Contribution of LTi and TH17 cells to B cell aggregate formation in the central nervous system in a mouse model of multiple sclerosis

Background In a subgroup of patients suffering from progressive multiple sclerosis (MS), which is an inflammation-mediated neurodegenerative disease of the central nervous system (CNS), B cell aggregates were discovered within the meninges. Occurrence of these structures was associated with a more severe disease course and cortical histopathology. We have developed the B cell-dependent MP4-induced experimental autoimmune encephalomyelitis (EAE) as a mouse model to mimic this trait of the human disease. The aim of this study was to determine a potential role of lymphoid tissue inducer (LTi) and TH17 cells in the process of B cell aggregate formation in the MP4 model. Methods We performed flow cytometry of cerebellar and splenic tissue of MP4-immunized mice in the acute and chronic stage of the disease to analyze the presence of CD3−CD5−CD4+RORγt+ LTi and CD3+CD5+CD4+RORγt+ TH17 cells. Myelin oligodendrocyte glycoprotein (MOG):35–55-induced EAE was used as B cell-independent control model. We further determined the gene expression profile of B cell aggregates using laser capture microdissection, followed by RNA sequencing. Results While we were able to detect LTi cells in the embryonic spleen and adult intestine, which served as positive controls, there was no evidence for the existence of such a population in acute or chronic EAE in neither of the two models. Yet, we detected CD3−CD5−CD4−RORγt+ innate lymphoid cells (ILCs) and TH17 cells in the CNS, the latter especially in the chronic stage of MP4-induced EAE. Moreover, we observed a unique gene signature in CNS B cell aggregates compared to draining lymph nodes of MP4-immunized mice and to cerebellum as well as draining lymph nodes of mice with MOG:35–55-induced EAE. Conclusion The absence of LTi cells in the cerebellum suggests that other cells might take over the function as an initiator of lymphoid tissue formation in the CNS. Overall, the development of ectopic lymphoid organs is a complex process based on an interplay between several molecules and signals. Here, we propose some potential candidates, which might be involved in the formation of B cell aggregates in the CNS of MP4-immunized mice

A comprehensive multi-domain dataset for mitotic figure detection

The prognostic value of mitotic figures in tumor tissue is well-established for many tumor types and automating this task is of high research interest. However, especially deep learning-based methods face performance deterioration in the presence of domain shifts, which may arise from different tumor types, slide preparation and digitization devices. We introduce the MIDOG++ dataset, an extension of the MIDOG 2021 and 2022 challenge datasets. We provide region of interest images from 503 histological specimens of seven different tumor types with variable morphology with in total labels for 11,937 mitotic figures: breast carcinoma, lung carcinoma, lymphosarcoma, neuroendocrine tumor, cutaneous mast cell tumor, cutaneous melanoma, and (sub)cutaneous soft tissue sarcoma. The specimens were processed in several laboratories utilizing diverse scanners. We evaluated the extent of the domain shift by using state-of-the-art approaches, observing notable differences in single-domain training. In a leave-one-domain-out setting, generalizability improved considerably. This mitotic figure dataset is the first that incorporates a wide domain shift based on different tumor types, laboratories, whole slide image scanners, and species

Tracking cell turnover in human brain using 15N-thymidine imaging mass spectrometry

Microcephaly is often caused by an impairment of the generation of neurons in the brain, a process referred to as neurogenesis. While most neurogenesis in mammals occurs during brain development, it thought to continue to take place through adulthood in selected regions of the mammalian brain, notably the hippocampus. However, the generality of neurogenesis in the adult brain has been controversial. While studies in mice and rats have provided compelling evidence for neurogenesis occurring in the adult rodent hippocampus, the lack of applicability in humans of key methods to demonstrate neurogenesis has led to an intense debate about the existence and, in particular, the magnitude of neurogenesis in the adult human brain. Here, we demonstrate the applicability of a powerful method to address this debate, that is, the in vivo labeling of adult human patients with 15N-thymidine, a non-hazardous form of thymidine, an approach without any clinical harm or ethical concerns. 15N-thymidine incorporation into newly synthesized DNA of specific cells was quantified at the single-cell level with subcellular resolution by Multiple-isotype imaging mass spectrometry (MIMS) of brain tissue resected for medical reasons. Two adult human patients, a glioblastoma patient and a patient with drug-refractory right temporal lobe epilepsy, were infused for 24 h with 15N-thymidine. Detection of 15N-positive leukocyte nuclei in blood samples from these patients confirmed previous findings by others and demonstrated the appropriateness of this approach to search for the generation of new cells in the adult human brain. 15N-positive neural cells were easily identified in the glioblastoma tissue sample, and the range of the 15N signal suggested that cells that underwent S-phase fully or partially during the 24 h in vivo labeling period, as well as cells generated therefrom, were detected. In contrast, within the hippocampus tissue resected from the epilepsy patient, none of the 2,000 dentate gyrus neurons analyzed was positive for 15N-thymidine uptake, consistent with the notion that the rate of neurogenesis in the adult human hippocampus is rather low. Of note, the likelihood of detecting neurogenesis was reduced because of (i) the low number of cells analyzed, (ii) the fact that hippocampal tissue was explored that may have had reduced neurogenesis due to epilepsy, and (iii) the labeling period of 24 h which may have been too short to capture quiescent neural stem cells. Yet, overall, our approach to enrich NeuN-labeled neuronal nuclei by FACS prior to MIMS analysis provides a promising strategy to quantify even low rates of neurogenesis in the adult human hippocampus after in vivo15N-thymidine infusion. From a general point of view and regarding future perspectives, the in vivo labeling of humans with 15N-thymidine followed by MIMS analysis of brain tissue constitutes a novel approach to study mitotically active cells and their progeny in the brain, and thus allows a broad spectrum of studies of brain physiology and pathology, including microcephaly

Congenital tracheal stenosis associated with left pulmonary artery sling accompanied by tracheal diverticula: A case report

We present the case of an eleven-year-old boy with history of stridor since birth with suspicion of tracheal stenosis due to a vascular ring. CT-evaluation revealed a pulmonary artery sling (PAS) and complete tracheal rings with a long segment funnel type tracheal stenosis. Histopathological evaluation additionally revealed a septum within the stenotic part dividing the trachea into a stenotic left part and a right part which turned out to only be an evagination like a small diverticulum. The long segment tracheal stenosis was opened in the middle and an anterior-posterior tracheal sliding plasty was performed after resecting the diseased part of the obstructive cartilage. The patient was extubated on the third postoperative day and the stridor disappeared immediately. To the best of our knowledge, this is the first report of tracheal stenosis with a pulmonary artery sling accompanied with tracheal diverticulum

Neurovascular Interface in Porcine Small Intestine: Specific for Nitrergic rather than Nonnitrergic Neurons

In the 1970s, by using classic histological methods, close topographical relationships between special areas of enteric ganglia and capillaries were shown in the pig. In this study, by application of double and triple immunohistochemistry, we confirmed this neurovascular interface and demonstrated that these zones are mainly confined to nitrergic neurons in the myenteric and the external submucosal plexus. In the upper small intestine of the pig, the respective neurons display type III morphology, i.e. they have long, slender and branched dendrites and a single axon. In another set of experiments, we prepared specimens for electron-microscopical analysis of these zones. Both ganglia and capillaries display continuous basement membranes, the smallest distances between them being 1,000 nm at the myenteric and 300 nm at the external submucosal level. The capillary endothelium was mostly continuous but, at the external submucosal level, scattered fenestrations were observed. This particular neurovascular relationship suggests that nitrergic neurons may require a greater amount of oxygen and/or nutrients. In guinea pig and mouse, previous ischemia/reperfusion experiments showed that nitrergic neurons are selectively damaged. Thus, a preferential blood supply of enteric nitrergic neurons may indicate that these neurons are more vulnerable in ischemia

Calbindin D28k-Immunoreactivity in Human Enteric Neurons

Calbindin (CALB) is well established as immunohistochemical marker for intrinsic primary afferent neurons in the guinea pig gut. Its expression by numerous human enteric neurons has been demonstrated but little is known about particular types of neurons immunoreactive for CALB. Here we investigated small and large intestinal wholemount sets of 26 tumor patients in order to evaluate (1) the proportion of CALB+ neurons in the total neuron population, (2) the colocalization of CALB with calretinin (CALR), somatostatin (SOM) and vasoactive intestinal peptide (VIP) and (3) the morphology of CALB+ neurons. CALB+ neurons represented a minority of myenteric neurons (small intestine: 31%; large intestine: 25%) and the majority of submucosal neurons (between 72 and 95%). In the submucosa, most CALB+ neurons co-stained for CALR and VIP (between 69 and 80%) or for SOM (between 20 and 3%). In the myenteric plexus, 85% of CALB+ neurons did not co-stain with the other markers investigated. An unequivocal correlation between CALB reactivity and neuronal morphology was found for myenteric type III neurons in the small intestine: uniaxonal neurons with long, slender and branched dendrites were generally positive for CALB. Since also other neurons displayed occasional CALB reactivity, this protein is not suited as an exclusive marker for type III neurons