Generation of a BAC transgenic mouse strain that expresses CreERT and a fluorescent protein under the transcriptional control of the Fzd5 locus.

BACKGROUND: The expression of FZD5 distinguishes immature human mesenchymal stem/stromal cells (MSC) in cultures, and the function of FZD5 is crucial for maintaining the proliferation and multilineage differentiation capacity of human MSC. We herein investigated whether Fzd5 expression also marks undifferentiated MSC in animals. METHODS: We generated a transgenic mouse strain (Fzd5-CreERT-tFP635) that expresses CreERT and the fluorescent protein, TurboFP635 (tFP635), under the transcriptional control of the Fzd5 gene using the BAC transgenic technique, and identified cells expressing tFP635 by flow cytometry. We also conducted lineage tracing with this strain. RESULTS: In the bone marrow of transgenic mice, tFP635 was preferentially expressed in MSC, Leptin receptor-expressing MSC (LepR+MSCs), and some Pdgfrα+ Sca1+ MSC (PαS). Inducible lineage tracing using the Fzd5-CreERT-tFP635; CAG-CAT-EGFP strain at the adult stage showed that Fzd5-expressing cells and their descendants labeled with GFP were progressively dominant in LepR+MSC and PαS, and GFP+ cells persisted for 1 year after the activation of CreERT. Adipocyte progenitor cells (APCs), osteoblast progenitor cells (OPCs), and Cd51+ stromal cells were also labeled with GFP. CONCLUSIONS: Our transgenic mouse marks two different types of MSC, LepR+MSC and PαS.

Local injection of CCL19-expressing mesenchymal stem cells augments the therapeutic efficacy of anti-PD-L1 antibody by promoting infiltration of immune cells.

BACKGROUND: Mesenchymal stem/stromal cells (MSC) accumulate and reside in tumor sites. METHODS: Taking advantage of this feature in anticancer therapy, immortalized murine MSC (iMSC) were genetically altered to produce chemokine (C-C motif) ligand 19 (iMSC/CCL19), which attracts dendritic cells (DC) and T lymphocytes. Thereafter, iMSC/CCL19 were examined for their therapeutic efficacy using a syngeneic CT26 colon carcinoma cell line. RESULTS: Co-injection of iMSC/CCL19 into mice significantly suppressed the in vivo growth of CT26 cells compared with that of CCL19-expressing immortalized fibroblasts (iFib/CCL19). This anticancer effect was not observed when injected in CT26-bearing nude mice. Co-injected iMSC/CCL19 survived longer than iFib/CCL19 in the tumor sites. In a therapeutic model, local injection of iMSC/CCL19 suppressed the tumor growth, and increased IFN (interferon)-γ+ CD8+ T cells and CCR7+ DC infiltration in tumor site was observed when treated with iMSC/CCL19, but not with iMSC. This antitumor effect was completely negated by depletion of CD4+ cells and partially negated by depletion of CD8+ cells. Furthermore, the antitumor effects induced by local injection of iMSC/CCL19 were augmented by additional therapy with anti-programmed death (PD)-ligand 1 (PD-L1) antibody, but not with anti-PD-1 antibody. This combination therapy cured most of the tumors in CT26-bearing mice. CONCLUSION: These results suggest that local therapy with iMSC/CCL19 can suppress tumor growth via effective recruitment of CCR7+ DC into tumor sites and increase IFN-γ+ CD8+ T cells, and that combination with anti-PD-L1 antibody therapy can be a powerful anticancer therapy.

Calcitonin gene-related peptide regulates type IV hypersensitivity through dendritic cell functions.

Dendritic cells (DCs) play essential roles in both innate and adaptive immune responses. In addition, mutual regulation of the nervous system and immune system is well studied. One of neuropeptides, calcitonin gene-related peptide (CGRP), is a potent regulator in immune responses; in particular, it has anti-inflammatory effects in innate immunity. For instance, a deficiency of the CGRP receptor component RAMP 1 (receptor activity-modifying protein 1) results in higher cytokine production in response to LPS (lipopolysaccharide). On the other hand, how CGRP affects DCs in adaptive immunity is largely unknown. In this study, we show that CGRP suppressed Th1 cell differentiation via inhibition of IL-12 production in DCs using an in vitro co-culture system and an in vivo ovalbumin-induced delayed-type hypersensitivity (DTH) model. CGRP also down-regulated the expressions of chemokine receptor CCR2 and its ligands CCL2 and CCL12 in DCs. Intriguingly, the frequency of migrating CCR2(+) DCs in draining lymph nodes of RAMP1-deficient mice was higher after DTH immunization. Moreover, these CCR2(+) DCs highly expressed IL-12 and CD80, resulting in more effective induction of Th1 differentiation compared with CCR2(-) DCs. These results indicate that CGRP regulates Th1 type reactions by regulating expression of cytokines, chemokines, and chemokine receptors in DCs.

Suppression of ovalbumin-induced allergic diarrhea by diminished intestinal peristalsis in RAMP1-deficient mice.

Recent studies have revealed that various neurotransmitters regulate the immune system via their receptors expressed on the immune cells. Calcitonin gene-related peptide (CGRP), a sensory nerve C-fiber neuropeptide, is also known to have the ability to modulate the functions of immune cells in vitro. However, the contribution of CGRP to the immune regulation in vivo remains to be fully elucidated. Here we report that mice deficient in receptor activity-modifying protein 1 (RAMP1), which is a subunit of the CGRP receptor, showed a significantly lower incidence of diarrhea compared with wild-type (WT) mice in the ovalbumin (OVA)-induced food allergic model. Serum OVA-specific IgE levels and the differentiation of T helper cells was comparable in WT mice and RAMP1-deficient mice. Moreover, there were no significant differences between recruitment and degranulation of mast cells in the small intestine of these mice. In contrast, significantly diminished intestinal peristalsis was observed by the allergy induction in RAMP1-deficient mice compared with WT mice. These results suggest that this suppression of allergic diarrhea is due to the diminished intestinal peristalsis in RAMP1-deficient mice.

High expression of a new marker PCA-1 in human prostate carcinoma.

PURPOSE: Identifying the genetic factors involved in prostate carcinogenesis is critical. Novel cancer-specific markers aid in early detection, in differentiating between cancer and nonmalignant disorders, and in monitoring clinical of prostate disease. We therefore examined differential gene displays in an attempt to identify genes that may be involved in prostate carcinogenesis. EXPERIMENTAL DESIGN: Applying fluorescent differential display analysis to human prostate carcinomas, we have identified and cloned several cDNA transcripts. Antisera were raised against synthetic peptides and used in Western blot and immunohistochemical analyses. The mRNAs were also analyzed by real-time reverse transcription-PCR. For functional analysis, we assessed methylmethane sulfonate (MMS)-induced toxicity in COS-7 cells after cDNA transfection. RESULTS: We identified a gene, designated prostate cancer antigen-1 (pca-1), which shows high mRNA expression in prostate carcinoma. Database analysis of the deduced amino acid sequence of PCA-1 indicated high similarity to Escherichia coli AlkB, a DNA alkylation damage repair enzyme. By immunohistochemical analysis, PCA-1 was expressed in a high number of both prostate carcinoma samples and in the atypical cells within high-grade prostatic intraepithelial neoplasias but not in benign prostatic hyperplasia or normal adjacent tissues. PCA-1-transfected COS-7 cells further showed resistance against MMS-induced cell death. CONCLUSIONS: These findings suggest that PCA-1 could be a useful diagnostic marker. Furthermore, because this human counterpart of AlkB exhibits a protective function against alkylation damage in mammalian cells, PCA-1 may also serve as a therapeutic target molecule for prostate cancer.