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Mechanistic target of rapamycin (mTOR)-signaling is one key driver of glioblastoma (GBM), facilitating tumor growth by promoting the shift to an anti-inflammatory, pro-cancerogenic microenvironment. Even though mTOR inhibitors such as rapamycin (RAPA) have been shown to interfere with GBM disease progression, frequently chaperoned toxic drug side effects urge the need for developing alternative or supportive treatment strategies. Importantly, previous work document that taste-immune associative learning with RAPA may be utilized to induce learned pharmacological placebo responses in the immune system. Against this background, the current study aimed at investigating the potential efficacy of a taste-immune associative learning protocol with RAPA in a syngeneic GBM rat model. Following repeated pairings of a novel gustatory stimulus with injections of RAPA, learned immune-pharmacological effects could be retrieved in GBM-bearing animals when re-exposed to the gustatory stimulus together with administering 10 % amount of the initial drug dose (0.5 mg/kg). These inhibitory effects on tumor growth were accompanied by an up-regulation of central and peripheral pro-inflammatory markers, suggesting that taste-immune associative learning with RAPA promoted the development of a pro-inflammatory anti-tumor microenvironment that attenuated GBM tumor growth to an almost identical outcome as obtained after 100 % (5 mg/kg) RAPA treatment. Together, our results confirm the applicability of taste-immune associative learning with RAPA in animal disease models where mTOR overactivation is one key driver. This proof-of-concept study may also be taken as a role model for implementing learning protocols as alternative or supportive treatment strategy in clinical settings, allowing the reduction of required drug doses and side effects without losing treatment efficacy.

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Purpose: Despite the high incidence of epidermal growth factor receptor (EGFR) gene amplification and rearrangement in glioblastomas, no suitable cell line exists that preserves these alterations in vitro and is tumorigenic in immunocompromised mice. On the basis of previous observations that glioblastoma cells cultured with serum lose the EGFR amplification rapidly and that EGF can inhibit the growth of EGFR-amplified tumor cells, we hypothesized that serum-free and EGF-free culture conditions could promote maintenance of the EGFR amplification.Experimental Design: Cells from EGFR-amplified glioblastomas were taken into culture using neural stem cell conditions with modifications, including varying oxygen concentrations and omission of routine EGF supplementation.Results: High-level EGFR amplification was rapidly lost in 5 glioblastoma cultures supplemented with EGF, whereas it was preserved in cultures from the same tumors established without EGF. Cultures from 2 glioblastomas developed into pairs of cell lines, with either stable maintenance or irreversible loss of high-level EGFR amplification in the majority of cells. One EGFR-amplified cell line preserved expression of the receptor variant EGFRvIII. Cell lines with high-level EGFR amplification/EGFRvIII expression formed highly aggressive tumors in nude mice, whereas nonamplified cell lines were either nontumorigenic or grew significantly more slowly. In contrast, nonamplified cell lines proliferated faster in vitro. All cell lines responded to erlotinib, with inhibition of receptor activation and proliferation but partly different effects on downstream signaling and migration.Conclusions: Isogenic glioblastoma cell lines maintaining stable differences in EGFR/EGFRvIII status can be derived by varying exposure to EGF ligand and reflect the intratumoral genetic heterogeneity. Clin Cancer Res; 18(7); 1901–13. ©2012 AACR.

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Recently, we found many immune cells including antigen presenting cells neurally hard wired in the T-cell zone of most lymphoid organs like amongst others, lymph nodes in rats, mice and humans. Single immune cells were reached by single neurites and enclosed with a dense neural meshwork. As it is well known that axons are always accompanied by glial cells, we were able to identify Schwann cells in the hilum, medullary and capsule region, like expected. Unexpected was the result, that we found oligodendrocyte-like cells in these regions, myelinating more than one axon. Likewise important was the finding, that one of the standard glial markers used, a polyclonal GFAP antibody equally bound to desmin and therefore marked nearly all stromal cells in cortical, paracortical and medullary cord regions. More detailed analysis showed that these results also appeared in many other non-lymphoid organs. Therefore, polyclonal GFAP antibodies are only conditionally usable for immunohistochemical analysis in peripheral tissues outside the central nervous system. It remains to be elucidated, if the binding of the GFAP antibody to desmin has its reason in a special desmin variant that can give stromal cells glial character.

Frontiers in immunology 13, 947961 (2022). doi:10.3389/fimmu.2022.947961, With growing molecular evidence for correlations between spatial arrangement of blood vasculature and fundamental immunological functions, carried out in distinct compartments of the subdivided lymph node, there is an urgent need for three-dimensional models that can link these aspects. We reconstructed such models at a 1.84 µm resolution by the means of X-ray phase-contrast imaging with a 2D Talbot array in a short time without any staining. In addition reconstructions are verified in immunohistochemistry staining as well as in ultrastructural analyses. While conventional illustrations of mammalian lymph nodes depict the hilus as a definite point of blood and lymphatic vessel entry and exit, our method revealed that multiple branches enter and emerge from an area that extends up to one third of the organ’s surface. This could be a prerequisite for the drastic and location-dependent remodeling of vascularization, which is necessary for lymph node expansion during inflammation. Contrary to corrosion cast studies we identified B-cell follicles exhibiting a two times denser capillary network than the deep cortical units of the T-cell zone. In addition to our observation of high endothelial venules spatially surrounding the follicles, this suggests a direct connection between morphology and B-cell homing. Our findings will deepen the understanding of functional lymph node composition and lymphocyte migration on a fundamental basis., Published by Frontiers Media, Lausanne

INTRODUCTION Recently, we found abundant innervation of antigen presenting cells that were reached and enclosed by single neurites. These neurally hard-wired antigen presenting cells (wAPC) could be observed in the T-cell zone of superficial cervical lymph nodes of rats and other mammalians, including humans. METHODS As a consequence, we investigated lymph nodes at many different anatomical positions as well as all primary and secondary lymphoid organs (SLO) in rodents for a similar morphology of innervation regarding antigen presenting cells known in those tissues. RESULTS As a result, we confirmed wAPC in lymph nodes independent from their draining areas and anatomical positions but also in all other T-cell zones of lymphoid organs, like Peyer's patches, NALT and BALT, as well as in the thymic medulla. Other cells were innervated in a similar fashion but with seemingly missing antigen presenting capacity. Both types of innervated immune cells were observed as being also present in the dermis of the skin. Only in the spleen wAPC could not be detected. Beyond this systematic finding, we also found another regular phenomenon: a dense network of neurites that stained for neurofilament always in antigen entrance areas of lymphoid organs (subsinoidal layer of lymph nodes, subepithelial dome of Peyer's patches, subsinoidal layer of the splenic white pulp, margins of NALT and BALT). Lastly, also thymic epithelial cells (TEC) restricted to the corticomedullary junction of the thymus showed similar neurofilament staining. CONCLUSIONS Therefore, we propose much more hard-wired and probably afferent connections between lymphoid organs and the central nervous system than is hitherto known.

Purpose: Despite the high incidence of epidermal growth factor receptor (EGFR) gene amplification and rearrangement in glioblastomas, no suitable cell line exists that preserves these alterations in vitro and is tumorigenic in immunocompromised mice. On the basis of previous observations that glioblastoma cells cultured with serum lose the EGFR amplification rapidly and that EGF can inhibit the growth of EGFR-amplified tumor cells, we hypothesized that serum-free and EGF-free culture conditions could promote maintenance of the EGFR amplification. Experimental Design: Cells from EGFR-amplified glioblastomas were taken into culture using neural stem cell conditions with modifications, including varying oxygen concentrations and omission of routine EGF supplementation. Results: High-level EGFR amplification was rapidly lost in 5 glioblastoma cultures supplemented with EGF, whereas it was preserved in cultures from the same tumors established without EGF. Cultures from 2 glioblastomas developed into pairs of cell lines, with either stable maintenance or irreversible loss of high-level EGFR amplification in the majority of cells. One EGFR-amplified cell line preserved expression of the receptor variant EGFRvIII. Cell lines with high-level EGFR amplification/EGFRvIII expression formed highly aggressive tumors in nude mice, whereas nonamplified cell lines were either nontumorigenic or grew significantly more slowly. In contrast, nonamplified cell lines proliferated faster in vitro. All cell lines responded to erlotinib, with inhibition of receptor activation and proliferation but partly different effects on downstream signaling and migration. Conclusions: Isogenic glioblastoma cell lines maintaining stable differences in EGFR/EGFRvIII status can be derived by varying exposure to EGF ligand and reflect the intratumoral genetic heterogeneity. Clin Cancer Res; 18(7); 1901–13. ©2012 AACR.

The neural hard-wired pathways in which the lymphoid organs are innervated by the nervous system is of special interest with respect to suggested afferent and sensory systems informing the central nervous system about the status of the immune system. Until today efferent also like afferent innervation seem to be unspecific, targeting many types of cells by affecting many cells at the same time. We for the first time show that antigen presenting cells (APC) are abundantly innervated in the T-cell enriched area, the subsinoidal layer and the cortical extrafollicular zone of lymph nodes in rats by a mesh of filamentous neurofilament positive structures originating from single nerve fibers and covering each single APC similar to a glass fishing float, so that we termed them “wired” APC (wAPC). These wAPC also found in humans seem to be restricted to the cell body, not to follow membranous extensions, they may be dynamic and receptive as MAP2 is expressed and axonal growth cones can be detected and they probably lack vesicular activity through missing synaptophysin expression. The specific innervation targeting single cells which show a distribution divided in several areas in one lymph node suggests a form of topographically organized afferent sensory system.