Age-dependent cellular reactions of the human immune system of humanized NOD scid gamma mice on LPS stimulus.

Despite sepsis being a life-threatening disease, targeted drugs that improve the therapy of affected patients are still lacking. Infants and adults differ in the maturity level of their immune system and this results in distinct reactions to Gram-negative bacteria. To study reactions of human immune cells in vivo, we used NOD scid gamma mice transplanted with human CD34+ stem cells to engraft a functional human immune system. Human cells undergo differentiation and maturation in these mice after transplantation and, accordingly, animals were divided into two groups: 8-13 wk and 15-22 wk after transplantation. Endotoxemia was induced by injecting LPS. Six h later, mice were euthanized. In both groups, LPS stimulation induced a decrease of CD14+ monocytes in peripheral blood, an up-regulation of activation markers on different cell subsets such as myeloid dendritic cells, and a release of the human cytokines TNF-α, IL-6 and IL-10. However, significant differences were detected with regard to the amounts of released cytokines, and 8-13-wk-old mice produced more IL-6, while PTX3 was mainly released by 15-22-wk-old animals. Thus, here we provide a potential model for preclinical research of sepsis in infants and adults.

Monitoring Disease Progression and Therapeutic Response in a Disseminated Tumor Model for Non-Hodgkin Lymphoma by Bioluminescence Imaging.

Xenograft tumor models are widely studied in cancer research. Our aim was to establish and apply a model for aggressive CD20-positive B-cell non-Hodgkin lymphomas, enabling us to monitor tumor growth and shrinkage in a noninvasive manner. By stably transfecting a luciferase expression vector, we created two bioluminescent human non-Hodgkin lymphoma cell lines, Jeko1(luci) and OCI-Ly3(luci), that are CD20 positive, a prerequisite to studying rituximab, a chimeric anti-CD20 antibody. To investigate the therapy response in vivo, we established a disseminated xenograft tumor model injecting these cell lines in NOD/SCID mice. We observed a close correlation of bioluminescence intensity and tumor burden, allowing us to monitor therapy response in the living animal. Cyclophosphamide reduced tumor burden in mice injected with either cell line in a dose-dependent manner. Rituximab alone was effective in OCI-Ly3(luci)-injected mice and acted additively in combination with cyclophosphamide. In contrast, it improved the therapeutic outcome of Jeko1(luci)-injected mice only in combination with cyclophosphamide. We conclude that well-established bioluminescence imaging is a valuable tool in disseminated xenograft tumor models. Our model can be translated to other cell lines and used to examine new therapeutic agents and schedules.

Genomewide RNAi screen identifies protein kinase Cb and new members of mitogen-activated protein kinase pathway as regulators of melanoma cell growth and metastasis.

A large-scale RNAi screen was performed for eight different melanoma cell lines using a pooled whole-genome lentiviral shRNA library. shRNAs affecting proliferation of transduced melanoma cells were negatively selected during 10 days of culture. Overall, 617 shRNAs were identified by microarray hybridization. Pathway analyses identified mitogen-activated protein kinase (MAPK) pathway members such as ERK1/2, JNK1/2 and MAP3K7 and protein kinase C ß (PKCß) as candidate genes. Knockdown of PKCß most consistently reduced cellular proliferation, colony formation and migratory capacity of melanoma cells and was selected for further validation. PKCß showed enhanced expression in human primary melanomas and distant metastases as compared with benign melanocytic nevi. Moreover, treatment of melanoma cells with PKCß-specific inhibitor enzastaurin reduced melanoma cell growth but had only small effects on benign fibroblasts. Finally, PKCß-shRNA significantly reduced lung colonization capacity of stably transduced melanoma cells in mice. Taken together, this study identified new candidate genes for melanoma cell growth and proliferation. PKCß seems to play an important role in these processes and might serve as a new target for the treatment of metastatic melanoma.

Melanoma cells use Thy-1 (CD90) on endothelial cells for metastasis formation.

The cell adhesion molecule Thy-1 (CD90) mediates the adhesion of melanoma cells to activated human endothelial cells (EC) via the interaction with the αvß3-integrin on the tumor cells in vitro. Here, we report a strong expression of Thy-1 on both blood vessel and lymphatic EC in melanoma and melanoma metastases. Vascular endothelial growth factor and tumor necrosis factor-α were identified as inducers of Thy-1 expression on EC in vitro. The physiological role of Thy-1 for lymphogenic and hematogenic metastasis of melanoma cells was substantiated in an experimental metastasis model using B16/F10 melanoma cells. Mice lacking Thy-1 showed markedly diminished experimental lung metastasis after injection of B16/F10 melanoma cells compared to wild-type littermate controls. In addition, on generation of a primary subcutaneous tumor, metastasis to regional lymph nodes was clearly reduced in Thy-1(-/-) mice. However, Thy-1 deletion did not affect subcutaneous primary tumor growth, tumor-induced recruitment of inflammatory cells or T cells, angiogenesis, or T-cell activation. In conclusion, Thy-1 contributes to metastasis of melanoma cells by mechanisms likely involving a Thy-1-mediated adhesion of melanoma cells to EC.

Comparison of hematopoietic stem cells derived from fresh and cryopreserved whole cord blood in the generation of humanized mice.

To study the function and maturation of the human hematopoietic and immune system without endangering individuals, translational human-like animal models are needed. We compare the efficiency of CD34(+) stem cells isolated from cryopreserved cord blood from a blood bank (CCB) and fresh cord blood (FCB) in generating highly engrafted humanized mice in NOD-SCID IL2Rγ(null) (NSG) rodents. Interestingly, the isolation of CD34(+) cells from CCB results in a lower yield and purity compared to FCB. The purity of CD34(+) isolation from CCB decreases with an increasing number of mononuclear cells that is not evident in FCB. Despite the lower yield and purity of CD34(+) stem cell isolation from CCB compared to FCB, the overall reconstitution with human immune cells (CD45) and the differentiation of its subpopulations e.g., B cells, T cells or monocytes is comparable between both sources. In addition, independent of the cord blood origin, human B cells are able to produce high amounts of human IgM antibodies and human T cells are able to proliferate after stimulation with anti-CD3 antibodies. Nevertheless, T cells generated from FCB showed increased response to restimulation with anti-CD3. Our study reveals that the application of CCB samples for the engraftment of humanized mice does not result in less engraftment or a loss of differentiation and function of its subpopulations. Therefore, CCB is a reasonable alternative to FCB and allows the selection of specific genotypes (or any other criteria), which allows scientists to be independent from the daily changing birth rate.