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We tested the effect of 6-(Methylsulfinyl)hexyl Isothiocyanate (6-MITC) and derivatives (I7447 and I7557) on the differentiation and maturation of human myeloid dendritic cells (DCs) in vitro, and skin transplantation in vivo. Triggering of CD14+ myeloid monocyte development toward myeloid DCs with and without 6-MITC and derivatives to examine the morphology, viability, surface marker expression, and cytokine production. Stimulatory activity on allogeneic naive T cells was measured by proliferation and interferon-γ production. The skin allograft survival area model was used to translate the 6-MITC and derivatives’ antirejection effect. All of the compounds had no significant effects on DC viability and reduced the formation of dendrites at concentrations higher than 10 μM. At this concentration, 6-MITC and I7557, but not I7447, inhibited the expression of CD1a and CD83. Both 6-MITC and I7557 exhibited T-cells and interferon-γ augmentation at lower concentrations and suppression at higher concentration. The 6-MITC and I7557 prolonged skin graft survival. Both the 6-MITC and I7557 treatment resulted in the accumulation of regulatory T cells in recipient rat spleens. No toxicity was evident in 6-MITC and I7557 treatment. The 6-MITC and I7557 induced human DC differentiation toward a tolerogenic phenotype and prolonged rat skin allograft survival. These compounds may be effective as immunosuppressants against transplant rejection.

The combined administration of programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) inhibitors might be considered as a treatment for poorly responsive cancer. We report a patient with brain metastatic lung adenocarcinoma in whom fatal myocarditis developed after sequential use of PD-1 and PD-L1 inhibitors. This finding was validated in syngeneic tumor-bearing mice. The mice bearing lung metastases of CT26 colon cancer cells treated with PD-1 and/or PD-L1 inhibitors showed that the combination of anti-PD-1 and anti-PD-L1, either sequentially or simultaneously administered, caused myocarditis lesions with myocyte injury and patchy mononuclear infiltrates in the myocardium. A significant increase of infiltrating neutrophils in myocytes was noted only in mice with sequential blockade, implying a role for the pathogenesis of myocarditis. Among circulating leukocytes, concurrent and subsequent treatment of PD-1 and PD-L1 inhibitors led to sustained suppression of neutrophils. Among tumor-infiltrating leukocytes, combinatorial blockade increased CD8+ T cells and NKG2D+ T cells, and reduced tumor-associated macrophages, neutrophils, and natural killer (NK) cells in the lung metastatic microenvironment. The combinatorial treatments exhibited better control and anti-PD-L1 followed by anti-PD-1 was the most effective. In conclusion, the combinatory use of PD-1 and PD-L1 blockade, either sequentially or concurrently, may cause fulminant cardiotoxicity, although it gives better tumor control, and such usage should be cautionary.

Esophageal cancer prognosis remains poor in current clinical practice. We previously reported that moscatilin can induce apoptosis and mitotic catastrophe in esophageal cancer cells, accompanied by upregulation of polo-like kinase 1 (Plk1) expression. We aimed to validate in vitro activity and Plk1 expression in vivo following moscatilin treatment and to examine the treatment&rsquo, s radiosensitizing effect. Human esophageal cancer cells were implanted in nude mice. Moscatilin was intraperitoneally (i.p.) injected into the mice. Tumor size, body weight, white blood cell counts, and liver and renal function were measured. Aberrant mitosis and Plk1 expression were assessed. Colony formation was used to measure survival fraction after radiation. Moscatilin significantly suppressed tumor growth in mice bearing human esophageal xenografts without affecting body weight, white blood cell counts, or liver and renal function. Moscatilin also induced aberrant mitosis and apoptosis. Plk1 expression was markedly upregulated in vivo. Moreover, moscatilin pretreatment enhanced CE81T/VGH and BE3 cell radioresponse in vitro. Moscatilin may inhibit growth of human esophageal tumors and sensitize esophageal cancer cells to radiation therapy.

Background: Neoadjuvant chemoradiation is the standard of care for locally advanced rectal cancer, however, pathological complete response (pCR) rate remains unsatisfactory. Tumor hypoxia in rectal cancer might be one factor of radioresistance. Mitomycin C (MMC), a hypoxia-activated prodrug (HAP), enhances radiosensitivity and is the standard adjunct to radiotherapy (RT) in anal cancer. Our study evaluated the effect of MMC and RT on rectal cancer and its tumor microenvironment (TME). Methods: In vitro hypoxia was induced by 1% O2 culture for CT26 rectal adenocarcinoma cells and HIF-1α expression was validated with Western blotting. Clonogenicity and MTT assays were used for radiation and drug survival. In syngeneic CT26 model, tumors were treated as follows: control, MMC (2 mg/kg/day), RT (2 Gy × 2 fractions), and combination with MMC 2 h prior to RT. Tumor volume, body weight, and white blood cell count were monitored for 1 month. Immune cells in TME were analyzed using flow cytometry. Results: MMC inhibited cell viability and enhanced radioresponse in CT 26 cells with delayed repair of DNA double strand break (P Ifnb1 was augmented by RT and upregulated with addition of MMC. In vivo treatment using MMC plus RT suppressed tumor growth with durable effect in comparison to MMC or RT alone (P Conclusions: The combination of MMC and RT may have synergistic therapeutic effect accompanied by modulation of TME towards favorable anti-tumor immunity.

e14115 Background: In clinical practice, the shifting use of PD-1 inhibitors and its ligand PD-L1 inhibitors due to ineffectiveness or toxicity is becoming more common and has distinct responses. To validate clinical observation, we examined the anti-cancer effect, tumor microenvironment (TME) modulation and toxicity of dual blockade of PD-1 and PD-L1 in mice. Case Presentation: Three patients with metastatic lung adenocarcinoma received nivolumab (5, 5 and 11 doses); after the initial response and subsequent progression, they were shifted to receive atezolimumab (1, 1 and 4 doses) at intervals of 70, 38 and 43 days, respectively. All 3 patients obtained partial response according to iRECIST. Two of the 3 patients developed pneumonitis at days 3 and 15 after the first dose of atezolimumab and pneumonitis subsided by methylprednisolone injection for 7 and 4 days. Methods: The Balb/c mice bearing lung metastasis of CT26 colon cancer cells were treated. Assessments included optical imaging for tumor burden, multi-parameter flow cytometry for TME, pathology and immuohistochemistry. Results: The pathology of lung showed that all the mice (100%, n = 3 for each group) treated with a combination of anti-PD-1 and anti-PD-L1, either sequential or simultaneous, developed severe clustered inflammatory infiltrates, mucus filling in bronchus and thrombi in small vessels. Among infiltrating leukocytes, combinatorial blockade increased LyC6+ inflammatory monocytes and NKG2D+ T cells and reduced tumor-associated macrophages in lung. Peripheral leukocytes in spleen had no such alteration. In lung metastatic foci, the expression of PD-1 was noted mainly within tumors, and this expression was reduced by combinatorial blockade. PD-L1 expression had no change. For control of lung metastasis, the combinatorial treatments exhibited better control, and anti-PD-L1 followed by anti-PD-1 was the most effective. No significant pneumonitis was noted in single blockade groups. Conclusions: The combination of PD-1 and PD-L1 blockade, either sequential or concurrent, may have better tumor control but cause severe pneumonitis. Dual blockade of PD-1 and PD-L1 should be used with caution.

Syncytin is an envelope protein of the human endogenous retrovirus family W (HERV-W). Syncytin is specifically expressed in the human placenta and mediates trophoblast cell fusion into the multinucleated syncytiotrophoblast layer. It is a polypeptide of 538 amino acids and is predicted to be posttranslationally cleaved into a surface (SU) subunit and a transmembrane (TM) subunit. Functional characterization of syncytin protein can aid understanding of the molecular mechanism underlying syncytin-mediated cell fusion. In this report, we studied the structure-function relationship of syncytin in 293T and HeLa cells transiently expressing wild-type syncytin or syncytin mutants generated by linker scanning and deletion mutagenesis. Of the 22 linker-inserted mutants, mutants InS51, InV139, InE156, InS493, InA506, and InL529 were fusogenic, suggesting that regions around amino acids S51, V139, and E156 in the SU subunit and S493, A506, and L529 in the cytoplasmic domain (CTM) of syncytin are flexible in conformation. Of the 17 deletion mutants, nine mutants with deletions in the region from amino acids 479 to 538 were fusogenic. The deletion mutant DelI480, containing only the first four amino acid residues in the cytoplasmic domain, had enhanced fusogenic activity in comparison with the wild-type. In addition, two heptad repeat regions (HRA and B) were defined in the TM subunit of syncytin. A peptide inhibitor derived from the C-terminal heptad repeat region (HRB) was shown to potently inhibit syncytin-mediated cell fusion. Our results suggest that the cytoplasmic domain of syncytin is not essential for syncytin-mediated fusion but may play a regulatory role, and an intramolecular interaction between HRA and B is involved in the fusion process.

Purpose: Zoledronic acid (ZOL), an effective nitrogen-containing bisphosphonate used to prevent excessive bone loss in clinical practice, has been shown to affect the development of dendritic cells by redirecting differentiation toward a state of atypical maturation. The study was aimed to examine whether ZOL can reduce acute rejection of skin allografts. Methods: A skin transplantation model using C57BL/6 to BALB/c mice was used. ZOL was injected intraperitoneally into transplant recipients post-surgically. Graft survival, body weight, leukocyte count, hepatic and renal functions were assessed. Results: ZOL treatment significantly prolonged skin allograft survival in mice. In terms of toxicity, there were no significant differences in body weight, leukocyte count, plasma alanine aminotransferase or creatinine levels between the ZOL-treated and control groups. Histopathology showed that the loss of skin integrity seen in control group was prevented by ZOL treatment. In draining lymph nodes and spleen, the number and clustering extent of mononuclear cells were markedly declined by ZOL treatment. The plasma IL-6 levels were reduced by treatment of ZOL. Conclusion: ZOL can prolong skin allograft survival without major toxicity.

BACKGROUND: Human placental mesenchymal stem cells (hPMCs) are thought to be multipotent, but their fate after in utero transplantation is not known. METHODS: hPMCs isolated from term placenta were assessed for their phenotype markers, mutilineage capacity, and immunomodulatory properties. Their engraftment potential was analyzed in a pregnant rat model after in utero transplantation at embryonic day 17. Immunohistochemistry, tracing of labeled cells, fluorescence in situ hybridization and real-time PCR were used to assess post-transplant chimerism. RESULTS: In vitro, lineage-negative, CD34-negative hPMCs differentiated into osteocytes, adipocytes, hepatocytes and endothelial cells with tube formation, and actively suppressed the rat lymphocyte proliferative response to allogeneic lymphocyte stimulation (P < 0.0001). After in utero transplantation into pregnant rats, a low level of engraftment was achieved in various fetal tissues. Engraftment occurred in more than 60% of the fetal rats. Cells persisted for at least 12 weeks after delivery and evidence was obtained to suggest differentiation into specific lineages, including hepatocytes and hematopoietic cells. However, a greater number of hPMCs migrated to the placenta than to the fetus, thus limiting the degree of cell engraftment in fetal organs. CONCLUSIONS: We conclude that hPMCs are mutipotent cells that can be engrafted long-term in immunocompetent rats after in utero transplantation.

Fusion of cytotrophoblasts into the multinucleated syncytiotrophoblast layer is essential for the development of a functional placenta. The envelope protein of a human endogenous retrovirus W (HERV-W) family member, syncytin 1, has been shown to mediate placental cell fusion. Recently, the envelope protein of another HERV family member (HERV-FRD), syncytin 2, has been identified and shown to be highly expressed in the placenta. To better understand the biology of syncytin 2, in this study we first investigated syncytin 2 gene expression in normal and preeclamptic placentas and then characterized the functions of syncytin 2. The expression of syncytin 2 gene was decreased in preeclamptic placentas and could be stimulated by the cAMP stimulant forskolin. The endoprotease furin was found to be involved in the posttranslational cleavage of syncytin 1 and 2 polypeptides into surface and transmembrane subunits. In addition, proper association of the subunits of syncytins 1 and 2 is probably required for the functional integrity of each protein, because subunit swapping of syncytins 1 and 2 failed to generate fusogenic chimeras. Finally, we demonstrated that the disulfide bridge-forming CX(2)C and CX(7)C motifs found in syncytins 1 and 2 are essential for their fusogenic activities, because mutations in the CX(2)C motif not only abolished fusogenesis but also functioned as dominant-negative mutants. Our results suggest that syncytin 2 may function as a second fusogenic protein for placental cell fusion.

The present study investigated the contributions of vagal and nonvagal sensory nerve fibers on neurogenic inflammation in rat bronchial airways. A surgical procedure was developed via the rat mediastinum ventral intercostal space to prepare an intercostal opening without causing pneumothorax for performing stellate ganglionectomy alone, thoracic vagus nerve section alone, and stellatectomy plus thoracic vagotomy, and for injecting capsaicin (2 microl, 10 mg/ml) and 6-hydroxydopamine (2 microl, 50 mg/ml) into the ganglion. One week later in our procedure, we investigated if neurogenic inflammation induced by an intravenous injection of capsaicin (300 nmol/ml/kg) and innervation density of substance P-immunoreactive sensory axons could be decreased after chronic denervation in the rat lower airways. The major findings were that surgical removal of the right stellate ganglion and local capsaicin application resulted in a significant attenuation of neurogenic plasma extravasation in the right bronchial tree evoked by systemic capsaicin application. Reduction of neurogenic plasma extravasation was totally abolished by combined stellatectomy and thoracic vagotomy. The number of substance P-containing axons was also greatly decreased following these surgical and capsaicin treatments. It is concluded that sensory nerve fibers from both vagal source and nonvagal (spinal) source, which associated with the stellate ganglion, contributed significantly to neurogenic inflammation in the bronchial airways with a slightly higher contribution from the vagus nerve.