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Abstract Background From 2014 to 2019, the Centre for Research Excellence in Integrated Quality Improvement (CRE-IQI) was evaluated as an innovation platform focusing on continuous quality improvement in Indigenous Australian primary healthcare. Although social network analysis (SNA) is a recognized method for evaluating the functioning, collaboration and effectiveness of innovation platforms, applied research is limited. This study applies SNA to evaluate the CRE-IQI’s functioning as an innovation platform. Methods Two surveys (2017, 2019) were conducted using social survey and network methods. Survey items covered respondent characteristics, their perceptions of the CRE-IQI’s performance, and its impact and sociometric relationships. Members’ relationship information was captured for the CRE-IQI at three time points, namely start (retrospectively), midpoint and final year, on three network types (knew, shared information, collaborated). SNA software was used to compute standard network metrics including diameter, density and centrality, and to develop visualizations. Survey and network results were addressed in a workshop held by members to develop improvement strategies. Results The response rate was 80% in 2017 and 65% in 2019 (n = 49 and 47, respectively). Between 2017 and 2019, respondents’ mean ratings of the CRE-IQI’s functioning and achievements in meeting its goals were sustained. They perceived the CRE-IQI as multidisciplinary, having effective management and governance, and incorporating Indigenous research leadership, representation and ways of working. Respondents recognized high levels of trust amongst members, rated “good communication and coordination with participants” highly, and “facilitating collaboration” as the CRE’s most strongly recognized achievement. In collaboration and information-sharing networks, average path length remained low in 2017 and 2019, indicating good small-world network properties for relaying information. On average, respondents shared information and collaborated with more CRE members in 2017 than 2019. However, in both 2017 and 2019 there were new collaborations and information-sharing outside of direct collaborations. CRE-IQI outcomes included: evidence generation; knowledge transfer and skills development in quality improvement; research capacity-building, career development; mentoring; grant support; development of new projects; health service support; and policy impact. Conclusions This study shows the utility of network analysis in evaluating the functioning, and collaboration, at the individual, organizational and health system levels, of an innovation platform, and adds to our understanding of factors enabling successful innovation platforms.

Objectives Though multidisciplinary research networks support the practice and effectiveness of continuous quality improvement (CQI) programmes, their characteristics and development are poorly understood. In this study, we examine publication outputs from a research network in Australian Indigenous primary healthcare (PHC) to assess to what extent the research network changed over time.Setting Australian CQI research network in Indigenous PHC from 2002 to 2019.Participants Authors from peer-reviewed journal articles and books published by the network.Design Coauthor networks across four phases of the network (2002–2004; 2005–2009; 2010–2014; 2015–2019) were constructed based on author affiliations and examined using social network analysis methods. Descriptive characteristics included organisation types, Indigenous representation, gender, student authorship and thematic research trends.Results We identified 128 publications written by 308 individual authors from 79 different organisations. Publications increased in number and diversity over each funding phase. During the final phase, publication outputs accelerated for organisations, students, project officers, Indigenous and female authors. Over time there was also a shift in research themes to encompass new clinical areas and social, environmental or behavioural determinants of health. Average degree (8.1), clustering (0.81) and diameter (3) indicated a well-connected network, with a core-periphery structure in each phase (p≤0.03) rather than a single central organisation (degree centralisation=0.55–0.65). Academic organisations dominated the core structure in all funding phases.Conclusion Collaboration in publications increased with network consolidation and expansion. Increased productivity was associated with increased authorship diversity and a decentralised network, suggesting these may be important factors in enhancing research impact and advancing the knowledge and practice of CQI in PHC. Publication diversity and growth occurred mainly in the fourth phase, suggesting long-term relationship building among diverse partners is required to facilitate participatory research in CQI. Despite improvements, further work is needed to address inequities in female authorship and Indigenous authorship.

Despite most acute myeloid leukemia (AML) patients entering remission following chemotherapy, outcomes remain poor due to surviving leukemic cells that contribute to relapse. The nature of these enduring cells is poorly understood. Here, through temporal single-cell transcriptomic characterization of AML hierarchical regeneration in response to chemotherapy, we reveal a cell population: AML regeneration enriched cells (RECs). RECs are defined by CD74/CD68 expression, and although derived from leukemic stem cells (LSCs), are devoid of stem/progenitor capacity. Based on REC in situ proximity to CD34-expressing cells identified using spatial transcriptomics on AML patient bone marrow samples, RECs demonstrate the ability to augment or reduce leukemic regeneration in vivo based on transfusion or depletion, respectively. Furthermore, RECs are prognostic for patient survival as well as predictive of treatment failure in AML cohorts. Our study reveals RECs as a previously unknown functional catalyst of LSC-driven regeneration contributing to the non-canonical framework of AML regeneration., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2024. Published by Elsevier Inc. All rights reserved.)

Overlapping principles of embryonic and tumor biology have been described, with recent multi-omics campaigns uncovering shared molecular profiles between human pluripotent stem cells (hPSCs) and adult tumors. Here, using a chemical genomic approach, we provide biological evidence that early germ layer fate decisions of hPSCs reveal targets of human cancers. Single-cell deconstruction of hPSCs-defined subsets that share transcriptional patterns with transformed adult tissues. Chemical screening using a unique germ layer specification assay for hPSCs identified drugs that enriched for compounds that selectively suppressed the growth of patient-derived tumors corresponding exclusively to their germ layer origin. Transcriptional response of hPSCs to germ layer inducing drugs could be used to identify targets capable of regulating hPSC specification as well as inhibiting adult tumors. Our study demonstrates properties of adult tumors converge with hPSCs drug induced differentiation in a germ layer specific manner, thereby expanding our understanding of cancer stemness and pluripotency., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

We systematically investigate functional and molecular measures of stemness in patients with acute myeloid leukemia (AML) using a cohort of 121 individuals. We confirm that the presence of leukemic stem cells (LSCs) detected through in vivo xenograft transplantation is associated with poor survival. However, the measurement of leukemic progenitor cells (LPCs) through in vitro colony-forming assays provides an even stronger predictor of overall and event-free survival. LPCs not only capture patient-specific mutations but also retain serial re-plating ability, demonstrating their biological relevance. Notably, LPC content represents an independent prognostic factor in multivariate analyses including clinical guidelines of risk stratification. Our findings suggest that LPCs provide a robust functional measure of AML, enabling quantitative and rapid assessment of a wide range of patients. This highlights the potential of LPCs as a valuable prognostic factor in AML management., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2023. Published by Elsevier Inc. All rights reserved.)

Screening of primary patient acute myeloid leukemia (AML) cells is challenging based on intrinsic characteristics of human AML disease and patient-specific conditions required to sustain AML cells in culture. This is further complicated by inter- and intra-patient heterogeneity, and "contaminating" normal cells devoid of molecular AML mutations. Derivation of induced pluripotent stem cells (iPSCs) from human somatic cells has provided approaches for the development of patient-specific models of disease biology and has recently included AML. Although reprogramming patient-derived cancer cells to pluripotency allows for aspects of disease modeling, the major limitation preventing applications and deeper insights using AML-iPSCs is the rarity of success and limited subtypes of AML disease that can be captured by reprogramming to date. Here, we tested and refined methods including de novo, xenografting, naïve versus prime states and prospective isolation for reprogramming AML cells using a total of 22 AML patient samples representing the wide variety of cytogenetic abnormalities. These efforts allowed us to derive genetically matched healthy control (isogenic) lines and capture clones found originally in patients with AML. Using fluorescently activated cell sorting, we revealed that AML reprogramming is linked to the differentiation state of diseased tissue, where use of myeloid marker CD33 compared to the stem cell marker, CD34, reduces reprogramming capture of AML+ clones. Our efforts provide a platform for further optimization of AML-iPSC generation, and a unique library of iPSC derived from patients with AML for detailed cellular and molecular study., (© The Author(s) 2023. Published by Oxford University Press.)

The generation of human hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) represents a major goal in regenerative medicine and is believed would follow principles of early development. HSCs arise from a type of endothelial cell called a "hemogenic endothelium" (HE), and human HSCs are experimentally detected by transplantation into SCID or other immune-deficient mouse recipients, termed SCID-Repopulating Cells (SRC). Recently, SRCs were detected by forced expression of seven transcription factors (TF) (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1, and SPI1) in hPSC-derived HE, suggesting these factors are deficient in hPSC differentiation to HEs required to generate HSCs. Here we derived PECAM-1-, Flk-1-, and VE-cadherin-positive endothelial cells that also lack CD45 expression (PFV CD45- ) which are solely responsible for hematopoietic output from iPSC lines reprogrammed from AML patients. Using HEs derived from AML patient iPSCs devoid of somatic leukemic aberrations, we sought to generate putative SRCs by the forced expression of 7TFs to model autologous HSC transplantation. The expression of 7TFs in hPSC-derived HE cells from an enhanced hematopoietic progenitor capacity was present in vitro, but failed to acquire SRC activity in vivo. Our findings emphasize the benefits of forced TF expression, along with the continued challenges in developing HSCs for autologous-based therapies from hPSC sources.

Natural products (NPs) encompass a rich source of bioactive chemical entities. Here, we used human cancer stem cells (CSCs) in a chemical genomics campaign with NP chemical space to interrogate extracts from diverse strains of actinomycete for anti-cancer properties. We identified a compound (McM25044) capable of selectively inhibiting human CSC function versus normal stem cell counterparts. Biochemical and molecular studies revealed that McM025044 exerts inhibition on human CSCs through the small ubiquitin-like modifier (SUMO) cascade, found to be hyperactive in a variety of human cancers. McM025044 impedes the SUMOylation pathway via direct targeting of the SAE1/2 complex. Treatment of patient-derived CSCs resulted in reduced levels of SUMOylated proteins and suppression of progenitor and stem cell capacity measured in vitro and in vivo. Our study overcomes a barrier in chemically inhibiting oncogenic SUMOylation activity and uncovers a unique role for SAE2 in the biology of human cancers., Competing Interests: Declaration of interests The authors declare no competing financial interests. However, some authors are inventors of a provisional patent related to unique targeting of human cancer cells using SAE2 and related probe compounds., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Microscopic colitis (MC) is a common cause of chronic secretory diarrhea with variable etiologies, including nonsteroidal anti-inflammatory drugs. As its name implies, the diagnosis requires consistent histopathologic findings that typically accompany normal-appearing mucosa. However, accumulating evidence suggests that the presence of distinct endoscopic features is associated with MC. We present a case of MC that highlights the importance of recognizing these macroscopic findings because they can aid in diagnosis and have significant management implications., (© 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of The American College of Gastroenterology.)

Identifying precise targets of individual cancers remains challenging. Chronic lymphocytic leukemia (CLL) represents the most common adult hematologic malignancy, and trisomy 12 (tri12) represents a quarter of CLL patients. We report that tri12 human pluripotent stem cells (hPSCs) allow for the identification of gene networks and targets specific to tri12, which are controlled by comparative normal PSCs. Identified targets are upregulated in tri12 leukemic cells from a cohort of 159 patients with monoclonal B cell lymphocytosis and CLL. tri12 signaling patterns significantly influence progression-free survival. Actionable targets are identified using high-content drug testing and functionally validated in an additional 44 CLL patient samples. Using xenograft models, interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor is potent and selective against human tri12 CLL versus healthy patient-derived xenografts. Our study uses hPSCs to uncover targets from genetic aberrations and apply them to cancer. These findings provide immediate translational potential as biomarkers and targets for therapeutic intervention., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone variants (HVs) are a subfamily of epigenetic regulators implicated in embryonic development, but their role in human stem cell fate remains unclear. Here, we reveal that the phosphorylation state of the HV H2A.X (γH2A.X) regulates self-renewal and differentiation of human pluripotent stem cells (hPSCs) and leukemic progenitors. As demonstrated by CRISPR-Cas deletion, H2A.X is essential in maintaining normal hPSC behavior. However, reduced levels of γH2A.X enhances hPSC differentiation toward the hematopoietic lineage with concomitant inhibition of neural development. In contrast, activation and sustained levels of phosphorylated H2A.X enhance hPSC neural fate while suppressing hematopoiesis. This controlled lineage bias correlates to occupancy of γH2A.X at genomic loci associated with ectoderm versus mesoderm specification. Finally, drug modulation of H2A.X phosphorylation overcomes differentiation block of patient-derived leukemic progenitors. Our study demonstrates HVs may serve to regulate pluripotent cell fate and that this biology could be extended to somatic cancer stem cell control., Competing Interests: Declaration of interests The authors declare no competing financial interests, (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The aberrant expression of dopamine receptors (DRDs) in acute myeloid leukemia (AML) cells has encouraged the repurposing of DRD antagonists such as thioridazine (TDZ) as anti-leukemic agents. Here, we access patient cells from a Phase I dose escalation trial to resolve the cellular and molecular bases of response to TDZ, and we extend these findings to an additional independent cohort of AML patient samples tested preclinically. We reveal that in DRD2 + AML patients, DRD signaling in leukemic progenitors provides leukemia-exclusive networks of sensitivity that spare healthy hematopoiesis. AML progenitor cell suppression can be increased by the isolation of the positive enantiomer from the racemic TDZ mixture (TDZ + ), and this is accompanied by reduced cardiac liability. Our study indicates that the development of DRD-directed therapies provides a targeting strategy for a subset of AML patients and potentially other cancers that acquire DRD expression upon transformation from healthy tissue., Competing Interests: The authors declare no competing interests., (Crown Copyright © 2021.)

Despite successful remission induction, recurrence of acute myeloid leukemia (AML) remains a clinical obstacle thought to be caused by the retention of dormant leukemic stem cells (LSCs). Using chemotherapy-treated AML xenografts and patient samples, we have modeled patient remission and relapse kinetics to reveal that LSCs are effectively depleted via cell-cycle recruitment, leaving the origins of relapse unclear. Post-chemotherapy, in vivo characterization at the onset of disease relapse revealed a unique molecular state of leukemic-regenerating cells (LRCs) responsible for disease re-growth. LRCs are transient, can only be detected in vivo, and are molecularly distinct from therapy-naive LSCs. We demonstrate that LRC features can be used as markers of relapse and are therapeutically targetable to prevent disease recurrence., (Copyright © 2018 Elsevier Inc. All rights reserved.)

We completed a phase 1 dose-escalation trial to evaluate the safety of a dopamine receptor D2 (DRD2) antagonist thioridazine (TDZ), in combination with cytarabine. Thirteen patients 55 years and older with relapsed or refractory acute myeloid leukemia (AML) were enrolled. Oral TDZ was administered at 3 dose levels: 25 mg (n = 6), 50 mg (n = 4), or 100 mg (n = 3) every 6 hours for 21 days. Intermediate-dose cytarabine was administered on days 6 to 10. Dose-limiting toxicities (DLTs) included grade 3 QTc interval prolongation in 1 patient at 25 mg TDZ and neurological events in 2 patients at 100 mg TDZ (gait disturbance, depressed consciousness, and dizziness). At the 50-mg TDZ dose, the sum of circulating DRD2 antagonist levels approached a concentration of 10 μM, a level noted to be selectively active against human AML in vitro. Eleven of 13 patients completed a 5-day lead-in with TDZ, of which 6 received TDZ with hydroxyurea and 5 received TDZ alone. During this period, 8 patients demonstrated a 19% to 55% reduction in blast levels, whereas 3 patients displayed progressive disease. The extent of blast reduction during this 5-day interval was associated with the expression of the putative TDZ target receptor DRD2 on leukemic cells. These preliminary results suggest that DRD2 represents a potential therapeutic target for AML disease. Future studies are required to corroborate these observations, including the use of modified DRD2 antagonists with improved tolerability in AML patients. This trial was registered at www.clinicaltrials.gov as #NCT02096289., (© 2018 by The American Society of Hematology.)

Human pluripotent stem cells (hPSCs) generate hematopoietic progenitor cells (HPCs) but fail to engraft xenograft models used to detect adult/somatic hematopoietic stem cells (HSCs) from donors. Recent progress to derive hPSC-derived HSCs has relied on cell-autonomous forced expression of transcription factors; however, the relationship of bone marrow to transplanted cells remains unknown. Here, we quantified a failure of hPSC-HPCs to survive even 24 hr post transplantation. Across several hPSC-HPC differentiation methodologies, we identified the lack of CXCR4 expression and function. Ectopic CXCR4 conferred CXCL12 ligand-dependent signaling of hPSC-HPCs in biochemical assays and increased migration/chemotaxis, hematopoietic progenitor capacity, and survival and proliferation following in vivo transplantation. This was accompanied by a transcriptional shift of hPSC-HPCs toward somatic/adult sources, but this approach failed to produce long-term HSC xenograft reconstitution. Our results reveal that networks involving CXCR4 should be targeted to generate putative HSCs with in vivo function from hPSCs., (Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.)

Acute myeloid leukaemia (AML) is distinguished by the generation of dysfunctional leukaemic blasts, and patients characteristically suffer from fatal infections and anaemia due to insufficient normal myelo-erythropoiesis. Direct physical crowding of bone marrow (BM) by accumulating leukaemic cells does not fully account for this haematopoietic failure. Here, analyses from AML patients were applied to both in vitro co-culture platforms and in vivo xenograft modelling, revealing that human AML disease specifically disrupts the adipocytic niche in BM. Leukaemic suppression of BM adipocytes led to imbalanced regulation of endogenous haematopoietic stem and progenitor cells, resulting in impaired myelo-erythroid maturation. In vivo administration of PPARγ agonists induced BM adipogenesis, which rescued healthy haematopoietic maturation while repressing leukaemic growth. Our study identifies a previously unappreciated axis between BM adipogenesis and normal myelo-erythroid maturation that is therapeutically accessible to improve symptoms of BM failure in AML via non-cell autonomous targeting of the niche.

Targeting of human cancer stem cells (CSCs) requires the identification of vulnerabilities unique to CSCs versus healthy resident stem cells (SCs). Unfortunately, dysregulated pathways that support transformed CSCs, such as Wnt/β-catenin signaling, are also critical regulators of healthy SCs. Using the ICG-001 and CWP family of small molecules, we reveal Sam68 as a previously unappreciated modulator of Wnt/β-catenin signaling within CSCs. Disruption of CBP-β-catenin interaction via ICG-001/CWP induces the formation of a Sam68-CBP complex in CSCs that alters Wnt signaling toward apoptosis and differentiation induction. Our study identifies Sam68 as a regulator of human CSC vulnerability., (Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.)

Increasing attention has been focused on the interactions between leukemia cells and their bone marrow (BM) microenvironment. We have recently shown that leukemic stem cells (LSCs) share common BM "niches" with their healthy counterparts. As a result of these shared niche requirements, human LSCs can be mobilized using existing pharmacological agents that facilitate competitive healthy reconstitution, leading to eradication of leukemia during BM transplantation.

Aberrant regulation of the canonical Wnt signaling pathway (Wnt-β-catenin-GSK3 axis) has been a prevalent theme in cancer biology since earlier observations until recent genetic discoveries gleaned from tumor genome sequencing. During the last few decades, a large body of work demonstrated the involvement of the Wnt-β-catenin-GSK3 signaling axis in the formation and maintenance of cancer stem cells (CSC) responsible for tumor growth in several types of human malignancies. Recent studies have elucidated epigenetic mechanisms that control pluripotency and stemness, and allow a first assessment on how embryonic and normal tissue stem cells are dysregulated in cancer to give rise to CSCs, and how canonical Wnt signaling might be involved. Here, we review emerging concepts highlighting the critical role of epigenetics in CSC development through abnormal canonical Wnt signaling. Finally, we refer to the characterization of novel and powerful inhibitors of chromatin organization machinery that, in turn, restore the Wnt-β-catenin-GSK3 signaling axis in malignant cells, and describe attempts/relevance to bring these compounds into preclinical and clinical studies., (©2014 American Association for Cancer Research.)

Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC-niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients., (© 2014 Boyd et al.)

Purpose of Review: Historically, studies of the hematopoietic stem cell (HSC) microenvironment in bone marrow have focused on the identification of individual supportive cell lineages likely to be responsible for maintaining HSCs in a self-renewing and regenerative state. More recently, awareness has developed regarding the broad and dynamic heterogeneity of nonhematopoietic cells that reside within the bone marrow space. We review recent insights that provide an emerging and complex context for understanding the spatially dependent regulation of HSC functional properties in the bone marrow and the collective inputs of multiple cell types., Recent Findings: Within the last 18 months, high-resolution imaging, xenograft modeling, and genetic mouse models have afforded innovative methods of detecting and interrogating HSCs with precision at the cellular level. Spatially distinct sites within the bone marrow house functionally divergent HSCs and progenitors, and these different habitats are becoming carefully characterized from a cellular and molecular perspective. This is critical toward understanding how bone marrow microenvironments adapt to accommodate cellular demands for hematopoiesis and how these mechanisms are disrupted in pathological conditions., Summary: The bone marrow is not a continuum but an integrated unit with complex trophic interactions. Emphasis on human data will become necessary as these concepts mature and develop translationally toward changing clinical practices in HSC transplantation and even in the treatment of leukemias.

Recent work has shown that leukemic stem cell self-renewal in chronic myeloid leukemia is dependent on cell-intrinsic hedgehog (Hh) signaling, and early clinical trials suggest that targeting this pathway is also therapeutic in patients with acute myeloid leukemia (AML). In this study, we aimed to better understand Hh signaling in normal hematopoiesis and AML by molecularly and functionally analyzing more than 200 primary human AML patient samples compared with nonleukemic controls. Gene expression analysis indicated that Hh pathway transcripts were similarly regulated in AML and nonleukemic controls, regardless of whether samples were purified based on primitive phenotypes. Consistent with these results, pharmacologic inhibition of Smoothened (SMO) did not preferentially reduce in vitro colony formation of AML versus normal progenitors. Using a unique analytic approach, messenger RNA expression of membrane receptor SMO was found to be unexpectedly rare within all hematopoietic samples analyzed, which is indicative of heterogeneity at the level of Hh signaling machinery. In contrast, abundant SMO expression could be readily detected in the nonhematopoietic fraction of human and murine bone marrow (BM) cells. Our predictions of increased SMO(+) cell frequencies within nonhematopoietic BM fractions were further supported by single-cell protein analyses. Although we did not find support for cell-autonomous sensitivity of AML cells to Hh pathway inhibition, we alternatively suggest that nonhematopoietic BM cells represent an indirect target through which primitive normal and leukemic cells can be modulated. These findings suggest current approaches to applying Hh inhibition should be carefully reevaluated to account for BM niche cell regulation that might be selectively Hh responsive., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Numerous studies have shown that the bone marrow (BM) niche plays a key role in mouse hematopoietic stem cell (HSC) function and involves contributions from a broad array of cell types. However, the composition and role of the human BM HSC niche have not been investigated. Here, using human bone biopsy specimens, we provide evidence of HSC propensity to localize to endosteal regions of the trabecular bone area (TBA). Through functional xenograft transplantation, we found that human HSCs localizing to the TBA have superior regenerative and self-renewal capacity and are molecularly distinct from those localizing to the long bone area (LBA). In addition, osteoblasts in the TBA possess unique characteristics and express a key network of factors that regulate TBA- versus LBA-localized human HSCs in vivo. Our study reveals that BM localization and architecture play a critical role in defining the functional and molecular properties of human HSCs., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Prevention of breast cancer can be achieved with a better understanding of the factors contributing to normal breast development. Because the breast develops postnatally, alterations in the development and lifetime activity of the neuroendocrine system may set up an environment that increases cancer risk. The present study examined how two neonatal experiences over the first 3 weeks of life influence normal and malignant mammary gland development in female BALB/c mice. Following puberty, both brief (15 minutes) and prolonged (4 hours) daily maternal separations of newborn mice accelerated mammary gland development relative to nonseparated mice. Despite similar mammary gland morphologies between mice exposed to these two neonatal separation experiences, only mice exposed to prolonged maternal separation bouts showed a higher incidence and faster onset of mammary tumorigenesis following adulthood carcinogen [7,12-dimethylbenz(a)anthracene] administration. Molecular analysis of estrogen receptor α (ERα) and p53, two proteins that have been implicated in breast cancer, revealed that for mice exposed to prolonged neonatal maternal separation bouts, mammary gland ERα protein levels were upregulated in a transcription-independent manner. On the other hand, p53 expression in mammary glands of adult mice was not differentially influenced by neonatal experiences. Our findings show that chronic, moderate psychosocial stress during the neonatal period increases the expression of ERα protein and promotes mammary tumorigenesis in adulthood., (©2010 AACR.)

Rosuvastatin is known to reduce low-density lipoprotein (LDL)-cholesterol and improve endothelial function. In addition to lipid-lowering, statins may exert pleiotropic (nonlipid lowering) effects on microvascular function. We compared the neurophysiological and vascular responses of dietary control and treatment with 10 mg of rosuvastatin in 16 subjects with neuropathy and established type 2 diabetes. Skin blood flow (SkBF) measurements were measured at baseline, after 18 weeks of diet, and after 18 weeks of diet and treatment with rosuvastatin in response to local warming and ischemia reperfusion. The study results show that total cholesterol (196.50 ± 8.02 to 134.88 ± 10.86 mg/dL) and LDL-cholesterol (114 ± 10.4 to 63.4 ± 8.48 mg/dL) decreased significantly after 18 weeks of rosuvastatin, but not after 18 weeks of diet. Neuropathy scores decreased from 8.34 ± 1.26 at baseline to 6.00 ± 0.90 after rosuvastatin treatment. Basal SkBF was significantly different from baseline, 6.81 ± 0.42 to 9.92 ± 0.78 after rosuvastatin treatment (P ≤ 0.001). These results indicate that rosuvastatin therapy positively changed basal SkBF and measures of neurovascular function. Although there was a profound lipid lowering, it is not clear that this mediated the increases in SkBF and decreases in neuropathy scores.

Background: Results of previous studies have suggested that involvement in religious activities may be associated with lower rates of smoking. We sought to determine whether frequent attendance at religious services is associated with less smoking among young adults., Methods: This prospective cohort study of 4569 adults aged 20 to 32 years included approximately equal numbers of blacks and whites and men and women from 4 cities in the United States who attended the 1987/1988 examination of the Coronary Artery Risk Development in Young Adults (CARDIA) study. Frequency of attendance at religious services and denominational affiliation were determined by self-report questionnaire in 1987/1988. Cigarette smoking was determined by interview at this time and again 3 years later., Results: Of 4544 participants who completed the tobacco questionnaire in 1987/1988, 34% (891/2598) who attended religious services less than once per month or never and 23% (451/1946) who attended religious services at least once per month reported current smoking (odds ratio [OR], 1.7; 95% confidence interval [CI], 1.5-2.0; P<.001). This association between less frequent attendance at religious services and current smoking was found in most denominations and remained significant after adjusting for potential confounding variables (OR, 1.5; 95% CI, 1.3-1.8; P<.001). During 3-year follow-up, nonsmokers who reported little or no religious involvement had an increased risk of smoking initiation (adjusted OR, 1.9; 95% CI, 1.3-2.7; P<.001)., Conclusions: Young adults who attend religious services have lower rates of current and subsequent cigarette smoking. The potential health benefits associated with religious involvement deserve further study.

The philosophical and ethical concept of autonomy is herein examined, ex post facto, using an existential lens to examine the process of a personal friend's dying. Anagogy, defined as interpretation of a word, passage, or text that finds beyond the literal, allegorical, and moral senses a fourth and ultimate spiritual or mystical sense, is intended to enlarge the understanding of the use of autonomy in this case. The idea of personhood linked inextricably to reason is, therefore, understood as empowering an individual to choose among various actions, to define and redefine life goals, and to give priority to selected values and moral tenants, which reveal a moral hermeneutic. Conditions and circumstances, existentially exposed, limit choice in unexpected ways, such that the predicted value of autonomy is vulnerable to misuse or misunderstanding. The intent to respect the dignity of every person is central to the philosophy of Respect for Persons ethics, and assumes that autonomy, as freedom of the moral agent, is a moral duty. Implicit reality of freedom is, in a practical sense, essential to being rational agents who can thereby exercise informed choice. The moral law, law of freedom, involves the autonomy of the will and an ultimate end to which all action is directed. Defined as the highest good, morality unites virtue and happiness by ascribing the ultimate end sought as God. The freedom to use rational will finds principles within its own rational nature. The ability to create maxims is autonomy of the will, which equates with the dignity of persons. My recent experience as a companion to a personal friend with a terminal illness inspired me to re-evaluate the concept of autonomy as it is too often interpreted in modern ethical discourse as a individualistic right of choice as opposed to the hermeneutic of dignity of person. This paper describes a shift of position in understanding the paradox of autonomy in this existential context.

Tracheobronchial stent use has been reported in the medical literature for more than 40 years. Silicone stents are the most widely used stents in therapy for varying tracheobronchial lesions at most centers. However, newer designs and modifications of stents are now available with delivery systems that have been designed to facilitate using fiberoptic bronchoscopy. We describe our initial experience placing 13 self-expandable metallic Wallstent stents, including the covered design, in a total of seven patients via a fiberoptic bronchoscope. All patients had benign or malignant obstructing lesions and one patient had an associated malignant tracheoesophageal fistula. The procedure was technically easy and was well tolerated. Following stenting there was a visible increase in airway diameter and a marked improvement in symptoms for all patients. Median survival after stent placement is currently 10 months.

Purpose: To introduce case management to a general medicine ward team of a teaching hospital to improve patient care and ensure comprehensive longitudinal care., Method: The Department of Veterans Affairs Medical Center is one of four hospitals used by University of Oklahoma School of Medicine residents. There are five medicine teams, each comprising a second- or third-year resident, one or two interns, two medical students, and a faculty physician. The case-management program was initiated in November 1994. No attempt was made to limit the residents assigned to the case-managed team (i.e., many residents who worked with the case-managed team subsequently rotated through the other teams). Patients were assigned to the teams by rotation, and no attempt was made to adjust for the severity of illness among admissions. The teams were separated as follows: pre-case-management teams (all five teams prior to the case-management program), non-case-management teams (the four teams without case managers after the program's initiation), and the case-management team. The study periods were January-July 1994 (pre-case management) and January-July 1995 (after case management)., Results: The numbers of patients treated by the three groups were 1,305, 1,139, and 289, respectively. The median length of stay for pre-case-management patients was 5 days (interquartile range, 3-9 days); for non-case-management patients, 5 days (range, 3-8 days); and for case-management patients, 5 days (range, 3-7 days). The cumulative distribution of lengths of stay for case-management patients was significantly different from those of the other study groups by the Kolmogorov-Smirnov test (p = .02). More case-management patients were discharged by day 7. Rates of readmission were not significantly different between the teams., Conclusion: In this study a case-management program was effectively implemented in a teaching hospital, resulting in reduced lengths of stay for patients. As academic health centers become more concerned with efficiency and cost, case management should be seriously considered as a way to deal with such issues.

The antisense activity and gene specificity of two classes of oligonucleotides (ONs) were directly compared in a highly controlled assay. One class of ONs has been proposed to act by targeting the degradation of specific RNAs through an RNase H-mediated mechanism and consists of C-5 propynyl pyrimidine phosphorothioate ONs (propyne-S-ON). The second class of antisense agents has been proposed to function by sterically blocking target RNA formation, transport or translation and includes sugar modified (2'-O-allyl) ONs and peptide nucleic acids (PNAs). Using a CV-1 cell based microinjection assay, we targeted antisense agents representing both classes to various cloned sequences localized within the SV40 large T antigen RNA. We determined the propyne-S-ON was the most potent and gene-specific agent of the two classes which likely reflected its ability to allow RNase H cleavage of its target. The PNA oligomer inhibited T Ag expression via an antisense mechanism, but was less effective than the propyne-S-ON; the lack of potency may have been due in part to the PNAs slow kinetics of RNA association. Interestingly, unlike the 2'-O-allyl ON, the antisense activity of the PNA was not restricted to the 5' untranslated region of the T Ag RNA. Based on these findings we conclude that PNAs could be effective antisense agents with additional chemical modification that will lead to more rapid association with their RNA target.

To delineate the mechanisms of bovine leukemia virus (BLV) pathogenesis, four full-length BLV clones, 1, 8, 9, and 13, derived from the transformed cell line FLK-BLV and a clone construct, pBLV913, were introduced into bovine spleen cells by microinjection. Microinjected cells exhibited cytopathic effects and produced BLV p24 and gp51 antigens and infectious virus. The construct, pBLV913, was selected for infection of two sheep by inoculation of microinjected cells. After 15 months, peripheral blood mononuclear cells from these sheep served as inocula for the transfer of infection to four additional sheep. All six infected sheep seroconverted to BLV and had detectable BLV DNA in peripheral blood mononuclear cells after amplification by polymerase chain reaction. Four of the six sheep developed altered B/T-lymphocyte ratios between 33 and 53 months postinfection. One sheep died of unrelated causes, and one remained hematologically normal. Two of the affected sheep developed B lymphocytosis comparable to that observed in animals inoculated with peripheral blood mononuclear cells from BLV-infected cattle. This expanded B-lymphocyte population was characterized by elevated expression of B-cell surface markers, spontaneous blastogenesis, virus expression in vitro, and increased, polyclonally integrated provirus. One of these two sheep developed lymphocytic leukemia-lymphoma at 57 months postinfection. Leukemic cells had the same phenotype and harbored a single, monoclonally integrated provirus but produced no virus after in vitro cultivation. The range in clinical response to in vivo infection with cloned BLV suggests an important role for host immune response in the progression of virus replication and pathogenesis.

The amazing pace of discovery within the field of molecular biology has expanded the understanding of as well as the potential for genetic engineering in laboratory and domestic animals. Recently, specific techniques have been perfected, including recombinant DNA, genetic cloning, analysis of gene expression, and the sequencing of nucleic acids and proteins. These molecular techniques combined with methods allowing transfer of the recombinant genes into living cells and fertilized eggs advent the production of transgenic mice and other animals, the application of which is unlimited.

Human diploid fibroblasts immortalized by SV40 T antigen provide an experimental system for studying the progression and synergism in transformation by secondary oncogenes. We have utilized the human fibroblast line HAL, which was immortalized with an orgin-defective SV40 genome encoding a temperature-sensitive T antigen, to study the cooperativity between SV40 T antigen and the ras oncogene in the progression of transformation. This study demonstrates that HAL cells possess characteristic growth patterns, requiring 10% serum, are anchorage dependent, and express a temperature-sensitive T antigen. HAL cells rely on the normal functioning of T antigen for continual growth and therefore do not proliferate at 39 degrees C. Three new derivatives of the HAL cell line were generated by microinjection of the ras oncogene. The cell line v-ras-HAL was derived by microinjection of HAL cells with v-Ha-ras DNA. The cell lines c-rasSVneo-HAL and c-rasLTRhygro-HAL were established by microinjection of HAL cells with the plasmids pSV2neoT24 or fpHVT24, respectively, wherein the ras gene is transcriptionally regulated by the cellular promoter and driven by either the SV40 enhancer or an upstream LTR enhancer. The three ras containing cell lines grow in reduced serum concentrations (0 to 5%), are anchorage independent, and express both T antigen and ras p21. The v-ras-HAL and c-rasSVneo-HAL cell lines are still dependent upon the normal functioning of T antigen for continual growth at 39 degrees C, however the c-rasLTRhygro-HAL cell line does proliferate at 39 degrees C in 10% serum-containing medium. Therefore, we propose that neither v-Ha-ras nor c-ras can replace T antigen at 39 degrees C; rather T antigen and ras cooperate in progressive stages of transformation of human fibroblasts.

Incubation of adherent cells derived from peripheral blood mononuclear cells of cattle naturally infected with bovine leukemia virus (BLV) led to the establishment of three, persistently infected, primary cell cultures. These cultures were obtained exclusively from animals exhibiting persistent lymphocytosis, and not from uninfected or infected, hematologically normal cattle. The cells contained monoclonally integrated, full length BLV provirus, indicating that each culture resulted from clonal expansion of a single cell. They expressed high levels of all BLV specific mRNAs and showed intracellular reactivity to antibodies directed to viral gag and env proteins. Viral particle morphogenesis was highly restricted as determined by low levels of reverse transcriptase activity in cell supernatants and the paucity of viral particles on the cell surface. Analysis of cellular antigenic determinants, using monoclonal antibodies to bovine leukocyte differentiation and major histocompatibility complex antigens, was inconclusive. Cytochemical, morphologic, and ultrastructural analyses were consistent with endothelial cells and they exhibited the distinctive functional capacity of endothelial cells derived from specialized postcapillary venules, which constitute sites of lymphocyte extravasation. These data suggest that infection of these endothelial cells may be involved in the development of persistent lymphocytosis in BLV-infected animals.

A series of independent proviral molecular clones of bovine immunodeficiency-like virus (BIV) obtained from a genomic library of BIV-infected bovine cell DNA were physically and biologically characterized. Heteroduplex mapping shows that two of these BIV clones (106 and 127) contain uninterrupted proviral sequences approximately 9.0 kb in length, flanked by nonhomologous bovine cellular sequences. Microinjection of purified DNA from BIV clone 106 or 127 into susceptible bovine cells produces virus-specific cytopathic effects, including syncytium induction, supernatant reverse transcriptase activity, and infectious virus particle formation, similar to the effects produced by parental virus stock. Using restriction enzyme mapping, it was determined that the two infectious clones share 13 of 14 sites mapped within the provirus; thus, based on this criterion, the two clones are nearly identical, with the exception of a single polymorphic site recognized in the 3' half of the genome. BIV appears to be an exogenous pathogenic virus, because Southern hybridization analyses detected no endogenous sequences related to BIV in DNA from a variety of uninfected bovine cells and tissues. Most of the BIV-related DNA found in cells 96 hr after infection is present as linear unintegrated viral DNA, although the presence of host flanking sequences in our proviral clones indicates that integration takes place. These biologically active clones of BIV will be of use in defining further the mechanisms of BIV pathogenesis and in engineering specific diagnostic reagents to determine the prevalence of BIV in cattle populations.

Several virion and nonvirion DNAs were tested for the ability to activate endogenous type C virus in BALB/c-derived mouse cells using the calcium precipitation technique. The DNAs from all herpesviruses tested activated xenotropic type C virus synthesis. These included DNAs from herpes simplex virus types 1 and 2, Epstein-Barr virus, human cytomegalovirus, SA8 virus, infectious bovine rhinotracheitis virus, pseudorabies virus, and herpes saimiri virus (M-DNA). In contrast, DNAs from vaccinia virus, simian virus 40, primate cells, bacteria, mycoplasma, and salmon sperm showed no ability to activate type C virus when tested under the same conditions. Several herpesviruses and vaccinia virus, which were highly infectious for the BALB/c cells used, were tested for their ability to activate type C virus after UV irradiation. All herpesviruses tested were positive, while vaccinia virus was negative. Unirradiated simian virus 40 also showed no ability to activate type C virus. Activation of type C virus by DNA from herpes simplex virus was observed after shearing or sonication of the DNA to an average size of 3 x 10(6) daltons, but was not observed with DNA sonicated to an average size of 1 x 10(6) daltons. Alkali denaturation of DNA from herpes simplex virus or treatment with DNase, but not RNase, destroyed its ability to activate type C virus, as did crosslinking of the DNA with 4,5',8-trimethylpsoralen (psoralen) and light.

A transformed mouse cell line (H238) was obtained following the infection of 238 mouse cells with ultraviolet (UV) irradiation-inactivated herpes simplex virus type 2 (HSV-2). The transformed cells produced tumors with a 100% incidence within 8 weeks in 6-week-old syngeneic BALB/c mice at an inoculum of 1 times 10(6). Indirect immunofluorescence (IF) tests revealed the presence of HSV antigens in the transformed cells. Antibodies to HSV-2 were found in the sera of tumor-bearing animals by neutralization and IF techniques. Neither HSV-2 infectious virus nor viral antigens could be detected by the transfer of transformed-cell DNA into permissive cells.

Water samples from the Monocacy River in Frederick County, Maryland, yielded twenty-four isolates which were resistant to tetracycline (TeR, 25 micrograms/ml). Although these organisms were not initially cultured on a coliform-selective medium, twenty-two of the isolates were Gram-negative and carriers of antibiotic resistance to five or more antibiotics; erythromycin, methicillin, novobiocin, penicillin and tetracycline. Of the isolates 45% were biochemically identified as Providencia stuartii; one isolate which contained a 29.4 kilobase plasmid carried the determinant for tetracycline resistance.

Treatment with dioctyltin dichloride (DOTC) induced increased mortality, atrophy of the thymus and suppression of responses to concanavalin A. Brown Norway (BN) rats were more susceptible than Lewis (LEW) to the lethal effects of DOTC and males were more susceptible than females. Increased mortality did not correlate with changes in the histological appearance of the thymus. LEW X BN F1 hybrids resembled the LEW parent in susceptibility to the lethal effects of DOTC and the BN parent in susceptibility to changes in the thymus.

BALB/c-derived 10E2 cells were made thymidine kinase(TK)-negative and one isolated clone (B2) was used for studying morphological and biochemical transformations by herpes simplex virus (HSV) type 1 (strain 10412). The B2 cells displayed a "normal" flat appearance and were nontumorigenic in nude mice when tested at frequent intervals over a period of 45 subcultures. B2 cells infected with UV-irradiated HSV (UV-HSV) and maintained in normal growth medium showed foci of spindle-shaped cells after one subculture. The cells from these morphologically transformed foci were tumorigenic in nude mice and were TK negative. B2 cells infected with UV-HSV or transfected with the HSV-1 TK gene and maintained in TK-selective medium showed discrete colonies of cells which displayed a normal flat appearance and expressed the viral TK enzyme. These biochemically transformed B2 cells were nontumorigenic in nude mice. The findings with B2 cells indicate that biochemical and morphological transformations by HSV-1 are independent events and suggest that the HSV-1 TK gene is a suitable vehicle for introducing non-TK genes into cells to assess their transforming potential.

An infectious proviral clone of the human immunodeficiency virus (HIV) was microinjected into the cell nucleus in six cell lines derived from caprine, ovine, bovine, or human solid tissue to study the utility of this method in effecting viral gene expression in nonlymphoid cells. Immunofluorescence assays for HIV demonstrated viral gene expression in only 5% of cells (100-200 cells per line) 24-48 h after microinjection; however, no reverse transcriptase activity was detectable, presumably due to a low level of virus release in this limited number of cells. Therefore, to indirectly assess infectious virus release, microinjected cells were cocultured with human T4 antigen-positive lymphocytes (H9) sensitive to HIV infection. Syncytia formation, electron microscopy, reverse transcriptase activity, and radioimmunoassay for HIV p24 were used to monitor viral gene expression in cocultures. HIV was efficiently recovered by cocultivating H9 with microinjected cells 48 h after microinjection, regardless of the tissue type or species of origin. H9 syncytia were visualized in some cocultures as early as day 5 but were readily apparent in all experiments on days 7-10. Syncytia induction in H9 was the earliest and most reliable indicator of infectious virus release. A recombinant construct containing a subgenomic envelope gene derived from the proviral clone of HIV was microinjected into human glioblastoma cells. Twenty-four to 48 h after manipulation, 5-20% of microinjected cells were found by immunofluorescence assay to express low levels of a putative gp120. These results suggest a possible approach to producing virus-free HIV envelope antigens in mammalian cells and may be relevant to subunit vaccine development.

Murine and hamster cell lines, each transformed with a different strain of herpes simplex virus (HSV), were examined for cross-reacting antigens by in vitro and in vivo assays. A comparative study by the indirect immunofluorescence technique detected common cross-reacting viral antigens. Cytoplasmic fluorescence patterns were observed in the 333-8-9 hamster line, the H238 murine line, and the H238 clonal lines; these patterns were identical to the fluorescence pattern of HSV -2-infected controls when reacted with HSV antiserum. Tumor rejection studies in the BALB/c host indicated that each cell line provided immunity against a tumorigenic challenge of transformed mouse cells. The H238 clone EC1 3 provided a 53% immunity against itself at an inoculum of 10(6); the 333-8-9 line supported a 26% immunity. These data demonstrate a common HSV antigenicity between the murine and hamster transformed lines and further indicate that the HSV genome is involved in transformation.

A BALB/c continuous cell line, 238, was transformed following infection with UV-inactivated herpes simplex virus type 2 (HSV-2) was designated H238. Several cell lines were derived from single cells of H238 by cloning in soft agar. The clonal lines express cytoplasmic antigens by immunofluorescence when reacted with rabbit HSV-2 antiserum and mouse tumor-bearer serum. The cloned cell lines induce tumors in immunocompetent mice by the subcutaneous route with a 100% incidence at an inoculum of 10(6) cells. These properties are consistent with those of the H238 parent cell line. In contrast to the parent line, however, the cloned cell lines do not elicit HSV neutralizing antibody in the tumor-bearing host.