Compartmentalization of small ruminant lentivirus between blood and colostrum in infected goats

The compartmentalization of small ruminant lentivirus (SRLV) subtype A (Maedi-Visna virus) and B (caprine arthritis-encephalitis virus) variants was analyzed in colostrum and peripheral blood mononuclear cells of four naturally infected goats. Sequence analysis of DNA and RNA encompassing the V4-V5 env regions showed a differential distribution of SRLV variants between the two compartments. Tissue-specific compartmentalization was demonstrated by phylogenetic analysis in three of the four cases. In these animals colostrum proviral sequences were clustered relative to the blood viral sequences. In one goat, the blood and colostrum-derived provirus sequences were intermingled, suggesting trafficking of virus between the two tissues or mirroring a recent infection. Surprisingly, the pattern of free virus variants in the colostrum of all animals corresponded only partially to that of the proviral form, suggesting that free viruses might not derive from infected colostral cells. The compartmentalization of SRLV between peripheral blood and colostrum indicates that lactogenic transmission may involve specific viruses not present in the proviral populations circulating in the blood.

Deregulated ephrin-B2 signaling in mammary epithelial cells alters the stem cell compartment and interferes with the epithelial differentiation pathway

Cancer most probably originates from stem/progenitor cells and exhibits a similar cell hierarchy as normal tissues. Moreover, there is growing evidence that only the stem cells are capable of metastasis formation. We have previously shown that overexpression of a dominant negative ephrin-B2 mutant interferes with mammary gland differentiation and confers a metastatic phenotype to NeuT-induced mammary tumors with an increase in cells with stem/progenitor characteristics. To investigate the role of ephrin-B2 in the control of the mammary stem cell niche, we analyzed the mammary stem and progenitor cell populations in transgenic mice overexpressing the mutant ephrin-B2. Quantification by FACS analysis revealed a significant increase of cells in the basal/alveolar cell-, the bi-potent progenitor- and the stem cell-enriched fractions. Moreover, the supposed precursors of estrogen receptor-positive cells were elevated in the stem cell-enriched fraction. In contrast, the epithelium from transgenic mice overexpressing the native ephrin-B2 gene showed an augmentation of the luminal cell- and the bi-potent progenitor-enriched fractions. Repopulation assays revealed that the epithelial cells of truncated ephrin-B2 transgenic epithelial cells have a higher regeneration capacity than those of controls and of native ephrin-B2 transgenic mice, confirming the augmentation of stem cells. Morphologically, these outgrowths exhibited impaired basal/luminal compartmentalization and epithelial polarization. These results demonstrate that deregulated ephrin-B2 expression interferes with the regulation of the stem cell niche and leads to a shift of the differentiation pathway and may thereby contribute to the acquisition of the metastatic phenotype long before carcinogenic growth becomes apparent.

Mice overexpressing BAFF develop a commensal flora-dependent, IgA-associated nephropathy

B cell activation factor of the TNF family (BAFF) is a potent B cell survival factor. BAFF overexpressing transgenic mice (BAFF-Tg mice) exhibit features of autoimmune disease, including B cell hyperplasia and hypergammaglobulinemia, and develop fatal nephritis with age. However, basal serum IgA levels are also elevated, suggesting that the pathology in these mice may be more complex than initially appreciated. Consistent with this, we demonstrate here that BAFF-Tg mice have mesangial deposits of IgA along with high circulating levels of polymeric IgA that is aberrantly glycosylated. Renal disease in BAFF-Tg mice was associated with IgA, because serum IgA was highly elevated in nephritic mice and BAFF-Tg mice with genetic deletion of IgA exhibited less renal pathology. The presence of commensal flora was essential for the elevated serum IgA phenotype, and, unexpectedly, commensal bacteria-reactive IgA antibodies were found in the blood. These data illustrate how excess B cell survival signaling perturbs the normal balance with the microbiota, leading to a breach in the normal mucosal-peripheral compartmentalization. Such breaches may predispose the nonmucosal system to certain immune diseases. Indeed, we found that a subset of patients with IgA nephropathy had elevated serum levels of a proliferation inducing ligand (APRIL), a cytokine related to BAFF. These parallels between BAFF-Tg mice and human IgA nephropathy may provide a new framework to explore connections between mucosal environments and renal pathology.

The habitat, double life, citizenship, and forgetfulness of IgA

Immunoglobulin A (IgA) is the main secretory immunoglobulin of mucous membranes and is powerfully induced by the presence of commensal microbes in the intestine. B cells undergo class switch recombination to IgA in the mucosa-associated lymphoid tissues, particularly mesenteric lymph nodes (MLNs) and Peyer's patches, through both T-dependent and T-independent pathways. IgA B cells primed in the mucosa traffic from the intestinal lymphoid structures, initially through the lymphatics and then join the bloodstream, to home back to the intestinal mucosa as IgA-secreting plasma cells. Once induced, anti-bacterial IgA can be extremely long-lived but is replaced if there is induction of additional IgA specificities by other microbes. The mucosal immune system is anatomically separated from the systemic immune system by the MLNs, which act as a firewall to prevent penetration of live intestinal bacteria to systemic sites. Dendritic cells sample intestinal bacteria and induce B cells to switch to IgA. In contrast, intestinal macrophages are adept at killing extracellular bacteria and are able to clear bacteria that have crossed the mucus and epithelial barriers. There is both a continuum between innate and adaptive immune mechanisms and compartmentalization of the mucosal immune system from systemic immunity that function to preserve host microbial mutualism.

Innate and adaptive immunity in host-microbiota mutualism

Healthy individuals live in peaceful co-existence with an immense load of intestinal bacteria. This symbiosis is advantageous for both the host and the bacteria. For the host it provides access to otherwise undigestible nutrients and colonization resistance against pathogens. In return the bacteria receive an excellent nutrient habitat. The mucosal immune adaptations to the presence of this commensal intestinal microflora are manifold. Although bacterial colonization has clear systemic consequences, such as maturation of the immune system, it is striking that the mutualistic adaptive (T and B cells) and innate immune responses are precisely compartmentalized to the mucosal immune system. Here we summarize the mechanisms of mucosal immune compartmentalization and its importance for a healthy host-microbiota mutualism.

Why is 11beta-hydroxysteroid dehydrogenase type 1 facing the endoplasmic reticulum lumen? Physiological relevance of the membrane topology of 11beta-HSD1

11Beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is essential for the local activation of glucocorticoid receptors (GR). Unlike unliganded cytoplasmic GR, 11beta-HSD1 is an endoplasmic reticulum (ER)-membrane protein with lumenal orientation. Cortisone might gain direct access to 11beta-HSD1 by free diffusion across membranes, indirectly via intracellular binding proteins or, alternatively, by insertion into membranes. Membranous cortisol, formed by 11beta-HSD1 at the ER-lumenal side, might then activate cytoplasmic GR or bind to ER-lumenal secretory proteins. Compartmentalization of 11beta-HSD1 is important for its regulation by hexose-6-phosphate dehydrogenase (H6PDH), which regenerates cofactor NADPH in the ER lumen and stimulates oxoreductase activity. ER-lumenal orientation of 11beta-HSD1 is also essential for the metabolism of the alternative substrate 7-ketocholesterol (7KC), a major cholesterol oxidation product found in atherosclerotic plaques and taken up from processed cholesterol-rich food. An 11beta-HSD1 mutant adopting cytoplasmic orientation efficiently catalyzed the oxoreduction of cortisone but not 7KC, indicating access to cortisone from both sides of the ER-membrane but to 7KC only from the lumenal side. These aspects may be relevant for understanding the physiological role of 11beta-HSD1 and for developing therapeutic interventions to control glucocorticoid reactivation.

Low prevalence of SV40 in Swiss mesothelioma patients after elimination of false-positive PCR results

The association of simian virus 40 (SV40) with malignant pleural mesothelioma is currently under debate. In some malignancies of viral aetiology, viral DNA can be detected in the patients' serum or plasma. To characterize the prevalence of SV40 in Swiss mesothelioma patients, we optimized a real-time PCR for quantitative detection of SV40 DNA in plasma, and used a monoclonal antibody for immunohistochemical detection of SV40 in mesothelioma tissue microarrays. Real-time PCR was linear over five orders of magnitude, and sensitive to a single gene copy. Repeat PCR determinations showed excellent reproducibility. However, SV40 status varied for independent DNA isolates of single samples. We noted that SV40 detection rates by PCR were drastically reduced by the implementation of strict room compartmentalization and decontamination procedures. Therefore, we systematically addressed common sources of contamination and found no cross-reactivity with DNA of other polyomaviruses. Contamination during PCR was rare and plasmid contamination was infrequent. SV40 DNA was reproducibly detected in only 4 of 78 (5.1%) plasma samples. SV40 DNA levels were low and not consistently observed in paired plasma and tumour samples from the same patient. Immunohistochemical analysis revealed a weak but reproducible SV40 staining in 16 of 341 (4.7%) mesotheliomas. Our data support the occurrence of non-reproducible SV40 PCR amplifications and underscore the importance of proper sample handling and analysis. SV40 DNA and protein were found at low prevalence (5%) in plasma and tumour tissue, respectively. This suggests that SV40 does not appear to play a major role in the development of mesothelioma.

Functions of lipid raft membrane microdomains at the blood-brain barrier

The blood-brain barrier (BBB) is a highly specialized structural and functional component of the central nervous system that separates the circulating blood from the brain and spinal cord parenchyma. Brain endothelial cells (BECs) that primarily constitute the BBB are tightly interconnected by multiprotein complexes, the adherens junctions and the tight junctions, thereby creating a highly restrictive cellular barrier. Lipid-enriched membrane microdomain compartmentalization is an inherent property of BECs and allows for the apicobasal polarity of brain endothelium, temporal and spatial coordination of cell signaling events, and actin remodeling. In this manuscript, we review the role of membrane microdomains, in particular lipid rafts, in the BBB under physiological conditions and during leukocyte transmigration/diapedesis. Furthermore, we propose a classification of endothelial membrane microdomains based on their function, or at least on the function ascribed to the molecules included in such heterogeneous rafts: (1) rafts associated with interendothelial junctions and adhesion of BECs to basal lamina (scaffolding rafts); (2) rafts involved in immune cell adhesion and migration across brain endothelium (adhesion rafts); (3) rafts associated with transendothelial transport of nutrients and ions (transporter rafts).

The liver may act as a firewall mediating mutualism between the host and its gut commensal microbiota.

A prerequisite for establishment of mutualism between the host and the microbial community that inhabits the large intestine is the stringent mucosal compartmentalization of microorganisms. Microbe-loaded dendritic cells trafficking through lymphatics are arrested at the mesenteric lymph nodes, which constitute the firewall of the intestinal lymphatic circulation. We show in different mouse models that the liver, which receives the intestinal venous blood circulation, forms a vascular firewall that captures gut commensal bacteria entering the bloodstream during intestinal pathology. Phagocytic Kupffer cells in the liver of mice clear commensals from the systemic vasculature independently of the spleen through the liver's own arterial supply. Damage to the liver firewall in mice impairs functional clearance of commensals from blood, despite heightened innate immunity, resulting in spontaneous priming of nonmucosal immune responses through increased systemic exposure to gut commensals. Systemic immune responses consistent with increased extraintestinal commensal exposure were found in humans with liver disease (nonalcoholic steatohepatitis). The liver may act as a functional vascular firewall that clears commensals that have penetrated either intestinal or systemic vascular circuits.

Dendritic spine morphology determines membrane-associated protein exchange between dendritic shafts and spine heads.

The purpose of this study was to examine whether variability in the shape of dendritic spines affects protein movement within the plasma membrane. Using a combination of confocal microscopy and the fluorescence loss in photobleaching technique in living hippocampal CA1 pyramidal neurons expressing membrane-linked GFP, we observed a clear correlation between spine shape parameters and the diffusion and compartmentalization of membrane-associated proteins. The kinetics of membrane-linked GFP exchange between the dendritic shaft and the spine head compartment were slower in dendritic spines with long necks and/or large heads than in those with short necks and/or small heads. Furthermore, when the spine area was reduced by eliciting epileptiform activity, the kinetics of protein exchange between the spine compartments exhibited a concomitant decrease. As synaptic plasticity is considered to involve the dynamic flux by lateral diffusion of membrane-bound proteins into and out of the synapse, our data suggest that spine shape represents an important parameter in the susceptibility of synapses to undergo plastic change.

Imaging of the spleen in malaria

Splenomegaly, albeit variably, is a hallmark of malaria; yet, the role of the spleen in Plasmodium infections remains vastly unknown. The implementation of imaging to study the spleen is rapidly advancing our knowledge of this so-called "blackbox" of the abdominal cavity. Not only has ex vivo imaging revealed the complex functional compartmentalization of the organ and immune effector cells, but it has also allowed the observation of major structural remodeling during infections. In vivo imaging, on the other hand, has allowed quantitative measurements of the dynamic passage of the parasite at spatial and temporal resolution. Here, we review imaging techniques used for studying the malarious spleen, from optical microscopy to in vivo imaging, and discuss the bright perspectives of evolving technologies in our present understanding of the role of this organ in infections caused by Plasmodium.