg complexed with specific IgG or as immune complex isolated from patients is better internalized compared with free HBsAg; (2) during antigen presentation the expression of adhesion (ICAM-1) and accessory (HLA-DR/Ia) molecules was significantly decreased in uraemic patients, especially in non-responders; and (3) impaired internalization of HBsAg as well as a decrease in ICAM-1 and HLA-DR/Ia expression, correlated well with the blunted proliferation of CD4+ T cells stimulated by autologous monocytes induced by HBsAg.

Summary Our experiments were directed towards the detection of the influence of interleukin-1 (IL-1); interleukin-3 (IL-3), and granulocyte-macrophage colonystimulating factor (GM-CSF) on the generation of granulocyte-macrophage progenitor cells. We also set out to examine whether this process is connected with changes within the early precursor cell compartment. Bone marrow suspension cultures (12 days) supplemented with these cytokines were tested for the presence of GM colony-forming cells (GM-CFC) in a colony-forming unit assay. The percentage of CD 34+ and HLA-DR+ as well as the number of blasts and promyelocytes were estimated cytofluorometrically and morphologically. The proliferative effect of GM-CSF was associated with a net increase of GM-CFC and HLA-DR+ myeloid cells and a decrease in the percentage of CD 34+ early precursor cells. IL-3 acted similarly and also caused an absolute decrease of CD 34+ cells in the cultures. IL-1 did not stimulate the generation of blasts or GM-CFC but elevated the number of CD 34- as well as HLA-DR-expressing cells in the cultures. These results imply that GM-CSF supported the maintenance of hematopoiesis in vitro. The transition from early precursor cells to committed myeloid progenitor cells (GM-CFC) and more mature precursor cells (G-CFC, M-CFC) may be supported by GM-CSF without affecting the self-renewing capacity of CD 34+ early precursors. In contrast, the blast-generating and proliferation-inducing action of IL-3 is associated with a drop in the total number of CD 34+ stem cells. An efficient renewal of this population obviously depends on the presence of IL-1.

Correction to: Leukemia (2017) 31, 491–495; doi:10.1038/leu.2016.276; published online 11 November 2016 Following the publication of this article, the authors noted that Dr Grimes’ name was incorrect. The complete and correct author names appear above.

Experience with unrelated allogeneic UCB stem cell transplantation in the non-myeloablative setting is limited because of concerns of graft failure due to limited cell content. Dual UCB graft infusion has been explored to overcome cell dose limitations, but influence on engraftment is unclear. This single institution phase I trial examined RIC followed by single UCB transplantation for patients unable to tolerate fully myeloablative regimens and lacking a matched adult donor. Conditioning consisted of TBI 200 cGy, fludarabine 175 mg/m², cyclophosphamide 2 gm/m², ATG 60 mg/kg. Trial design specified selection of UCB grafts to be matched at 4/6 HLA loci or better with one unit infused if cryopreserved nucleated cell dose exceeded 2.5 x 107/kg recipient weight. If no adequate single UCB unit to meet these criteria was available, study patients received 2 UCB units containing a combined cell dose exceeding 1.5 x 107/kg. Grafts were analyzed via flow cytometry post thaw for stem cell and lymphocyte expression of surface antigens, among them CD4, CD8, CD7, CD34, CD38, CD45RA, CD45RO. 23 patients have been enrolled, the majority diagnosed with AML (n=17). Median age was 47 (range 25–68) years. Single UCB units meeting stated criteria were identified for all but one study patient, who was excluded from graft analysis. The time to engraftment was measured from the date of transplantation to the date of 3 successive days ANC≥500/μL attained. In addition, we assessed time to peripheral blood donor lymphocyte chimerism ≥ 60%. Patients were censored at the date of last follow-up or the date of death. 94% (95%CI: 77-.99%) of patients achieved ANC≥500/μL by T+36 with median of 26.5 ( range 11–55) days. 68% (95%CI: 48–85%) of patients (n=15) achieved >60% donor derived chimerism by median of 22 (range 14–35) days. Median cryopreserved and infused total nucleated cell dose was 2.85 x 107 and 2.5 x 107 cells/kg, respectively. Median infused CD34 cell dose was 1.71 (range 0.21–5.39) x 105 cells/kg. By univariate analysis the dose of infused UCB graft T-cells including CD4 and CD8 T-cells co-expressing CD45RO, and CD34 stem cells co-expressing CD7 and CD38 correlated with neutrophil recovery, p=0.046, 0.008, and 0.033, respectively. Median overall survival at this early interim analysis has not been reached; with 86% and 65% survival at 3 and 24 months, respectively. Event free survival is 78% and 44.5% at 3 and 24 months, respectively. In summary, in this heavily pretreated group of advanced age patients, RIC and single unit unrelated allogeneic UCB stem cell transplantation is safe. Identification of a single UCB graft of HLA match 4/6 meeting cell dose requirement 2.5x107/kg is identified in the vast majority of full size adult patients. Engraftment and survival rates are higher than reported in single UCB transplants following administration of myeloablative regimens and similar to recent reports for RIC with double UCB graft infusion. The potential benefit of infusion of 1 vs. 2 UCB units after RIC in adult patients remains to be fully evaluated.

Ineffective erythropoiesis in MDS is associated with impaired EPO-Receptor activation of STAT5 [Hoefsloot et al. Blood1997; 89:1690]. However, identification of regulatory molecules that impede the EPO-R signal remains elusive. CD45 is a transmembrane PTP expressed in hematopoietic cells that co-localizes with the EPO-R and attenuates signaling by dephosphorylating tyrosines on Jak-2 and Lyn to abolish kinase activity. Expression within the erythroid lineage is limited to CD71Hi erythroblasts and is down-regulated with differentiation. CD45 is comprised of several isoforms generated by alternate splicing. In lymphoid cells, dimerization of the smallest isoform, CD45RO, which predominates in normal erythroids, suppresses PTP activity, whereas larger isoforms such as CD45RA or -RB, favor monomeric distribution and primed PTP activity. We recently found that lenalidomide augments EPO-R:STAT5 activation in non-del5q MDS CD71Hi cells by inhibiting the CD45 PTP, implying inherent CD45 PTP over-activity. To investigate CD45 regulation of the EPO-R:STAT5 signal, we characterized CD45 isoform distribution in CD71Hi erythroids from normal [N=7] and MDS marrow specimens [N=9], and evaluated the relationship to EPO-R:STAT-5 activation and PTP activity. Flow quantitation of EPO-induced STAT-5 phosphorylation (PHN) confirmed that PHN capacity is significantly impaired in lower risk, non-del5q MDS compared to normal erythroid precursors (median [SD] log10 geometric pSTAT5 MFI EPO:isotype ratio, 0.435 [0.21] MDS vs. 1.284 [0.06] Normal; P<0.0001). Comparison of CD45 isoform distribution in showed that CD45-RO predominates in normal CD71Hi cells (mean [SD] RA:RO ratio, 0.33[0.23); RB:RO, 0.04[0.03)), whereas CD45-RA and -RB are preferentially expressed in MDS (RA:RO 1.9[0.54]; RB:RO 0.28[0.12]; P<0.0001). Regression analysis of the relationship between CD45 isoform distribution and STAT-5 PHN after EPO stimulation showed that cellular RA and RB fraction was highly correlated (r=0.927, P<0.001) and PHN capacity inversely correlated with CD45-RA or -RB proportion (P<0.0001). In contrast, the magnitude of lenalidomide enhancement of EPO:STAT5 PHN directly correlated with CD45RA and -RB isoform fraction (P=0.0008), and was significantly higher in MDS compared to normal erythroids (median log10 geometric MFI [SD] pSTAT5 EPO+CC-5013:EPO, 0.989 [0.25] MDS vs. 0.0195 [0.07] Normal; P=0.0005). Genomic DNA genotyping for CD45 polymorphisms by allele-specific PCR & sequencing showed no skewing in SNP distribution (exon 6, A138G; exon 4, C77G & C59A) among MDS patients [N=101] compared to normal controls [N=44]. These data provide the first evidence that CD45 PTP over-activity impairs the EPO-R signal in MDS. Our investigations show that the magnitude of EPO-R:pSTAT5 signal reduction directly correlates with the proportion of larger CD45 isoforms in erythroid precursors and is restored by CD45 PTP inhibition. These findings suggest that erythroid CD45 isoform profile may identify patients potentially responsive to rhu-EPO or selective PTP inhibitors.

Early clinical studies have demonstrated benefit in patients receiving cellular therapy utilizing bone marrow derived (BM)-CD133+ hematopoietic stem cells (HSC) for treatment of cardiovascular disease. Umbilical cord blood (UCB) is a potential source of CD133+ cells for use as an allogeneic cell source for therapeutic angiogenesis, however, the benefits must be weighed against potential adverse immunologic responses. We tested the hypothesis that allogeneic CD133+ cells derived from UCB may be defective as professional antigen-presenting-cells (APC) and thus may mediate TH2 T-cell immune response. CD133+ cells were isolated from UCB mononuclear cells (MNC) by magnetic autoMACs bead selection (Miltenyi Biotech, Auburn CA) and analyzed for purity and surface expression of MHC and co-stimulatory antigens by flow cytometry. We have demonstrated induced immune reactivity by mixed lymphocyte reactions (MLR) using healthy adult peripheral blood MNC as responders stimulated by irradiated CD133+ from UCB and BM (ratio 3:1) with both 3H-thymidine and CFSE staining. Surface expression of both MHC class I (57.6±17.2%) and MHC class II (66.5±12.5%) are present on the majority of UCB CD133+ cells. To test the ability of CD133+ cells to function as APC, a modified MLR was performed. Briefly, isolated UCB CD133+ cells were cultured for 96h in the presence of adult peripheral blood (AB) derived-MNC or isolated CD3 AB T-cells as responders at a 3:1 ratio (responder:effector) in RPMI 1640 supplemented with 10% FBS and 1% L-glutamine. CD133+ cells induced proliferation in both non-selected AB MNC (21.7±6.4e3cpm) and selected AB T-cell (85.6±15.1e3 cpm) cultures as measured by 3H-thymidine incorporation. UCB CD133+ cells were noted to lack surface expression of co-stimulatory antigens including: CD40 (0.93±1.0%), CD80 (0.85±0.15%), and CD86 (0.88±0.64%). Because primary T-cell receptor stimulation in the absence of a co-stimulatory response is known to induce TH2 responses we compared UCB MNC and CD133+ derived from the same unit as stimulators for responding HLA mismatched allogeneic AB MNC. Preliminary results demonstrate elevated levels of IL-4 and IL-10 produced by responder AB MNC stimulated by CD133+ cells as compared to UCB MNC, with measured increases of 964±538pg/mL (IL-10) and 141±45.7pg/mL (IL-4). In conclusion, though CD133+ cells derived from UCB express both MHC class I and II they lack surface expression of co-stimulatory receptors, and are defective as professional APC. Additionally, UCB CD133+ cells induce elevated levels of IL-4 and IL-10 by responding allogeneic adult MNC in modified mixed lymphocyte in vitro cultures. MNC stimulation by selected UCB CD133+ cells elicit elevated levels of tolerance-associated cytokines IL-4 and IL-10 as compared to UCB MNC used as stimulator cells. Taken together, UCB CD133 lack co-stimulatory receptor expression and elicit TH2 T-lymphocyte responses, and may allow for immune tolerance responses when used as a source of allogeneic stem cells for therapeutic use in vasculogenesis.

Early clinical studies have demonstrated benefit in patients receiving cellular therapy utilizing bone marrow derived (BM)-CD133+hematopoietic stem cells (HSC) for treatment of cardiovascular disease. Umbilical cord blood (UCB) is a potential source of CD133+cells for use as an allogeneic cell source for therapeutic angiogenesis, however, the benefits must be weighed against potential adverse immunologic responses. We tested the hypothesis that allogeneic CD133+cells derived from UCB may be defective as professional antigen-presenting-cells (APC) and thus may mediate TH2T-cell immune response. CD133+cells were isolated from UCB mononuclear cells (MNC) by magnetic autoMACs bead selection (Miltenyi Biotech, Auburn CA) and analyzed for purity and surface expression of MHC and co-stimulatory antigens by flow cytometry. We have demonstrated induced immune reactivity by mixed lymphocyte reactions (MLR) using healthy adult peripheral blood MNC as responders stimulated by irradiated CD133+from UCB and BM (ratio 3:1) with both 3H-thymidine and CFSE staining. Surface expression of both MHC class I (57.6±17.2%) and MHC class II (66.5±12.5%) are present on the majority of UCB CD133+cells. To test the ability of CD133+cells to function as APC, a modified MLR was performed. Briefly, isolated UCB CD133+cells were cultured for 96h in the presence of adult peripheral blood (AB) derived-MNC or isolated CD3 AB T-cells as responders at a 3:1 ratio (responder:effector) in RPMI 1640 supplemented with 10% FBS and 1% L-glutamine. CD133+cells induced proliferation in both non-selected AB MNC (21.7±6.4e3cpm) and selected AB T-cell (85.6±15.1e3 cpm) cultures as measured by 3H-thymidine incorporation. UCB CD133+cells were noted to lack surface expression of co-stimulatory antigens including: CD40 (0.93±1.0%), CD80 (0.85±0.15%), and CD86 (0.88±0.64%). Because primary T-cell receptor stimulation in the absence of a co-stimulatory response is known to induce TH2responses we compared UCB MNC and CD133+derived from the same unit as stimulators for responding HLA mismatched allogeneic AB MNC. Preliminary results demonstrate elevated levels of IL-4 and IL-10 produced by responder AB MNC stimulated by CD133+cells as compared to UCB MNC, with measured increases of 964±538pg/mL (IL-10) and 141±45.7pg/mL (IL-4). In conclusion, though CD133+cells derived from UCB express both MHC class I and II they lack surface expression of co-stimulatory receptors, and are defective as professional APC. Additionally, UCB CD133+cells induce elevated levels of IL-4 and IL-10 by responding allogeneic adult MNC in modified mixed lymphocyte in vitrocultures. MNC stimulation by selected UCB CD133+cells elicit elevated levels of tolerance-associated cytokines IL-4 and IL-10 as compared to UCB MNC used as stimulator cells. Taken together, UCB CD133 lack co-stimulatory receptor expression and elicit TH2T-lymphocyte responses, and may allow for immune tolerance responses when used as a source of allogeneic stem cells for therapeutic use in vasculogenesis.

Ineffective erythropoiesis in MDS is associated with impaired EPO-Receptor activation of STAT5 [Hoefsloot et al. Blood 1997; 89:1690]. However, identification of regulatory molecules that impede the EPO-R signal remains elusive. CD45 is a transmembrane PTP expressed in hematopoietic cells that co-localizes with the EPO-R and attenuates signaling by dephosphorylating tyrosines on Jak-2 and Lyn to abolish kinase activity. Expression within the erythroid lineage is limited to CD71Hierythroblasts and is down-regulated with differentiation. CD45 is comprised of several isoforms generated by alternate splicing. In lymphoid cells, dimerization of the smallest isoform, CD45RO, which predominates in normal erythroids, suppresses PTP activity, whereas larger isoforms such as CD45RA or -RB, favor monomeric distribution and primed PTP activity. We recently found that lenalidomide augments EPO-R:STAT5 activation in non-del5q MDS CD71Hicells by inhibiting the CD45 PTP, implying inherent CD45 PTP over-activity. To investigate CD45 regulation of the EPO-R:STAT5 signal, we characterized CD45 isoform distribution in CD71Hierythroids from normal [N=7] and MDS marrow specimens [N=9], and evaluated the relationship to EPO-R:STAT-5 activation and PTP activity. Flow quantitation of EPO-induced STAT-5 phosphorylation (PHN) confirmed that PHN capacity is significantly impaired in lower risk, non-del5q MDS compared to normal erythroid precursors (median [SD] log10 geometric pSTAT5 MFI EPO:isotype ratio, 0.435 [0.21] MDS vs. 1.284 [0.06] Normal; P<0.0001). Comparison of CD45 isoform distribution in showed that CD45-RO predominates in normal CD71Hicells (mean [SD] RA:RO ratio, 0.33[0.23); RB:RO, 0.04[0.03)), whereas CD45-RA and -RB are preferentially expressed in MDS (RA:RO 1.9[0.54]; RB:RO 0.28[0.12]; P<0.0001). Regression analysis of the relationship between CD45 isoform distribution and STAT-5 PHN after EPO stimulation showed that cellular RA and RB fraction was highly correlated (r=0.927, P<0.001) and PHN capacity inversely correlated with CD45-RA or -RB proportion (P<0.0001). In contrast, the magnitude of lenalidomide enhancement of EPO:STAT5 PHN directly correlated with CD45RA and -RB isoform fraction (P=0.0008), and was significantly higher in MDS compared to normal erythroids (median log10 geometric MFI [SD] pSTAT5 EPO+CC-5013:EPO, 0.989 [0.25] MDS vs. 0.0195 [0.07] Normal; P=0.0005). Genomic DNA genotyping for CD45 polymorphisms by allele-specific PCR & sequencing showed no skewing in SNP distribution (exon 6, A138G; exon 4, C77G & C59A) among MDS patients [N=101] compared to normal controls [N=44]. These data provide the first evidence that CD45 PTP over-activity impairs the EPO-R signal in MDS. Our investigations show that the magnitude of EPO-R:pSTAT5 signal reduction directly correlates with the proportion of larger CD45 isoforms in erythroid precursors and is restored by CD45 PTP inhibition. These findings suggest that erythroid CD45 isoform profile may identify patients potentially responsive to rhu-EPO or selective PTP inhibitors.

Experience with unrelated allogeneic UCB stem cell transplantation in the non-myeloablative setting is limited because of concerns of graft failure due to limited cell content. Dual UCB graft infusion has been explored to overcome cell dose limitations, but influence on engraftment is unclear. This single institution phase I trial examined RIC followed by single UCB transplantation for patients unable to tolerate fully myeloablative regimens and lacking a matched adult donor. Conditioning consisted of TBI 200 cGy, fludarabine 175 mg/m², cyclophosphamide 2 gm/m², ATG 60 mg/kg. Trial design specified selection of UCB grafts to be matched at 4/6 HLA loci or better with one unit infused if cryopreserved nucleated cell dose exceeded 2.5 x 107/kg recipient weight. If no adequate single UCB unit to meet these criteria was available, study patients received 2 UCB units containing a combined cell dose exceeding 1.5 x 107/kg. Grafts were analyzed via flow cytometry post thaw for stem cell and lymphocyte expression of surface antigens, among them CD4, CD8, CD7, CD34, CD38, CD45RA, CD45RO. 23 patients have been enrolled, the majority diagnosed with AML (n=17). Median age was 47 (range 25–68) years. Single UCB units meeting stated criteria were identified for all but one study patient, who was excluded from graft analysis. The time to engraftment was measured from the date of transplantation to the date of 3 successive days ANC≥500/μL attained. In addition, we assessed time to peripheral blood donor lymphocyte chimerism ≥ 60%. Patients were censored at the date of last follow-up or the date of death. 94% (95%CI: 77-.99%) of patients achieved ANC≥500/μL by T+36 with median of 26.5 ( range 11–55) days. 68% (95%CI: 48–85%) of patients (n=15) achieved >60% donor derived chimerism by median of 22 (range 14–35) days. Median cryopreserved and infused total nucleated cell dose was 2.85 x 107and 2.5 x 107cells/kg, respectively. Median infused CD34 cell dose was 1.71 (range 0.21–5.39) x 105cells/kg. By univariate analysis the dose of infused UCB graft T-cells including CD4 and CD8 T-cells co-expressing CD45RO, and CD34 stem cells co-expressing CD7 and CD38 correlated with neutrophil recovery, p=0.046, 0.008, and 0.033, respectively. Median overall survival at this early interim analysis has not been reached; with 86% and 65% survival at 3 and 24 months, respectively. Event free survival is 78% and 44.5% at 3 and 24 months, respectively. In summary, in this heavily pretreated group of advanced age patients, RIC and single unit unrelated allogeneic UCB stem cell transplantation is safe. Identification of a single UCB graft of HLA match 4/6 meeting cell dose requirement 2.5x107/kg is identified in the vast majority of full size adult patients. Engraftment and survival rates are higher than reported in single UCB transplants following administration of myeloablative regimens and similar to recent reports for RIC with double UCB graft infusion. The potential benefit of infusion of 1 vs. 2 UCB units after RIC in adult patients remains to be fully evaluated.

The myelodysplastic syndromes (MDS) represent a heterogeneous group of disorders with different underlying disease biologies and prognoses. Low-risk MDS, defined as patients with refractory anemia (RA) or refractory anemia with ringed sideroblasts (RARS), with International Prognostic Scoring System (IPSS) scores of 0–1.0, are associated with relatively good prognosis. No study has evaluated outcomes in this population comparing treatment with growth factors (GF) to chemotherapy (Chemo). METHODS: A MEDLINE search on the keywords MDS, RA, RARS, treatment, growth factors and chemotherapy was performed. Only original articles with pathologically confirmed RA or RARS that included the patient characteristics and effect of therapy in individual cases were included. Treatments were categorized as either GF (erythropoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor) or Chemo (differentiating agents, immunomudulators, cytotoxic agents). IPSS was calculated if not reported, when possible. Outcomes were categorized as response, disease progression, or death. Responses were standardized and recalibrated according to the International Working Group (IWG) criteria, as complete response, partial response, or hematologic improvement. T-tests were performed for continuous variables, chi-square for categorical, and a Cox proportional hazards model for survival controlling for known risk factors. RESULTS: Of 653 potential articles from 1985 to 2005, 137 papers representing 2,301 patients with low-risk MDS were reviewed. Individual patient data was available in 799 cases. Baseline characteristics were (mean, standard deviation): age 67 ± 12, hemoglobin 8.4 ± 8.57, ANC 1.55 ± 4.78, platelet count 116 ± 144, erythropoietin level 236 ± 968. Ninety-one percent of patients were transfusion-dependent, requiring a median of 3 pRBC transfusions per month (range 0–6). Patients had IPSS scores from 0–1.0 in 90% and were diagnosed a median of 12 months prior to therapy initiation. Neither of those two variables differed between the GF and Chemo groups. Four hundred and seventeen patients were received Chemo, amongst whom 39 deaths were recorded, and 382 patients received GF (5 died). Of those treated with GF, 45% responded, compared to 41.5% for those treated with chemo (p=0.31). The average response time for Chemo and GF was 10.8 months (n=141) vs 8.0 months (n=112, p<0.01). There was no difference in unadjusted survival time between those treated with Chemo and GF patients (n=799, hazard ratio=2.07 favoring GF, p=0.14) as shown below: Figure Figure After controlling for age and baseline transfusion needs, the Cox proportional hazards model found no difference for the survival between the two groups (n=607, hazard ratio=2.25, p=0.14). CONCLUSIONS: For patients with low-risk MDS, response rates to GF and Chemo are similar. Despite a significantly prolonged response time to Chemo compared to GF, there was no difference in survival, even when controlling for differences in age and transfusion needs. Decision tools need to be developed to determine which therapy to choose in patients with low-risk MDS.

This study was undertaken to evaluate the monocyte function of uraemic non-responders to hepatitis B vaccination. Therefore, some parameters concerning antigen processing by monocytes (Mo) as antigen presenting cells (APC) were analysed. It was found that in uraemic non-responders, (1) the internalization of HBsAg by monocytes was significantly decreasjed—HBsAg complexed with specific IgG or as immune complex isolated from patients is better internalized compared with free HBsAg; (2) during antigen presentation the expression of adhesion (ICAM-1) and accessory (HLA-DR/Ia) molecules was significantly decreased in uraemic patients, especially in non-responders; and (3) impaired internalization of HBsAg as well as a decrease in ICAM-1 and HLA-DR/Ia expression, correlated well with the blunted proliferation of CD4+ T cells stimulated by autologous monocytes induced by HBsAg.