Conserved meningeal lymphatic drainage circuits in mice and humans.

Meningeal lymphatic vessels (MLVs) were identified in the dorsal and caudobasal regions of the dura mater, where they ensure waste product elimination and immune surveillance of brain tissues. Whether MLVs exist in the anterior part of the murine and human skull and how they connect with the glymphatic system and extracranial lymphatics remained unclear. Here, we used light-sheet fluorescence microscopy (LSFM) imaging of mouse whole-head preparations after OVA-A555 tracer injection into the cerebrospinal fluid (CSF) and performed real-time vessel-wall (VW) magnetic resonance imaging (VW-MRI) after systemic injection of gadobutrol in patients with neurological pathologies. We observed a conserved three-dimensional anatomy of MLVs in mice and humans that aligned with dural venous sinuses but not with nasal CSF outflow, and we discovered an extended anterior MLV network around the cavernous sinus, with exit routes through the foramina of emissary veins. VW-MRI may provide a diagnostic tool for patients with CSF drainage defects and neurological diseases.

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 °C. Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Anatomy and function of the vertebral column lymphatic network in mice.

Cranial lymphatic vessels (LVs) are involved in the transport of fluids, macromolecules and central nervous system (CNS) immune responses. Little information about spinal LVs is available, because these delicate structures are embedded within vertebral tissues and difficult to visualize using traditional histology. Here we show an extended vertebral column LV network using three-dimensional imaging of decalcified iDISCO+-clarified spine segments. Vertebral LVs connect to peripheral sensory and sympathetic ganglia and form metameric vertebral circuits connecting to lymph nodes and the thoracic duct. They drain the epidural space and the dura mater around the spinal cord and associate with leukocytes. Vertebral LVs remodel extensively after spinal cord injury and VEGF-C-induced vertebral lymphangiogenesis exacerbates the inflammatory responses, T cell infiltration and demyelination following focal spinal cord lesion. Therefore, vertebral LVs add to skull meningeal LVs as gatekeepers of CNS immunity and may be potential targets to improve the maintenance and repair of spinal tissues.

Strength training improves body composition, muscle strength and increases CD4+ T lymphocyte levels in people living with HIV/AIDS.

The establishment of physical training programs for people living with HIV/AIDS (PLWHA) has several benefits. The study aimed to analyze the effect of resistance training using prediction of intensity by subjective perception of effort (SPE) on body composition, muscle strength, and TCD4+ lymphocyte levels in PLWHA. This a randomized controlled trial study. Participants (11 men and 8 women), were divided in two groups: exercise group (EG) and control group (CG). The EG was submitted to 12 weeks of a resistance-training program based in the prediction of intensity by SPE. Body mass percentages were evaluated using tetrapolar bioelectrical impedance and skinfold methods. We used Flow Cytometry to quantify CD4+ T lymphocytes. Patients showed significant changes in Body Fat Percentage (Δ%=-6.23%), Lean Body Mass (Δ%=2.45%), and CD4T lymphocytes levels (Δ%=15.77%). They also showed significant increase in muscular strength presented in the test for one repetition maximum in all the evaluated exercises. Our data suggest that exercising program prescribed by SPE is capable of improving immune function, body composition, and muscular strength in PLWHA.

The availability of the embryonic TGF-ß protein Nodal is dynamically regulated during glioblastoma multiforme tumorigenesis.

BACKGROUND: Glioblastoma (GBM) is the most common primary brain tumor presenting self-renewing cancer stem cells. The role of these cells on the development of the tumors has been proposed to recapitulate programs from embryogenesis. Recently, the embryonic transforming growth factor-ß (TGF-ß) protein Nodal has been shown to be reactivated upon tumor development; however, its availability in GBM cells has not been addressed so far. In this study, we investigated by an original approach the mechanisms that dynamically control both intra and extracellular Nodal availability during GBM tumorigenesis. METHODS: We characterized the dynamics of Nodal availability in both stem and more differentiated GBM cells through morphological analysis, immunofluorescence of Nodal protein and of early (EEA1 and Rab5) and late (Rab7 and Rab11) endocytic markers and Western Blot. Tukey's test was used to analyze the prevalent correlation of Nodal with different endocytic markers inside specific differentiation states, and Sidak's multiple comparisons test was used to compare the prevalence of Nodal/endocytic markers co-localization between two differentiation states of GBM cells. Paired t test was used to analyze the abundance of Nodal protein, in extra and intracellular media. RESULTS: The cytoplasmic distribution of Nodal was dynamically regulated and strongly correlated with the differentiation status of GBM cells. While Nodal-positive vesicle-like particles were symmetrically distributed in GBM stem cells (GBMsc), they presented asymmetric perinuclear localization in more differentiated GBM cells (mdGBM). Strikingly, when subjected to dedifferentiation, the distribution of Nodal in mdGBM shifted to a symmetric pattern. Moreover, the availability of both intracellular and secreted Nodal were downregulated upon GBMsc differentiation, with cells becoming elongated, negative for Nodal and positive for Nestin. Interestingly, the co-localization of Nodal with endosomal vesicles also depended on the differentiation status of the cells, with Nodal seen more packed in EEA1/Rab5 + vesicles in GBMsc and more in Rab7/11 + vesicles in mdGBM. CONCLUSIONS: Our results show for the first time that Nodal availability relates to GBM cell differentiation status and that it is dynamically regulated by an endocytic pathway during GBM tumorigenesis, shedding new light on molecular pathways that might emerge as putative targets for Nodal signaling in GBM therapy.