Cerebellar projections to the red nucleus and inferior olive originate from separate populations of neurons in the rat: A non-fluorescent double labeling study

In the rat, the extent of collateralization of projections from the cerebellar nuclei to the red nucleus and inferior olive was investigated using a retrograde double labeling technique. The combination of tracers selected, cholera toxin-β-subunit and WGA-BSA-gold, not only enabled the use of small injection sites but also resulted in clearly distinguishable and permanently stained neurons that could be analyzed in counterstained sections

A retrograde double-labeling technique for light microscopy A combination of axonal transport of cholera toxin B-subunit and a gold-lectin conjugate

A light microscopical, non-fluorescent, retrograde double-labeling technique is described. Cholera toxin B-subunit (CTb) and a conjugate of wheatgerm agglutinin and bovine serum albumin coupled to 10 nm gold particles (gold-lectin) are both excellent retrograde tracers and, when visualized by means of immunohistochemistry and silver intensification, respectively, may be readily identified within the same cell. This light microscopical retrograde double-labeling technique is illustrated in rat with experiments designed to investigate the collat

GATA3 haploinsufficiency causes a rapid deterioration of distortion product otoacoustic emissions (DPOAEs) in mice

Human HDR (hypoparathyroidism, deafness and renal dysplasia)-syndrome is caused by haploinsufficiency of zinc-finger transcription factor GATA3. The hearing loss due to GATA3 haploinsufficiency has been shown to be peripheral in origin, but it is unclear to what extent potential aberrations in the outer hair cells (OHCs) contribute to this disorder. To further elucidate the pathophysiological mechanism underlying the hearing defect in HDR-syndrome, we investigated the OHCs in heterozygous Gata3-knockout mice at both the functional and morphological level. While the signal-to-noise ratios of distortion product otoacoustic emissions (DPOAE) in wild type mice did not change significantly during the first half-year of live, those in the heterozygous Gata3 mice decreased dramatically. In addition, both light microscopic and transmission electron microscopic analyses showed that the number of OHCs containing vacuoles was increased in the mutants. Together, these findings indicate that outer hair cell malfunctioning plays a major role in the hearing loss in HDR-syndrome

Investigating the on-demand service characteristics: an empirical study

Purpose: Technological developments and new customer expectations of immediacy have driven businesses to adopt on-demand service models. The purpose of this paper is to study the characteristics of a range of on-demand services in order to better understand the meaning of “on-demand” and its implications for service management. This enables the on-demand service logic to be applied to other service contexts, where it may add value for customers. Design/methodology/approach: The study starts with a focused literature review and continues with a multiple case study methodology, as the on-demand service concept is in the early stages of theory development. Seven cases were studied, based on a maximum variation sampling strategy. Findings: The results show that on-demand services are characterized by three interrelated characteristics: being highly available, responsive and scalable. Analysis further reveals that on-demand services display differences within the conceptual boundaries of these characteristics, i.e. they vary in terms of their availability, responsiveness and scalability. Originality/value:

Monitoring regulatory T cells in clinical samples: consensus on an essential marker set and gating strategy for regulatory T cell analysis by flow cytometry

Regulatory T cell (Treg)-mediated immunosuppression is considered a major obstacle for successful cancer immunotherapy. The association between clinical outcome and Tregs is being studied extensively in clinical trials, but unfortunately, no consensus has been reached about (a) the markers and (b) the gating strategy required to define human Tregs in this context, making it difficult to draw final conclusions. Therefore, we have organized an international workshop on the detection and functional testing of Tregs with leading experts in the field, and 40 participants discussing different analyses and the importance of different markers and context in which Tregs were analyzed. This resulted in a rationally composed ranking list of “Treg markers”. Subsequently, the proposed Treg markers were tested to get insight into the overlap/differences between the most frequently used Treg definitions and their utility for Treg detection in various human tissues. Here, we conclude that the CD3, CD4, CD25, CD127, and FoxP3 markers are the minimally required markers to define human Treg cells. Staining for Ki67 and CD45RA showed to provide additional information on the activation status of Tregs. The use of markers was validated in a series of PBMC from healthy donors and cancer patients, as well as in tumor-draining lymph nodes and freshly isolated tumors. In conclusion, we propose an essential marker set comprising antibodies to CD3, CD4, CD25, CD127, Foxp3, Ki67, and CD45RA and a corresponding robust gating strategy for the context-dependent analysis of Tregs by flow cytometry

Classification of current anticancer immunotherapies

During the past decades, anticancer immunotherapy has evolved from a promising therapeutic option to a robust clinical reality. Many immunotherapeutic regimens are now approved by the US Food and Drug Administration and the European Medicines Agency for use in cancer patients, and many others are being investigated as standalone therapeutic interventions or combined with conventional treatments in clinical studies. Immunotherapies may be subdivided into “passive” and “active” based on their ability to engage the host immune system against cancer. Since the anticancer activity of most passive immunotherapeutics (including tumor-targeting monoclonal antibodies) also relies on the host immune system, this classification does not properly reflect the complexity of the drug-host-tumor interaction. Alternatively, anticancer immunotherapeutics can be classified according to their antigen specificity. While some immunotherapies specifically target one (or a few) defined tumor-associated antigen(s), others operate in a relatively non-specific manner and boost natural or therapy-elicited anticancer immune responses of unknown and often broad specificity. Here, we propose a critical, integrated classification of anticancer immunotherapies and discuss the clinical relevance of these approaches