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Decentralizing childhood tuberculosis services, including diagnosis, is now recommended by the WHO and could contribute to increasing tuberculosis detection in high burden countries. However, implementing microbiological tests and clinical evaluation could be challenging for health care workers (HCWs) in Primary Health Centers (PHCs) and even District Hospitals (DHs). We sought to assess the acceptability of decentralizing a comprehensive childhood tuberculosis diagnosis package from HCWs' perspective. We conducted implementation research nested within the TB-Speed Decentralization study. HCWs from two health districts of Cambodia, Cameroon, Côte d'Ivoire, Mozambique, Sierra Leone, and Uganda implemented systematic screening, nasopharyngeal aspirates (NPA) and stool sample collection with molecular testing, clinical evaluation and chest X-ray (CXR) interpretation. We investigated their experiences and perceptions in delivering the diagnostic package components in 2020-21 using individual semi-structured interviews. We conducted thematic analysis, supported by the Theoretical Framework of Acceptability. HCWs (n = 130, 55% female, median age 36 years, 53% nurses, 72% PHC-based) perceived that systematic screening, although increasing workload, was beneficial as it improved childhood tuberculosis awareness. Most HCWs shared satisfaction and confidence in performing NPA, despite procedure duration, need to involve parents/colleagues and discomfort for children. HCWs shared positive attitudes towards stool sample-collection but were frustrated by delayed stool collection associated with cultural practices, transport and distance challenges. Molecular testing, conducted by nurses or laboratory technicians, was perceived as providing quality results, contributing to diagnosis. Clinical evaluation and diagnosis raised self-efficacy issues and need for continuous training and clinical mentoring. HCWs valued CXR, however complained that technical and logistical problems limited access to digital reports. Referral from PHC to DH was experienced as burdensome. HCWs at DH and PHC-levels perceived and experienced decentralized childhood tuberculosis diagnosis as acceptable. Implementation however could be hampered by feasibility issues, and calls for innovative referral mechanisms for patients, samples and CXR., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Joshi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Opisthorchis viverrini is a carcinogenic parasite that can cause bile duct cancer called cholangiocarcinoma. A study of the immune response of this parasite in susceptible and non-susceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamsters and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between 1 and 2 weeks post-infection, whereas it was positive between 2 and 4 weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice reacts strongly with the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted with a single protein band. Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host reacts quicker and stronger than the susceptible host.

Gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) is a poorly understood disease with limited treatment options. A better understanding of this disease would greatly benefit from the availability of representative preclinical models. Here, we present the potential of tumor organoids, three-dimensional cultures of tumor cells, to model GEP-NEC. We established three GEP-NEC organoid lines, originating from the stomach and colon, and characterized them using DNA sequencing and immunohistochemistry. Organoids largely resembled the original tumor in expression of synaptophysin, chromogranin and Ki-67. Models derived from tumors containing both neuroendocrine and non-neuroendocrine components were at risk of overgrowth by non-neuroendocrine tumor cells. Organoids were derived from patients treated with cisplatin and everolimus and for the three patients studied, organoid chemosensitivity paralleled clinical response. We demonstrate the feasibility of establishing NEC organoid lines and their potential applications. Organoid culture has the potential to greatly extend the repertoire of preclinical models for GEP-NEC, supporting drug development for this difficult-to-treat tumor type., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Dijkstra, van den Berg, Weeber, van de Haar, Velds, Kaing, Peters, Eskens, de Groot, Tesselaar and Voest.)

Clinical implementation of tumor organoids for personalized medicine requires that pure tumor organoids can be reliably established. Here, we present our experience with organoid cultures from >70 non-small cell lung cancer (NSCLC) samples. We systematically evaluate several methods to identify tumor purity of organoids established from intrapulmonary tumors. Eighty percent of organoids from intrapulmonary lesions have a normal copy number profile, suggesting overgrowth by normal airway organoids (AOs). This is further supported by the failure to detect mutations found in the original tumor in organoids. Histomorphology alone is insufficient to determine tumor purity, but when combined with p63 immunostaining, tumor and normal AOs can be distinguished. Taking into account overgrowth by normal AOs, the establishment rate of pure NSCLC organoids is 17%. Therefore, current methods are insufficient to establish pure NSCLC organoids from intrapulmonary lesions. We discourage their use unless steps are taken to prevent overgrowth by normal AOs., Competing Interests: Declaration of Interests H.C. declares the following management/advisory relationships: distinguished visiting professor, University of Hong Kong; member of the Scientific Advisory Board of Surrozen, San Francisco, California, USA; member of the Scientific Advisory Board of Kallyope, New York, USA; member of the Scientific Advisory Board of Merus, Utrecht, the Netherlands; scientific adviser to Life Science Partners, Amsterdam, the Netherlands; non-executive board member of Roche Holding Ltd., Basel, Switzerland., (Copyright © 2020 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cells are key players in cancer immunotherapy, but strategies to expand tumor-reactive cells and study their interactions with tumor cells at the level of an individual patient are limited. Here we describe the generation and functional assessment of tumor-reactive T cells based on cocultures of tumor organoids and autologous peripheral blood lymphocytes. The procedure consists of an initial coculture of 2 weeks, in which tumor-reactive T cells are first expanded in the presence of (IFNγ-stimulated) autologous tumor cells. Subsequently, T cells are evaluated for their capacity to carry out effector functions (IFNγ secretion and degranulation) after recognition of tumor cells, and their capacity to kill tumor organoids. This strategy is unique in its use of peripheral blood as a source of tumor-reactive T cells in an antigen-agnostic manner. In 2 weeks, tumor-reactive CD8 + T-cell populations can be obtained from ~33-50% of samples from patients with non-small-cell lung cancer (NSCLC) and microsatellite-instable colorectal cancer (CRC). This enables the establishment of ex vivo test systems for T-cell-based immunotherapy at the level of the individual patient.

There is a clear and unmet clinical need for biomarkers to predict responsiveness to chemotherapy for cancer. We developed an in vitro test based on patient-derived tumor organoids (PDOs) from metastatic lesions to identify nonresponders to standard-of-care chemotherapy in colorectal cancer (CRC). In a prospective clinical study, we show the feasibility of generating and testing PDOs for evaluation of sensitivity to chemotherapy. Our PDO test predicted response of the biopsied lesion in more than 80% of patients treated with irinotecan-based therapies without misclassifying patients who would have benefited from treatment. This correlation was specific to irinotecan-based chemotherapy, however, and the PDOs failed to predict outcome for treatment with 5-fluorouracil plus oxaliplatin. Our data suggest that PDOs could be used to prevent cancer patients from undergoing ineffective irinotecan-based chemotherapy., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Chemotherapy-resistance remains a major obstacle to effective anti-cancer treatment. We previously showed that platinum analogs cause the release of two fatty acids. These platinum-induced fatty acids (PIFAs) induced complete chemoresistance in mice, whereas co-administration of a COX-1 inhibitor, indomethacin, prevented PIFA release and significantly enhanced chemosensitivity. To assess the safety of combining indomethacin with platinum-based chemotherapy, and to explore its efficacy and associated PIFA levels, a multi-center phase I trial was conducted., Methods: The study was comprised of two arms: oxaliplatin plus capecitabine (CAPOX, arm I) and cisplatin plus gemcitabine, capecitabine or 5FU (arm II) in patients for whom these regimens were indicated as standard care. Indomethacin was escalated from 25 to 75 mg TID, using a standard 3 × 3 design per arm, and was administered orally 8 days around chemo-infusion from cycle two onwards. PIFA levels were measured before and after treatment initiation, with and without indomethacin., Results: Thirteen patients were enrolled, of which ten were evaluable for safety analyses. In arm I, no dose-limiting toxicities were observed, and all indomethacin dose levels were well-tolerated. Partial responses were observed in three patients (30%). Indomethacin lowered plasma levels of 12-S-hydroxy-5,8,10-heptadecatrienoic acid (12-S-HHT), whereas 4,7,10,13-hexadecatetraenoic acid (16:4(n-3)) levels were not affected. Only one patient was included in arm II; renal toxicity led to closure of this cohort., Conclusions: Combined indomethacin and CAPOX treatment is safe and reduces the concentrations of 12-S-HHT, which may be associated with improved chemosensitivity. The recommended phase II dose is 75 mg indomethacin TID given 8 days surrounding standard dosed CAPOX.

Background: Alzheimer's disease (AD) is the most common form of dementia. AD has a multifactorial disease etiology and is currently untreatable. Multiple genes and molecular mechanisms have been implicated in AD, including ß-amyloid deposition in the brain, neurofibrillary tangle accumulation of hyper-phosphorylated Tau, synaptic failure, oxidative stress and inflammation. Relatively little is known about the role of the blood-brain barriers, especially the blood-cerebrospinal fluid barrier (BCSFB), in AD. The BCSFB is involved in cerebrospinal fluid (CSF) production, maintenance of brain homeostasis and neurodegenerative disorders., Results: Using an Agilent platform with common reference design, we performed a large scale gene expression analysis and functional annotation of the Choroid Plexus Epithelium (CPE), which forms the BCSFB. We obtained 2 groups of freshly frozen Choroid Plexus (CP) of 7 human donor brains each, with and without AD: Braak stages (0-1) and (5-6). We cut CP cryo-sections and isolated RNA from cresyl-violet stained, laser dissected CPE cells. Gene expression results were analysed with T-tests (R) and the knowledge-database Ingenuity. We found statistically significantly altered gene expression data sets, biological functions, canonical pathways, molecular networks and functionalities in AD-affected CPE. We observed specific cellular changes due to increased oxidative stress, such as the unfolded protein response, E1F2 and NRF2 signalling and the protein ubiquitin pathway. Most likely, the AD-affected BCSFB barrier becomes more permeable due to downregulation of CLDN5. Finally, our data also predicted down regulation of the glutathione mediated detoxification pathway and the urea cycle in the AD CPE, which suggest that the CPE sink action may be impaired. Remarkably, the expression of a number of genes known to be involved in AD, such as APP, PSEN1, PSEN2, TTR and CLU is moderate to high and remains stable in both healthy and affected CPE. Literature labelling of our new functional molecular networks confirmed multiple previous (molecular) observations in the AD literature and revealed many new ones., Conclusions: We conclude that CPE failure in AD exists. Combining our data with those of the literature, we propose the following chronological and overlapping chain of events: increased Aß burden on CPE; increased oxidative stress in CPE; despite continuous high expression of TTR: decreased capability of CPE to process amyloid; (pro-) inflammatory and growth factor signalling by CPE; intracellular ubiquitin involvement, remodelling of CPE tight junctions and, finally, cellular atrophy. Our data corroborates the hypothesis that increased BCSFB permeability, especially loss of selective CLDN5-mediated paracellular transport, altered CSF production and CPE sink action, as well as loss of CPE mediated macrophage recruitment contribute to the pathogenesis of AD.

Introduction: This study aimed to develop a simple and inexpensive ex vivo model to teach students the use of electronic apex locators in a preclinical setting., Methods: Using 27 extracted human teeth, the Raypex 5 (VDW, Munich, Germany) and Dentaport ZX (J. Morita Co, Kyoto, Japan) were tested in three different media (ie, alginate, sugar-free gelatin, and 0.9% sodium chloride solution). Working lengths determined by these models were compared with those obtained by digital radiography and direct visualization using a linear mixed modeling statistical approach., Results: Raypex 5 exhibited a higher percentage of measurements accurate to ± 0.5 mm and ± 1.0 mm of the control across all three media in all tooth types. In multirooted teeth, alginate showed the highest accuracy., Conclusions: The most accurate EAL/embedding medium combination was Raypex 5/alginate to both ± 0.5 mm and ± 1.0 mm of the control. The model tested in this study was accurate, easy to assemble, and cost-effective, making it suitable for teaching purposes., (Copyright © 2011 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Objective: Repair of myelin injury in multiple sclerosis may fail, resulting in chronic demyelination, axonal loss, and disease progression. As cellular pathways regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN; eg, phosphatidylinositol-3-kinase [PI-3K]) have been reported to enhance axon regeneration and oligodendrocyte maturation, we investigated potentially beneficial effects of Pten loss of function in the oligodendrocyte lineage on remyelination., Methods: We characterized oligodendrocyte numbers and myelin sheath thickness in mice with conditional inactivation of Pten in oligodendrocytes, Olig2-cre, Pten(fl/fl) mice. Using a model of central nervous system demyelination, lysolecithin injection into the spinal cord white matter, we performed short- and long-term lesioning experiments and quantified oligodendrocyte maturation and myelin sheath thickness in remyelinating lesions., Results: During development, we observed dramatic hypermyelination in the corpus callosum and spinal cord. Following white matter injury, however, there was no detectable improvement in remyelination. Moreover, we observed progressive myelin sheath abnormalities and massive axon degeneration in the fasciculus gracilis of mutant animals, as indicated by ultrastructure and expression of SMI-32, amyloid precursor protein, and caspase 6., Interpretation: These studies indicate adverse effects of chronic Pten inactivation (and by extension, activation PI-3K signaling) on myelinating oligodendrocytes and their axonal targets. We conclude that PTEN function in oligodendrocytes is required to regulate myelin thickness and preserve axon integrity. In contrast, PTEN is dispensable during myelin repair, and its inactivation confers no detectable benefit.

The progressive loss of CNS myelin in patients with multiple sclerosis (MS) has been proposed to result from the combined effects of damage to oligodendrocytes and failure of remyelination. A common feature of demyelinated lesions is the presence of oligodendrocyte precursors (OLPs) blocked at a premyelinating stage. However, the mechanistic basis for inhibition of myelin repair is incompletely understood. To identify novel regulators of OLP differentiation, potentially dysregulated during repair, we performed a genome-wide screen of 1040 transcription factor-encoding genes expressed in remyelinating rodent lesions. We report that approximately 50 transcription factor-encoding genes show dynamic expression during repair and that expression of the Wnt pathway mediator Tcf4 (aka Tcf7l2) within OLPs is specific to lesioned-but not normal-adult white matter. We report that beta-catenin signaling is active during oligodendrocyte development and remyelination in vivo. Moreover, we observed similar regulation of Tcf4 in the developing human CNS and lesions of MS. Data mining revealed elevated levels of Wnt pathway mRNA transcripts and proteins within MS lesions, indicating activation of the pathway in this pathological context. We show that dysregulation of Wnt-beta-catenin signaling in OLPs results in profound delay of both developmental myelination and remyelination, based on (1) conditional activation of beta-catenin in the oligodendrocyte lineage in vivo and (2) findings from APC(Min) mice, which lack one functional copy of the endogenous Wnt pathway inhibitor APC. Together, our findings indicate that dysregulated Wnt-beta-catenin signaling inhibits myelination/remyelination in the mammalian CNS. Evidence of Wnt pathway activity in human MS lesions suggests that its dysregulation might contribute to inefficient myelin repair in human neurological disorders.

Within the motoneuron precursor (pMN) domain of the developing spinal cord, the bHLH transcription factor, Olig2, plays critical roles in pattern formation and the generation of motor neuron and oligodendrocyte precursors. How are the multiple functions of Olig2 regulated? We have isolated a large BAC clone encompassing the human OLIG2 locus that rescues motor neuron and oligodendrocyte development but not normal pattern formation in Olig2(-/-) embryos. Within the BAC clone, we identified a conserved 3.6 kb enhancer sub-region that directs reporter expression specifically in the motor neuron lineage but not oligodendrocyte lineage in vivo. Our findings indicate complex regulation of Olig2 by stage- and lineage-specific regulatory elements. They further suggest that transcriptional regulation of Olig2 is involved in segregation of pMN neuroblasts.

In the vertebrate brain, much remains to be understood concerning the origin of glial cell diversity and the potential lineage relationships between the various types of glia. Besides astrocytes and myelin-forming oligodendrocytes, other macroglial cell populations are found in discrete areas of the central nervous system (CNS). They share functional features with astrocytes and oligodendrocytes but also display specific characteristics. Such specialized cells, called pituicytes, are located in the neurohypophysis (NH). Our work focuses on the lineage of the pituicytes during rodent development. First, we show that cells identified with a combination of oligodendrocyte precursor cell (OPC) markers are present in the developing rat NH. In culture, neonatal NH progenitors also share major functional characteristics with OPCs, being both migratory and bipotential, i.e. able to give rise to type 2 astrocytes and oligodendrocytes. We then observe that, either in vitro or after transplantation into myelin-deficient Shiverer brain, pieces of NH generate myelinating oligodendrocytes, confirming the oligodendrogenic potentiality of NH cells. However, no mature oligodendrocyte can be found in the NH. This led us to hypothesize that the OPCs present in the developing NH might be generating other glial cells, especially the pituicytes. Consistent with this hypothesis, the OPCs appear during NH development before pituicytes differentiate. Finally, we establish a lineage relationship between olig1+ cells, most likely OPCs, and the pituicytes by fate-mapping experiments using genetically engineered mice. This constitutes the first demonstration that OPCs generate glial cells other than oligodendrocytes in vivo., ((c) 2005 Wiley-Liss, Inc.)

We investigated whether co-expression of Neurog 1 and Atoh 1 in common neurosensory precursors could explain the loss of hair cells in Neurog 1 null mice. Analysis of terminal mitosis, using BrdU, supports previous findings regarding timing of exit from cell cycle. Specifically, we show that cell cycle exit occurs in spiral sensory neurons in a base-to-apex progression followed by cell cycle exit of hair cells in the organ of Corti in an apex-to-base progression, with some overlap of cell cycle exit in the apex for both hair cells and spiral sensory neurons. Hair cells in Neurog 1 null mice show cell cycle exit in an apex-to-base progression about 1-2 days earlier. Atoh 1 is expressed in an apex-to-base progression rather then a base-to-apex progression as in wildtype littermates. We tested the possible expression of Atoh1 in neurosensory precursors using two Atoh 1-Cre lines. We show Atoh 1-Cre mediated beta-galactosidase expression in delaminating sensory neuron precursors as well as undifferentiated epithelial cells at E11 and E12.5. PCR analysis shows expression of Atoh 1 in the otocyst as early as E10.5, prior to any histology-based detection techniques. Combined, these data suggest that low levels of Atoh 1 exist much earlier in precursors of hair cells and sensory neurons, possibly including neurosensory precursors. Analysis of Atoh 1-Cre expression in E18.5 embryos and P31 mice reveal beta-galactosidase stain in all hair cells but also in vestibular and cochlear sensory neurons and some supporting cells. A similar expression of Atoh 1-LacZ exists in postnatal and adult vestibular and cochlear sensory neurons, and Atoh 1 expression in vestibular sensory neurons is confirmed with RT-PCR. We propose that the absence of NEUROG 1 protein leads to loss of sensory neuron formation through a phenotypic switch of cycling neurosensory precursors from sensory neuron to hair cell fate. Neurog 1 null mice show a truncation of clonal expansion of hair cell precursors through temporally altered terminal mitosis, thereby resulting in smaller sensory epithelia., (Developmental Dynamics 234:633-650, 2005. (c) 2005 Wiley-Liss, Inc.)

Olig1 and Olig2 are closely related basic helix-loop-helix (bHLH) transcription factors that are expressed in myelinating oligodendrocytes and their progenitor cells in the developing central nervous system (CNS). Olig2 is necessary for the specification of oligodendrocytes, but the biological functions of Olig1 during oligodendrocyte lineage development are poorly understood. We show here that Olig1 function in mice is required not to develop the brain but to repair it. Specifically, we demonstrate a genetic requirement for Olig1 in repairing the types of lesions that occur in patients with multiple sclerosis.

Leptomeningeal glioneuronal heterotopias are a focal type of cortical dysplasia in which neural cells migrate aberrantly into superficial layers of the cerebral cortex and meninges. These heterotopias are frequently observed as microscopic abnormalities in the brains of individuals with central nervous system (CNS) malformations and epilepsy. Previous work has demonstrated that the function of Emx2, which encodes a homeodomain transcription factor, is essential for development of the cortical preplate, which gives rise to the marginal zone and subplate. However, transcriptional targets of EMX2 during CNS development are unknown. We report that leptomeningeal glioneuronal heterotopias form in Emx2(-/-) mice that are equivalent to human lesions. Additionally, we observed ectopic expression of Wnt1 in the embryonic roofplate organizer region and dorsal telencephalon. To determine the phenotypic consequences of such Wnt1 misexpression, we deleted a putative EMX2 DNA-binding site from the Wnt1 enhancer and used this to misexpress Wnt1 in the developing murine CNS. Heterotopias were detected in transgenic mice as early as 13.5 days postcoitum, consistent with a defect of preplate development during early phases of radial neuronal migration. Furthermore, we observed diffuse abnormalities of reelin- and calretinin-positive cell populations in the marginal zone and subplate similar to those observed in Emx2-null animals. Taken together, these findings indicate that EMX2 is a direct repressor of Wnt1 expression in the developing mammalian telencephalon. They further suggest that EMX2-Wnt1 interactions are essential for normal development of preplate derivatives in the mammalian cerebral cortex.

Background: Organizing signals such as Sonic hedgehog are thought to specify neuronal subtype identity by regulating the expression of homeodomain proteins in progenitors of the embryonic neural tube. One of these, Nkx2.2, is necessary and sufficient for the development of V3 interneurons., Results: We report that Olig genes, encoding basic helix-loop-helix (bHLH) proteins, are expressed in a subset of Nkx2.2 progenitors before the establishment of interneurons and oligodendroglial precursors. Gain-of-function analysis in transgenic mouse embryos indicates that Olig genes specifically inhibit the establishment of Sim1-expressing V3 interneurons. Moreover, coexpression of Olig2 with Nkx2.2 in the chick neural tube generated cells expressing Sox10, a marker of oligodendroglial precursors. Colocalization of Olig and Nkx2.2 proteins at the dorsal extent of the Nkx2.2 expression domain is consistent with regulatory interactions that define the potential of progenitor cells in the border region., Conclusions: Interactions between homeodomain and Olig bHLH proteins evidently regulate neural cell fate acquisition and diversification in the ventral neural tube. In particular, interactions between Olig and Nkx2.2 proteins inhibit V3 interneuron development and promote the formation of alternate cell types, including those expressing Sox10.