Association of hematopoietic cell transplantation-specific comorbidity index with resource utilization after allogeneic transplantation.

Comorbidities affect clinical outcomes and costs in medicine. The hematopoietic cell transplantation (HCT)-specific comorbidity index (HCT-CI) predicts mortality risk after HCT. Its association with resource utilization (RU) is unknown. In this single-center, retrospective study, we examined the association of HCT-CI with RU (readmissions, length of hospital stay (LOS) and days out of hospital alive (DOHA)) in first 100 days (n=328) and 1 year (n=226) in allogeneic HCT patients from January 2010 to June 2014. Age, disease risk, conditioning and use of antithymocyte globulin were significantly different in the four groups with HCT-CI 0 to1 (n=138), 2 (n=56), 3 (n=55) or ⩾4 (n=79). Although the readmissions were higher in the first 100 days for patients with HCT-CI >0-1 (P=0.03), they were not significantly different in patients over 1 year (P=0.13). In the multivariable analysis, patients with HCT-CI score of >0 to 1 had increased LOS and fewer DOHA in both 100 days and 1 year after HCT. In this exploratory analysis, we found that HCT-CI >0 to 1 is associated with increased RU after allogeneic HCT. Recognizing predictors of RU can identify patients at risk of high utilization and help understand what drives health-care costs.

Stable insulin-secreting ducts formed by reprogramming of cells in the liver using a three-gene cocktail and a PPAR agonist.

With the long-term aim of developing a new type of therapy for diabetes, we have investigated the reprogramming of liver cells in normal mice toward a pancreatic phenotype using the gene combination Pdx1, Ngn3, MafA. CD1 mice were rendered diabetic with streptozotocin and given a single dose of Ad-PNM, an adenoviral vector containing all three genes. Ad-PNM induced hepatocytes of the liver to produce insulin, and the blood glucose became normalized. But over several weeks, the insulin-positive cells were lost and the blood glucose rose back to diabetic levels. Simultaneous administration of a peroxisome-proliferator-activated receptor agonist, WY14643, caused remission of diabetes at a lower dose of Ad-PNM and also caused the appearance of a population of insulin-secreting ductal structures in the liver. The insulin-positive ducts were stable and were able to relieve diabetes in the long term. We show that the effect of WY14643 is associated with the promotion of cell division of the ductal cells, which may increase their susceptibility to being reprogrammed toward a beta cell fate.

Inhibition of Hes1 activity in gall bladder epithelial cells promotes insulin expression and glucose responsiveness.

The biliary system has a close developmental relationship with the pancreas, evidenced by the natural occurrence of small numbers of biliary-derived beta-cells in the biliary system and by the replacement of biliary epithelium with pancreatic tissue in mice lacking the transcription factor Hes1. In normal pancreatic development, Hes1 is known to repress endocrine cell formation. Here we show that glucose-responsive insulin secretion can be induced in biliary epithelial cells when activity of the transcription factor Hes1 is antagonised. We describe a new culture system for adult murine gall bladder epithelial cells (GBECs), free from fibroblast contamination. We show that Hes1 is expressed both in adult murine gall bladder and in cultured GBECs. We have created a new dominant negative Hes1 (DeltaHes1) by removal of the DNA-binding domain, and show that it antagonises Hes1 function in vivo. When DeltaHes1 is introduced into the GBEC it causes expression of insulin RNA and protein. Furthermore, it confers upon the cells the ability to secrete insulin following exposure to increased external glucose. GBEC cultures are induced to express a wider range of mature beta cell markers when co-transduced with DeltaHes1 and the pancreatic transcription factor Pdx1. Introduction of DeltaHes1 and Pdx1 can therefore initiate a partial respecification of phenotype from biliary epithelial cell towards the pancreatic beta cell.

Use of adenovirus for ectopic gene expression in Xenopus.

We show that replication defective adenovirus can be used for localized overexpression of a chosen gene in Xenopus tadpoles. Xenopus contains two homologs of the Coxsackie and Adenovirus Receptor (xCAR1 and 2), both of which can confer sensitivity for adenovirus infection. xCAR1 mRNA is present from the late gastrula stage and xCAR2 throughout development, both being widely expressed in the embryo and tadpole. Consistent with the expression of the receptors, adenovirus will infect a wide range of Xenopus tissues cultured in vitro. It will also infect early embryos when injected into the blastocoel or archenteron cavities. Furthermore, adenovirus can be delivered by localized injection to tadpoles and will infect a patch of cells around the injection site. The expression of green fluorescent protein in infected cells persists for several weeks. This new gene delivery method complements the others that are already available. Developmental Dynamics 238:1412-1421, 2009. (c) 2009 Wiley-Liss, Inc.

Metaplasia and somatic cell reprogramming.

The nature and occurrence of metaplasia is briefly reviewed. A theory of how metaplasia is initiated is presented, depending on the idea that it represents an alteration in the combination of developmental transcription factors that are expressed. Two examples of experimental metaplasia, provoked by over-expression of specific transcription factors, are presented: the transformation of B lymphocytes to macrophages, and of pancreatic exocrine cells to hepatocytes. The formation of induced pluripotential stem cells (iPS cells) is considered an example of the same process, in which the destination state is the embryonic stem cell. It is noted that iPS cell production is a stochastic process, depending on selection to obtain the desired cell type. It is proposed that analogous technology, using the appropriate transcription factors, could be used to bring about transformation to cell types other than embryonic stem cells.

Origin of pancreatic endocrine cells from biliary duct epithelium.

We describe an explant culture system to study the formation of pancreatic-type endocrine cells by the biliary tract. In this model, beta-cells and other endocrine cells appear in the biliary duct epithelium and their number increases. Evidence for an origin from the duct epithelium is threefold. Firstly, differentiating cells transiently co-express insulin and bind Dolichos lectin. Secondly, beta-cells in cultures isolated from Alb-Cre-R26R-LacZ mice are beta-galactosidase positive. Thirdly, co-culture of biliary epithelium and ROSA26 pancreatic buds shows that endocrine cells do not migrate from the pancreas. The expression of the pancreatic transcription factors Pdx1, HNF6 and Sox9 is widespread, as is Hes1, which represses endocrine development, while that of Ngn3, which is a proendocrine transcription factor, is transient, consistent with an early stage of endocrine cell differentiation. Nicotinamide will increase the number of beta-cells formed, while EGF+LIF completely inhibits their formation.

[Expression of foreign gene by cysteine proteinase null recombinant baculovirus].

The baculovirus expression vector systems (BEVS) are broadly used for producing foreign proteins in lepidopteran larvae. Most commercial BEVS are engineered to insert foreign genes into the polyhedrin (polh) locus and lack the polh gene. These viruses cannot produce occlusion bodies and are inconvenient for per os inoculation of larvae. Current knowledge in baculovirus genomics makes it possible to engineer BEVS into other parts of the virus genome. In our work, we have expressed recombinant M-HBsAg (middle surface antigen of human hepatitis B) in the baculovirus construct, rBmNPV-Deltav-cath-M-HBsAg, inserting foreign gene into the v-cath locus of the Bombyx mori nucleopolyhedrovirus (BmNPV) such that the v-cath gene is deleted and the native polh gene is retained. Silkworm larvae were infected per os and M-HBsAg was observed to be abundantly produced at a very late stage of infection.

Efficacy of decitabine in the treatment of patients with chronic myelomonocytic leukemia (CMML).

Chronic myelomonocytic leukemia (CMML) characterized by cytopenias, bone marrow and peripheral blood cell dysplasia is notoriously hard to treat. Recent reclassification of CMML as a myelodysplastic/myeloproliferative (MDS/MPS) disease rather than a myelodysplastic syndrome (MDS) by the World Health Organisation (WHO) has led to a review of CMML patients treated with decitabine. Overall response rates (ORR) (complete response [CR]+partial response [PR]) in the subset of patients with CMML in one pivotal phase 3 trial (D-0007) and two phase 2 trials (PCH 95-11, PCH 97-19) decitabine were reviewed. For consistency across trials, all decitabine-treated patients were evaluated using the phase 2 response criteria (CR was defined by normocellular bone marrow with <5% blasts and normal Hgb, WBC, and platelet counts, and PR required 50% decrease in blast count, increases in Hgb by >1.5 mmol/L, WBC count by >1000, and platelet count by >50,000). A total of 31 patients diagnosed with CMML are included in this review. Similar demographics and disease characteristics were observed in all three studies, with an average age of 70.2 years and 71% of patients male. Baseline WBC of >20,000 were observed in 8/28 (29%) patients and baseline bone marrow blasts >5% in 11/28 (39%) patients. All clinical responses were centrally reviewed. The ORR was 25% (14% CR+11% PR). Hematologic improvement was observed in 11% of patients and stable disease in 39% of patients. The decitabine adverse event profile seen in CMML patients was similar to observations in other hematologic patient populations, with myelosuppression and related infectious complications. These data demonstrate encouraging activity for decitabine in CMML, and suggest that studies in other myeloproliferative diseases may be warranted.

The Xenopus tadpole: a new model for regeneration research.

The Xenopus tadpole is a favourable organism for regeneration research because it is suitable for a wide range of micromanipulative procedures and for a wide range of transgenic methods. Combination of these techniques enables genes to be activated or inhibited at specific times and in specific tissue types to a much higher degree than in any other organism capable of regeneration. Regenerating systems include the tail, the limb buds and the lens. The study of tail regeneration has shown that each tissue type supplies the cells for its own replacement: there is no detectable de-differentiation or metaplasia. Signalling systems needed for regeneration include the BMP and Notch signalling pathways, and perhaps also the Wnt and FGF pathways. The limb buds will regenerate completely at early stages, but not once they are fully differentiated. This provides a good opportunity to study the loss of regenerative ability using transgenic methods.

Betacellulin inhibits amylase and glucagon production and promotes beta cell differentiation in mouse embryonic pancreas.

AIMS/HYPOTHESIS: Betacellulin, a member of the epidermal growth factor family, is expressed in the pancreas and is thought to regulate differentiation of beta cells during development. The aim of the present study was to investigate the effects of exogenous betacellulin on the development of the mouse embryonic pancreas. MATERIALS AND METHODS: We used an in vitro culture model system based on the isolation and culture of the dorsal embryonic pancreas from day 11.5 embryos. Cultures were treated for up to 10 days with 10 ng/ml betacellulin and then analysed for changes in the expression of pancreatic exocrine, endocrine and ductal markers. RESULTS: Pancreases developed in culture and expressed the full complement of exocrine (both acinar and ductal) and endocrine cell types. Betacellulin enhanced branching morphogenesis and the proliferation of mesenchyme, increased Pdx1 and insulin production and inhibited the production of the exocrine cell marker amylase and the endocrine hormone glucagon. CONCLUSIONS/INTERPRETATION: These results suggest betacellulin has distinct and separate effects on exocrine, endocrine and ductal differentiation. In the future, betacellulin could perhaps be utilised to increase the production of beta cells from embryonic pancreatic tissue for therapeutic transplantation.

Relapse of acute promyelocytic leukemia with PML-RARalpha mutant subclones independent of proximate all-trans retinoic acid selection pressure.

Relapse of acute promyelocytic leukemia (APL) following all-trans retinoic acid (ATRA) therapy has been associated with the acquisition of mutations in the high-affinity ATRA binding site in PML-RARalpha, but little information is available about the selection dynamics of the mutation-harboring subclones. In this study, 6/18 patients treated with sequential ATRA and chemotherapy on protocol INT0129 relapsed with complete replacement of the nonmutant pretreatment APL cell population by a PML-RARalpha mutant subclone. Two patients relapsed in proximity of ATRA treatment; however, in four patients there was a 6-48 month hiatus between the last ATRA treatment and relapse. The mutant subclones were not detectable in samples tested > or = 3 months before relapse at > or = 1 in 10(2) (10(-2)) sensitivity. In one patient, a functionally weak mutation was detected at 10(-4) sensitivity before therapy but only limited pre-relapse enrichment of the mutant subclone was observed on subsequent ATRA therapy. These results indicate that proximate ATRA selection pressure is frequently not the main determinant for the emergence of strongly dominant PML-RARalpha mutant subclones and suggest that APL subclones harboring PML-RARalpha mutations are predisposed to the acquisition of secondary genetic/epigenetic alterations that result in a growth/survival advantage.

Cellular and molecular mechanisms of regeneration in Xenopus.

We have employed transgenic methods combined with embryonic grafting to analyse the mechanisms of regeneration in Xenopus tadpoles. The Xenopus tadpole tail contains a spinal cord, notochord and segmented muscles, and all tissues are replaced when the tail regenerates after amputation. We show that there is a refractory period of very low regenerative ability in the early tadpole stage. Tracing of cell lineage with the use of single tissue transgenic grafts labelled with green fluorescent protein (GFP) shows that there is no de-differentiation and no metaplasia during regeneration. The spinal cord, notochord and muscle all regenerate from the corresponding tissue in the stump; in the case of the muscle the satellite cells provide the material for regeneration. By using constitutive or dominant negative gene products, induced under the control of a heat shock promoter, we show that the bone morphogenetic protein (BMP) and Notch signalling pathways are both essential for regeneration. BMP is upstream of Notch and has an independent effect on regeneration of muscle. The Xenopus limb bud will regenerate completely at the early stages but regenerative ability falls during digit differentiation. We have developed a procedure for making tadpoles in which one hindlimb is transgenic and the remainder wild-type. This has been used to introduce various gene products expected to prolong the period of regenerative capacity, but none has so far been successful.

Acute renal failure requiring dialysis after allogeneic blood and marrow transplantation identifies very poor prognosis patients.

We examined the incidence, risk factors and associated mortality of acute renal failure requiring dialysis (Renal Bearman Grade [BG] 3) in a 3-year cohort of 97 consecutive allogeneic blood and marrow transplantation (alloBMT) patients. In all, 20 (21%) developed Renal BG3 (all died by day +132) and 77 (79%) developed renal insufficiency (Renal BG1-2). Renal BG3 was a contributing or primary cause of death in 18 (90%) patients who continued to require dialysis at time of death. The two Renal BG3 patients whose deaths were not related to renal failure died on day +103 of hemorrhage and day +132 of underlying disease. By univariate analysis, age, unrelated donor, veno-occlusive disease (VOD) and grade III-IV acute graft-versus-host disease with hepatic involvement were significantly associated with Renal BG3. The multivariate model of time to Renal BG3 determined only a prior diagnosis of severe acute GVHD (RR=4.1, 95% CI 1.6-10.3, P=0.003) and VOD (RR=9.1, 95% CI 3.5-23.7, P<0.001) as significant independent predictors. Renal BG3 is generally considered a conditioning regimen-related toxicity. This study demonstrates that Renal BG3 is most commonly a complication of hepatic co-morbidities after allogeneic blood and marrow transplantation and identifies patients with a very poor prognosis.

Use of nonvolume-reduced (unmanipulated after thawing) umbilical cord blood stem cells for allogeneic transplantation results in safe engraftment.

Volume reduction of umbilical cord blood (UCB) units before infusion is standard in most transplant centers. We examined 26 patients who underwent transplantation from May 1997 to December 2001 with unmanipulated (n=18) or volume-reduced (n=8) UCB units for engraftment. Of 18 unmanipulated UCBT patients, 16 achieved ANC >500/mm(3), a median of 26 days (range, 16-104) post-UCBT; two died before engraftment on days +2 and +14. Of 18 unmanipulated UCBT patients, 10 achieved platelet recovery, a median of 60.5 days (range, 41-144) post-UCBT; eight patients died before platelet recovery +2 to +255 days post-UCBT. These results are similar to several reported studies and our series utilizing volume-reduced UCB units for UCBT. At a median follow-up of 29.5 months, the 100-day and 3-year overall survivals of unmanipulated UCBT were 61.1% (95% CI, 38.6-83.6) and 48.6% (95% CI, 24.8-72.4) and of volume-reduced UCBT were 60% (95% CI, 24.4-95.6) and 22.5% (95% CI, 0-58.7). There was no serious toxicity from UCB infusion using unmanipulated UCB units. We conclude that unmanipulated UCB units may be infused safely into UCBT patients with adequate engraftment and survival.

Opportunities for Trisenox (arsenic trioxide) in the treatment of myelodysplastic syndromes.

Arsenic trioxide (ATO) has a long history of efficacy as an antileukemic agent. However, with the advent of modern therapy, it had been relegated to a historical footnote. In the 1990s, investigators in China reported that ATO was safe and had dramatic efficacy in patients with acute promyelocytic leukemia (APL). Preclinical investigations indicate that the biological targets of this novel drug extend to a variety of malignancies other than APL and include induction of apoptosis, nonterminal differentiation, and suppression of proliferation and angiogenesis. The myelodysplastic syndromes (MDSs) present a particular therapeutic challenge. Ineffective hematopoiesis predominates in patients with low-grade prognostic scores. The survival of those patients with high-grade disease is compromised by a high risk of leukemia transformation. Although a number of therapeutic options have been investigated, none has emerged as being broadly efficacious and having an acceptable toxicity profile. No drug has yet received approval by the Food and Drug Administration for this indication. Biologic features of MDS, which include accelerated apoptotic potential, limited maturation capacity, and medullary neovascularity, create a strong scientific rationale for the investigation of ATO in MDS. This report describes the history and scientific basis for ATO treatment of hematologic malignancies, enumerates the potential benefits of ATO in MDS, and discusses the direction of ongoing trials of this novel antineoplastic agent.

HLA-DR antigen-negative acute myeloid leukemia.

Human leukocyte antigen (HLA) Class II antigens are variably expressed on acute myeloid leukemia (AML) blasts. The biological and clinical significance of HLA Class II antigen expression by AML cells is not known. Therefore, we sought to characterize cases of AML without detectable HLA-DR expression. Samples from 248 consecutive adult AML patients were immunophenotyped by multiparameter flow cytometry at diagnosis. HLA-DR antigens were not detected on AML cells from 43 patients, including 20 with acute promyelocytic leukemia (APL), and 23 with other subtypes of AML. All APL cases had t(15;17), but there were no characteristic chromosome abnormalities in non-APL cases. No direct expression of other antigens was identified in HLA-DR-negative APL and non-APL cases. Interestingly, cells from three HLA-DR-negative non-APL patients had similar morphology to that of the hypogranular variant of APL. This morphology, however, was not present in any HLA-DR-positive AML cases. Treatment response was similar in the 23 HLA-DR-negative non-APL and the 205 HLA-DR-positive patients. Finally, relapse was infrequently associated with changes in HLA-DR antigen expression, as the HLA-DR antigen was lost at relapse in only 4% of HLA-DR-positive cases, and was gained at relapse in only 17% of HLA-DR-negative cases. We conclude that HLA-DR-negative AML includes approximately equal numbers of APL and non-APL cases, and that the morphology of HLA-DR-negative non-APL cases can mimic the hypogranular variant of APL. The diagnosis of APL cannot be based on morphology and lack of HLA-DR antigen expression; rather, it requires cytogenetic or molecular confirmation.

Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARalpha mRNA using real-time reverse-transcription polymerase chain reaction.

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARalpha, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARalpha plasmid DNA. PML-RARalpha transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 microg of RNA from PML-RARalpha-negative cells. Using 1.0 to 2.5 microg of input RNA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.