Changes in nuclear 3,5,3'-triiodothyronine receptor expression in rat dorsal root ganglia and sciatic nerve during development: comparison with regeneration.

The action of the thyroid hormones on responsive cells in the peripheral nervous system requires the presence of nuclear triiodothyronine receptors (NT3R). These nuclear receptors, including both the alpha and beta subtypes of NT3R, were visualized by immunocytochemistry with the specific 2B3 monoclonal antibody. In the dorsal root ganglia (DRG) of rat embryos, NT3R immunoreactivity was first discretely revealed in a few neurons at embryonic day 14 (E14), then strongly expressed by all neurons at E17 and during the first postnatal week; all DRG neurons continued to possess clear NT3R immunostaining, which faded slightly with age. The peripheral glial cells in the DRG displayed a short-lived NT3R immunoreaction, starting at E17 and disappearing from the satellite and Schwann cells by postnatal days 3 and 7 respectively. In the developing sciatic nerve, Schwann cells also exhibited transient NT3R immunoreactivity restricted to a short period ranging from E17 to postnatal day 10; the NT3R immunostaining of the Schwann cells vanished proximodistally along the sciatic nerve, so that the Schwann cells rapidly became free of detectable NT3R immunostaining. However, after the transection or crushing of an adult sciatic nerve, the NT3R immunoreactivity reappeared in the Schwann cells adjacent to the lesion by 2 days, then along the distal segment in which the axons were degenerating, and finally disappeared by 45 days, when the regenerating axons were allowed to re-occupy the distal segment.(ABSTRACT TRUNCATED AT 250 WORDS)

NLRP3 Inflammasome Is Expressed and Functional in Mouse Brain Microglia but Not in Astrocytes.

Neuroinflammation is the local reaction of the brain to infection, trauma, toxic molecules or protein aggregates. The brain resident macrophages, microglia, are able to trigger an appropriate response involving secretion of cytokines and chemokines, resulting in the activation of astrocytes and recruitment of peripheral immune cells. IL-1β plays an important role in this response; yet its production and mode of action in the brain are not fully understood and its precise implication in neurodegenerative diseases needs further characterization. Our results indicate that the capacity to form a functional NLRP3 inflammasome and secretion of IL-1β is limited to the microglial compartment in the mouse brain. We were not able to observe IL-1β secretion from astrocytes, nor do they express all NLRP3 inflammasome components. Microglia were able to produce IL-1β in response to different classical inflammasome activators, such as ATP, Nigericin or Alum. Similarly, microglia secreted IL-18 and IL-1α, two other inflammasome-linked pro-inflammatory factors. Cell stimulation with α-synuclein, a neurodegenerative disease-related peptide, did not result in the release of active IL-1β by microglia, despite a weak pro-inflammatory effect. Amyloid-β peptides were able to activate the NLRP3 inflammasome in microglia and IL-1β secretion occurred in a P2X7 receptor-independent manner. Thus microglia-dependent inflammasome activation can play an important role in the brain and especially in neuroinflammatory conditions.

Inflammation in schizophrenia: A question of balance.

In the past decade, there has been renewed interest in immune/inflammatory changes and their associated oxidative/nitrosative consequences as key pathophysiological mechanisms in schizophrenia and related disorders. Both brain cell components (microglia, astrocytes, and neurons) and peripheral immune cells have been implicated in inflammation and the resulting oxidative/nitrosative stress (O&NS) in schizophrenia. Furthermore, down-regulation of endogenous antioxidant and anti-inflammatory mechanisms has been identified in biological samples from patients, although the degree and progression of the inflammatory process and the nature of its self-regulatory mechanisms vary from early onset to full-blown disease. This review focuses on the interactions between inflammation and O&NS, their damaging consequences for brain cells in schizophrenia, the possible origins of inflammation and increased O&NS in the disorder, and current pharmacological strategies to deal with these processes (mainly treatments with anti-inflammatory or antioxidant drugs as add-ons to antipsychotics).

A novel gene expression signature in peripheral blood mononuclear cells for early detection of colorectal cancer.

BACKGROUND: Early detection and treatment of colorectal adenomatous polyps (AP) and colorectal cancer (CRC) is associated with decreased mortality for CRC. However, accurate, non-invasive and compliant tests to screen for AP and early stages of CRC are not yet available. A blood-based screening test is highly attractive due to limited invasiveness and high acceptance rate among patients. AIM: To demonstrate whether gene expression signatures in the peripheral blood mononuclear cells (PBMC) were able to detect the presence of AP and early stages CRC. METHODS: A total of 85 PBMC samples derived from colonoscopy-verified subjects without lesion (controls) (n = 41), with AP (n = 21) or with CRC (n = 23) were used as training sets. A 42-gene panel for CRC and AP discrimination, including genes identified by Digital Gene Expression-tag profiling of PBMC, and genes previously characterised and reported in the literature, was validated on the training set by qPCR. Logistic regression analysis followed by bootstrap validation determined CRC- and AP-specific classifiers, which discriminate patients with CRC and AP from controls. RESULTS: The CRC and AP classifiers were able to detect CRC with a sensitivity of 78% and AP with a sensitivity of 46% respectively. Both classifiers had a specificity of 92% with very low false-positive detection when applied on subjects with inflammatory bowel disease (n = 23) or tumours other than CRC (n = 14). CONCLUSION: This pilot study demonstrates the potential of developing a minimally invasive, accurate test to screen patients at average risk for colorectal cancer, based on gene expression analysis of peripheral blood mononuclear cells obtained from a simple blood sample.

Statin use and peripheral blood progenitor cells mobilization in patients with multiple myeloma.

PURPOSE: Statins have beneficial effects in patients after myocardial infarction and at least part of the benefit results from mobilization of marrow endothelial progenitors to repopulate damaged myocardial tissues. This study examines if statins may have the same effect in mobilizing marrow progenitors to be harvested and subsequently used in high-dose chemotherapy with progenitor cell rescue in multiple myeloma. METHODS: From 2006 to 2012, 86 patients with multiple myeloma were mobilized with the use of G-CSF and were retrospectively analyzed. Patients with other malignancies or mobilized with the use of chemotherapy or with plerixafor were excluded. RESULTS: The median age of the patients was 60 years. 72 patients had received one line of chemotherapy and 14 patients two or more lines of chemotherapy. Twenty patients were taking statins at the time of the harvest while 66 patients were not. In the group of patients taking statins the success rate of first leukapheresis (obtaining the target number of 4 × 10(6) CD34+ cells/kg) was 85 % while in the group not taking statins this rate was 63.6 %. Despite the comparatively small number of patients this difference approached statistical significance (χ (2) = 0.07). CONCLUSION: This retrospective analysis of 86 patients shows for the first time a possible benefit of statins for peripheral blood progenitor cells mobilization in patients with multiple myeloma. Larger studies would be required to clarify the issue. If their effectiveness is confirmed, statins could be a safe and cheaper addition to chemotherapy and plerixafor for peripheral hematopoietic stem cell mobilization.

Adipose-derived stem cells enhance peripheral nerve regeneration.

Traumatic injuries resulting in peripheral nerve lesions often require a graft to bridge the gap. Although autologous nerve auto-graft is still the first-choice strategy in reconstructions, it has the severe disadvantage of the sacrifice of a functional nerve. Cell transplantation in a bioartificial conduit is an alternative strategy to create a favourable environment for nerve regeneration. We decided to test new fibrin nerve conduits seeded with various cell types (primary Schwann cells and adult stem cells differentiated to a Schwann cell-like phenotype) for repair of sciatic nerve injury. Two weeks after implantation, the conduits were removed and examined by immunohistochemistry for axonal regeneration (evaluated by PGP 9.5 expression) and Schwann cell presence (detected by S100 expression). The results show a significant increase in axonal regeneration in the group of fibrin seeded with Schwann cells compared with the empty fibrin conduit. Differentiated adipose-derived stem cells also enhanced regeneration distance in a similar manner to differentiated bone marrow mesenchymal stem cells. These observations suggest that adipose-derived stem cells may provide an effective cell population, without the limitations of the donor-site morbidity associated with isolation of Schwann cells, and could be a clinically translatable route towards new methods to enhance peripheral nerve repair.

Cutting edge: IL-7 regulates the peripheral pool of adult ROR gamma+ lymphoid tissue inducer cells.

During fetal life, CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch development in mice. In adult animals, CD4(+)CD3(-) cells are found in low numbers in lymphoid organs. Whether adult CD4(+)CD3(-) cells are LTi cells and are generated and maintained through cytokine signals has not been directly addressed. In this study we show that adult CD4(+)CD3(-) cells adoptively transferred into neonatal CXCR5(-/-) mice induced the formation of intestinal lymphoid tissues, demonstrating for the first time their bona fide LTi function. Increasing IL-7 availability in wild-type mice either by IL-7 transgene expression or treatment with IL-7/anti-IL-7 complexes increased adult LTi cell numbers through de novo generation from bone marrow cells and increased the survival and proliferation of LTi cells. Our observations demonstrate that adult CD4(+)lineage(-) cells are LTi cells and that the availability of IL-7 determines the size of the adult LTi cell pool.

Collagen (NeuraGen®) nerve conduits and stem cells for peripheral nerve gap repair.

Collagen nerve guides are used clinically for peripheral nerve defects, but their use is generally limited to lesions up to 3 cm. In this study we combined collagen conduits with cells as an alternative strategy to support nerve regeneration over longer gaps. In vitro cell adherence to collagen conduits (NeuraGen(®) nerve guides) was assessed by scanning electron microscopy. For in vivo experiments, conduits were seeded with either Schwann cells (SC), SC-like differentiated bone marrow-derived mesenchymal stem cells (dMSC), SC-like differentiated adipose-derived stem cells (dASC) or left empty (control group), conduits were used to bridge a 1cm gap in the rat sciatic nerve and after 2-weeks immunohistochemical analysis was performed to assess axonal regeneration and SC infiltration. The regenerative cells showed good adherence to the collagen walls. Primary SC showed significant improvement in distal stump sprouting. No significant differences in proximal regeneration distances were noticed among experimental groups. dMSC and dASC-loaded conduits showed a diffuse sprouting pattern, while SC-loaded showed an enhanced cone pattern and a typical sprouting along the conduits walls, suggesting an increased affinity for the collagen type I fibrillar structure. NeuraGen(®) guides showed high affinity of regenerative cells and could be used as efficient vehicle for cell delivery. However, surface modifications (e.g. with extracellular matrix molecule peptides) of NeuraGen(®) guides could be used in future tissue-engineering applications to better exploit the cell potential.

Transforming growth factor beta 1 production by CD4+ CD25+ regulatory T cells in peripheral blood mononuclear cells from healthy subjects stimulated with Leishmania guyanensis.

Transforming growth factor beta (TGF-beta) has been shown to be a central immunomodulator used by leishmaniae to escape effective mechanisms of protection in human and murine infections with these parasites. However, all the information is derived from studies of established infection, while little is known about TGF-beta production in response to Leishmania stimulation in healthy subjects. In this study, TGF-beta1 production was demonstrated in peripheral blood mononuclear cells from healthy subjects never exposed to leishmaniae in response to live Leishmania guyanensis, and the TGF-beta1-producing cells were described as a distinct subpopulation of CD4(+) CD25(+) regulatory T cells. The suppressive properties of CD4(+) CD25(+) T cells were demonstrated in vitro by their inhibition of production of interleukin 2 (IL-2) and IL-10 by CD4(+) CD25(-) T cells in the presence of either anti-CD3 or L. guyanensis. Although neutralization of TGF-beta1 did not reverse the suppressive activity of CD4(+) CD25(+) T cells activated by anti-CD3, it reversed the suppressive activity of CD4(+) CD25(+) T cells activated by L. guyanensis. Altogether our data demonstrated that TGF-beta1 is involved in the suppressive activity of L. guyanensis-stimulated CD4(+) CD25(+) T cells from healthy controls.

Direct presentation of a melanocyte-associated antigen in peripheral lymph nodes induces cytotoxic CD8+ T cells.

Encounter of self-antigens in the periphery by mature T cells induces tolerance in the steady-state. Hence, it is not understood why the same peripheral antigens are also promiscuously expressed in the thymus to mediate central tolerance. Here, we analyzed CD8(+) T-cell tolerance to such an antigen constituted by ovalbumin under the control of the tyrosinase promoter. As expected, endogenous CD8(+) T-cell responses were altered in the periphery of transgenic mice, resulting from promiscuous expression of the self-antigen in mature medullary epithelial cells and deletion of high-affinity T cells in the thymus. In adoptive T-cell transfer experiments, we observed constitutive presentation of the self-antigen in peripheral lymph nodes. Notably, this self-antigen presentation induced persisting cytotoxic cells from high-affinity CD8(+) T-cell precursors. Lymph node resident melanoblasts expressing tyrosinase directly presented the self-antigen to CD8(+) T cells, independently of bone marrow-derived antigen-presenting cells. This peripheral priming was independent of the subcellular localization of the self-antigen, indicating that this mechanism may apply to other melanocyte-associated antigens. Hence, central tolerance by promiscuous expression of peripheral antigens is a mandatory, rather than a superfluous, mechanism to counteract the peripheral priming, at least for self-antigens that can be directly presented in lymph nodes. The peripheral priming by lymph node melanoblasts identified here may constitute an advantage for immunotherapies based on adoptive T-cell transfer.

A role of peripheral myelin protein 2 in lipid homeostasis of myelinating Schwann cells.

Peripheral myelin protein 2 (Pmp2, P2 or Fabp8), a member of the fatty acid binding protein family, was originally described together with myelin basic protein (Mbp or P1) and myelin protein zero (Mpz or P0) as one of the most abundant myelin proteins in the peripheral nervous system (PNS). Although Pmp2 is predominantly expressed in myelinated Schwann cells, its role in glia is currently unknown. To study its function in PNS biology, we have generated a complete Pmp2 knockout mouse (Pmp2(-/-) ). Comprehensive characterization of Pmp2(-/-) mice revealed a temporary reduction in their motor nerve conduction velocity (MNCV). While this change was not accompanied by any defects in general myelin structure, we detected transitory alterations in the myelin lipid profile of Pmp2(-/-) mice. It was previously proposed that Pmp2 and Mbp have comparable functions in the PNS suggesting that the presence of Mbp can partially mask the Pmp2(-/-) phenotype. Indeed, we found that Mbp lacking Shi(-/-) mice, similar to Pmp2(-/-) animals, have preserved myelin structure and reduced MNCV, but this phenotype was not aggravated in Pmp2(-/-) /Shi(-/-) mutants indicating that Pmp2 and Mbp do not substitute each other's functions in the PNS. These data, together with our observation that Pmp2 binds and transports fatty acids to membranes, uncover a role for Pmp2 in lipid homeostasis of myelinating Schwann cells.

Selective accumulation of differentiated FOXP3(+) CD4 (+) T cells in metastatic tumor lesions from melanoma patients compared to peripheral blood

Precise identification of regulatory T cells is crucial in the understanding of their role in human cancers. Here, we analyzed the frequency and phenotype of regulatory T cells (Tregs), in both healthy donors and melanoma patients, based on the expression of the transcription factor FOXP3, which, to date, is the most reliable marker for Tregs, at least in mice. We observed that FOXP3 expression is not confined to human CD25(+/high) CD4(+) T cells, and that these cells are not homogenously FOXP3(+). The circulating relative levels of FOXP3(+) CD4(+) T cells may fluctuate close to 2-fold over a short period of observation and are significantly higher in women than in men. Further, we showed that FOXP3(+) CD4(+) T cells are over-represented in peripheral blood of melanoma patients, as compared to healthy donors, and that they are even more enriched in tumor-infiltrated lymph nodes and at tumor sites, but not in normal lymph nodes. Interestingly, in melanoma patients, a significantly higher proportion of functional, antigen-experienced FOXP3(+) CD4(+) T was observed at tumor sites, compared to peripheral blood. Together, our data suggest that local accumulation and differentiation of Tregs is, at least in part, tumor-driven, and illustrate a reliable combination of markers for their monitoring in various clinical settings.

TCR-MHC class II interaction is required for peripheral expansion of CD4 cells in a T cell-deficient host.

It is well established that T cell-deficient nude and SCID mice can be reconstituted by i.v. injection of small numbers of purified peripheral CD4+ T cells; however, the requirements for expansion of the transferred T cells in such systems are not clear. We show here that blood and lymphoid organs of MHC class II-deficient mice (which selectively lack mature CD4+ T cells) cannot be reconstituted by transfer of purified splenic CD4+ T cells, whereas TCRalpha-deficient mice (which lack both CD4+ and CD8+ mature T cells) are readily reconstituted. The failure of CD4+ T cell reconstitution in MHC class II-deficient mice was not due to the presence of CD8+ T cells, since similar results were obtained in TCRalpha-MHC class II double-deficient mice. Consistent with most previous studies CD4+ T cells in reconstituted TCRalpha-deficient mice had a diverse TCR Vbeta repertoire and were predominantly of an activated/memory (CD44high) phenotype. Collectively our data demonstrate that the expansion of peripheral CD4+ T cells in a T cell-deficient host is dependent upon interactions of the TCR with MHC class II.

The cells and peripheral representation of sodium taste in mice.

Salt taste in mammals can trigger two divergent behavioural responses. In general, concentrated saline solutions elicit robust behavioural aversion, whereas low concentrations of NaCl are typically attractive, particularly after sodium depletion. Notably, the attractive salt pathway is selectively responsive to sodium and inhibited by amiloride, whereas the aversive one functions as a non-selective detector for a wide range of salts. Because amiloride is a potent inhibitor of the epithelial sodium channel (ENaC), ENaC has been proposed to function as a component of the salt-taste-receptor system. Previously, we showed that four of the five basic taste qualities-sweet, sour, bitter and umami-are mediated by separate taste-receptor cells (TRCs) each tuned to a single taste modality, and wired to elicit stereotypical behavioural responses. Here we show that sodium sensing is also mediated by a dedicated population of TRCs. These taste cells express the epithelial sodium channel ENaC, and mediate behavioural attraction to NaCl. We genetically engineered mice lacking ENaCalpha in TRCs, and produced animals exhibiting a complete loss of salt attraction and sodium taste responses. Together, these studies substantiate independent cellular substrates for all five basic taste qualities, and validate the essential role of ENaC for sodium taste in mice.