Timothy A. Steele

Human reserve pluripotent mesenchymal stem cells are present in the connective tissues of skeletal muscle and dermis derived from fetal, adult, and geriatric donors

This study details the profile of 13 cell surface cluster differentiation markers on human reserve stem cells derived from connective tissues. Stem cells were isolated from the connective tissues of dermis and skeletal muscle derived from fetal, mature, and geriatric humans. An insulin/dexamethasone phenotypic bioassay was used to determine the identity of the stem cells from each population. All populations contained lineage‐committed myogenic, adipogenic, chondrogenic, and osteogenic progenitor stem cells as well as lineage‐uncommitted pluripotent stem cells capable of forming muscle, adipocytes, cartilage, bone, fibroblasts, and endothelial cells. Flow cytometric analysis of adult stem cell populations revealed positive staining for CD34 and CD90 and negative staining for CD3, CD4, CD8, CD11c, CD33, CD36, CD38, CD45, CD117, Glycophorin‐A, and HLA DR‐II. Anat Rec 264:51–62, 2001.

Adult reserve stem cells and their potential for tissue engineering

Tissue restoration is the process whereby multiple damaged cell types are replaced to restore the histoarchitecture and function to the tissue. Several theories, have been proposed to explain the phenomenon of tissue restoration in amphibians and in animals belonging to higher order. These theories include dedifferentiation of damaged tissues, transdifferentiation of lineage-committed progenitor cells, and activation of reserve, precursor cells. Studies by Young et al. and others demonstrated that connective tissue compartments throughout postnatal individuals contain reserve precursor cells. Subsequent repetitive single cell-cloning and cell-sorting studies revealed that these reserve precursor cells consisted of multiple populations of cells, including, tissue-specific progenitor cells, germ-layer lineage stem cells, and pluripotent stem cells. Tissue-specific progenitor cells display various capacities for differentiation, ranging from unipotency (forming a single cell type) to multipotency (forming multiple cell types). However, all progenitor cells demonstrate a finite life span of 50 to 70 population doublings before programmed cell senescence and cell death occurs. Germ-layer lineage stem cells can form a wider range of cell types than a progenitor cell. An individual germ-layer lineage stem cell can form all cells types within its respective germ-layer lineage (i.e., ectoderm, mesoderm, or endoderm). Pluripotent stem cells can form a wider range of cell types than a single germ-layer lineage stem cell. A single pluripotent stem cell can form cells belonging to all three germ layer lineages. Both germ-layer lineage stem cells and pluripotent stem cells exhibit extended capabilities for self-renewal, far surpassing the limited life span of progenitor cells (50–70 population doublings). The authors propose that the activation of quiescent tissue-specific progenitor cells, germ-layer lineage stem cells, and/or pluripotent stem cells may be a potential explanation, along with dedifferentiation and transdifferentiation, for the process of tissue restoration. Several model systems are currently being investigated to determine the possibilities of using these adult quiescent reserve precursor cells for tissue engineering.

Phosphatidylinositol metabolism accompanies early activation events in tumor target cell-stimulated human natural killer cells

This study examined the role of phospholipid metabolism in human natural killer (NK) cells upon activation by tumor target cells(TC). The effector cell (EC) population consisted of peripheral blood lymphocytes enriched for NK cells. Upon a 5-min exposure of EC to the NK-sensitive tumor TC K562 and U937, nearly four- and threefold increases in the incorporation of 32P into phosphatidylinositol (PI) occurred, respectively. In contrast, no increase in 32P incorporation into PI was seen when two NK-resistant TC were used. In addition, little or no change in the incorporation of 32P into phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine took place with any of the above TC. Depletion of Leu 11b-positive cells abolished the increase in 32P incorporation into PI when K562 were used in the phospholipid assay. Furthermore, labeling kinetics of this phospholipid turnover showed that it occurred less than 5 min following exposure to NK-sensitive TC and that phosphatidic acid, a breakdown product of phosphoinositides, was produced during this 5-min period. These results indicated that metabolism of a phosphoinositide took place and that it occurred in association with early activation events in NK cells. Quercetin and dibutyryladenosine-cyclic monophosphate (dbcAMP) plus theophylline exerted profound inhibitory effects on both NK activity and PI metabolism, suggesting a linkage between the two events. The inhibitors had no effect on target cell-binding capacity, indicating that the inhibition occurred postbinding. PI metabolism took place in the absence of extracellular calcium even though NK activity was completely abolished under the same conditions. Thus, we have shown PI metabolism, but not other phospholipids, to occur in human NK cells upon exposure to NK-sensitive TC, in association with early activation events. This event was independent of extracellular calcium and could be inhibited by quercetin or dbcAMP plus theophylline.

Chemotherapy-induced immunosuppression and reconstitution of immune function

In the cancer patient, there are a variety of factors that favor the suppression of the immune system, including the cancer itself (especially, malignancies of B and T lymphocytes), high dose chemotherapeutic agents, and corticosteroids. In addition, by its very nature, the myeloablative conditioning regimen used for stem-cell transplantation is immunosuppressive. It is important that immune function is restored so that the patient can resist infection, but it may be equally important to reconstitute immunity due to the potential added benefit of an anti-leukemic response. It is the intent of this commentary/review to discuss the effects of chemotherapy on selected immune cell populations and approaches to reconstitute immune function following chemotherapy. In this issue, Slater et al. [1] illustrate the double-edged sword of high dose chemotherapy. Their laboratory investigations demonstrate the cytotoxic efficacy, yet immunosuppressive effects, of high dose topoisomerase II inhibitor VP-16. As a result, treating L1210 tumor-bearing mice with 20 mg/kg VP-16 resulted in a relatively high 60 days survival rate, however, cured mice could not resist a L1210 tumor challenge. In contrast, when tumor-bearing mice were treated with 3 or 5 mg/kg VP-16, correspondingly fewer mice survived, but most survivors were able to reject a tumor challenge. Mitogenic assays revealed a suppressed proliferation potential of splenic lymphocytes treated with the higher dose of VP-16. These investigations exemplify the opposing effects of high dose chemotherapy, i.e. tumor cytotoxic efficacy coupled with immunosuppression. In discussing the effects of chemotherapy on immune function, it is important to differentiate between the terms immunosuppression and myelosuppression [2]. Many chemotherapeutic agents are myelosuppressive in that they inhibit bone marrow production of blood cells and platelets

Desferrioxamine treatment increases the genomic stability of Ataxia-telangiectasia cells

Ataxia-telangiectasia (AT) is an autosomal recessive disorder characterized by genomic instability, chronic oxidative damage, and increased cancer incidence. Compared to normal cells, AT cells exhibit unusual sensitivity to exogenous oxidants, including t-butyl hydroperoxide (t-BOOH). Since ferritin releases labile iron under oxidative stress (which is chronic in AT) and labile iron mediates the toxic effects of t-butyl hydroperoxide, we hypothesized that chelation of intracellular labile iron would increase the genomic stability of AT cells, with and without exogenous oxidative stress. Here we report that desferrioxamine treatment increases the plating efficiency of AT, but not normal cells, in the colony forming-efficiency assay (a method often used to measure genomic stability). Additionally, desferrioxamine increases AT, but not normal cell resistance, to t-butyl hydroperoxide in this assay. Last, AT cells exhibit increased sensitivity to the toxic effects of FeCl(2) in the colony forming-efficiency assay and fail to demonstrate a FeCl(2)-induced G(2) checkpoint response when compared to normal cells. Our data indicates that: (1) chelation of labile iron increases genomic stability in AT cells, but not normal cells; and (2) AT cells exhibit deficits in their responses to iron toxicity. While preliminary, our findings suggest that AT might be, in part, a disorder of iron metabolism and treatment of individuals with AT with desferrioxamine might have clinical efficacy.

Chlorpromazine inhibits human natural killer cell activity and antibody-dependent cell-mediated cytotoxicity

The phenothiazine antipsychotic drug chlorpromazine, a potent calmodulin antagonist, inhibited human natural killer cell-mediated cytotoxicity in a dose-dependent manner. This inhibition of natural killer cell activity was not due to interference with conjugate formation, nor was it due to toxicity to the natural killer cells or nonspecific alteration of the target cells. Chlorpromazine also suppressed antibody-dependent cell-mediated cytotoxicity in a dose-dependent fashion, suggesting that the two activities share a calmodulin-dependent step. Finally, it was not possible to overcome chlorpromazine-mediated inhibition of natural killer cell activity by first treating the cells with interleukin-2 or Beta-interferon.

Statins inhibit proliferation and cytotoxicity of a human leukemic natural killer cell line

BackgroundNatural killer cells comprise the body’s first line of defense against virus-infected cells. As is true of all lymphocytes, natural killer cell malignancies can develop, however natural killer cell leukemias can be very difficult to treat due to their intrinsic resistance to chemotherapeutic agents. With the recent understanding that statin drugs may have anti-cancer properties, our investigations have focused on the ability of statins to inhibit the growth and cytotoxicity of the YT-INDY natural killer cell leukemia cell line.ResultsOur findings indicate that several statin compounds can inhibit YT-INDY proliferation disrupt cell cycle progression and abrogate natural killer cell cytotoxicity. Since natural killer cell leukemia cytotoxicity may play a role in the pulmonary damage seen in these patients, this is an important finding. Cytotoxicity, proliferation and cell cycle progression could be restored by the addition of mevalonate, signifying that the statin effects are brought about through HMG CoA reductase inhibition. The mevalonate pathway intermediate geranylgeranyl pyrophosphate, but not other intermediates in the mevalonate pathway, partially reversed statin-induced inhibition of YT-INDY proliferation and cytotoxicity. These results suggest that blockage of products made in the latter part of the mevalonate pathway may account for the observed inhibitory effects on YT-INDY proliferation and cytotoxicity. However, geranylgeranyl pyrophosphate could not reverse the statin-induced inhibition of the cell cycle.ConclusionsThese results suggest that the statin drugs should be investigated as a potential therapeutic strategy for human natural killer cell leukemias possibly in combination with chemotherapeutic agents.

Elimination of EL-4 and L1210 Murine Tumors by 1,3-bis (2-Chloroethyl)-1-Nitrosourea Requires an Intact Immune Response:

Abstract The chemotherapeutic agent 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) is commonly used to treat several types of human cancers. Recent investigations have suggested that elimination of tumor by BCNU is dependent on more than the cytotoxic activity of the drug. We have extended those findings by showing that cyclosporin A (CS) can inhibit the BCNU-mediated rejection of EL-4 or L1210 tumors in mice. It was shown that mice could be cured of EL-4 or L1210 ascites tumors with a single intraperitoneal injection of BCNU. When CS, an inhibitor of the activation of T lymphocytes, was administered to mice that had received either EL-4 or L1210 tumor and were treated with BCNU, nearly all the mice died by Day 60. When CS was administered to BCNU-treated mice starting at 1, 2, or 3 weeks after tumor injection, inhibition of the BCNU therapy did not occur. Finally, the ability of animals that had been cured of tumor by the BCNU therapy to reject a lethal challenge dose of homologous tumor was shown to be CS insensitive. These results suggest that the BCNU-mediated elimination of tumor from mice requires a functional immune response in addition to the cytotoxic activity of this chemotherapeutic agent.

The Role of Interferon- α in a Successful Murine Tumor Therapy

Combination therapy using reovirus type 3 and the chemotherapeutic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) is sufficient to cure approximately 80% of EL-4 lymphoma tumor–bearing BD2F1 male mice. Cured animals can be challenged with the EL-4 tumor, in the absence of the therapy, to yield 100% survival, whereas those challenged with heterologous tumor produce 0% survival. These results strongly suggest that a host-immune response is responsible for the observed therapeutic effect. Reovirus, a double-stranded RNA virus, is an efficient inducer of type I interferon. In an effort to determine the role of virus in this therapy, we substituted interferon-a (IFN-α) for reovirus in the therapy. Doses of IFN-α from 1000–10,000 U were capable of replacing reovirus to produce cure rates similar to reovirus. Spleen cells isolated from therapy-treated animals demonstrated high levels of cytotoxicity against the natural killer cell–sensitive cell line YAC-1, but not against EL-4 tumor. In vitro stimulation of isolated spleen cells by IFN-α resulted in a high level of natural killer cell activity, but no cytotoxicity against the EL-4 tumor. A significant antiproliferative effect against the EL-4 tumor in cell culture was demonstrated by IFN-α, Finally, therapy-treated, tumor-bearing mice that were injected with anti–IFN-α + -β antibodies had similar survival levels as control mice, indicating that other cytokines might also play a role in promoting tumor killing. These investigations suggest that IFN-α may be a mediator of antitumor activity in the reovirus therapy system.

Possible Role of Retinoids in Hepatitis B Virus-Associated Liver Damage

Liver damage following hepatitis B virus (HBV) infection may be due to the action of retinoids as modulators of viral replication. The reduced rate of survival of liver grafts in patients with HBV infection could also be due to the continued presence of the virus, stimulated by retinoids in the graft tissue. Subject to obtaining empirical support for this hypothesis, the use of retinoid-blocking agents could be explored to reduce the risk of liver damage in HBV infection and to enhance the survival of liver grafts. Continued use of such agents may need to be used in conjunction with anti-viral modalities such as HBV hyperimmune globulin and lamivudine to prevent recurrent liver damage and to increase the long-term viability of the graft.