Intraperitoneal murine Echinococcus multilocularis infection induces differentiation of TGF-beta-expressing DCs that remain immature

Intraperitoneal larval infection (alveolar echinococcosis, AE) with Echinococcus multilocularis in mice impairs host immunity. Metacestode metabolites may modulate immunity putatively via dendritic cells. During murine AE, a relative increase of peritoneal DCs (pe-DCs) in infected mice (AE-pe-DCs; 4% of total peritoneal cells) as compared to control mice (naive pe-DCs; 2%) became apparent in our study. The differentiation of AE-pe-DCs into TGF-beta-expressing cells and the higher level of IL-4 than IFN-gamma/IL-2 mRNA expression in AE-CD4+pe-T cells indicated a Th2 orientation. Analysis of major accessory molecule expression on pe-DCs from AE-infected mice revealed that CD80 and CD86 were down-regulated on AE-pe-DCs, while ICAM-1(CD54) remained practically unchanged. Moreover, AE-pe-DCs had a weaker surface expression of MHC class II (Ia) molecules as compared to naive pe-DCs. The gene expression level of molecules involved in MHC class II (Ia) synthesis and formation of MHC class II (Ia)-peptide complexes were down-regulated. In addition, metacestodes excreted/secreted (E/S) or vesicle-fluid (V/F) antigens were found to alter MHC class II molecule expression on the surface of BMDCs. Finally, conversely to naive pe-DCs, an increasing number of AE-pe-DCs down-regulated Con A-induced proliferation of naive CD4+pe-T cells. These findings altogether suggested that TGF-beta-expressing immature AE-pe-DCs might play a significant role in the generation of a regulatory immune response within the peritoneal cavity of AE-infected mice.

Absence of the calcium-binding protein calretinin, not of calbindin D-28k, causes a permanent impairment of murine adult hippocampal neurogenesis

Calretinin (CR) and calbindin D-28k (CB) are cytosolic EF-hand Ca(2+)-binding proteins and function as Ca(2+) buffers affecting the spatiotemporal aspects of Ca(2+) transients and possibly also as Ca(2+) sensors modulating signaling cascades. In the adult hippocampal circuitry, CR and CB are expressed in specific principal neurons and subsets of interneurons. In addition, CR is transiently expressed within the neurogenic dentate gyrus (DG) niche. CR and CB expression during adult neurogenesis mark critical transition stages, onset of differentiation for CR, and the switch to adult-like connectivity for CB. Absence of either protein during these stages in null-mutant mice may have functional consequences and contribute to some aspects of the identified phenotypes. We report the impact of CR- and CB-deficiency on the proliferation and differentiation of progenitor cells within the subgranular zone (SGZ) neurogenic niche of the DG. Effects were evaluated (1) two and four weeks postnatally, during the transition period of the proliferative matrix to the adult state, and (2) in adult animals (3 months) to trace possible permanent changes in adult neurogenesis. The absence of CB from differentiated DG granule cells has no retrograde effect on the proliferative activity of progenitor cells, nor affects survival or migration/differentiation of newborn neurons in the adult DG including the SGZ. On the contrary, lack of CR from immature early postmitotic granule cells causes an early loss in proliferative capacity of the SGZ that is maintained into adult age, when it has a further impact on the migration/survival of newborn granule cells. The transient CR expression at the onset of adult neurogenesis differentiation may thus have two functions: (1) to serve as a self-maintenance signal for the pool of cells at the same stage of neurogenesis contributing to their survival/differentiation, and (2) it may contribute to retrograde signaling required for maintenance of the progenitor pool.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

SUMMARY: In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of approximately 67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Short- and long-term cultivation of embryonic and neonatal murine keratinocytes

Studies using cultured cells allow one to dissect complex cellular mechanisms in greater detail than when studying living organisms alone. However, before cultured cells can deliver meaningful results they must accurately represent the in vivo situation. Over the last three to four decades considerable effort has been devoted to the development of culture media which improve in vitro growth and modeling accuracy. In contrast to earlier large-scale, non-specific screening of factors, in recent years the development of such media has relied increasingly on a deeper understanding of the cell's biology and the selection of growth factors to specifically activate known biological processes. These new media now enable equal or better cell isolation and growth, using significantly simpler and less labor-intensive methodologies. Here we describe a simple method to isolate and cultivate epidermal keratinocytes from embryonic or neonatal skin on uncoated plastic using a medium specifically designed to retain epidermal keratinocyte progenitors in an undifferentiated state for improved isolation and proliferation and an alternative medium to support terminal differentiation.

HIV-1 Nef interferes with T-lymphocyte circulation through confined environments in vivo

HIV-1 negative factor (Nef) elevates virus replication and contributes to immune evasion in vivo. As one of its established in vitro activities, Nef interferes with T-lymphocyte chemotaxis by reducing host cell actin dynamics. To explore Nef's influence on in vivo recirculation of T lymphocytes, we assessed lymph-node homing of Nef-expressing primary murine lymphocytes and found a drastic impairment in homing to peripheral lymph nodes. Intravital imaging and 3D immunofluorescence reconstruction of lymph nodes revealed that Nef potently impaired T-lymphocyte extravasation through high endothelial venules and reduced subsequent parenchymal motility. Ex vivo analyses of transendothelial migration revealed that Nef disrupted T-lymphocyte polarization and interfered with diapedesis and migration in the narrow subendothelial space. Consistently, Nef specifically affected T-lymphocyte motility modes used in dense environments that pose high physical barriers to migration. Mechanistically, inhibition of lymph node homing, subendothelial migration and cell polarization, but not diapedesis, depended on Nef's ability to inhibit host cell actin remodeling. Nef-mediated interference with in vivo recirculation of T lymphocytes may compromise T-cell help and thus represents an important mechanism for its function as a HIV pathogenicity factor.

Low levels of mannan-binding lectin or ficolins are not associated with an increased risk of cytomegalovirus disease in HIV-infected patients

Background In HIV-infected patients, prediction of Cytomegalovirus (CMV) disease remains difficult. A protective role of mannan-binding lectin (MBL) and ficolins against CMV disease has been reported after transplantation, but the impact in HIV-infected patients is unclear. Methods In a case-control study nested within the Swiss HIV Cohort Study, we investigated associations between plasma levels of MBL/ficolins and CMV disease. We compared HIV-infected patients with CMV disease (cases) to CMV-seropositive patients without CMV disease (controls) matched for CD4 T-cells, sampling time, and use of combination antiretroviral therapy. MBL and M-ficolin, L-ficolin, and H-ficolin were quantified using ELISA. Results We analysed 105 cases and 105 matched controls. CMV disease was neither associated with MBL (odds ratio [OR] 1.03 per log10 ng/mL increase (95% CI 0.73–1.45)) nor with ficolins (OR per log10 ng/mL increase 0.66 (95% CI 0.28–1.52), 2.34 (95% CI 0.44–12.36), and 0.89 (95% CI 0.26–3.03) for M-ficolin, L-ficolin, and H-ficolin, respectively). We found no evidence of a greater association between MBL and CMV disease in patients with low CD4 counts; however in the multivariable analysis, CMV disease was more likely in patients with an increased HIV RNA (OR 1.53 per log10 copies/mL; 95% CI 1.08–2.16), or a shorter duration of HIV-infection (OR 0.91 per year; 95% CI 0.84–0.98). Conclusions CMV disease is not associated with low levels of MBL/ficolins, suggesting a lack of a protective role in HIV-infected patients.

Interleukin-17A stimulates granulocyte-macrophage colony-stimulating factor release by murine osteoblasts in the presence of 1,25-dihydroxyvitamin D(3) and inhibits murine osteoclast development in vitro

OBJECTIVE To investigate the effects of interleukin-17A (IL-17A) on osteoclastogenesis in vitro. METHODS Bone marrow cells (BMCs) were isolated from the excised tibia and femora of wild-type C57BL/6J mice, and osteoblasts were obtained by sequential digestion of the calvariae of ddY, C57BL/6J, and granulocyte-macrophage colony-stimulating factor-knockout (GM-CSF(-/-)) mice. Monocultures of BMCs or cocultures of BMCs and osteoblasts were supplemented with or without 1,25-dihydroxyvitamin D(3)(1,25[OH](2)D(3)), recombinant human macrophage colony-stimulating factor (M-CSF), RANKL, and IL-17A. After 5-6 days, the cultures were fixed with 4% paraformaldehyde and subsequently stained for the osteoclast marker enzyme tartrate-resistant acid phosphatase (TRAP). Osteoprotegerin (OPG) and GM-CSF expression were measured by enzyme-linked immunosorbent assay, and transcripts for RANK and RANKL were detected by real-time polymerase chain reaction. RESULTS In both culture systems, IL-17A alone did not affect the development of osteoclasts. However, the addition of IL-17A plus 1,25(OH)(2)D(3) to cocultures inhibited early osteoclast development within the first 3 days of culture and induced release of GM-CSF into the culture supernatants. Furthermore, in cocultures of GM-CSF(-/-) mouse osteoblasts and wild-type mouse BMCs, IL-17A did not affect osteoclast development, corroborating the role of GM-CSF as the mediator of the observed inhibition of osteoclastogenesis by IL-17A. CONCLUSION These findings suggest that IL-17A interferes with the differentiation of osteoclast precursors by inducing the release of GM-CSF from osteoblasts.

Charakteristika schwer essgestörter Patientinnen in einer universitären Einrichtung und therapeutische Richtlinien

Patientinnen und Patienten mit einer schweren Essstörung (Anorexie, Bulimie, weitere) finden einerseits selten Eingang in systematische Studien, sind andererseits aber häufig auf ein stationäres Behandlungssetting in einem tertiären Zentrum angewiesen. Die kürzlich veröffentlichte S3-Leitlinie zur Behandlung von Essstörungen erlaubt eine klarere Einschätzung der Hospitalisationsbedürftigkeit schwer Essgestörter als bisher. In der vorliegenden Arbeit wurden 26 Patientinnen und Patienten mit einer schweren Essstörung, die konsekutiv auf einer spezialisierten psychosomatisch/internistischen universitären Einrichtung hospitalisiert wurden, retrospektiv hinsichtlich ihrer biologischen, psychologischen und sozialen Merkmale charakterisiert und in Bezug zur S3-Leitlinie gestellt. Die biopsychosozialen Charakteristika der untersuchten Population zeigen, dass die Hospitalisierung schwer Essgestörter im tertiärmedizinischen Setting mit einem multiprofessionellen Behandlungsteam evidenzbasiert erfolgt.