Cerebral angiogenesis ameliorates pathological disorders in Nemo-deficient mice with small-vessel disease.

Cerebral small-vessel diseases (SVDs) often follow a progressive course. Little is known about the function of angiogenesis, which potentially induces regression of SVDs. Here, we investigated angiogenesis in a mouse model of incontinentia pigmenti (IP), a genetic disease comprising features of SVD. IP is caused by inactivating mutations of Nemo, the essential component of NF-κB signaling. When deleting Nemo in the majority of brain endothelial cells (NemobeKO mice), the transcriptional profile of vessels indicated cell proliferation. Brain endothelial cells expressed Ki67 and showed signs of DNA synthesis. In addition to cell proliferation, we observed sprouting and intussusceptive angiogenesis in NemobeKO mice. Angiogenesis occurred in all segments of the vasculature and in proximity to vessel rarefaction and tissue hypoxia. Apparently, NEMO was required for productive angiogenesis because endothelial cells that had escaped Nemo inactivation showed a higher proliferation rate than Nemo-deficient cells. Therefore, newborn endothelial cells were particularly vulnerable to ongoing recombination. When we interfered with productive angiogenesis by inducing ongoing ablation of Nemo, mice did not recover from IP manifestations but rather showed severe functional deficits. In summary, the data demonstrate that angiogenesis is present in this model of SVD and suggest that it may counterbalance the loss of vessels.

Altered B-cell subsets and functional B-cell defects in selective IgM deficiency.

Primary selective IgM deficiency (sIgM) is characterized by diminished serum IgM, infections and autoimmunity. Although there is some evidence of B-cell defects the pathogenesis of sIgM is poorly understood. We determined peripheral B-cell subsets and IgM-expression levels in 31 adult sIgM patients by flow cytometry. In a subset of patients B-cell subset alterations and antibody-secreting cells were determined by flow cytometry and ELISpot assay after in vitro differentiation.Patients had significantly increased transitional, decreased IgM only, switched and non-switched memory B cells and decreased membrane IgM-expression levels on memory B-cell subsets compared to healthy controls. A strongly diminished B-cell differentiation and expansion capacity was observed in 5/6 investigated patients. Severely reduced IgM-secreting capacity was detected in 2/6 patients.Taken together, our results show altered B-cell subsets and severe functional B-cell defects in sIgM. This may provide a diagnostic tool and basis for subclassification of patients to study the pathogenetic background.

Monitoring the dynamics of clonal tumour evolution in vivo using secreted luciferases.

Tumours are heterogeneous cell populations that undergo clonal evolution during tumour progression, metastasis and response to therapy. Short hairpin RNAs (shRNAs) generate stable loss-of-function phenotypes and are versatile experimental tools to explore the contribution of individual genetic alterations to clonal evolution. In these experiments tumour cells carrying shRNAs are commonly tracked with fluorescent reporters. While this works well for cell culture studies and leukaemia mouse models, fluorescent reporters are poorly suited for animals with solid tumours--the most common tumour types in cancer patients. Here we develop a toolkit that uses secreted luciferases to track the fate of two different shRNA-expressing tumour cell clones competitively, both in vitro and in vivo. We demonstrate that secreted luciferase activities can be measured robustly in the blood stream of tumour-bearing mice to accurately quantify, in a minimally invasive manner, the dynamic evolution of two genetically distinct tumour subclones in preclinical mouse models of tumour development, metastasis and therapy.