Independent genomic polymorphisms in the PknH serine threonine kinase locus during evolution of the Mycobacterium tuberculosis Complex affect virulence and host preference.

Species belonging to the Mycobacterium tuberculosis Complex (MTBC) show more than 99% genetic identity but exhibit distinct host preference and virulence. The molecular genetic changes that underly host specificity and infection phenotype within MTBC members have not been fully elucidated. Here, we analysed RD900 genomic region across MTBC members using whole genome sequences from 60 different MTBC strains so as to determine its role in the context of MTBC evolutionary history. The RD900 region comprises two homologous genes, pknH1 and pknH2, encoding a serine/threonine protein kinase PknH flanking the tbd2 gene. Our analysis revealed that RD900 has been independently lost in different MTBC lineages and different strains, resulting in the generation of a single pknH gene. Importantly, all the analysed M. bovis and M. caprae strains carry a conserved deletion within a proline rich-region of pknH, independent of the presence or absence of RD900. We hypothesized that deletion of pknH proline rich-region in M. bovis may affect PknH function, having a potential role in its virulence and evolutionary adaptation. To explore this hypothesis, we constructed two M. bovis 'knock-in' strains containing the M. tuberculosis pknH gene. Evaluation of their virulence phenotype in mice revealed a reduced virulence of both M. bovis knock-in strains compared to the wild type, suggesting that PknH plays an important role in the differential virulence phenotype of M. bovis vs M. tuberculosis.

Supporting Evidence of Human Enteric Nervous System Adult Neurogenesis: Presence of Primary Cilia and Adult Neurogenesis Markers.

Adult neurogenesis has been profusely studied in central nervous system. However, its presence in enteric nervous system remains elusive although it has been recently demonstrated in mice and intimately linked to glial cells. Moreover, primary cilium is an important organelle in central adult neurogenesis. In the present study, we analysed some parallelisms between central and enteric nervous system (ENS) in humans based on ultrastructural and immunohistochemical techniques. Thus, we described the presence of primary cilia in some subtypes of glial cells and Interstitial Cells of Cajal (ICCs) and we performed 3-D reconstructions to better characterise their features. Besides, we studied the expression of several adult neurogenesis-related proteins. Immature neuron markers were found in human ENS, supporting the existence of adult neurogenesis. However, only ICCs showed proliferation markers. Hence, we propose a new paradigm where ICCs would constitute the original neural stem cells which, through asymmetrical cell division, would generate the new-born neurons.

Evidence of p75 Neurotrophin Receptor Involvement in the Central Nervous System Pathogenesis of Classical Scrapie in Sheep and a Transgenic Mouse Model.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

Hedgehog signalling pathway activation in gastrointestinal stromal tumours is mediated by primary cilia.

BACKGROUND: Gastrointestinal stromal tumour (GIST) is a mesenchymal cancer which derives from interstitial cells of Cajal. To determine whether a relationship between Hedgehog (Hh) signalling pathway and primary cilia exists in GIST tumours is intended here. METHODS: Immunohistochemical, immunofluorescence and ultrastructural techniques were performed in this study. RESULTS: We show that GIST cells present primary cilia (an antenna-like structure based on microtubules). But, moreover, we prove Hedgehog signalling pathway activation in these tumours (a pathway related with tumoural features such as proliferation, migration or stemness) and we show for the first time that this signalling pathway activation in GIST is mediated by primary cilia, likely in a paracrine way. CONCLUSION: Thus, primary cilia and Hedgehog signalling would be fundamental in tumoural microenvironment control of GIST cells for their maintenance, differentiation and proliferation.

Neuroimmune Response in Natural Preclinical Scrapie after Dexamethasone Treatment.

A recently published report on chronic dexamethasone treatment for natural scrapie supported the hypothesis of the potential failure of astroglia in the advanced stage of disease. Herein, we aimed to extend the aforementioned study on the effect of this anti-inflammatory therapy to the initial phase of scrapie, with the aim of elucidating the natural neuroinflammatory process occurring in this neurodegenerative disorder. The administration of this glucocorticoid resulted in an outstanding reduction in vacuolation and aberrant protein deposition (nearly null), and an increase in glial activation. Furthermore, evident suppression of IL-1R and IL-6 and the exacerbation of IL-1α, IL-2R, IL-10R and IFNγR were also demonstrated. Consequently, the early stage of the disease is characterized by an intact neuroglial response similar to that of healthy individuals attempting to re-establish homeostasis. A complex network of neuroinflammatory markers is involved from the very early stages of this prion disease, which probably becomes impaired in the more advanced stages. The in vivo animal model used herein provides essential observations on the pathogenesis of natural scrapie, as well as the possibility of establishing neuroglia as potential target cells for anti-inflammatory therapy.

Assessment of Glial Activation Response in the Progress of Natural Scrapie after Chronic Dexamethasone Treatment.

Neuroinflammation has been correlated with the progress of neurodegeneration in many neuropathologies. Although glial cells have traditionally been considered to be protective, the concept of them as neurotoxic cells has recently emerged. Thus, a major unsolved question is the exact role of astroglia and microglia in neurodegenerative disorders. On the other hand, it is well known that glucocorticoids are the first choice to regulate inflammation and, consequently, neuroglial inflammatory activity. The objective of this study was to determine how chronic dexamethasone treatment influences the host immune response and to characterize the beneficial or detrimental role of glial cells. To date, this has not been examined using a natural neurodegenerative model of scrapie. With this aim, immunohistochemical expression of glial markers, prion protein accumulation, histopathological lesions and clinical evolution were compared with those in a control group. The results demonstrated how the complex interaction between glial populations failed to compensate for brain damage in natural conditions, emphasizing the need for using natural models. Additionally, the data showed that modulation of neuroinflammation by anti-inflammatory drugs might become a research focus as a potential therapeutic target for prion diseases, similar to that considered previously for other neurodegenerative disorders classified as prion-like diseases.

Pulmonary but Not Subcutaneous Delivery of BCG Vaccine Confers Protection to Tuberculosis-Susceptible Mice by an Interleukin 17-Dependent Mechanism.

Some of the most promising novel tuberculosis vaccine strategies currently under development are based on respiratory vaccination, mimicking the natural route of infection. In this work, we have compared pulmonary and subcutaneous delivery of BCG vaccine in the tuberculosis-susceptible DBA/2 mouse strain, a model in which parenterally administered BCG vaccine does not protect against tuberculosis. Our data show that intranasally but not subcutaneously administered BCG confers robust protection against pulmonary tuberculosis challenge. In addition, our results indicate that pulmonary vaccination triggers a Mycobacterium tuberculosis-specific mucosal immune response orchestrated by interleukin 17A (IL-17A). Thus, IL-17A neutralization in vivo reduces protection and abrogates M. tuberculosis-specific immunoglobulin A (IgA) secretion to respiratory airways and lung expression of polymeric immunoglobulin receptor induced following intranasal vaccination. Together, our results demonstrate that pulmonary delivery of BCG can overcome the lack of protection observed when BCG is given parenterally, suggesting that respiratory tuberculosis vaccines could have an advantage in tuberculosis-endemic countries, where intradermally administered BCG has inefficient effectiveness against pulmonary tuberculosis.

Biogenesis of a new type of extracellular vesicles in gastrointestinal stromal tumors: ultrastructural profiles of spheresomes.

Extracellular vesicles (EVs) have emerged as an intercellular communication mediator in cancer. They seem to be involved in tumor processes by means of transformation of surrounding cells previous to metastasis by transferring miRNAs and oncogenic proteins. It is known that EVs, depending on their source, can be exosomes or ectosomes. Although the first type constitutes a specific population formed from the endosomal system, via multivesicular bodies, the ectosome biogenesis is not yet well known. In this study, we report a new type of EVs which has been termed spheresomes. While exosomes come from multivesicular bodies and ectosomes from direct budding of plasma membrane, spheresomes present a new mechanism of shedding from a spherical membrane structure which we have named multivesicular spheres. These EVs are first described in gastrointestinal stromal tumor cells in the present study. But moreover, these new membrane spherical structures appear not only next to tumoral cells but also different distances from them. Since some other authors have evidenced oncogenic KIT-containing EVs, it is also suggested here that surrounding cells uptake of these first described EVs, GIST-derived spheresomes, could induce tumor invasiveness. That is why the prevention of signaling processes developed by these new EVs may represent an alternative approach for GIST treatment.

Ultrastructural changes in the progress of natural Scrapie regardless fixation protocol.

Because few studies regarding ultrastructural pathological changes associated with natural prion diseases have been performed, the present study primarily intended to determine consistent lesions at the subcellular level and to demonstrate whether these changes are evident regardless of the fixation protocol. Thus far, no assessment method has been developed for classifying the possible variations according to the disease stage, although such an assessment would contribute to clarifying the pathogenesis of this neurodegenerative disease. Therefore, animals at different disease stages were included here. This study presents the first description of lesions associated with natural Scrapie in the cerebellum. Vacuolation, which preferentially occurs around Purkinje cells and which displays a close relation with glial cells, is one of the most novel observations provided in this study. The disruption of hypolemmal cisterns in this neuronal type and the presence of a primary cilium in the granular layer both represent the first findings concerning prion diseases. The possibility of including samples regardless of their fixation protocol is confirmed in this work. Therefore, a high proportion of tissue bank samples that are currently being wasted can be included in ultrastructural studies, which constitute a valuable source for information regarding physiological and pathological samples.

Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie.

BACKGROUND: Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. RESULTS: In the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. CONCLUSIONS: The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease.

Morphological approach to assess the involvement of astrocytes in prion propagation.

Transmissible Spongiform Encephalopathies (TSEs) are a group of neurodegenerative disorders affecting animals and humans and for which no effective treatment is available to date. Vacuolation, neuronal/neurite degeneration, deposition of pathological prion protein (PrPsc) and gliosis are changes typically found in brains from TSE affected individuals. However, the actual role of this last feature, microgliosis and astrocytosis, has not been precisely determined. The overall objective of this work is to assess the involvement of glial cells as components of the host protective system in prion propagation; specifically, to analyze the behavior of astroglial cells in prion progression. To achieve this aim, histopathological and immunohistochemical techniques were carried out on samples from cerebella using Scrapie as the prototype of natural TSEs as this made it possible to assess different stages of the disease; specifically, ages and genotypes from Scrapie-affected animals corresponding to different sources, by using optical, confocal and electron microscopy. The results provided in the present study demonstrate the indisputable involvement of astroglia in prion progression by showing specific changes of this glial population matching up to the evolution of the disease. Moreover, cerebellar lesions mainly associated to Purkinje cells that have not previously been reported in animal prion diseases in natural transmission are described here. The close relationship between PrPsc and GFAP hiperimmunoreactivity and Purkinje cells, alongside the evident thickening of their neurites at terminal stages demonstrated in this study, suggest that these neurons are the main target of this neurodegenerative disease.

Factors Involved in the Immunological Protection against Rabies Virus in Dogs in Spain.

Rabies, a viral disease spread by infected animal bites that causes encephalitis in humans and other mammals, is a neglected infectious disease present on all continents except Antarctica. Spain has been free of terrestrial rabies since 1978. However, due to its geographical situation, it represents a bridge for imported cases from an endemic continent such as Africa to Europe. Rabies vaccination in dogs is an essential preventive tool against this zoonosis. The aim of this study was to determine the state of the immune response against rabies virus in dogs in Spain and to demonstrate whether several factors that have been previously related to the influence of the seroprevalence of this species are involved here. The seroconversion level of this zoonotic virus was assessed in a total of 1060 animals. Indirect ELISA was used to obtain data for statistical analysis to evaluate the studied variables. Working under the concept of One Health, this study provides relevant information to be taken into consideration not only to prevent re-emergence in countries free of this disease but also for prevention and control in endemic countries.

Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality.

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

A comparative study of modified confirmatory techniques and additional immuno-based methods for non-conclusive autolytic bovine spongiform encephalopathy cases.

BACKGROUND: In the framework of the Bovine Spongiform Encephalopathy (BSE) surveillance programme, samples with non-conclusive results using the OIE confirmatory techniques have been repeatedly found. It is therefore necessary to question the adequacy of the previously established consequences of this non-conclusive result: the danger of failing to detect potentially infected cattle or erroneous information that may affect the decision of culling or not of an entire bovine cohort. Moreover, there is a very real risk that the underreporting of cases may possibly lead to distortion of the BSE epidemiological information for a given country.In this study, samples from bovine nervous tissue presenting non-conclusive results by conventional OIE techniques (Western blot and immunohistochemistry) were analyzed. Their common characteristic was a very advanced degree of autolysis. All techniques recommended by the OIE for BSE diagnosis were applied on all these samples in order to provide a comparative study. Specifically, immunohistochemistry, Western blotting, SAF detection by electron microscopy and mouse bioassay were compared. Besides, other non confirmatory techniques, confocal scanning microscopy and colloidal gold labelling of fibrils, were applied on these samples for confirming and improving the results. RESULTS: Immunocytochemistry showed immunostaining in agreement with the positive results finally provided by the other confirmatory techniques. These results corroborated the suitability of this technique which was previously developed to examine autolysed (liquified) brain samples. Transmission after inoculation of a transgenic murine model TgbovXV was successful in all inocula but not in all mice, perhaps due to the very scarce PrPsc concentration present in samples.Electron microscopy, currently fallen into disuse, was demonstrated to be, not only capable to provide a final diagnosis despite the autolytic state of samples, but also to be a sensitive diagnostic alternative for resolving cases with low concentrations of PrPsc. CONCLUSIONS: Demonstration of transmission of the disease even with low concentrations of PrPsc should reinforce that vigilance is required in interpreting results so that subtle changes do not go unnoticed. To maintain a continued supervision of the techniques which are applied in the routine diagnosis would prove essential for the ultimate eradication of the disease.

Spheresomes are the main extracellular vesicles in low-grade gliomas.

Cancer progression and its impact on treatment response and prognosis is deeply regulated by tumour microenvironment (TME). Cancer cells are in constant communication and modulate TME through several mechanisms, including transfer of tumour-promoting cargos through extracellular vesicles (EVs) or oncogenic signal detection by primary cilia. Spheresomes are a specific EV that arise from rough endoplasmic reticulum-Golgi vesicles. They accumulate beneath cell membrane and are released to the extracellular medium through multivesicular spheres. This study describes spheresomes in low-grade gliomas using electron microscopy. We found that spheresomes are more frequent than exosomes in these tumours and can cross the blood-brain barrier. Moreover, the distinct biogenesis processes of these EVs result in unique cargo profiles, suggesting different functional roles. We also identified primary cilia in these tumours. These findings collectively contribute to our understanding of glioma progression and metastasis.

Diagnosis in Scrapie: Conventional Methods and New Biomarkers.

Scrapie, a naturally occurring prion disease affecting goats and sheep, comprises classical and atypical forms, with classical scrapie being the archetype of transmissible spongiform encephalopathies. This review explores the challenges of scrapie diagnosis and the utility of various biomarkers and their potential implications for human prion diseases. Understanding these biomarkers in the context of scrapie may enable earlier prion disease diagnosis in humans, which is crucial for effective intervention. Research on scrapie biomarkers bridges the gap between veterinary and human medicine, offering hope for the early detection and improved management of prion diseases.

Involvement of astrocytes in transmissible spongiform encephalopathies: a confocal microscopy study.

Astroglial proliferation associated with pathological prion protein (PrPsc) deposition is widely described in Transmissible Spongiform Encephalopathies (TSEs). However, little is known of the actual role played by glia in their pathogenesis. The aim of the study has been to determine whether PrPsc is located exclusively in neurons or in both neurons and glial cells present in the central nervous system in a natural Scrapie model. Samples of cerebellum from 25 Scrapie sheep from various flocks were sectioned. Following epitope retrieval with formic acid, proteinase K and heat treatment, primary antibody L42 and primary antibodies against glial fibrillary acidic protein were applied as prion- and astrocytic-specific markers, respectively. For visualization, a suitable mixture of fluorochrome-conjugated secondary antibodies was used. Relevant controls were processed in the same manner. As determined by confocal microscopy, PrPsc deposits co-localized with glial cells in all samples. Our results suggest that these cells can sustain active prion propagation, in agreement with similar findings from other studies of primary cell cultures and inoculated mice. Furthermore, despite ongoing debate regarding whether varied TSE sources show differences in their tropism for different cell lineages in the brains of affected animals, no differences in co-localization results were seen.

Changes induced by natural scrapie in the calretinin-immunopositive cells and fibres of the sheep cerebellar cortex.

Calretinin (CR)-immunopositive cells and fibres in the cerebellar cortex (vermal archicerebellum and neocerebellum) of scrapie-affected, ARQ/ARQ, Rasa Aragonesa breed sheep were studied in comparison with healthy, young and aged, ARQ/ARQ, Rasa Aragonesa animals and with Manchega breed sheep. The scrapie-affected sheep showed signs of both cellular involution and hypertrophic/hyperimmunoreactive responses in all neuronal subtypes; the distribution of the neuronal subtypes in the archi- and neocerebellum, however, did not change compared with controls. The results suggest that the different CR expression and/or CR content of cerebellar cortical neurons in scrapie-affected sheep are more related to their specific functions than any neuroprotective response. The reduction in the cell density of some CR-immunopositive neuronal subsets (i.e. unipolar brush cells) is contradictory to the supposed neuroprotective role of the calcium binding protein CR. However, the hyperimmunoreactivity of many CR-immunopositive neuronal subsets (e.g. the Purkinje cells) suggests the involvement of an over-expression of CR (transitory or restricted to selected neurons) as an adaptative mechanism to fight against the neurodegeneration caused by this prion disease. The changes in the number of immunopositive cells and the hypertrophic/hyperimmunoreactive response seen in scrapie-affected and aged sheep suggests that some different and some similar mechanisms are at work in this disease and aging.

Stromal cells of giant cell tumor of bone show primary cilia in giant cell tumor of bone.

Giant cell tumor of bone (GCTB) is a locally aggressive primary bone neoplasm composed by tumoral stromal cells (SCs) and a reactive component that consists of monocytic/histiocytic cells that give rise by fusion to osteoclast-like multinucleated cells. Recently, specific Histone 3.3 mutations have been demonstrated in SCs of GCTB. Many of the pathways related to bone proliferation and regulation depend on the primary cilium, a microtubule-based organelle that protrudes outside the cell and acts as a sensorial antenna. In the present work, we aimed to study the presence and role of primary cilia in GCTB. Ultrastructural, immunohistochemical, and immunofluorescence studies were performed in order to demonstrate, for the first time, that the primary cilium is located in spindle-shaped SCs of GCTB. Moreover, we showed Hedgehog (Hh) signaling pathway activation in these cells. Hence, primary cilia may play a relevant role in GCTB tumorogenesis through Hh signaling activation in SCs. RESEARCH HIGHLIGHTS: Transmission electron microscopy allows describing and differentiating cellular subpopulations in giant cell tumor of bone (GCTB). The primary cilium is present in some tumoral stromal cells of GCTB. Hedgehog signalling is activated in these cells.

Novel Morphological Glial Alterations in the Spectrum of Prion Disease Types: A Focus on Common Findings.

Human prion diseases are a group of rare fatal neurodegenerative diseases with sporadic, genetic, and acquired forms. They are neuropathologically characterized by pathological prion protein accumulation, neuronal death, and vacuolation. Classical immunological response has long been known not to play a major in prion diseases; however, gliosis is known to be a common feature although variable in extent and poorly described. In this investigation, astrogliosis and activated microglia in two brain regions were assessed and compared with non-neurologically affected patients in a representative sample across the spectrum of Creutzfeldt-Jakob disease (CJD) forms and subtypes in order to analyze the influence of prion strain on pathological processes. In this report, we choose to focus on features common to all CJD types rather than the diversity among them. Novel pathological changes in both glial cell types were found to be shared by all CJD types. Microglial activation correlated to astrogliosis. Spongiosis, but not pathological prion protein deposition, correlated to both astrogliosis and microgliosis. At the ultrastructural level, astrocytic glial filaments correlated with pathological changes associated with prion disease. These observations confirm that neuroglia play a prominent role in the neurodegenerative process of prion diseases, regardless of the causative prion type.