In vitro induction of lymph node cell proliferation by mouse bone marrow dendritic cells following stimulation with different Echinococcus multilocularis antigens

The immune response of mice experimentally infected with Echinococcus multilocularis metacestodes becomes impaired so as to allow parasite survival and proliferation. Our study tackled the question on how different classes of E. multilocularis antigens (crude vesicular fluid (VF); purified proteinic rec-14-3-3; purified carbohydrate Em2(G11)) are involved in the maturation process of bone-marrow-derived dendritic cells (BMDCs) and subsequent exposure to lymph node (LN) cells. In our experiments, we used BMDCs cultivated from either naïve (control) or alveolar echinococcosis (AE)-infected C57BL/6 mice. We then tested surface markers (CD80, CD86, MHC class II) and cytokine expression levels (interleukin (IL)-10, IL-12p40 and tumour necrosis factor (TNF)-α) of non-stimulated BMDCs versus BMDCs stimulated with different Em-antigens or lipopolysaccharide (LPS). While LPS and rec-14-3-3-antigen were able to induce CD80, CD86 and (to a lower extent) MHC class II surface expression, Em2(G11) and, strikingly, also VF-antigen failed to do so. Similarly, LPS and rec-14-3-3 yielded elevated IL-12, TNF-α and IL-10 expression levels, while Em2(G11) and VF-antigen didn't. When naïve BMDCs were loaded with VF-antigen, they induced a strong non-specific proliferation of uncommitted LN cells. For both, BMDCs or LN cells, isolated from AE-infected mice, proliferation was abrogated. The most striking difference, revealed by comparing naïve with AE-BMDCs, was the complete inability of LPS-stimulated AE-BMDCs to activate lymphocytes from any LN cell group. Overall, the presenting activity of BMDCs from AE-infected mice seemed to trigger unresponsiveness in T cells, especially in the case of VF-antigen stimulation, thus contributing to the suppression of clonal expansion during the chronic phase of AE infection.

Systemically transferred hematopoietic stem cells home to the subretinal space and express RPE-65 in a mouse model of retinal pigment epithelium damage

Stem cell regeneration of damaged tissue has recently been reported in many different organs. Since the loss of retinal pigment epithelium (RPE) in the eye is associated with a major cause of visual loss - specifically, age-related macular degeneration - we investigated whether hematopoietic stem cells (HSC) given systemically can home to the damaged subretinal space and express markers of RPE lineage. Green fluorescent protein (GFP) cells of bone marrow origin were used in a sodium iodate (NaIO(3)) model of RPE damage in the mouse. The optimal time for adoptive transfer of bone marrow-derived stem cells relative to the time of injury and the optimal cell type [whole bone marrow, mobilized peripheral blood, HSC, facilitating cells (FC)] were determined by counting the number of GFP(+) cells in whole eye flat mounts. Immunocytochemistry was performed to identify the bone marrow origin of the cells in the RPE using antibodies for CD45, Sca-1, and c-kit, as well as the expression of the RPE-specific marker, RPE-65. The time at which bone marrow-derived cells were adoptively transferred relative to the time of NaIO(3) injection did not significantly influence the number of cells that homed to the subretinal space. At both one and two weeks after intravenous (i.v.) injection, GFP(+) cells of bone marrow origin were observed in the damaged subretinal space, at sites of RPE loss, but not in the normal subretinal space. The combined transplantation of HSC+FC cells appeared to favor the survival of the homed stem cells at two weeks, and RPE-65 was expressed by adoptively transferred HSC by four weeks. We have shown that systemically injected HSC homed to the subretinal space in the presence of RPE damage and that FC promoted survival of these cells. Furthermore, the RPE-specific marker RPE-65 was expressed on adoptively transferred HSC in the denuded areas.

Tie2 receptor expression and phosphorylation in cultured cells and mouse tissues

Accumulating experimental evidence indicates that endothelial cell growth and blood vessel morphogenesis are processes that are governed by the activity of specifically expressed receptor tyrosine kinases (RTKs). We have used two new rat monoclonal antibodies (mAbs) to study the expression and phosphorylation of one such receptor, mouse Tie2 (tyrosine kinase that contains immunoglobulin-like loops and epidermal-growth-factor-similar domains 2]), in transfected cells, endothelioma cell lines and mouse tissues. The Tie2 receptor was found to be constitutively autophosphorylated when over-expressed in COS7 cells. In contrast, the endogenous Tie2 protein was not phosphorylated in endothelioma cell lines. However, in these cell lines, Tie2 could be induced to become tyrosine phosphorylated, and this activation was found to be independent of Tie1. Studying Tie2 receptor activity during angiogenesis in mouse development, the receptor was only weakly phosphorylated in the early postnatal mouse brain whereas a stronger activation could be detected in mouse embryos at day 10.5 post coitum.

Multiparameter flow cytometry characterization of MHC class I negative mouse bone marrow cells

BACKGROUND: MHC-I down-regulation was described in foetal liver progenitors, and two different subsets of adult bone marrow derived stem cells. These cells, namely, MHC-I-/Thy1+ bone marrow derived liver stem cells (BMDLSC) and the multipotent adult progenitors (MAPC) differentiated into functioning hepatocytes. The aim of this paper was to characterize the MHC-I negative bone marrow compartment as it pertains to BMDLSC and MAPC. MATERIAL/METHODS: We performed multiparameter flow-cytometry analyses of the MHC-I negative compartment using hematopoietic (CD45, Ter119), and stem cell markers (Thy1.2, c-Kit, IL-3R, CD34) in adult mice. RESULTS: When analysing CD45 and Ter119 expression, the MHC-I negative bone marrow compartment divides into four sub-populations: 1. CD45-/Ter119+: 86.0+/-4.4%; 2. CD45+/Ter119+: 0.2+/-0.1%; 3. CD45+/Ter119-: 11.6+/-3.0%; 4. CD45-/Ter119-: 2.0+/-2.1%. Stem cells markers were only expressed on MHC-I negative/ CD45+/Ter119- cells. In vivo, MAPC (Ter119-/CD45- cells) are composed of MHC-I negative (24%) and MHC-I positive cells and do not express any of the stem cell markers tested. CONCLUSIONS: In conclusion, mouse BMDLSC and MAPC are two distinct stem cell populations. Down-regulation of MHC-I was the only common characteristic found between BMDLSC and MAPC suggesting that selection of MHC-I negative cells might represent an efficient strategy to enrich for bone marrow stem cells with liver developmental potential.

Isolation of sphere-forming stem cells from the mouse inner ear

The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.

Electrophysiological properties of mouse and epitope-tagged human cardiac sodium channel Na v1.5 expressed in HEK293 cells

Background: The pore-forming subunit of the cardiac sodium channel, Na v1.5, has been previously found to be mutated in genetically determined arrhythmias. Na v1.5 associates with many proteins that regulate its function and cellular localisation. In order to identify more in situ Na v1.5 interacting proteins, genetically-modified mice with a high-affinity epitope in the sequence of Na v1.5 can be generated. Methods: In this short study, we (1) compared the biophysical properties of the sodium current (I Na) generated by the mouse Na v1.5 (mNa v1.5) and human Na v1.5 (hNa v1.5) constructs that were expressed in HEK293 cells, and (2) investigated the possible alterations of the biophysical properties of the human Na v1.5 construct that was modified with specific epitopes. Results: The biophysical properties of mNa v1.5 were similar to the human homolog. Addition of epitopes either up-stream of the N-terminus of hNa v1.5 or in the extracellular loop between the S5 and S6 transmembrane segments of domain 1, significantly decreased the amount of I Na and slightly altered its biophysical properties. Adding green fluorescent protein (GFP) to the N-terminus did not modify any of the measured biophysical properties of hNa v1.5. Conclusions: These findings have to be taken into account when planning to generate genetically-modified mouse models that harbour specific epitopes in the gene encoding mNa v1.5.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma

PURPOSE In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFNγ production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain

Latrepirdine (Dimebon; dimebolin) is a neuroactive compound that was associated with enhanced cognition, neuroprotection and neurogenesis in laboratory animals, and has entered phase II clinical trials for both Alzheimer's disease and Huntington's disease (HD). Based on recent indications that latrepirdine protects cells against cytotoxicity associated with expression of aggregatable neurodegeneration-related proteins, including Aβ42 and γ-synuclein, we sought to determine whether latrepirdine offers protection to Saccharomyces cerevisiae. We utilized separate and parallel expression in yeast of several neurodegeneration-related proteins, including α-synuclein (α-syn), the amyotrophic lateral sclerosis-associated genes TDP43 and FUS, and the HD-associated protein huntingtin with a 103 copy-polyglutamine expansion (HTT gene; htt-103Q). Latrepirdine effects on α-syn clearance and toxicity were also measured following treatment of SH-SY5Y cells or chronic treatment of wild-type mice. Latrepirdine only protected yeast against the cytotoxicity associated with α-syn, and this appeared to occur via induction of autophagy. We further report that latrepirdine stimulated the degradation of α-syn in differentiated SH-SY5Y neurons, and in mouse brain following chronic administration, in parallel with elevation of the levels of markers of autophagic activity. Ongoing experiments will determine the utility of latrepirdine to abrogate α-syn accumulation in transgenic mouse models of α-syn neuropathology. We propose that latrepirdine may represent a novel scaffold for discovery of robust pro-autophagic/anti-neurodegeneration compounds, which might yield clinical benefit for synucleinopathies including Parkinson's disease, Lewy body dementia, rapid eye movement (REM) sleep disorder and/or multiple system atrophy, following optimization of its pro-autophagic and pro-neurogenic activities.

The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions

Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions.

Postnatal Notch1 activation induces T‑cell malignancy in conditional and inducible mouse models

The Notch1 signaling pathway is essential for hematopoietic development. However, the effects of postnatal activation of Notch1 signaling on hematopoietic system is not yet fully understood. We previously generated ZEG‑IC‑Notch1 transgenic mice that have a floxed β‑geo/stop signal between a CMV promoter and intracellular domain of Notch1 (IC‑Notch1). Constitutively active IC‑Notch1 is silent until the introduction of Cre recombinase. In this study, endothelial/hematopoietic specific expression of IC‑Notch1 in double transgenic ZEG‑IC‑Notch1/Tie2‑Cre embryos induced embryonic lethality at E9.5 with defects in vascular system but not in hematopoietic system. Inducible IC‑Notch1 expression in adult mice was achieved by using tetracycline regulated Cre system. The ZEG‑IC‑Notch1/Tie2‑tTA/tet‑O‑Cre triple transgenic mice survived embryonic development when maintained on tetracycline. Post‑natal withdrawal of tetracycline induced expression of IC‑Notch1 transgene in hematopoietic cells of adult mice. The triple transgenic mice displayed extensive T‑cell infiltration in multiple organs and T‑cell malignancy of lymph nodes. In addition, the protein levels of p53 and alternative reading frame (ARF) were decreased in lymphoma‑like neoplasms from the triple transgenic mice while their mRNA expression remained unchanged, suggesting that IC‑Notch1 might repress ARF‑p53 pathway by a post‑transcriptional mechanism. This study demonstrated that activation of constitutive Notch1 signaling after embryonic development alters adult hematopoiesis and induces T‑cell malignancy.

Splicing changes in SMA mouse motoneurons and SMN-depleted neuroblastoma cells: evidence for involvement of splicing regulatory proteins.

Spinal Muscular Atrophy (SMA) is caused by deletions or mutations in the Survival Motor Neuron 1 (SMN1) gene. The second gene copy, SMN2, produces some, but not enough, functional SMN protein. SMN is essential to assemble small nuclear ribonucleoproteins (snRNPs) that form the spliceosome. However, it is not clear whether SMA is caused by defects in this function that could lead to splicing changes in all tissues, or by the impairment of an additional, less well characterized, but motoneuron-specific SMN function. We addressed the first possibility by exon junction microarray analysis of motoneurons (MNs) isolated by laser capture microdissection from a severe SMA mouse model. This revealed changes in multiple U2-dependent splicing events. Moreover, splicing appeared to be more strongly affected in MNs than in other cells. By testing mutiple genes in a model of progressive SMN depletion in NB2a neuroblastoma cells, we obtained evidence that U2-dependent splicing changes occur earlier than U12-dependent ones. As several of these changes affect genes coding for splicing regulators, this may acerbate the splicing response induced by low SMN levels and induce secondary waves of splicing alterations.

CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis

Current therapies to treat inflammatory bowel diseases have limited efficacy, significant side effects, and often wane over time. Little is known about the cellular and molecular mechanisms operative in the process of mucosal healing from colitis. To study such events, we developed a new model of reversible colitis in which adoptive transfer of CD4(+)CD45RB(hi) T cells into Helicobacter typhlonius-colonized lymphopenic mice resulted in a rapid onset of colonic inflammation that was reversible through depletion of colitogenic T cells. Remission was associated with an improved clinical and histopathological score, reduced immune cell infiltration to the intestinal mucosa, altered intestinal gene expression profiles, regeneration of the colonic mucus layer, and the restoration of epithelial barrier integrity. Notably, colitogenic T cells were not only critical for induction of colitis but also for maintenance of disease. Depletion of colitogenic T cells resulted in a rapid drop in tumor necrosis factor α (TNFα) levels associated with reduced infiltration of inflammatory immune cells to sites of inflammation. Although neutralization of TNFα prevented the onset of colitis, anti-TNFα treatment of mice with established disease failed to resolve colonic inflammation. Collectively, this new model of reversible colitis provides an important research tool to study the dynamics of mucosal healing in chronic intestinal remitting-relapsing disorders.Mucosal Immunology advance online publication 16 September 2015; doi:10.1038/mi.2015.93.