David A. Hess

Bone marrow-derived stem cells initiate pancreatic regeneration.

We show that transplantation of adult bone marrow–derived cells expressing c-kit reduces hyperglycemia in mice with streptozotocin-induced pancreatic damage. Although quantitative analysis of the pancreas revealed a low frequency of donor insulin-positive cells, these cells were not present at the onset of blood glucose reduction. Instead, the majority of transplanted cells were localized to ductal and islet structures, and their presence was accompanied by a proliferation of recipient pancreatic cells that resulted in insulin production. The capacity of transplanted bone marrow–derived stem cells to initiate endogenous pancreatic tissue regeneration represents a previously unrecognized means by which these cells can contribute to the restoration of organ function.

High aldehyde dehydrogenase and expression of cancer stem cell markers selects for breast cancer cells with enhanced malignant and metastatic ability.

Cancer stem cells (CSCs) have recently been identified in leukaemia and solid tumours; however, the role of CSCs in metastasis remains poorly understood. This dearth of knowledge about CSCs and metastasis is due largely to technical challenges associated with the use of primary human cancer cells in pre‐clinical models of metastasis. Therefore, the objective of this study was to develop suitable pre‐clinical model systems for studying stem‐like cells in breast cancer metastasis, and to test the hypothesis that stem‐like cells play a key role in metastatic behaviour. We assessed four different human breast cancer cell lines (MDA‐MB‐435, MDA‐MB‐231, MDA‐MB‐468, MCF‐7) for expression of prospective CSC markers CD44/CD24 and CD133, and for functional activity of aldehyde dehydrogenase (ALDH), an enzyme involved in stem cell self‐protection. We then used fluorescence‐activated cell sorting and functional assays to characterize differences in malignant/metastatic behaviour in vitro (proliferation, colony‐forming ability, adhesion, migration, invasion) and in vivo (tumorigenicity and metastasis). Sub‐populations of cells demonstrating stem‐cell‐like characteristics (high expression of CSC markers and/or high ALDH) were identified in all cell lines except MCF‐7. When isolated and compared to ALDHlowCD44low/– cells, ALDHhiCD44+CD24− (MDA‐MB‐231) and ALDHhiCD44+CD133+ (MDA‐MB‐468) cells demonstrated increased growth (P < 0.05), colony formation (P < 0.05), adhesion (P < 0.001), migration (P < 0.001) and invasion (P < 0.001). Furthermore, following tail vein or mammary fat pad injection of NOD/SCID/IL2γ receptor null mice, ALDHhiCD44+CD24− and ALDHhiCD44+CD133+ cells showed enhanced tumorigenicity and metastasis relative to ALDHlowCD44low/– cells (P < 0.05). These novel results suggest that stem‐like ALDHhiCD44+CD24− and ALDHhiCD44+CD133+ cells may be important mediators of breast cancer metastasis.

Revascularization of ischemic limbs after transplantation of human bone marrow cells with high aldehyde dehydrogenase activity

The development of cell therapies to treat peripheral vascular disease has proven difficult because of the contribution of multiple cell types that coordinate revascularization. We characterized the vascular regenerative potential of transplanted human bone marrow (BM) cells purified by high aldehyde dehydrogenase (ALDH(hi)) activity, a progenitor cell function conserved between several lineages. BM ALDH(hi) cells were enriched for myelo-erythroid progenitors that produced multipotent hematopoietic reconstitution after transplantation and contained nonhematopoietic precursors that established colonies in mesenchymal-stromal and endothelial culture conditions. The regenerative capacity of human ALDH(hi) cells was assessed by intravenous transplantation into immune-deficient mice with limb ischemia induced by femoral artery ligation/transection. Compared with recipients injected with unpurified nucleated cells containing the equivalent of 2- to 4-fold more ALDH(hi) cells, mice transplanted with purified ALDH(hi) cells showed augmented recovery of perfusion and increased blood vessel density in ischemic limbs. ALDH(hi) cells transiently recruited to ischemic regions but did not significantly integrate into ischemic tissue, suggesting that transient ALDH(hi) cell engraftment stimulated endogenous revascularization. Thus, human BM ALDH(hi) cells represent a progenitor-enriched population of several cell lineages that improves perfusion in ischemic limbs after transplantation. These clinically relevant cells may prove useful in the treatment of critical ischemia in humans.

Widespread nonhematopoietic tissue distribution by transplanted human progenitor cells with high aldehyde dehydrogenase activity

Transplanted adult progenitor cells distribute to peripheral organs and can promote endogenous cellular repair in damaged tissues. However, development of cell‐based regenerative therapies has been hindered by the lack of preclinical models to efficiently assess multiple organ distribution and difficulty defining human cells with regenerative function. After transplantation into β‐glucuronidase (GUSB)‐deficient NOD/SCID/mucopolysaccharidosis type VII mice, we characterized the distribution of lineage‐depleted human umbilical cord blood‐derived cells purified by selection using high aldehyde dehydrogenase (ALDH) activity with CD133 coexpression. ALDHhi or ALDHhiCD133+ cells produced robust hematopoietic reconstitution and variable levels of tissue distribution in multiple organs. GUSB+ donor cells that coexpressed human leukocyte antigen (HLA‐A,B,C) and hematopoietic (CD45+) cell surface markers were the primary cell phenotype found adjacent to the vascular beds of several tissues, including islet and ductal regions of mouse pancreata. In contrast, variable phenotypes were detected in the chimeric liver, with HLA+/CD45+ cells demonstrating robust GUSB expression adjacent to blood vessels and CD45−/HLA− cells with diluted GUSB expression predominant in the liver parenchyma. However, true nonhematopoietic human (HLA+/CD45−) cells were rarely detected in other peripheral tissues, suggesting that these GUSB+/HLA−/CD45− cells in the liver were a result of downregulated human surface marker expression in vivo, not widespread seeding of nonhematopoietic cells. However, relying solely on continued expression of cell surface markers, as used in traditional xenotransplantation models, may underestimate true tissue distribution. ALDH‐expressing progenitor cells demonstrated widespread and tissue‐specific distribution of variable cellular phenotypes, indicating that these adult progenitor cells should be explored in transplantation models of tissue damage.

Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization.

Transplanted murine bone marrow (BM) progenitor cells recruit to the injured pancreas and induce endogenous beta cell proliferation to improve islet function. To enrich for analogous human progenitor cell types that stimulate islet regeneration, we purified human BM based on high-aldehyde dehydrogenase activity (ALDH(hi)), an enzymatic function conserved in hematopoietic, endothelial, and mesenchymal progenitor lineages. We investigated the contributions of ALDH(hi) mixed progenitor cells or culture-expanded, ALDH-purified multipotent stromal cell (MSC) subsets to activate endogenous programs for islet regeneration after transplantation into streptozotocin-treated NOD/SCID mice. Intravenous injection of uncultured BM ALDH(hi) cells improved systemic hyperglycemia and augmented insulin secretion by increasing islet size and vascularization, without increasing total islet number. Augmented proliferation within regenerated endogenous islets and associated vascular endothelium indicated the induction of islet-specific proliferative and pro-angiogenic programs. Although cultured MSC from independent human BM samples showed variable capacity to improve islet function, and prolonged expansion diminished hyperglycemic recovery, transplantation of ALDH-purified regenerative MSC reduced hyperglycemia and augmented total beta cell mass by stimulating the formation of small beta cell clusters associated with the ductal epithelium, without evidence of increased islet vascularization or Ngn3(+) endocrine precursor activation. Thus, endogenous islet recovery after progenitor cell transplantation can occur via distinct regenerative mechanisms modulated by subtypes of progenitor cells administered. Further, understanding of how these islet regenerative and pro-angiogenic programs are activated by specific progenitor subsets may provide new approaches for combination cellular therapies to combat diabetes.

Cytotoxicity of sulfonamide reactive metabolites: apoptosis and selective toxicity of CD8+ cells by the hydroxylamine of sulfamethoxazole

Treatment with sulfonamide antibiotics in HIV‐infected patients is associated with a high incidence (> 40%) of adverse drug events, including severe hypersensitivity reactions. Sulfonamide reactive metabolites have been implicated in the pathogenesis of these adverse reactions. Sulfamethoxazole hydroxylamine (SMX‐HA) induces lymphocyte toxicity and suppression of proliferation in vitro; the mechanism(s) of these immunomodulatory effects remain unknown. We investigated the cytotoxicity of SMX‐HA via apoptosis on human peripheral blood mononuclear cells and purified cell subpopulations in vitro. CD19+, CD4+, and CD8+ cells were isolated from human peripheral blood by positive selection of cell surface molecules by magnetic bead separation. SMX‐HA induced significant CD8+ cell death (67 ± 7%) at 100 µM SMX‐HA, with only minimal CD4+ cell death (8 ± 4%). No significant subpopulation toxicity was shown when incubated with parent drug (SMX). Flow cytometry measuring phosphatidylserine externalization 24 h after treatment with 100 µM and 400 µM SMX‐HA revealed 14.1 ± 0.7% and 25.6 ± 4.2% annexin‐positive cells, respectively, compared to 3.7 ± 1.2% in control PBMCs treated with 400 µM SMX. Internucleosomal DNA fragmentation was observed in quiescent and stimulated PBMCs 48 h after incubation with SMX‐HA. Our data show that CD8+ cells are highly susceptible to the toxic effects of SMX‐HA through enhanced cell death by apoptosis.—Hess, D. A., Sisson, M. E., Suria, H., Wijsman, J., Puvanesasingham, R., Madrenas, J., Rieder, M. J. Cytotoxicity of sulfonamide reactive metabolites: apoptosis and selective toxicity of CD8+ cells by the hydroxylamine of sulfamethoxazole. FASEB J. 13, 1688–1698 (1999)

The Role of Reactive Drug Metabolites in Immune-Mediated Adverse Drug Reactions

OBJECTIVE: To highlight recent advances in the understanding of adverse drug reactions (ADRs), with a focus on models outlining interactions between drug metabolism, disease processes, and immunity. Specific mechanisms that identify the metabolic pathways responsible for drug bioactivation to reactive drug metabolites (RDMs) involved in the initiation and propagation of specific immune-mediated hypersensitivity reactions are discussed. Drug classes well known to be associated with immune-mediated ADRs are reviewed and the clinical implications of current research are discussed. DATA SOURCES: Original experimental research and immunologic review articles relevant to ADR diagnosis and etiology. DATA EXTRACTION: Results of relevant in vitro experiments and clinical reactions to drug therapy were compiled and reviewed. Critical discoveries concerning the identification of RDMs involved in ADRs were highlighted, with respect to RDM involvement in the production of an immune response to drug haptens. DATA SYNTHESIS: Drug adverse effects are classified according to clinical characteristics, immune interactions, and mechanistic similarities. Cytochrome P450 bioactivation of drug molecules to RDMs is a prerequisite to many ADRs. An electrophilic metabolite may react with cellular macromolecules (i.e., lipids, proteins, nucleic acids), resulting in direct cellular damage and organ toxicity. Covalent binding of an RDM to cellular macromolecules may also result in the formation of a hapten that is capable of eliciting a cellular or humoral immune response against drug or protein epitopes, culminating in the characteristic symptoms of hypersensitivity reactions. Mechanistic details concerning the identification of stable protein-metabolite conjugates and their interaction with the immune system remain unclear. Genetic imbalance between bioactivation and detoxification pathways, as well as reduced cellular defense against RDMs due to disease or concomitant drug therapy, act as risk factors to the onset and severity of ADRs. CONCLUSIONS: Adverse reactions to drug therapy cause significant morbidity and mortality. Identification of the pathways involved in drug bioactivation and detoxification may elucidate the potential of chemical agents to induce immune-mediated ADRs. Understanding the mechanisms of ADRs to current xenobiotics is helpful in the prevention and management of ADRs, and may prove useful in the design of novel therapeutic agents with reduced incidence of severe adverse events.

Human progenitor cells with high aldehyde dehydrogenase activity efficiently engraft into damaged liver in a novel model

Human cord blood stem cells (hCBSCs) have been reported to generate hepatocyte‐like cells and thus hold promise for repairing damaged liver. However, the frequency of hCBSC‐derived hepatocytes varies tremendously between different studies, and it is still controversial as to whether hCBSC‐derived cells can transdifferentiate into hepatocytes or simply fuse to recipient hepatocytes. We used the β‐glucuronidase–deficient nonobese diabetic/severe combined immunodeficient/mucopolysaccharidosis type VII (NOD/SCID/MPSVII) mouse model for better identification of engrafted cells. We transplanted lineage‐depleted human umbilical cord blood–derived cells with high aldehyde dehydrogenase activity (ALDHhiLin−) into irradiated NOD/SCID/MPSVII mice followed by carbon tetrachloride administration to induced liver damage. ALDHhiLin− cells were efficiently engrafted in the recipient mouse livers and improved recovery of the mice from toxic insult. The percentage of human cells in these livers ranged between 3% and 14.2% using quantitative real‐time polymerase chain reaction. Furthermore, human‐originated cells expressing liver‐specific α1‐antitrypsin messenger RNA, albumin and hepatocyte nuclear factor 1 protein were detected in the recipient livers. Interestingly, human versus murine centromeric fluorescent in situ hybridization analysis on the liver sections demonstrated that most human cells were not fused to mouse cells. However, the majority of the human originated albumin‐expressing cells also carried mouse genetic material, hence were the product of cell fusion. Conclusion: hCBSCs or their progeny may home to the injured liver and release trophic factors that hasten tissue repair, whereas fusion of these cells with hepatocytes may occur rarely and contribute to a lesser extent to liver repair. (HEPATOLOGY 2009.)

Pretreatment with glucocorticoids enhances T-cell effector function: possible implication for immune rebound accompanying glucocorticoid withdrawal.

Glucocorticoids (GCs) exert their immunosuppressive/antiproliferative effects largely through inhibition of cytokine expression, and paradoxically upregulate the expression of (proinflammatory) cytokine receptors on select nonlymphoid cells. Clinically, withdrawal of GCs was frequently associated with worsening of the outcome of heightened immunity disorders, thereby implicating enhanced cytokine and cytokine receptor expression as a possible consequence of acute/short-term GCs withdrawal. In view of the significance of this complication of GC therapy, we addressed the effect of GC withdrawal on cytokine receptor expression and subsequent T-cell effector function, using the proliferation of human T cells as biological readout. To mimic GC withdrawal, T cells were treated with GCs or controls, stimulated, and incubated for 16–20 h at 37°C, washed, and reactivated for a further 4–48 h. Surface marker expression was assessed by FACS analysis, and proliferation was determined by measuring the cellular uptake of tritiated thymidine. Dexamethasone (DEX) and prednisolone (PRED), in a concentration-dependent manner, inhibited T-cell proliferation induced by anti-CD28 Ab + PMA. However, pretreatment of T cells activated with mitogens, cross-linking antibodies, or PMA + ionomycin (“CD3-bypass” stimulation regimen), but not resting T cells, with DEX or PRED resulted in a marked increase in IL-1R, IL-2Rα, and IL-6R expression, which was accompanied by a significant enhancement in T-cell proliferation. This effect of GCs was neither stimulus specific nor did it result from alteration in cell viability, and was paralleled by augmentation in cytokine (rIL-2) effects on DEX-pretreated and preactivated T cells. Taken together, our results underline the dual effects of GCs in regulating T-cell activation and cytokine expression. In essence, GCs directly inhibited T-cell proliferation by suppressing cytokine production, and, by enhancing cytokine receptor expression, pretreatment with GCs augmented T-cell proliferation.

Human cord blood progenitors with high aldehyde dehydrogenase activity improve vascular density in a model of acute myocardial infarction

Human stem cells from adult sources have been shown to contribute to the regeneration of muscle, liver, heart, and vasculature. The mechanisms by which this is accomplished are, however, still not well understood. We tested the engraftment and regenerative potential of human umbilical cord blood-derived ALDHhiLin-, and ALDHloLin- cells following transplantation to NOD/SCID or NOD/SCID β2m null mice with experimentally induced acute myocardial infarction. We used combined nanoparticle labeling and whole organ fluorescent imaging to detect human cells in multiple organs 48 hours post transplantation. Engraftment and regenerative effects of cell treatment were assessed four weeks post transplantation. We found that ALDHhiLin- stem cells specifically located to the site of injury 48 hours post transplantation and engrafted the infarcted heart at higher frequencies than ALDHloLin- committed progenitor cells four weeks post transplantation. We found no donor derived cardiomyocytes and few endothelial cells of donor origin. Cell treatment was not associated with any detectable functional improvement at the four week endpoint. There was, however, a significant increase in vascular density in the central infarct zone of ALDHhiLin- cell-treated mice, as compared to PBS and ALDHloLin- cell-treated mice.ConclusionsOur data indicate that adult human stem cells do not become a significant part of the regenerating tissue, but rapidly home to and persist only temporarily at the site of hypoxic injury to exert trophic effects on tissue repair thereby enhancing vascular recovery.