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Highlights • Oncolytic adenovirus targeting a transactivator PAX3-FOXO1 was newly developed. • Promoter of MYOG can be a potential candidate for controlling adenovirus for RMS. • Mutation in MEF2 binding site enhances the specificity and safety of the virus., The PAX3-FOXO1 fusion gene functions as a transactivator and increases expression of many cancer-related genes. These lead to metastases and other unfavorable outcomes for alveolar rhabdomyosarcoma (ARMS) patients. In order to target ARMS with the PAX3-FOXO1 transactivator, we developed an Oncolytic Adenovirus (OAd) regulated by the myogenin (pMYOG) promoter with a mutation in the Myocyte Enhancer Factor-2 binding site (mMEF2) in this study. The expression of MYOG in the two RMS cell lines (Rh30; PAX3-FOXO1-positive, RD; PAX3-FOXO1-negative) is about 1,000 times higher than normal skeletal muscle cell (SkMC). Ad5/3-pMYOG(S)-mMEF2 (short-length pMYOG-controlled OAd with mMEF2) showed strong replication and cytocidal effect in Rh30, but to a much lesser extent in RD. Ad5/3-pMYOG(S) (pMYOG-controlled OAd with native pMYOG) showed similar effects in RD and Rh30. Neither virus killed SkMC, indicating that Ad5/3-pMYOG(S)-mMEF2 selectively replicates and kills cells with PAX3-FOXO1. Additionally, Ad5/3-pMYOG(S)-mMEF2 showed replication and spread in vitro as well as tumor growth suppression and intratumoral viral spread in vivo, selectively in Rh30 not in RD. Our findings revealed that Ad5/3-pMYOG(S)-mMEF2 shows a promise as a safe and potent therapy to improve treatment in PAX3-FOXO1-positive ARMSs.

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

β-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.

Oncolytic adenoviruses (Ad) are promising tools for cancer therapeutics. Most Ad-based therapies utilize species C serotypes, with Adenovirus type 5 (Ad5) most commonly employed. Prior clinical trials demonstrated low efficiency of oncolytic Ad5 vectors, mainly due to the absence of Ad5 primary receptor (Coxsackie and Adenovirus Receptor, CAR) on cancer cells. Engineering serotype chimeric vectors (Ad5/3) to utilize Adenovirus type 3 (Ad3) receptors has greatly improved their oncolytic potential. Clinical translation of these infectivity-enhanced vectors has been challenging due to a lack of replication permissive animal models. In this study, we explored pigs as a model to study the performance of fiber-modified Ad5/3 chimeric vectors. As a control, the Ad5 fiber-unmodified virus was used. We analyzed binding, gene transfer, replication, and cytolytic ability of Ad5 and Ad5/3 in various non-human cell lines (murine, hamster, canine, porcine). Among all tested cell lines only porcine cells supported active binding and replication of Ad5/3. Syrian hamster cells supported Ad5 replication but showed no evidence of productive viral replication after infection with Ad5/3 vectors. Transduction and replication ability of Ad5/3 in porcine cells outperformed Ad5, a phenomenon often observed in human cancer cell lines. Replication of Ad5 and Ad5/3 was subsequently evaluated in vivo in immunocompetent pigs. Quantitative PCR analyses 7 days post infection revealed Ad5 and Ad5/3 DNA and replication-dependent luciferase activity in the swine lungs and spleen indicating active replication in these tissues. These studies demonstrated the flaws in using Syrian hamsters for testing serotype chimeric Ad5/3 vectors. This is the first report to validate the pig as a valuable model for preclinical testing of oncolytic adenoviruses utilizing Adenovirus type 3 receptors. We hope that these data will help to foster the clinical translation of oncolytic adenoviruses including those with Ad3 retargeted tropism.

Scientific Reports 6: Article number: 21094; published online: 19 February 2016; updated: 20 April 2016. The original version of this Article contained errors in the spelling of the authors Jana Ninkovic, Vidhu Anand, Raini Dutta, Lisa Koodie, Santanu Banerjee and Sabita Roy which were incorrectly given as Ninkovic Jana, Anand Vidhu, Dutta Raini, Koodie Lisa, Banerjee Santanu and Roy Sabita.

Opioid drug abusers have a greater susceptibility to gram positive (Gram (+)) bacterial infections. However, the mechanism underlying opioid modulation of Gram (+) versus Gram (−) bacterial clearance has not been investigated. In this study, we show that opioid treatment resulted in reduced phagocytosis of Gram (+), when compared to Gram (−) bacteria. We further established that LPS priming of chronic morphine treated macrophages leads to potentiated phagocytosis and killing of both Gram (+) and Gram (−) bacteria in a P-38 MAP kinase dependent signaling pathway. In contrast, LTA priming lead to inhibition of both phagocytosis and bacterial killing. This study demonstrates for the first time the differential effects of TLR4 and TLR2 agonists on morphine induced inhibition of phagocytosis. Our results suggest that the incidence and severity of secondary infections with Gram (+) bacteria would be higher in opioid abusers.

Recently, the use of oncolytic viruses encoding the sodium-iodide symporter (NIS) has become an attractive approach to achieve radionuclide imaging of cancer. However, the potential of virus-induced NIS expression to facilitate therapy with radioactive 131I has not been fully explored. We designed a tumor-selective, infectivity-improved oncolytic adenovirus (Ad5/3-Cox2-NIS-ADP) modified to express high quantities of NIS. This virus induced radioisotope uptake in prostate and lung cancer cells and facilitated SPECT/CT imaging, but more importantly, supported the effective therapy with 131I. In our original vector, enhanced oncolysis was mediated by overexpression of adenoviral death protein (ADP). Although this structure was operative in detection and therapeutic regimens, we were concerned that the cytolytic effect of ADP may affect NIS membrane localization, and diminish its effectiveness as a theranostic tool. We therefore designed an identical Ad5/3-Cox2-NIS-ADP-deleted virus {ADP(-)} and assessed the impact of ADP on NIS expression and radioisotope uptake. Western blot and immunocytochemical analyses of pancreatic cancer cells infected with ADP(-) demonstrated higher NIS levels when compared to the ADP(+) counterpart. This correlated with an improved 125I uptake in vitro. SPECT/CT imaging studies assessing 99mTcO4- accumulation in mice to visualize pancreatic adenocarcinoma (PDAC) using patient-derived xenografts (PDX) showed that a single dose of ADP(-) accumulated far greater levels of 99mTcO4- when compared to ADP(+). Remarkably, ADP(-) produced stronger signal that was maintained up until day 32, and this outlasted ADP(+) and the control vector, replication-deficient AdCMV-NIS, currently employed in a clinical trial. Notably, within the PDAC, ADP(-) showed a distinct NIS cell membrane distribution pattern as it co-localized with cell membrane bound-cytokeratin-4. Unlike ADP(-), ADP(+) produced a punctate NIS staining pattern, with little to no cell membrane localization. These results support our hypothesis that ADP-cytolytic effect reduces NIS membrane localization, subsequently affecting radionuclide uptake. To evaluate the therapeutic potential of NIS-based radiotherapy, we treated mice bearing PDAC tumors with virotherapy alone or in combination with 131I. NIS-expressing OAds in combination with 131I significantly reduced tumor growth when compared to viro- or radiotherapy alone. The therapeutic effect in mice treated with ADP(-) combined with 131I outperformed that with ADP(+) or AdCMV-NIS vectors. Quantitative analyses of 131I uptake in tumor tissues with a gamma counter showed a clear trend where ADP(-) retained higher 131I uptake than ADP(+). These findings support the clinical applicability of the ADP-deleted OAd as the more sensitive tool for NIS-based cancer diagnosis and therapy. We are currently investigating the biodistributon and toxicity of our vectors in a pig model. Citation Format: Lisa Koodie, Eriko Iguchi Kawakami, Kari Jacobsen, Zuzan Cayci, Andrew Taylor, Edward W. Greeno, Robert J. Schumacher, John C. Morris, Martin E. Fernandez-Zapico, Julia Davydova. A novel oncolytic adenovirus for radioiodine therapy and imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5925.

Oncolytic adenoviruses (Ad) are promising tools in the development of cancer therapeutics. A majority of Ad-based therapies utilize serotypes of species C, with Adenovirus type 5 (Ad5) being the most commonly employed. Previously, clinical trials have demonstrated the low efficiency of Ad5 vectors, mainly due to absence of the Ad5 primary receptor (Coxsackie Adenovirus Receptor, CAR) in cancer cells. Engineering Ad vectors utilizing the species B (Ad3, Ad35, Ad11) receptors have greatly improved the oncolytic potential of Ad-based therapies. However, the lack of a viable animal model has impeded clinical translation of these tropism-modified vectors. Mouse models are insufficient because Ad does not replicate in murine tissue. Non-human apes are not feasible due to availability and cost. Cotton rats and Syrian hamsters, although permissive of Ad5 replication, are not suitable for Ad3-, Ad35-, and Ad11-retargeted vectors due to the lack of species B primary receptors (CD46 and desmoglein 2) in rodent systems. In this study, we explored pigs as a model to study performance of the group B oncolytic adenoviruses by employing the fiber-modified Ad5/Ad3 chimeric vector. As a control, the Ad5 fiber-unmodified virus was used. First, we demonstrated the ability of swine cell lines to support replication of both Ad5 and Ad5/Ad3. Second, we analyzed binding, gene transfer, cytolytic, and replication ability of Ad5 and Ad5/Ad3 in various non-human cell lines (swine, hamster, murine, rat, bovine, canine). Our data confirmed that while hamster cell lines (HP1 and HapT1) were able to support binding and replication of Ad5-based vectors, they failed to support that with the Ad3-retargeted vectors. Additionally, we showed that among all tested cell lines, only porcine cells (PK15 and PTK75) were supportive of both binding and replication of the Ad3-retargeted virus. Of note, Ad5/Ad3 outperformed Ad5 in its cytolytic effect in porcine cell lines. These in vitro results prompted evaluation of the vectors in vivo. Immunocompetent Yorkshire pigs were systemically injected with a single dose of Ad5- and Ad5/3- expressing luciferase (Luc) from the Ad E3 region. Quantitative PCR analyses of the primary organs collected 7 days post-infection revealed Ad5 and Ad5/3 viral DNA in the lungs and spleen. Replication-dependent Luc expression was also observed in these tissue samples suggesting active viral replication. The quantity of viral DNA in other tissue such as the kidneys, liver, and pancreas was negligible. The results of these in vitro and in vivo studies indicate that pigs are a promising model to assess unmodified and tropism-modified adenoviral vectors. Citation Format: Malavika Chandrashekar, Lisa Koodie, Michele Dunning, George Ruth, Richard Bianco, Masato Yamamoto, Julia Davydova. The porcine model for onocolytic adenovirus-based therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5914.

In mucopolysaccharidosis-I (MPS-I), α-L-iduronidase deficiency leads to progressive heparan sulfate (HS) and dermatan sulfate (DS) glycosaminoglycan (GAG) accumulation. The functional consequences of these accumulated molecules are unknown. HS critically influences tissue morphogenesis by binding to and modulating the activity of several cytokines (eg, fibroblast growth factors [FGFs]) involved in developmental patterning. We recently isolated a multipotent progenitor cell from postnatal human bone marrow, which differentiates into cells of all 3 embryonic lineages. The availability of multipotent progenitor cells from healthy volunteers and patients with MPS-I (Hurler syndrome) provides a unique opportunity to directly examine the functional effects of abnormal HS on cytokine-mediated stem-cell proliferation and survival. We demonstrate here that abnormally sulfated HS in Hurler multipotent progenitor cells perturb critical FGF-2–FGFR1-HS interactions, resulting in defective FGF-2–induced proliferation and survival of Hurler multipotent progenitor cells. Both the mitogenic and survival-promoting activities of FGF-2 were restored by substitution of Hurler HS by normal HS. This perturbation of critical HS–cytokine receptor interactions may represent a mechanism by which accumulated HS contributes to the developmental pathophysiology of Hurler syndrome. Similar mechanisms may operate in the pathogenesis of other diseases where structurally abnormal GAGs accumulate.

Tumor cells secrete factors that stimulate the migration of peripheral blood leukocytes and enhance tumor progression by affecting angiogenesis. In these studies, we investigated the effect of morphine, a known immunosuppressant, on leukocyte migration and recruitment to conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells. Our results indicate that morphine treatment reduced the migration and recruitment of tumor-infiltrating leukocytes into Matrigel plugs and polyvinyl alcohol sponges containing conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells when compared with placebo. A reciprocal increase in peripheral blood leukocytes was observed at the time of plug or sponge removal in morphine-treated mice. Decreased angiogenesis was observed in conditioned media derived from long-term cultures of mouse Lewis lung carcinoma cells Matrigel plugs taken from morphine-treated wild-type mice when compared with placebo but was abolished in morphine-treated μ-opioid receptor knockout mice. In addition, in vitro studies using trans-well and electric cell substrate impedance sensing system studies reveal for the first time morphine's inhibitory effects on leukocyte migration and their ability to transmigrate across an activated endothelial monolayer. Taken together, these studies indicate that morphine treatment can potentially decrease leukocyte transendothelial migration and reduce angiogenesis associated with tumor growth. The use of morphine for cancer pain management may be beneficial through its effects on angiogenesis.

In this study, we assess our novel Oncolytic Adenovirus (OAd) expressing a dual function therapeutic and imaging transgene, the sodium-iodide symporter (NIS) as a promising alternative for pancreatic cancer treatment. We hypothesized that NIS expression in pancreatic cancer will induce uptake of radioiodine and allow noninvasive SPECT/CT imaging with 123I. We designed our OAd-NIS to overcome the low efficacy of previous vectors. These viruses have a modified Ad5/Ad3 fiber-knob shown to improve the poor transduction of pancreatic cancer cells. Viral replication is controlled under the Cox2 promoter allowing specific delivery of viral genes, and most of adenoviral E3 genes are deleted and replaced with NIS and Adenovirus Death Protein (ADP) genes. We have previously demonstrated significantly improved ability of ADP-overexpressing OAds to enhance viral release and cytolytic activity. Although improved oncolysis enhances viral release and improve the therapeutic effect in solid tumors, it can potentially reduce radioiodide tracer uptake. To test this possibility we designed and compared identical vectors with ADP (OAd-NIS-ADP) and without ADP (OAd-NIS-noADP) in vitro and in vivo. We first compared virus killing ability in pancreatic cancer cell lines. The ADP+ vector was significantly more cytolytic that no-ADP counterpart and showed improved viral replication and spread. Cox2-controlled OAd did not produce cell death in Cox2-negative control, confirming selectivity. We next assessed OAds ability to induce NIS protein expression and radioiodine uptake. NIS-OAds efficiently produce glycosylated NIS multimers as early as 2-days post infection. Infection with no-ADP vectors resulted in a significantly greater radioiodine uptake (125I) compared to ADP+ viruses. Importantly, ex vivo uptake test in human patient tissues confirmed the high level of NIS in pancreatic adenocarcinoma samples, and revealed no 125I uptake in normal pancreas. We further assessed the ability of our vectors to visualize human pancreatic cancer xenografts in a mouse model by monitoring 99mTcO4− accumulation with SPECT-CT. The OAd-NIS-noADP showed an earlier and more sustained radioisotope uptake when compared to ADP+ supporting its use as a more sensitive diagnostic tool for pancreatic cancer. Of contrary, the ADP+ vector significantly outperformed OAd-NIS-noADP in tumor shrinkage. These results suggest that while OAd-NIS-noADP produces a greater radiotracer uptake and can be used as a diagnostic tool, its ADP+ counterpart results in a better therapeutic effect when applied as a monotherapy. It is now essential to estimate the therapeutic ability of both vectors upon combination with I-131 (ongoing study). Further investigation will include the imaging and therapeutic studies in patient-derived xenografts. Ultimately, the goal of our research is to design a multimodal therapy with radiation and oncolytic virus for diagnosis and therapy of cancers.

Morphine has been recognized as a highly potent analgesic agent used in cancer and non-cancer (neuropathic, surgical) pain management. Cancer patients may be prescribed morphine at different stages of the disease, during neoplastic growth and progression, during surgical resection and even in end stage palliative care. Morphine has been shown to suppress immune cell activation, ­functionality and cytokine secretion. While the initial infiltration of immune cells during tumor growth can be beneficial in destroying stressed tumor cells, ­prolonged accumulation results in a dampened immune response, enhanced angiogenesis, tumor growth and thus metastasis. The aim of this chapter is summarize the ­immunosuppressive effects of morphine as it relates to metastasis. We describe the effects of morphine as it pertains to tumor cell proliferation and growth, immune cell contribution to angiogenesis and extracellular matrix remodeling within the tumor microenvironment.

Infection rate among intravenous drug users (IDU) is higher than the general public, and is the major cause of morbidity and hospitalization in the IDU population. Epidemiologic studies provide data on increased prevalence of opportunistic bacterial infections such as TB and pneumonia, and viral infections such as HIV-1 and hepatitis in the IDU population. An important component in the intravenous drug abuse population and in patients receiving medically indicated chronic opioid treatment is opioid withdrawal. Data on bacterial virulence in the context of opioid withdrawal suggest that mice undergoing withdrawal had shortened survival and increased bacterial load in response to Salmonella infection. As the body of evidence in support of opioid dependency and its immunosuppressive effects is growing, it is imperative to understand the mechanisms by which opioids exert these effects and identify the populations at risk that would benefit the most from the interventions to counteract opioid immunosuppressive effects. Thus, it is important to refine the existing animal model to closely match human conditions and to cross-validate these findings through carefully controlled human studies. Better understanding of the mechanisms will facilitate the search for new therapeutic modalities to counteract adverse effects including increased infection rates. This review will summarize the effects of morphine on innate and adaptive immunity, identify the role of the mu opioid receptor in these functions and the signal transduction activated in the process. The role of opioid withdrawal in immunosuppression and the clinical relevance of these findings will also be discussed.

Morphine, a highly potent analgesic agent, is frequently prescribed for moderate to severe cancer pain. In this study, morphine was administered at a clinically relevant analgesic dose to assess tumor cell-induced angiogenesis and subcutaneous tumor growth in nude mice using mouse Lewis lung carcinoma cells (LLCs). Implantation of mice with a continuous slow-release morphine pellet achieved morphine plasma levels within 250–400 ng/ml (measured using a radioimmunoassay, Coat-A-Count Serum Morphine) and was sufficient to significantly reduce tumor cell-induced angiogenesis and tumor growth when compared with placebo treatment. Morphometric analysis for blood vessel formation further confirmed that morphine significantly reduced blood vessel density (P < 0.003), vessel branching (P < 0.05), and vessel length (P < 0.002) when compared with placebo treatment. Morphine’s effect was abolished in mice coadministered the classical opioid receptor antagonist, naltrexone, and in mu-opioid receptor knockout mice, supporting the involvement of the classical opioid receptors in vivo. Morphine’s inhibitory effect is mediated through the suppression of the hypoxia-induced mitochondrial p38 mitogen-activated protein kinase (MAPK) pathway. Our results suggest that in vitro morphine treatment of LLCs inhibits the hypoxia-induced nuclear translocation of hypoxia-inducible transcription factor 1α to reduce vascular endothelial growth factor transcription and secretion, in a manner similar to pharmacological blockade with the p38 MAPK-specific inhibitor, SB203585. These studies indicate that morphine, in addition to its analgesic function, may be exploited for its antiangiogenic potential.

Patients prescribed morphine for the management of chronic pain, and chronic heroin abusers, often present with complications such as increased susceptibility to opportunistic infections and inadequate healing of wounds. We investigated the effect of morphine on wound-healing events in the presence of an infection in an in vivo murine model that mimics the clinical manifestations seen in opioid user and abuser populations. We show for the first time that in the presence of an inflammatory inducer, lipopolysaccharide, chronic morphine treatment results in a marked decrease in wound closure, compromised wound integrity, and increased bacterial sepsis. Morphine treatment resulted in a significant delay and reduction in both neutrophil and macrophage recruitment to the wound site. The delay and reduction in neutrophil reduction was attributed to altered early expression of keratinocyte derived cytokine and was independent of macrophage inflammatory protein 2 expression, whereas suppression of macrophage infiltration was attributed to suppressed levels of the potent macrophage chemoattractant monocyte chemotactic protein-1. When the effects of chronic morphine on later wound healing events were investigated, a significant suppression in angiogenesis and myofibroblast recruitment were observed in animals that received chronic morphine administration. Taken together, our findings indicate that morphine treatment results in a delay in the recruitment of cellular events following wounding, resulting in a lack of bacterial clearance and delayed wound closure.

The outcome of microbial infection in an organism is a dynamic process that depends on factors derived from both the microorganism and the host. In chronic human infections, the kind of immune response made in response to pathogens may be of vital importance to host defense. An inappropriate immune response may not only result in lack of protection, but even contribute to disease severity. Chronic morphine use and abuse has been documented to result in severe immune consequence (Roy and Loh 1996; Dinda et al. 2005; Friedman and Eisenstein 2004) and thus may pose a significant risk factor to opportunistic infection. It is therefore not surprising that epidemiological studies show increased prevalence of opportunistic infections such as tuberculosis, HIV infection, and pneumonia in opioid abusers (Quaglio et al. 2002; Nath et al. 2002; Georges et al. 1999). Besides the sharing of unsterilized, contaminated needles, the occurrence of infections in these patients has been attributed to the immune modulatory effect of morphine. In animal studies where confounding variables such as nutritional status, environmental influences, history of drug use, and genetic variability can be controlled, morphine treatment resulted in significant immune deficits. Defense against microbes is mediated by the early reactions of innate immunity and the later response of adaptive immunity. Chronic morphine has been shown to affect both these arms of immune defense (Vallejo et al. 2004). This review on the immunological effects of morphine summarizes the effects of this drug on innate and adaptive immunity, identifies the role of the mu opioid receptor in these functions, and finally discusses how changes in these parameters increase the risk for opportunistic infection.

The estrogen signaling pathway induces proliferation in non-small cell lung cancer (NSCLC) and represents a novel therapeutic target for lung cancer. Estrogen receptor β (ERβ) is the predominant ER isoform in lung cancer and we have previously shown that high cytoplasmic ERβ combined with low progesterone receptor (PR) expression is a poor prognostic marker for NSCLC patients. To further elucidate the biological differences between ERβ high/PR low versus ERβ low/PR high expressing lung tumors, a mRNA microarray analysis using the Illumina HT-12 V4 Bead Chip platform was performed using RNA extracted from 64 cases. 165 genes were differentially expressed between these two groups at P Citation Format: Laura P. Stabile, Natalie J. Rothenberger, Marjorie Romkes, Lisa Koodie, Mariya Farooqui, Sanja Dacic, Jill M. Siegfried. Estrogen modulation of fibroblast growth factor signaling in non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1861. doi:10.1158/1538-7445.AM2015-1861

We have derived from normal human, mouse, and rat postnatal bone marrow primitive, multipotent adult progenitor cells (MAPCs) that can differentiate into most mesodermal cells and neuroectodermal cells in vitro and into all embryonic lineages in vivo. Here, we show that MAPCs can also differentiate into hepatocyte-like cells in vitro. Human, mouse, and rat MAPCs, cultured on Matrigel with FGF-4 and HGF, differentiated into epithelioid cells that expressed hepatocyte nuclear factor-3beta (HNF-3beta), GATA4, cytokeratin 19 (CK19), transthyretin, and alpha-fetoprotein by day 7, and expressed CK18, HNF-4, and HNF-1alpha on days 14-28. Virtually all human, as well as a majority of rodent cells stained positive for albumin and CK18 on day 21; 5% (rodent) to 25% (human) cells were binucleated by day 21. These cells also acquired functional characteristics of hepatocytes: they secreted urea and albumin, had phenobarbital-inducible cytochrome p450, could take up LDL, and stored glycogen. MAPCs, which can be expanded in vitro and maintained in an undifferentiated state for more than 100 population doublings, can thus differentiate into cells with morphological, phenotypic, and functional characteristics of hepatocytes. MAPCs may therefore be an ideal cell for in vivo therapies for liver disorders or for use in bioartificial liver devices. ispartof: Journal of Clinical Investigation vol:109 issue:10 pages:1291-302 ispartof: location:United States status: published

It is here reported that mesenchymal stem cells known to give rise to limb-bud mesoderm can, at the single-cell level, also differentiate into cells of visceral mesoderm and can be expanded extensively by means of clinically applicable methods. These cells were named mesodermal progenitor cells (MPCs). MPCs were selected by depleting bone marrow mononuclear cells from more than 30 healthy human donors of CD45+/glycophorin-A (GlyA)+ cells. Cells were cultured on fibronectin with epidermal growth factor and platelet-derived growth factor BB and 2% or less fetal calf serum. It was found that 1/5 × 103CD45−GlyA− cells, or 1/106 bone marrow mononuclear cells, gave rise to clusters of small adherent cells. Cell-doubling time was 48 to 72 hours, and cells have been expanded in culture for more than 60 cell doublings. MPCs are CD34−, CD44low, CD45−, CD117 (cKit)−, class I–HLA−, and HLA-DR−. MPCs differentiated into cells of limb-bud mesoderm (osteoblasts, chondrocytes, adipocytes, stroma cells, and skeletal myoblasts) as well as visceral mesoderm (endothelial cells). Retroviral marking was used to definitively prove that single MPCs can differentiate into cells of limb bud and visceral mesoderm. Thus, MPCs that proliferate without obvious senescence under clinically applicable conditions and differentiate at the single-cell level not only into mesenchymal cells but also cells of visceral mesoderm may be an ideal source of stem cells for treatment of genetic or degenerative disorders affecting cells of mesodermal origin.

Tumor cells initiate angiogenesis within the tumor micro-environment. The maintenance of these newly formed vessels is highly dependent on the migration and recruitment of immune cells. In previous studies, we have demonstrated that chronic morphine administration suppresses tumor cell induced angiogenesis to inhibit tumor growth in mice. Here we tested the hypothesis that a known immunosuppressant as morphine also regulates immune cell recruitment into- and migration towards a tumor microenvironment. To mimic a tumor microenvironment, matrigel was admixed with conditioned media derived from Lewis lung carcinoma cells (LLCCM) and implanted subcutaneous in WT or Mu-Opioid Receptor knockout mice (MORKO) treated with saline or morphine through pellet implantation. Interestingly, plugs taken from placebo treated mice (day 7) showed significant leukocyte infiltration, greater than that compared to plugs from morphine treated mice (p