RESUMO
Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.
Assuntos
Linhagem da Célula , Células Germinativas , Ovário , Diferenciação Sexual , Análise de Célula Única , Testículo , Animais , Cromatina/genética , Cromatina/metabolismo , Feminino , Células Germinativas/citologia , Células Germinativas/metabolismo , Células da Granulosa/citologia , Células da Granulosa/metabolismo , Humanos , Imunoglobulinas , Macrófagos/metabolismo , Masculino , Glicoproteínas de Membrana , Proteínas de Membrana , Camundongos , Microscopia de Fluorescência , Ovário/citologia , Ovário/embriologia , Fator de Transcrição PAX8 , Gravidez , Primeiro Trimestre da Gravidez , Segundo Trimestre da Gravidez , Receptores Imunológicos , Diferenciação Sexual/genética , Testículo/citologia , Testículo/embriologia , TranscriptomaRESUMO
Inosine has robust neuroprotective effects, but it is unclear if inosine acts as direct ligand of adenosine receptors or if it triggers metabolic effects indirectly modifying the activity of adenosine receptors. We now combined radioligand binding studies with electrophysiological recordings in hippocampal slices to test how inosine controls synaptic transmission and plasticity. Inosine was without effect at 30 µM and decreased field excitatory post-synaptic potentials by 14% and 33% at 100 and 300 µM, respectively. These effects were prevented by the adenosine A1 receptor antagonist DPCPX. Inosine at 300 (but not 100) µM also decreased the magnitude of long-term potentiation (LTP), an effect prevented by DPCPX and by the adenosine A2A receptor antagonist SCH58261. Inosine showed low affinity towards human and rat adenosine receptor subtypes with Ki values of > 300 µM; only at the human and rat A1 receptor slightly higher affinities with Ki values of around 100 µM were observed. Affinity of inosine at the rat A3 receptor was higher (Ki of 1.37 µM), while it showed no interaction with the human orthologue. Notably, the effects of inosine on synaptic transmission and plasticity were abrogated by adenosine deaminase and by inhibiting equilibrative nucleoside transporters (ENT) with dipyridamole and NBTI. This shows that the impact of inosine on hippocampal synaptic transmission and plasticity is not due to a direct activation of adenosine receptors but is instead due to an indirect modification of the tonic activation of these adenosine receptors through an ENT-mediated modification of the extracellular levels of adenosine.
Assuntos
Adenosina , Nucleosídeos , Ratos , Humanos , Animais , Adenosina/metabolismo , Nucleosídeos/metabolismo , Receptor A1 de Adenosina/metabolismo , Transmissão Sináptica/fisiologia , Antagonistas de Receptores Purinérgicos P1/farmacologia , Inosina/farmacologia , Hipocampo/metabolismoRESUMO
BACKGROUND: B-cell affinity maturation in germinal center relies on regulated actin dynamics for cell migration and cell-to-cell communication. Activating mutations in the cytoskeletal regulator Wiskott-Aldrich syndrome protein (WASp) cause X-linked neutropenia (XLN) with reduced serum level of IgA. OBJECTIVE: We investigated the role of B cells in XLN pathogenesis. METHODS: We examined B cells from 6 XLN patients, 2 of whom had novel R268W and S271F mutations in WASp. By using immunized XLN mouse models that carry the corresponding patient mutations, WASp L272P or WASp I296T, we examined the B-cell response. RESULTS: XLN patients had normal naive B cells and plasmablasts, but reduced IgA+ B cells and memory B cells, and poor B-cell proliferation. On immunization, XLN mice had a 2-fold reduction in germinal center B cells in spleen, but with increased generation of plasmablasts and plasma cells. In vitro, XLN B cells showed reduced immunoglobulin class switching and aberrant cell division as well as increased production of immunoglobulin-switched plasma cells. CONCLUSIONS: Overactive WASp predisposes B cells for premature differentiation into plasma cells at the expense of cell proliferation and immunoglobulin class switching.
Assuntos
Linfócitos B , Neutropenia , Proteína da Síndrome de Wiskott-Aldrich , Animais , Linfócitos B/citologia , Divisão Celular , Doenças Genéticas Ligadas ao Cromossomo X , Humanos , Imunoglobulina A , Camundongos , Neutropenia/genética , Plasmócitos/patologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
Alzheimer's disease (AD), which predominantly affects women, involves at its onset a metabolic deregulation associated with a synaptic failure. Here, we performed a behavioral, neurophysiological and neurochemical characterization of 9-month-old female APPswe/PS1dE9 (APP/PS1) mice as a model of early AD. These animals showed learning and memory deficits in the Morris water maze, increased thigmotaxis and anxiety-like behavior and showed signs of fear generalization. Long-term potentiation (LTP) was decreased in the prefrontal cortex (PFC), but not in the CA1 hippocampus or amygdala. This was associated with a decreased density of sirtuin-1 in cerebrocortical synaptosomes and a decreased density of sirtuin-1 and sestrin-2 in total cerebrocortical extracts, without alterations of sirtuin-3 levels or of synaptic markers (syntaxin, synaptophysin, SNAP25, PSD95). However, activation of sirtuin-1 did not affect or recover PFC-LTP deficit in APP/PS1 female mice; instead, inhibition of sirtuin-1 increased PFC-LTP magnitude. It is concluded that mood and memory dysfunction in 9-month-old female APP/PS1 mice is associated with a parallel decrease in synaptic plasticity and in synaptic sirtuin-1 levels in the prefrontal cortex, although sirtiun1 activation failed to restore abnormal cortical plasticity.
Assuntos
Doença de Alzheimer , Córtex Pré-Frontal , Sirtuína 1 , Animais , Feminino , Camundongos , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Aprendizagem em Labirinto , Camundongos Transgênicos , Córtex Pré-Frontal/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismoRESUMO
BACKGROUND: Advances in three-dimensional culture technologies have led to progression in systems used to model the gonadal microenvironment in vitro. Despite demonstrating basic functionality, tissue organisation is often limited. We have previously detailed a three-dimensional culture model termed the three-layer gradient system to generate rat testicular organoids in vitro. Here we extend the model to human first-trimester embryonic gonadal tissue. RESULTS: Testicular cell suspensions reorganised into testis-like organoids with distinct seminiferous-like cords situated within an interstitial environment after 7 days. In contrast, tissue reorganisation failed to occur when mesonephros, which promotes testicular development in vivo, was included in the tissue digest. Organoids generated from dissociated female gonad cell suspensions formed loosely organised cords after 7 days. In addition to displaying testis-specific architecture, testis-like organoids demonstrated evidence of somatic cell differentiation. Within the 3-LGS, we observed the onset of AMH expression in the cytoplasm of SOX9-positive Sertoli cells within reorganised testicular cords. Leydig cell differentiation and onset of steroidogenic capacity was also revealed in the 3-LGS through the expression of key steroidogenic enzymes StAR and CYP17A1 within the interstitial compartment. While the 3-LGS generates a somatic cell environment capable of supporting germ cell survival in ovarian organoids germ cell loss was observed in testicular organoids. CONCLUSION: The 3-LGS can be used to generate organised whole gonadal organoids within 7 days. The 3-LGS brings a new opportunity to explore gonadal organogenesis and contributes to the development of more complex in vitro models in the field of developmental and regenerative medicine.
Assuntos
Células de Sertoli , Testículo , Animais , Colágeno , Combinação de Medicamentos , Feminino , Gônadas , Humanos , Laminina , Masculino , Proteoglicanas , Ratos , SuspensõesRESUMO
Theobromine is a caffeine metabolite most abundant in dark chocolate, of which consumption is linked with a lower risk of cognitive decline. However, the mechanisms through which theobromine affects neuronal function remain ill-defined. Using electrophysiological recordings in mouse hippocampal synapses, we now characterized the impact of a realistic concentration of theobromine on synaptic transmission and plasticity. Theobromine (30 µM) facilitated synaptic transmission while decreasing the magnitude of long-term potentiation (LTP), with both effects being blunted by adenosine deaminase (2 U/mL). The pharmacological blockade of A1R with DPCPX (100 nM) eliminated the theobromine-dependent facilitation of synaptic transmission, whereas the A2AR antagonist SCH58261 (50 nM), as well as the genetic deletion of A2AR, abrogated the theobromine-induced impairment of LTP. Furthermore, theobromine prevented LTP deficits and neuronal loss, respectively, in mouse hippocampal slices and neuronal cultures exposed to Aß1-42 peptides, considered a culprit of Alzheimer's disease. Overall, these results indicate that theobromine affects information flow via the antagonism of adenosine receptors, normalizing synaptic plasticity and affording neuroprotection in dementia-related conditions in a manner similar to caffeine.
Assuntos
Adenosina Desaminase , Cafeína , Adenosina Desaminase/metabolismo , Animais , Cafeína/metabolismo , Cafeína/farmacologia , Hipocampo/metabolismo , Camundongos , Plasticidade Neuronal , Receptor A2A de Adenosina/metabolismo , Sinapses/metabolismo , Teobromina/farmacologiaRESUMO
Adenosine A2A receptors (A2AR) control fear memory and the underlying processes of synaptic plasticity in the amygdala. In other brain regions, A2AR activation is ensured by ATP-derived extracellular adenosine formed by ecto-5'-nucleotidase or CD73. We now tested whether CD73 is also responsible to provide for the activation of A2AR in controlling fear memory and amygdala long-term potentiation (LTP). The bilateral intracerebroventricular injection of the CD73 inhibitor αß-methylene ADP (AOPCP, 1 nmol/ventricle/day) phenocopied the effect of the A2AR blockade by decreasing the expression of fear memory, an effect disappearing in CD73-knockout (KO) mice and in forebrain neuronal A2AR-KO mice. In the presence of PPADS (20 µM) to eliminate any modification of ATP/ADP-mediated P2 receptor effects, both AOPCP (100 µM) and the A2AR antagonist, SCH58261 (50 nM), decreased LTP magnitude in synapses of projection from the external capsula into the lateral amygdala, an effect eliminated in slices from both forebrain neuronal A2AR-KO mice and CD73-KO mice. These data indicate a key role of CD73 in the process of A2AR-mediated control of fear memory and underlying synaptic plasticity processes in the amygdala, paving the way to envisage CD73 as a new therapeutic target to interfere with abnormal fear-like emotional processing.
Assuntos
5'-Nucleotidase , Receptor A2A de Adenosina , Camundongos , Animais , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Adenosina/metabolismo , Camundongos Endogâmicos C57BL , Tonsila do Cerebelo/metabolismo , Camundongos Knockout , Medo/fisiologia , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismoRESUMO
We performed a retrospective study of coronavirus disease 2019 (COVID-19) in people with human immunodeficiency virus (PWH). PWH with COVID-19 demonstrated severe lymphopenia and decreased CD4+ T cell counts. Levels of inflammatory markers, including C-reactive protein, fibrinogen, D-dimer, interleukin 6, interleukin 8, and tumor necrosis factor α were commonly elevated. In all, 19 of 72 hospitalized individuals (26.4%) died and 53 (73.6%) recovered. PWH who died had higher levels of inflammatory markers and more severe lymphopenia than those who recovered. These findings suggest that PWH remain at risk for severe manifestations of COVID-19 despite antiretroviral therapy and that those with increased markers of inflammation and immune dysregulation are at risk for worse outcomes.
Assuntos
COVID-19/imunologia , COVID-19/virologia , Infecções por HIV/imunologia , Infecções por HIV/virologia , Idoso , COVID-19/sangue , COVID-19/mortalidade , Feminino , Infecções por HIV/sangue , Infecções por HIV/mortalidade , HIV-1/isolamento & purificação , Hospitalização/estatística & dados numéricos , Humanos , Inflamação/sangue , Inflamação/imunologia , Inflamação/virologia , Mediadores da Inflamação/sangue , Mediadores da Inflamação/imunologia , Contagem de Linfócitos , Linfopenia/virologia , Masculino , Pessoa de Meia-Idade , New York/epidemiologia , Estudos Retrospectivos , Fatores de Risco , SARS-CoV-2/isolamento & purificaçãoRESUMO
Calcium (Ca2+ ) is essential for the normal functioning of the brain: Ca2+ inflow into synaptic compartments is a major trigger of neurotransmitter release and of long-term plastic changes of synaptic efficiency. Ca2+ influx depends on the gradient for this ion across the plasma membrane, and slight fluctuations of extracellular Ca2+ concentration have significant impact on axonal excitability, synaptic transmission, and plasticity. The work by Forsberg et al. reports a concentration of physiologically active Ca2+ in human cerebrospinal fluid (CSF) that is half of that normally used in artificial CSF in electrophysiological brain slice experiments. By studying the impact of such difference of extracellular Ca2+ concentration, the authors reported significant differences in terms of neuronal excitability and long-term potentiation in rat hippocampal slices. We now discuss these new findings in the context of the spatial organization of synapses, on the role of astrocytes in filtering the synaptic content and on the complexity of plasticity in a broad context of metaplastic modifications of synaptic efficiency.
Assuntos
Cálcio , Células Piramidais , Animais , Hipocampo , Humanos , Potenciação de Longa Duração , Ratos , Sinapses , Transmissão SinápticaRESUMO
Alzheimer's disease (AD) begins with a deficit of synaptic function and adenosine A2A receptors (A2AR) are mostly located in synapses controlling synaptic plasticity. The over-activation of adenosine A2A receptors (A2AR) causes memory deficits and the blockade of A2AR prevents memory damage in AD models. We now enquired if this prophylactic role of A2AR might be extended to a therapeutic potential. We used the triple transgenic model of AD (3xTg-AD) and defined that the onset of memory dysfunction occurred at 4â¯months of age in the absence of locomotor or emotional alterations. At the onset of memory deficits, 3xTg mice displayed a decreased density of markers of excitatory synapses (10.6⯱â¯3.8% decrease of vGluT1) without neuronal or glial overt damage and an increase of synaptic A2AR in the hippocampus (130⯱â¯22%). After the onset of memory deficits in 3xTg-AD mice, a three weeks treatment with the selective A2AR antagonist normalized the up-regulation of hippocampal A2AR and restored hippocampal-dependent reference memory, as well as the decrease of hippocampal synaptic plasticity (60.0⯱â¯3.7% decrease of long-term potentiation amplitude) and the decrease of global (syntaxin-I) and glutamatergic synaptic markers (vGluT1). These findings show a therapeutic-like ability of A2AR antagonists to recover synaptic and memory dysfunction in early AD.
Assuntos
Antagonistas do Receptor A2 de Adenosina/uso terapêutico , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Transtornos da Memória/genética , Transtornos da Memória/metabolismo , Plasticidade Neuronal/fisiologia , Antagonistas do Receptor A2 de Adenosina/farmacologia , Doença de Alzheimer/tratamento farmacológico , Animais , Modelos Animais de Doenças , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Aprendizagem em Labirinto/fisiologia , Transtornos da Memória/tratamento farmacológico , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plasticidade Neuronal/efeitos dos fármacos , Projetos Piloto , Receptor A2A de Adenosina/metabolismoRESUMO
BACKGROUND AIMS: The therapeutic application of CD34+ circulating progenitor cells (which includes endothelial progenitor cells) has been hampered by the quantity and quality of isolated circulating CD34(+) cells from the patient's peripheral blood. Our group had previously established a suspension culture system for human CD34(+) cells, with increased quantity and quality (QQ) of the angiogenic cell product. We successfully scaled up the expansion process with the use of culture bags because there is the need to move toward a dynamic and fully controlled bioreactor system to meet Good Manufacturing Practice (GMP) standards and attain clinically meaningful cell doses in a time- and cost-effective way. METHODS: CD34(+) cells isolated from mobilized peripheral blood of healthy donors were expanded ex vivo for 7 days in QQ medium (serum-free) in cell culture bags (30 mL) and pre- and post-expansion cells were characterized by means of flow cytometry and quantitative polymerase chain reaction; angiogenic potential was assessed by use of the in vitro tube formation assay. RESULTS: Our data show effective expansion of the cultured population (7-fold) while maintaining the stem/progenitor content and increasing the endothelial population. Moreover, post-expanded cells showed higher tube formation capacity compared with pre-expanded cells. In addition, an upregulation of the anti-inflammatory gene expression and a downregulation of pro-inflammatory genes were observed, which suggests that the increase in angiogenic potential is not paired with an increase in the inflammatory profile. CONCLUSIONS: The QQ expansion method was successfully scaled up to cell culture bags and was able to meet GMP standards, with a higher in vitro angiogenic profile.
Assuntos
Antígenos CD34/metabolismo , Meios de Cultura Livres de Soro/farmacologia , Células Progenitoras Endoteliais/metabolismo , Inflamação/imunologia , Neovascularização Fisiológica/fisiologia , Reatores Biológicos , Técnicas de Cultura de Células , Ciclo Celular , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Voluntários Saudáveis , Humanos , Inflamação/genética , Regulação para CimaRESUMO
Potassium iodide has demonstrated several therapeutic applications over time, being the choice for shielding the thyroid during radiation emergencies involving radioiodine release. Amidst the ongoing military conflict between Ukraine and Russia and the growing concern regarding the potential deployment of nuclear weapons, there has been a surge in the demand for potassium iodide across Europe. This work aimed to comprehensively review the current knowledge regarding the pharmacology, physiology, adverse effects, the protective role in reducing the risk of thyroid cancer and recommendations for potassium iodide use during radiation emergencies. Evidence on adverse effects is scarce, as potassium iodide is generally well-tolerated. Guidelines for thyroid blocking with potassium iodide during radiation emergencies suggest that, among populations vulnerable to radioiodine exposure, the benefits of potassium iodide outweigh the risks of adverse effects. Controversial topics surrounding the utilization of potassium iodide in radiation emergencies include the prophylaxis in iodine-deficient regions and following the detonation of dirty bombs, whether granule formulations versus tablets should be used and mental health concerns. Although the rise in demand seems to be a justified security measure, it is essential to recognize that potassium iodide protects the thyroid from radioiodine and does not impact the body's absorption of other radioactive materials or defend against external radiation exposure.
Assuntos
Iodeto de Potássio , Iodeto de Potássio/uso terapêutico , Humanos , Ucrânia , Europa (Continente) , Glândula Tireoide/efeitos da radiação , Glândula Tireoide/efeitos dos fármacos , Radioisótopos do Iodo/efeitos adversos , Protetores contra Radiação/uso terapêutico , Neoplasias da Glândula Tireoide , Lesões por Radiação , Animais , Armas NuclearesRESUMO
Human primordial germ cells (hPGCs), the precursors of eggs and sperm, start their complex development shortly after specification and during their migration to the primitive gonads. Here, we describe protocols for specifying hPGC-like cells (hPGCLCs) from resetting precursors and progressing them with the support of human hindgut organoids. Resetting hPGCLCs (rhPGCLCs) are specified from human embryonic stem cells (hESCs) transitioning from the primed into the naive state of pluripotency. Hindgut organoids are also derived from hESCs after a sequential differentiation into a posterior endoderm/hindgut fate. Both rhPGCLCs and hindgut organoids are combined and co-cultured for 25 d. The entire procedure takes ~1.5 months and can be successfully implemented by a doctoral or graduate student with basic skills and experience in hESC cultures. The co-culture system supports the progression of rhPGCLCs at a developmental timing analogous to that observed in vivo. Compared with previously developed hPGCLC progression protocols, which depend on co-cultures with mouse embryonic gonadal tissue, our co-culture system represents a developmentally relevant model closer to the environment that hPGCs first encounter after specification. Together with the potential for investigations of events during hPGC specification and early development, these protocols provide a practical approach to designing efficient models for in vitro gametogenesis. Notably, the rhPGCLC-hindgut co-culture system can also be adapted to study failings in hPGC migration, which are associated with the etiology of some forms of infertility and germ cell tumors.
Assuntos
Endoderma , Sêmen , Humanos , Masculino , Animais , Camundongos , Células Germinativas , Diferenciação Celular , OrganoidesRESUMO
Malnutrition is considered one of the major public health problems worldwide and negatively affects the growth, development and learning of schoolchildren. This study developed and evaluated a fermented milk drink with added Umbu (Spondias tuberosa) pulp in the weight gain and renutrition of mice submitted to malnutrition by calorie restriction, and in malnourished children. The supplementation with this fermented milk drink contributed to an increase of 7.2 % in body weight, and 64.3 % in albumin, and a reduction of 35 % in cholesterol in malnourished mice. In humans, a group of nine malnourished children consumed a daily 200 mL serving of the milk drink (for 60 days). For humans, the fermented milk drink allowed an increase of 16.5 % in body weight, and 20.9 % in body mass index in malnourished children. In conclusion, fermented milk drink has a positive effect on the re-nutrition of malnourished mice and helps to improve the nutritional status of malnourished children.
Assuntos
Anacardiaceae , Desnutrição , Criança , Humanos , Animais , Camundongos , Soro do Leite , Leite , Estado Nutricional , Proteínas do Soro do Leite , Aumento de Peso , Peso CorporalRESUMO
STUDY QUESTION: Can human pre- and peri-pubertal testicular cells obtained from childhood cancer patients, previously treated with chemotherapy, form testicular organoids (TOs)? SUMMARY ANSWER: Organoid formation from testicular tissue collected from childhood cancer patients positively correlates with SRY-Box transcription factor 9 (SOX9) expression in Sertoli cells, which in turn negatively correlates with previous exposure to alkylating chemotherapy. WHAT IS KNOWN ALREADY: Pre- and peri-pubertal boys exposed to highly gonadotoxic therapies can only safeguard their fertility potential through testicular tissue cryopreservation. Today, there is no established clinical tool to restore fertility using these testicular samples. Organoids hold promise in providing fundamental early insights in creating such platforms. However, the generation of TOs that closely resemble the innate testis, to enable a thorough monitoring of the necessary steps for germ cell differentiation and somatic functionalities, remains a challenge. STUDY DESIGN SIZE DURATION: We used a Matrigel-based three-layer gradient culture system to generate human TOs and to reveal whether chemotherapy exposure affects TO formation capacity and the functionality of pre- and peri-pubertal testicular somatic cells. Testicular cells of 11 boys (aged 7.7 ± 4.1 (mean ± SD) years) were assessed for TO formation in relation to previous chemotherapy exposure and SOX9 expression in histological sections of paraffin-embedded testicular tissue samples collected on the day of biopsy and compared with testicular tissue samples obtained from 28 consecutive patients (aged 6.9 ± 3.8 (mean ± SD) years). All 39 patients were part of the fertility preservation project NORDFERTIL; an additional 10 samples (from boys aged 5.5 ± 3.5 (mean ± SD) years, without an underlying pathology) in an internal biobank collection were used as controls. PARTICIPANTS/MATERIALS SETTING METHODS: We obtained 49 testicular tissue samples from boys aged 0.8-13.4 years. Fresh samples (n = 11) were dissociated into single-cell suspensions and applied to a three-layer gradient culture system for organoid formation. Histological sections of another 28 samples obtained as part of the fertility preservation project NORDFERTIL, and 10 samples from a sample collection of a pathology biobank were used to evaluate the effects of prior exposure to alkylating agents on testicular samples. Testicular organoid formation was defined based on morphological features, such as compartmentalized structures showing cord formation, and protein expression of testicular cell-specific markers for germ and somatic cells was evaluated via immunohistochemical staining. Hormone secretion was analysed by specific enzyme-linked immunosorbent assays for testosterone and anti-Müllerian hormone (AMH) production. MAIN RESULTS AND THE ROLE OF CHANCE: Our results revealed that 4 out of 11 prepubertal testicular samples formed TOs that showed compartmentalized cord-like structures surrounded by interstitial-like areas and increasing levels of both testosterone as well as AMH over a 7-day culture period. We observed that SOX9 expression was correlated positively with TO formation. Moreover, exposure to alkylating agents before biopsy was inversely correlated with SOX9 expression (P = 0.006). LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Due to the limited amount of material available, only 11 out of the 39 pre- and peri-pubertal testicular tissue samples could be used for the organoid formation experiments. The testicular tissue samples obtained from a sample collection of the internal biobank of Department of Pathology, Karolinska University Hospital were considered normal and included in the study if no testicular pathology was reported. However, detailed information regarding previous medical treatments and/or testicular volumes of the patients included in this biobank was not available. WIDER IMPLICATIONS OF THE FINDINGS: Our observations suggest that SOX9 expression may serve as a putative indicator of TO formation, indicating a critical role of Sertoli cells in promoting organoid formation, seminiferous tubule integrity, and testicular function in pre- and peri-pubertal testicular tissue. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Swedish Childhood Cancer Foundation (PR2019-0123; PR2022-0115; TJ2020-0023) (J.-B.S.), Finnish Cancer Society (K.J.), Finnish Foundation for Paediatric Research (K.J.), Swedish Research Council (2018-03094; 2021-02107) (J.-B.S.), and Birgitta and Carl-Axel Rydbeck's Research Grant for Paediatric Research (2020-00348; 2020-00335; 2021-00073; 2022-00317) (J.-B.S. and K.J.). Y.C. and Y.Y. received a scholarship from the Chinese Scholarship Council. J.P.A-L. was supported by a Starting Grant in Medicine and Health (2022-01467) from the Swedish Research Council. R.T.M. was supported by a UKRI Future Leaders Fellowship (MR/S017151/1). The MRC Centre for Reproductive Health was supported by an MRC Centre Grant (MR/N022556/1). The authors declare no competing interests.
RESUMO
Cardiovascular disease is among the main causes of mortality and morbidity worldwide. Despite significant advances in medical and interventional therapy, the prognosis of conditions such as ischemic heart disease is still dismal. There is thus a need to investigate new therapeutic tools, one of which is stem cell therapy. Hematopoietic stem cells are the most studied type, and the fact that their biology is relatively well understood has led to their being used in preclinical research and clinical trials. However, the results of some of these studies have been controversial, which has opened the way for studies on other cell types, such as mesenchymal stem cells. These cells have immunomodulatory properties which suggest that they have therapeutic potential in cardiology. In the present article, the authors review the state of the art regarding mesenchymal stem cells, from basic and translational research to their use in clinical trials on ischemic heart disease, heart failure and arrhythmias, and discuss possible future uses.
Assuntos
Cardiopatias/cirurgia , Transplante de Células-Tronco Mesenquimais , HumanosRESUMO
The study evaluated the reliability and repeatability of the force and surface electromyography activity (EMG) outcomes obtained through voluntary and electrically evoked contractions of knee extensors in females (n = 18) and males (n = 20) and compared these data between sexes. Maximal isometric voluntary contractions (iMVCs) of knee extensors associated with electrical stimulation of the femoral nerve were performed over 4 days (48-h interval), with the first day involving familiarization procedures, the second involving three trials (1-h interval), and the third and fourth involving just one trial. The intraclass correlation coefficient (ICC), coefficient of variation (CV), and repeatability of outcomes from within- and between-day trials were determined for each sex. Females presented lower maximal voluntary force during iMVC (iMVCForce) and associated vastus lateralis EMG activity (root mean square, RMSVL), force evoked by potentiated doublet high-frequency (Db100Force) and single stimuli (Qtw), and M-wave amplitude than males (P ≤ 0.01, partial eta squared ≥0.94). Voluntary activation (VA) and RMSVL/M-wave amplitude did not differ between sexes. iMVCForce, VA, Db100Force, Qtw, and M-wave amplitude were the most reliable outcomes in within-day trials, with similar results between sexes (ICC > 0.62; CV < 6.4%; repeatability: 12.2%-22.6%). When investigating between-day trials, the iMVCForce, VA, Db100Force, and Qtw were the most reliable (ICC > 0.66; CV < 7.5%; repeatability: 13.2%-33.45%) with similar results between sexes. In conclusion, females presented lower iMVCForce and evoked response than males. Although reliability and repeatability statistics vary between trials, data (e.g., from EMG or force signal), and sexes, most of the outcomes obtained through this technique are reliable in females and males.NEW & NOTEWORTHY Although reliability and repeatability of knee extensors vary according to the type of neuromuscular function outcome (e.g., from force or EMG responses), the trial intervals (i.e., hours or days), and the sex of the participant, most force and EMG outcomes obtained through these neuromuscular assessment protocols present ICC > 0.75, very good CV (<10%), and repeatability <25% in within- and between-day trials in both sexes.
Assuntos
Contração Isométrica , Joelho , Masculino , Humanos , Feminino , Reprodutibilidade dos Testes , Eletromiografia , Joelho/fisiologia , Contração Isométrica/fisiologia , Músculo Quadríceps/fisiologia , Músculo Esquelético/fisiologia , Contração Muscular/fisiologia , Fadiga Muscular/fisiologiaRESUMO
The intracerebroventricular (icv) injection of amyloid peptides (Aß) models Alzheimer's disease (AD) in mice, as typified by the onset within 15 days of deficits of memory and of hippocampal long-term potentiation (LTP) that are prevented by the blockade of adenosine A2A receptors (A2AR). Since A2AR overfunction is sufficient to trigger memory deficits, we tested if A2AR were upregulated in hippocampal synapses before the onset of memory deficits to support the hypothesis that A2AR overfunction could be a trigger of AD. Six to eight days after Aß-icv injection, mice displayed no alterations of hippocampal dependent memory; however, they presented an increased excitability of hippocampal synapses, a slight increase in LTP magnitude in Schaffer fiber-CA1 pyramid synapses and an increased density of A2AR in hippocampal synapses. A2AR blockade with SCH58261 (50 nM) normalized excitability and LTP in hippocampal slices from mice sacrificed 7-8 days after Aß-icv injection. Fifteen days after Aß-icv injection, mice displayed evident deficits of hippocampal-dependent memory deterioration, with reduced hippocampal CA1 LTP but no hyperexcitability and a sustained increase in synaptic A2AR, which blockade restored LTP magnitude. This shows that the upregulation of synaptic A2AR precedes the onset of deterioration of memory and of hippocampal synaptic plasticity, supporting the hypothesis that the overfunction of synaptic A2AR could be a trigger of memory deterioration in AD.
Assuntos
Doença de Alzheimer , Animais , Camundongos , Regulação para Cima , Doença de Alzheimer/induzido quimicamente , Receptor A2A de Adenosina , Plasticidade Neuronal , Adenosina , Transtornos da Memória/induzido quimicamenteRESUMO
Adenosine operates a modulation system fine-tuning the efficiency of synaptic transmission and plasticity through A1 and A2A receptors (A1R, A2AR), respectively. Supramaximal activation of A1R can block hippocampal synaptic transmission, and the tonic engagement of A1R-mediated inhibition is increased with increased frequency of nerve stimulation. This is compatible with an activity-dependent increase in extracellular adenosine in hippocampal excitatory synapses, which can reach levels sufficient to block synaptic transmission. We now report that A2AR activation decreases A1R-medated inhibition of synaptic transmission, with particular relevance during high-frequency-induced long-term potentiation (LTP). Thus, whereas the A1R antagonist DPCPX (50 nM) was devoid of effects on LTP magnitude, the addition of an A2AR antagonist SCH58261 (50 nM) allowed a facilitatory effect of DPCPX on LTP to be revealed. Additionally, the activation of A2AR with CGS21680 (30 nM) decreased the potency of the A1R agonist CPA (6-60 nM) to inhibit hippocampal synaptic transmission in a manner prevented by SCH58261. These observations show that A2AR play a key role in dampening A1R during high-frequency induction of hippocampal LTP. This provides a new framework for understanding how the powerful adenosine A1R-mediated inhibition of excitatory transmission can be controlled to allow the implementation of hippocampal LTP.
Assuntos
Potenciação de Longa Duração , Receptor A2A de Adenosina , Adenosina/farmacologia , Hipocampo/metabolismo , Receptor A2A de Adenosina/metabolismo , Transmissão Sináptica , Animais , CamundongosRESUMO
Caffeic acid is a polyphenolic compound present in a vast array of dietary components. We previously showed that caffeic acid reduces the burden of brain ischemia joining evidence by others that it can attenuate different brain diseases. However, it is unknown if caffeic acid affects information processing in neuronal networks. Thus, we now used electrophysiological recordings in mouse hippocampal slices to test if caffeic acid directly affected synaptic transmission, plasticity and dysfunction caused by oxygen-glucose deprivation (OGD), an in vitro ischemia model. Caffeic acid (1-10 µM) was devoid of effect on synaptic transmission and paired-pulse facilitation in Schaffer collaterals-CA1 pyramidal synapses. Also, the magnitude of either hippocampal long-term potentiation (LTP) or the subsequent depotentiation were not significantly modified by 10 µM caffeic acid. However, caffeic acid (10 µM) increased the recovery of synaptic transmission upon re-oxygenation following 7 min of OGD. Furthermore, caffeic acid (10 µM) also recovered plasticity after OGD, as heralded by the increased magnitude of LTP after exposure. These findings show that caffeic acid does not directly affect synaptic transmission and plasticity but can indirectly affect other cellular targets to correct synaptic dysfunction. Unraveling the molecular mechanisms of action of caffeic acid may allow the design of hitherto unrecognized novel neuroprotective strategies.