Graft-versus-host disease (GVHD) is a major obstacle in allogeneic hematopoietic cell transplantation. Given the dynamic changes in immune cell subsets and tissue organization, which occur in GVHD, localization and timing of critical immunological events in vivo may reveal basic pathogenic mechanisms. To this end, we transplanted luciferase-labeled allogeneic splenocytes and monitored tissue distribution by in vivo bioluminescence imaging. High-resolution analyses showed initial proliferation of donor CD4 ؉ T cells followed by CD8 ؉ T cells in secondary lymphoid organs with subsequent homing to the intestines, liver, and skin. Transplantation of purified naive T cells caused GVHD that was initiated in secondary lymphoid organs followed by target organ manifestation in gut, liver, and skin. IntroductionAllogeneic hematopoietic cell transplantation (HCT) has proven to be an effective therapy for a variety of life-threatening malignancies. 1 The beneficial effects of HCT are due to the graft-versustumor reaction, which is capable of destroying residual tumor cells that persist after chemotherapy or radiation therapy. 2 However, allogeneic HCT is limited by the immunologic recognition and destruction of host tissues, termed graft-versus-host disease (GVHD). Acute GVHD continues to be a major source of morbidity and mortality following HCT, which limits treatment of a broader spectrum of diseases, such as autoimmune diseases or organ transplant rejection. 3,4 Tissue-specific destruction of GVHD target organs, as gastrointestinal tract, liver, and skin, underlines the importance of migration capacities of alloreactive T lymphocytes. 5,6 In the current study we aimed to determine the time points of organ infiltration and focused on the role of different lymphoid organs in initiating acute GVHD. We used in vivo bioluminescence imaging (BLI) to analyze the migration pattern of whole splenocytes after transplantation into allogeneic recipients. BLI has already proven to be a sensitive and accurate means of characterizing engraftment patterns of hematopoietic stem cells, of monitoring tumor cell growth, and of assessing response to conventional and biological therapies. [7][8][9] We also aimed to clarify the role of different T-cell subsets during GVHD development. It is reported in the literature [10][11][12] that CD4 ϩ effector memory T (T EM ) cells do not cause GVHD. This prompted us to characterize their trafficking and proliferation pattern in vivo, while comparing it to purified naive CD4 ϩ T lymphocytes. Materials and methods MiceFVB/N (H-2 q , Thy1.1) mice and Balb/c mice (H-2 d , Thy1.2) were purchased from Jackson Laboratory (Bar Harbor, ME). The luciferaseexpressing (luc ϩ ) transgenic FVB/N line was generated as previously described. 9 Female heterozygous luc ϩ offspring of the transgenic founder line FVB-L2G85 were used for all transplantation experiments. All animal studies were performed under institutional approval. Flow cytometric cell purification and analysisThe following antibodies were purchased from...

The objective of the study was to track the distribution and differentiation of mesenchymal stem cells (MSCs) in tumor-bearing mice. The 4T1 murine breast cancer cells were labeled with renilla luciferase-monomeric red fluorescence protein (rLuc-mRFP) reporter gene. The MSCs labeled with firefly luciferase-enhanced green fluorescence protein (fLuc-eGFP) reporter gene (MSCs-R) were isolated from L2G85 transgenic mice that constitutively express fLuc-eGFP reporter gene. To study the tumor tropism of MSCs, we established both subcutaneous and lung metastasis models. In lung metastasis tumor mice, we injected MSCs-R intravenously either on the same day or 4 days after 4T1 tumor cell injection. In subcutaneous tumor mice, we injected MSCs-R intravenously 7 days after subcutaneous 4T1 tumor inoculation. The tumor growth was monitored by rLuc bioluminescence imaging (BLI). The fate of MSCs-R was monitored by fLuc BLI. The localization of MSCs-R in tumors was examined histologically. The osteogenic and adipogenic differentiation of MSCs-R was investigated by alizarin red S and oil red O staining, respectively. The mechanism of the dissimilar differentiation potential of MSCs-R under different tumor microenvironments was investigated. We found that the 4T1 cells were successfully labeled with rLuc-mRFP. The MSCs-R isolated from L2G85 transgenic mice constitutively express fLuc-eGFP reporter gene. When injected intravenously, MSCs-R survived, proliferated, and differentiated in tumor sites but not elsewhere. The localization of GFP+ MSCs-R in tumor lesions was confirmed ex vivo. In conclusion, the MSCs-R can selectively localize, survive, and proliferate in both subcutaneous tumor and lung metastasis as evidenced by noninvasive bioluminescence imaging and ex vivo validation. The MSCs-R migrated to lung tumor differentiated into osteoblasts, whereas the MSCs-R targeting subcutaneous tumor differentiated into adipocytes.

Background. Laboratory data suggests a reduction of Faecalibacterium prausnitzii (F. prausnitzii) is confirmed both in fecal samples in inflammatory bowel disease (IBD) patients. Numerous observational studies have suspected dysbiosis, an imbalance between protective and harmful bacteria to be relevant to the etiology and pathogenesis of IBD. Methods. Medline, EMBASE, Pubmed, and others. were searched by 2 independent reviewers. Of 48 abstracts reviewed, 11 studies met our inclusion criteria (subject N = 1180). Meta-analysis was performed with Review Manager 5.2. Results. The bacterial count of F. prausnitzii in IBD patients was significantly lower (6.7888 ± 1.8875) log10 CFU/g feces than healthy controls (7.5791 ± 1.5812) log10 CFU/g feces; P < 0.0001. The Standardization Mean Difference of F. prausnitzii in IBD patients was −0.94 (95% confidence interval [CI]: −1.07–−0.80). Subgroup analyses revealed a trend toward a greater effect for CD (SMD: −1.13, 95% CI: −1.32–−0.94) when compared to UC (SMD: −0.78, 95% CI: −0.97–−0.60). Conclusions. The abundance of F. prausnitzii was decreased in IBD patients compared with healthy controls. Furthermore, the reduction of F. prausnitzii and misbalance of the intestinal microbiota are particularly higher in CD patients with ileal involvement.

Recombinant adeno-associated viruses (rAAV) are promising gene transfer vectors that produce long-term expression without toxicity. To investigate future approaches for in utero gene delivery, the efficacy and safety of prenatal administration of rAAV were determined. Using luciferase as a reporter, expression was assessed by whole-body imaging and by analysis of luciferase activity in tissue extracts, at the time of birth and monthly thereafter. Transgene expression was detected in all injected animals. Highest levels of luciferase activity were detected at birth in the peritoneum and liver, while the heart, brain, and lung demonstrated low-level expression. In vivo luciferase imaging revealed persistent peritoneal expression for 18 months after in utero injection and provided a sensitive whole-body assay, useful in identifying tissues for subsequent analyses. There was no detectable hepatocellular injury. Antibodies that reacted with either luciferase or rAAV were not found. AAV sequences were not detected in germ-line tissues of injected animals or in tissues of their progeny. In utero AAV-mediated gene transfer in this animal model demonstrates that novel therapeutic vectors and strategies can be rapidly tested in vivo and that rAAV may be developed to ameliorate genetic diseases with perinatal morbidity and mortality.