Combination therapy using reovirus type 3 and the chemo-therapeutic 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-alpha (IFN-alpha) for reovirus in the therapy. Doses of IFN-alpha 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-alpha 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-alpha. Finally, therapy-treated, tumor-bearing mice that were injected with anti-IFN-alpha + -beta 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-alpha may be a mediator of antitumor activity in the reovirus therapy system.

Undifferentiated cells have been identified in the prenatal blastocyst, inner cell mass, and gonadal ridges of rodents and primates, including humans. After isolation these cells express molecular and immunological markers for embryonic cells, capabilities for extended self-renewal, and telomerase activity. When allowed to differentiate, embryonic stem cells express phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. When implanted in vivo, undifferentiated noninduced embryonic stem cells formed teratomas. In this report we describe a cell clone isolated from postnatal rat skeletal muscle and derived by repetitive single-cell clonogenic analysis. In the undifferentiated state it consists of very small cells having a high ratio of nucleus to cytoplasm. The clone expresses molecular and immunological markers for embryonic stem cells. It exhibits telomerase activity, which is consistent with its extended capability for self-renewal. When induced to differentiate, it expressed phenotypic markers for tissues of ectodermal, mesodermal, and endodermal origin. The clone was designated as a postnatal pluripotent epiblastic-like stem cell (PPELSC). The undifferentiated clone was transfected with a genomic marker and assayed for alterations in stem cell characteristics. No alterations were noted. The labeled clone, when implanted into heart after injury, incorporated into myocardial tissues undergoing repair. The labeled clone was subjected to directed lineage induction in vitro, resulting in the formation of islet-like structures (ILSs) that secreted insulin in response to a glucose challenge. This study suggests that embryonic-like stem cells are retained within postnatal mammals and have the potential for use in gene therapy and tissue engineering.

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 FeCl2 in the colony forming-efficiency assay and fail to demonstrate a FeCl2-induced G2 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.

Zinc oxide nanoparticles (ZnO-NPs) have many exciting properties that make their use in a continuous increase in various biomedical, industrial, and agricultural applications. This is associated with accumulation in the aquatic ecosystems and fish exposure with consequent deleterious effects. To determine the potential of thymol to counteract the immunotoxic effects of ZnO-NPs, Oreochromis niloticus was exposed to ZnO-NPs (⅕ LC50 =1.14 mg/L, for 28 days) with or without feeding a thymol-incorporated diet (1 or 2 g/kg diet). Our data demonstrated a reduction of aquaria water quality, leukopenia, and lymphopenia with a decrease in serum total protein, albumin, and globulin levels in exposed fish. At the same time, the stress indices (cortisol and glucose) were elevated in response to ZnO-NPs exposure. The exposed fish also revealed a decline in serum immunoglobulins, nitric oxide, and the activities of lysozyme and myeloperoxidase, in addition to reduced resistance to the Aeromonas hydrophila challenge. The RT-PCR analysis showed downregulation of antioxidant (SOD) superoxide dismutase and (CAT) catalase gene expression in the liver tissue with overexpression of the immune-related genes (TNF-α and IL-1β). Importantly, we found that thymol markedly protected against ZnO-NPs-induced immunotoxicity in fish co-supplemented with thymol (1 or 2 g/kg diet) in a dose-dependent manner. Our data confirm the immunoprotective and antibacterial effects of thymol in ZnO-NPs exposed fish, supporting the potential utility of thymol as a possible immunostimulant agent.

Sarcoidosis is a multisystemic disease of unknown etiology characterized by the formation of immune granulomas in involved organs. The cytokine profile in inflamed lesions of sarcoidosis is mainly determined by T helper 1 (Th1) cells. Interleukin 18 (IL-18) is primarily a monocyte/macrophage-derived cytokine. IL-18 has been recently identified as an IFNγ-inducing factor. The cytokine plays an important role in the induction of Th1 response and it may be responsible for sarcoidosis progression.The aim of the study was to assess the usefulness of IL-18 estimation in the sarcoidosis diagnosis and the disease course prognosis.The diagnosis of sarcoidosis was established in 88 patients (the mean age of 38.1±10.8 years). We measured IL-18 level in plasma and bronchoalveolar lavage fluid (BALF) cell culture supernatant (CCS) using the enzyme-linked immunoassay technique (ELISA). We also performed the flow cytometric analysis of BALF lymphocyte phenotype. Statistica 5.0 and non-parametric tests: the Mann–Whitney U-test and the Spearman correlation test, were used for statistical analysis.The patient group consisted of 55 subjects without acute symptoms of sarcoidosis, 14 patients with acute Löfgren syndrome and 19 subjects with Löfgren syndrome in the past. Lung hilar lymphadenopathy was diagnosed in 49 patients and lung interstitial changes in 39 subjects. After 6-month-observation, 49 patients were in remission, 20 subjects manifested persistent disease and 19 patients had sarcoidosis progression. Plasma IL-18 level was significantly (P<0.0001) higher in sarcoidosis patients (383±250 pg/ml) than in control subjects (146±72 pg/ml). Plasma IL-18 level was similar both in subjects with Löfgren syndrome and in other patients. However, IL-18 level in BALF CCS was significantly (P<0.05) lower in Löfgren syndrome patients than in subjects without acute manifestation of the disease. The highest IL-18 level in plasma was found in patients with disease progression, in subjects with lung interstitial changes and in patients with extrapulmonary manifestation of the disease. We observed a positive correlation between plasma IL-18 level and the percentage of BALF lymphocytes (R=0.202, P=0.06) as well as the percentage of activated HLA DR+T cells (R=0.23, P<0.05). There was a negative correlation between the IL-18 level in BALF CCS and the percentage of BALF CD3-positive and CD4-positive lymphocytes (R=−0.27, −0.23, P<0.05).IL-18 may play a significant role in the prolongation of sarcoidosis course. Its estimation may become a good prognostic factor, which should be analyzed together with other factors useful in sarcoidosis monitoring.

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.

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. © 2001 Wiley‐Liss, Inc.

Cancer is one of the leading causes of death in the United States. Although there has been significant progress in the areas of cancer etiology, diagnostic techniques, and cancer prevention, adequate therapeutic approaches for many cancers have lagged behind. One promising line of investigation is the virus therapy of cancer. This approach entails the use of viruses, such as retroviruses, adenovirus, and vaccinia virus, to modify tumor cells so that they become more susceptible to being killed by the host immune response, chemotherapeutic agents, or programmed cell death. This review discusses recent advances in the virus therapy of cancer from both basic science and clinical perspectives. Given the potential of viruses to kill tumor cells directly or transduce desired gene products to allow a vigorous host antitumor immune response, the virus therapy of cancer holds great promise in the treatment of cancer.

Adult hematopoietic differentiation is a developmental process that employs many of the same molecular mechanisms as embryogenesis. To explore the possibility that hedgehog signaling is involved in the control of hematopoietic differentiation, we screened a panel of human leukemia cell lines for the expression of Patched1 and Smoothened, the receptor and coreceptor for hedgehog ligands. Expression was found in multiple cell lines, and Patched1 expression was detected in normal marrow. Induction of myeloid differentiation in cell lines downregulated expression of both genes. When normal marrow mononuclear cells were grown in semisolid medium in the presence of 10 microM cyclopamine, development of colonies of granulocytic/monocytic lineage was unaffected in terms of both number and morphology. The number of erythroid colonies, however, was significantly reduced (P < 0.01). Furthermore, hemoglobinization was substantially delayed relative to controls in those erythroid colonies that did form. Incubation of hematopoietic progenitors with Shh-N and GM-CSF resulted in increased granulocyte/monocyte colonies (P < 0.01); the increase was blocked by cyclopamine. Incubation of hematopoietic progenitors with Shh-N and stem cell factor resulted in larger erythroid colonies. These results suggest that elements of the hedgehog signaling pathway are involved in the control of hematopoietic differentiation.

Each year millions of people suffer tissue loss or end-stage organ failure. While allogeneic therapies have saved and improved countless lives, they remain imperfect solutions. These therapies are limited by critical donor shortages, long-term morbidity, and mortality. A wide variety of transplants, congenital malformations, elective surgeries, and genetic disorders have the potential for treatment with autologous stem cells as a source of HLA-matched donor tissue. Our current research is aimed at characterizing cell surface cluster differentiation (CD) markers on human progenitor and pluripotent cells to aid in isolating comparatively purified populations of these cells. This study examined human pluripotent and progenitor cells isolated from fetal, mature, and geriatric individuals for the possible presence of 15 CD markers. The response to insulin and dexamethasone revealed that the cell isolates were composed of lineage-committed progenitor cells and lineage-uncommitted pluripotent cells. Flow cytometry showed cell populations positive for CD10, CD13, CD56, and MHC Class-I markers and negative for CD3, CD5, CD7, CD11b, CD14, CD15, CD16, CD19, CD25, CD45, and CD65 markers. Northern analysis revealed that CD13 and CD56 were actively transcribed at time of cell harvest. We report the first identification of CD10, CD13, CD56, and MHC Class-I cell surface antigens on these human cells.

ABSTRACT: To explore the possibility that bone morphogenetic proteins (BMPs) are autocrine/paracrine regulators of hematopoietic differentiation and function, we screened a panel of human cell lines encompassing the hematopoietic lineages for expression of members of this family of genes. Expression of BMP-2, BMP-4, BMP-6, BMP-7, Growth and Differentiation Factor-1 (GDF-1), Placental Bone Morphogenetic Protein (PLAB), and Transforming Growth Factor-β3 (TGF-β3) was detected in one or more cell lines. BMP-2, BMP-4, BMP-7, and TGF-β3 expression was also found in normal hematopoietic tissue. Expression of BMP-5 and BMP-8 was not seen. Lineage-restricted patterns of expression were found for BMP-4 (T-lymphoid), BMP-7 (lymphoid), PLAB (macrophage/monocyte), and GDF-1 (myeloid). Expression of BMP-2, GDF-1, and PLAB could be modulated by treatment with differentiating agents. Marked variations in the levels of BMP-4, BMP-7, and PLAB expression were encountered, indicating that disorders in BMP signaling pathways may play a role in the development of hematopoietic neoplasia.

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.

Reovirus type 3 is a double-stranded RNA virus that is a potent inducer of murine natural killer (NK) cell cytotoxicity, most likely as a result of virus-induced interferon production. We determined that reovirus was an effective inducer of high levels of NK cytotoxicity. A single injection of cyclosporine (CS), administered concurrently with reovirus or delayed until three days after injecting CS, significantly inhibited NK cytotoxicity. CS significantly suppressed reovirus-induced NK cytotoxicity when added directly to the chromium-release assay. We determined that CS inhibited the ability of murine spleen cells to form conjugates with YAC-1 tumor target cells. Finally. CS was shown to directly inhibit reovirus replication in vitro. Our results demonstrate that CS is an effective inhibitor of reovirus-induced NK cytotoxicity and suggest that inhibition occurs through multiple mechanisms including direct effects on the NK cells and direct inhibition of virus replication.

We have previously shown that the combined modalities of reovirus type 3 and the chemotherapeutic agent 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) synergize to cause the rejection of various murine tumors. Resistance of surviving animals to challenge with homologous but not heterologous tumor suggested that tumor was eliminated through an immune-mediated mechanism. In this study, we showed that mice undergoing therapy-mediated rejection of tumor were able to reject subsequent weekly challenges with EL-4 but not L1210 tumor cells. The mechanism underlying this therapy was investigated using cyclosporine (CS) to suppress immune responsiveness. A dose-related inhibition of therapy was observed with total inhibition occurring at 30 mg/kg/day. Delayed administration of CS at day 14 or later after tumor administration resulted in little or no inhibitory effect. The resistance of cured mice to EL-4 tumor challenge was not affected by CS, which is consistent with the reduced ability of CS to affect secondary immune responses. In addition, CS did not alter natural killer cell activity in mice receiving the BCNU/reovirus therapy. These results suggest that there is an obligatory immune response produced by the BCNU/reovirus therapy which arises early after the administration of the therapy.

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.

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.