Injected MSC-IFN-beta cells suppressed the growth of pulmonary metastases, presumably through the local production of IFN-beta in the tumor microenvironment. MSC may be an effective platform for the targeted delivery of therapeutic proteins to cancer sites.

Leptin is secreted by bone marrow (BM) adipocytes and stromal cells and was shown to stimulate myeloid proliferation. We here report that primary acute promyelocytic leukemia (APL) cells express high levels of the leptin-receptor (OB-R) long isoform. In cells with regulated promyelocytic leukemia-retinoic acid receptor (PML-RAR␣) expression, inducing PML-RAR␣ was found to increase OB-R levels. We then investigated the effects of leptin produced by BM adipocytes on APL cells using a coculture system with mesenchymal stem cell (MSC)-derived adipocytes. In PML-RAR␣-expressing cells, all-trans retinoic acid (ATRA)-and doxorubicininduced apoptosis were significantly reduced by coculture with adipocytedifferentiated MSCs. This antiapoptotic effect required direct cell-to-cell interactions, was associated with phosphorylation of signal transducer and activator of transcription-3 (STAT3) and mitogenactivated protein kinase (MAPK), and was reduced by blocking OB-R. This report provides a mechanistic basis for the BM adipocyte-leukemia cell interaction and suggests that OB-R receptor blockade may have therapeutic use in APL. IntroductionLeptin is a 16-kDa protein produced by adipocytes and was originally identified as a cytokine that regulates fat metabolism. 1 Adipocytes are abundant in the bone marrow (BM) microenvironment and might be responsible for the high concentration of leptin in BM. 2 The leptin receptor (OB-R) has recently been detected on human hematopoietic progenitor cells expressing the CD34 antigen, and leptin was reported to induce proliferation and differentiation in these cells. 3,4 Multiple isoforms of OB-R have been identified, including a long isoform and several isoforms with short cytoplasmic domains. 5,6 Although all isoforms share an identical extracellular ligand-binding domain with homology to the class I cytokine receptor family, they differ at the C-terminus. 6 Only the long OB-R isoform, which has the longest (303 amino acids) cytoplasmic domain and encodes all protein motifs, has the signaling capabilities of interleukin-6 (IL-6)-type cytokine receptors and can activate the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction pathway. [7][8][9] We have reported that leptin induces the proliferation and enhances the survival of primary leukemic cells from patients with acute myeloid leukemia (AML). 10 We have further demonstrated the presence of leptin receptors (OB-Rs) on primary AML cells, with the highest expression of OB-R long isoform on acute promyelocytic leukemia (APL) cells, which was absent on normal promyelocytes. 10 Given that APL is associated with increased body mass index (BMI) 11 and leptin serum levels correlate with body fat percentage, 12,13 leptin may play a role in the pathophysiology of APL. Alternatively, leptin secretion by BM adipocytes in the vicinity of leukemic cells could play a major role in the proliferation and survival of APL cells through paracrine interactions in the marrow microenvironment. The aims of this study we...

In many viral infections the host cell carries the viral genome without producing viral particles, a phenomenon known as viral latency. The cellular mechanisms by which viral latency is maintained or viral replication is induced are not known. The modulation of intracellular calcium concentrations by calcium ionophores induced Epstein-Barr viral antigens in lymphoblastoid cell lines that carry the virus. When calcium ionophores were used in conjunction with direct activators of protein kinase C (12-O-tetradecanoyl phorbol-13-acetate and a synthetic diacylglycerol), a greater induction of viral antigens was observed than with either agent alone. Activation of protein kinase C may be required for the expression of the viral genome.

The intracellular localization of Shc proteins was analyzed by immunofluorescence and immunoelectron microscopy in normal cells and cells expressing the epidermal growth factor receptor or the EGFR/erbB2 chimera. In unstimulated cells, the immunolabeling was localized in the central perinuclear area of the cell and mostly associated with the cytosolic side of rough endoplasmic reticulum membranes. Upon epidermal growth factor treatment and receptor tyrosine kinase activation, the immunolabeling became peripheral and was found to be associated with the cytosolic surface of the plasma membrane and endocytic structures, such as coated pits and endosomes, and with the peripheral cytosol. Receptor activation in cells expressing phosphorylation-defective mutants of Shc and erbB-2 kinase showed that receptor autophosphorylation, but not Shc phosphorylation, is required for redistribution of Shc proteins. The rough endoplasmic reticulum localization of Shc proteins in unstimulated cells and their massive recruitment to the plasma membrane, endocytic structures, and peripheral cytosol following receptor tyrosine kinase activation could account for multiple putative functions of the adaptor protein.The Shc locus is highly conserved throughout evolution and codes for three overlapping proteins of 66, 52, and 46 kDa (p66 shc have a common carboxy-terminal SH2 domain, flanked by a glycine-and proline-rich region of approximately 145 amino acids (collagen homologous region 1 [CH1]) (38). At the N terminus, adjacent to the CH1 region, they possess a region with functional properties of a phosphotyrosine-binding domain (PTB) (4,6,24,25,47,54). The binding specificities of the Shc PTB and SH2 domains differ (3,24,44,54). The unique amino-terminal portion of p66 shc contains an additional collagen homologous region (CH2 region). The CH1 and CH2 regions are putative SH3 binding sites (34).Shc proteins are phosphorylated by all activated receptor tyrosine kinases (RTKs) tested to date: epidermal growth factor receptor (EGFR) (38, 39), platelet-derived growth factor receptor (55), hepatocyte growth factor receptor (37, 39), erbB-2 receptor (47), insulin receptor (23,26,40,41,48), fibroblast growth factor receptor (53), nerve growth factor receptor (7, 50), and sea receptor (13).

Ultraviolet irradiation is capable of affecting skin surface lipids, especially squalene and cholesterol, both in vitro and in vivo, with generation of active lipoperoxides. The photodecomposition of the skin lipid component was carefully evaluated by capillary gas-chromatography. The effects of UV-induced lipoperoxides on human keratinocytes in culture and on guinea pig ear slices were compared with those of synthetic lipoperoxides, i.e. cumene hydroperoxide and 13-hydroperoxylinoleate. A time- and dose-dependent effect on protein synthesis and mitotic activity was observed. In cell culture low concentrations (0.05-5 micrograms/ml) of peroxidated squalene and synthetic lipoperoxides stimulated the incorporation of radiolabelled thymidine and phenylalanine, while higher concentrations (greater than 10 micrograms/ml), or longer periods of treatment, induced cellular damage. In guinea pig ear slices, the lipoperoxides (5-50 micrograms/ml) increased aminoacid incorporation and the number of epidermal pigment cells; higher concentrations (greater than 100 micrograms/ml) caused a derangement of epidermal structure. The results suggest that UV irradiation of skin generates lipoperoxides from the surface lipids which, in vitro, are capable of producing a number of changes in epidermal cells.

Many ovarian cancers originate from ovarian surface epithelium, where they develop from cysts intermixed with stroma. The stromal layer is critical to the progression and survival of the neoplasm and consequently is recruited into the tumor microenvironment. Using both syngenic mouse tumors (ID8-R), and human xenograft (OVCAR3, SKOV3) tumor models, we first confirmed intraperitoneally-circulating MSC could target, preferentially engraft and differentiate into α-SMA+ myofibroblasts, suggesting their role as “reactive stroma” in ovarian carcinoma development and confirming their potential as a targeted delivery vehicle for the intratumoral production of interferon-beta (IFNβ). Then, mice with ovarian carcinomas received weekly IP injections of IFNβ expressing MSC, resulting in complete eradication of tumors in 70% of treated OVCAR3 mice (P = 0.004) and an increased survival of treated SKOV3 mice compared with controls (P = 0.01). Similar tumor growth control was observed using murine IFNβ delivered by murine MSC in ID8-R ovarian carcinoma. As a potential mechanism of tumor killing, MSC produced IFNβ induced caspase-dependent tumor cell apoptosis. Our results demonstrate that ovarian carcinoma engraft MSC to participate in myofibrovascular networks and that IFNβ produced by MSC intratumorally modulates tumor kinetics, resulting in prolonged survival.

A peculiar characteristic of cells infected with human herpesvirus 6 (HHV6) is the absence of viral glycoproteins on the plasma membrane, which may reflect an atypical intracellular transport of the virions and/or the viral glycoproteins, different from that of the other members of the herpesvirus family. To investigate the maturation pathway of HHV-6 in the human T lymphoid cell line HSB-2, we used lectin cytochemistry and immunogold labeling combined with several electron microscopical techniques, such as ultrathin frozen sections, postembedding, and fracture-label. Immunolabeling with anti-gp116 and anti-gp82-gp105 monoclonal antibodies revealed that the viral glycoproteins are undetectable on nuclear membranes and that at the inner nuclear membrane nucleocapsids acquire a primary envelope lacking viral glycoproteins. After de-envelopment, cytoplasmic nucleocapsids acquire a thick tegument and a secondary envelope with viral glycoproteins at the level of neo-formed annulate lamellae or at the cis-side of the Golgi complex. Cytochemical labeling using helix pomatia lectin revealed that the newly acquired secondary viral envelopes contain intermediate forms of glycocomponents, suggesting a sequential glycosylation of the virions during their transit through the Golgi area before their final release into the extracellular space. Immunogold labeling also showed that the viral glycoproteins, which are not involved in the budding process, reach and accumulate in the endosomal/lysosomal compartment. Pulse-chase analysis indicated degradation of the gp116, consistent with its endosomal localization and with the absence of viral glycoproteins on the cell surface of the infected cells.

Several reports have suggested an association of human herpesvirus 6 (HHV-6) with multiple sclerosis. Autoreactive T lymphocytes directed against myelin components seem to contribute to the pathogenesis of the disease. It has been suggested that molecular mimicry between viral and self-antigens might be one of the mechanisms that determine the onset of several autoimmune diseases. Following this hypothesis, the purpose of the present study was to evaluate if HHV-6 could play a role in activating T cells capable of cross-reaction with an important myelin component, the myelin basic protein. T cell lines were established from 22 multiple sclerosis patients and from 16 healthy controls, and their capability to react to both virus and myelin basic protein antigens was compared. The analysis of T cell cross-reactivity in patients and controls did not show significant differences in the HHV-6 ability to activate myelin basic protein-reactive T cells. Similarly, the evaluation of the humoral immune response to HHV-6 in patients and controls did not mirror any abnormality in the HHV-6 status in multiple sclerosis patients. Therefore, although the findings of activity in vitro of T cell lines with dual specificity are consistent with the hypothesis of molecular mimicry, the lack of differences in cross-reactivity between patients and controls do not support molecular mimicry as an important mechanism in the physiopathology of this disease.