The dermis contains langerin+ dendritic cells that develop and function independently of epidermal Langerhans cells.

Langerhans cells (LCs) constitute a subset of dendritic cells (DCs) that express the lectin langerin and that reside in their immature state in epidermis. Paradoxically, in mice permitting diphtheria toxin (DT)-mediated ablation of LCs, epidermal LCs reappeared with kinetics that lagged behind that of their putative progeny found in lymph nodes (LNs). Using bone marrow (BM) chimeras, we showed that a major fraction of the langerin(+), skin-derived DCs found in LNs originates from a developmental pathway that is independent from that of epidermal LCs. This pathway, the existence of which was unexpected, originates in the dermis and gives rise to langerin(+) dermal DCs (DDCs) that should not be confused with epidermal LCs en route to LNs. It explains that after DT treatment, some langerin(+), skin-derived DCs reappear in LNs long before LC-derived DCs. Using CD45 expression and BrdU-labeling kinetics, both LCs and langerin(+) DDCs were found to coexist in wild-type mice. Moreover, DT-mediated ablation of epidermal LCs opened otherwise filled niches and permitted repopulation of adult noninflammatory epidermis with BM-derived LCs. Our results stress that the langerin(+) DC network is more complex than originally thought and have implications for the development of transcutaneous vaccines and the improvement of humanized mouse models.

Treatment of intestinal Behçet's syndrome with chimeric tumour necrosis factor alpha antibody

Few patients with Behçet's syndrome have gastrointestinal ulceration. Such patients are difficult to treat and have a higher mortality. Faced with refractory symptoms in two patients with intestinal Behçet's, we used the tumour necrosis factor alpha (TNF-alpha) monoclonal antibody infliximab to induce remission. Both women (one aged 27 years, the other 30 years) presented with orogenital ulceration, pustular rash, abdominal pain, bloody diarrhoea due to colonic ulceration, weight loss, and synovitis. One had thrombophlebitis, digital vasculitis, perianal fistula, and paracolic abscess; the other had conjunctivitis and an ulcer in the natal cleft. Treatment with prednisolone, methyl prednisolone, and thalidomide in one and prednisolone, colchicine, and cyclosporin in the other was ineffective. After full discussion, infliximab (3 mg/kg, dose reduced because of recent sepsis in one, and 5 mg/kg in the other) was administered. Within 10 days the ulcers healed, with resolution of bloody diarrhoea and all extraintestinal manifestations. A second infusion of infliximab was necessary eight weeks later in one case, followed by sustained (>15 months) remission on low dose thalidomide. Remission was initially sustained for 12 months in the other but thalidomide had to be stopped due to intolerance, and a good response to retreatment lasted only 12 weeks without immunosuppression, before a third infusion. The cause of Behçet's syndrome is unknown but peripheral blood CD45 gammadelta T cells in Behçet's produce >50-fold more TNF-alpha than controls when stimulated with phorbol myristate acetate and anti-CD3. Infliximab could have a role for inducing remission in Behçet's syndrome.

Isolation of a bone marrow-derived stem cell line with high proliferation potential and its application for preventing acute fatal liver failure

Transplantation of hepatocytes or hepatocyte-like cells of extrahepatic origin is a promising strategy for treatment of acute and chronic liver failure. We examined possible utility of hepatocyte-like cells induced from bone marrow cells for such a purpose. Clonal cell lines were established from the bone marrow of two different rat strains. One of these cell lines, rBM25/S3 cells, grew rapidly (doubling time, approximately 24 hours) without any appreciable changes in cell properties for at least 300 population doubling levels over a period of 300 days, keeping normal diploid karyotype. The cells expressed CD29, CD44, CD49b, CD90, vimentin, and fibronectin but not CD45, indicating that they are of mesenchymal cell origin. When plated on Matrigel with hepatocyte growth factor and fibroblast growth factor-4, the cells efficiently differentiated into hepatocyte-like cells that expressed albumin, cytochrome P450 (CYP) 1A1, CYP1A2, glucose 6-phosphatase, tryptophane-2,3-dioxygenase, tyrosine aminotransferase, hepatocyte nuclear factor (HNF)1 alpha, and HNF4alpha. Intrasplenic transplantation of the differentiated cells prevented fatal liver failure in 90%-hepatectomized rats. In conclusion, a clonal stem cell line derived from adult rat bone marrow could differentiate into hepatocyte-like cells, and transplantation of the differentiated cells could prevent fatal liver failure in 90%-hepatectomized rats. The present results indicate a promising strategy for treating human fatal liver diseases.

Efficient differentiation into skin cells of bone marrow cells recovered in a pellet after density gradient fractionation

We had previously demonstrated the participation of whole bone marrow cells from adult mice in the reconstitution of skin, including the epidermis and hair follicles. To get an insight into cell populations that give rise to the epithelial components of the reconstituted skin, we fractionated bone marrow cells derived from green fluorescent protein-transgenic mice by density gradient. Unexpectedly, we found that a substantial amount of mononucleated cells (approximately 30%) was recovered in the pellet fraction and that the cells in the pellet fraction preferentially differentiated into epithelial components of skin, rather than the cells in the mononuclear cell fraction. The pellet fraction contained more CD45-negative (thus uncommitted to the hematopoietic cell lineage) cells than the mononuclear cell fraction. These results indicate that density gradient fractionation results in significant loss of specific progenitor cells into the usually discarded pellet fraction.

Posterior capsule opacification in mice

Objective: To present a new model of posterior capsule opacification (PCO) in mice. Methods: An extracapsular lens extraction was performed in 28 consecutive mice. Animals were humanely killed 0 and 24 hours and 3 and 14 days after surgery. Eyes were enucleated and processed for light microscopy and immunohistochemistry. Results: In 20 animals (71%), the eye appeared well healed before death. In 8 animals (29%), postoperative complications were noted. All animals developed PCO 2 weeks after surgery. Immediately after extracapsular lens extraction, lens epithelial cells were present in the inner surface of the anterior capsule and at the lens bow. At 24 hours, lens epithelial cells started to migrate toward the center of the posterior capsule. At 3 days, multilayered lens epithelial cells throughout the lens capsule and capsular wrinkling were apparent. Lens fibers and Soemmerring ring formation were observed 14 days after surgery. CD45 and CD11b macrophages were found in greater numbers 24 hours and 3 days after surgery (CD45 , P = .04 and P <.001, respectively; and CD11b , P = .01 and P = .004, respectively). The number of CD45 cells remained statistically significantly higher (P = .04) 14 days after surgery. Conclusion: In mice, PCO occurs following extracapsular lens extraction and is associated with low-grade but significant macrophage response. Clinical Relevance: The use of genetically modified mice to evaluate the pathogenic mechanisms of PCO and search for new therapeutic modalities to prevent or treat PCO is now possible.

Culture and Characterization of Microglia from the Adult Murine Retina

Purpose. To develop a protocol for isolating and culturing murine adult retinal microglia and to characterize the phenotype and function of the cultured cells. Method. Retinal single-cell suspensions were prepared from adult MF1 mice. Culture conditions including culture medium, growth factors, seeding cell density, and purification of microglia from the mixed cultures were optimised. Cultured retinal microglial cells were phenotyped using the surface markers CD45, CD11b, and F4/80. Their ability to secrete proinflammatory cytokines in response to lipopolysaccharide (LPS) stimulation was examined using cytometric bead array (CBA) assay. Results. Higher yield was obtained when retinal single-cell suspension was cultured at the density of cells per cm2 in Dulbecco’s modified Eagle medium (DMEM)/F12 + Glutamax supplement with 20% fetal calf serum (FCS) and 20% L929 supernatant. We identified day 10 to be the optimum day of microglial isolation. Over 98% of the cells isolated were positive for CD45, CD11b, and F4/80. After stimulating with LPS they were able to secrete proinflammatory cytokines such as IL-6 and TNF-α and express CD86, CD40, and MHC-II. Conclusion. We have developed a simple method for isolating and culturing retinal microglia from adult mice.