RESUMO
The presence of an autocrine factor in milk that can trigger mammary gland involution was proposed more than 50 yr ago. To provide evidence for the existence of one or more autocrine factors, 10 multiparous cows in late lactation were quarter-milked for 7 d. Following this baseline period, the right front quarter of each cow was left unmilked, and the other quarters were milked for 7 d. Before the last milking of that period, milk (mammary secretions) was collected aseptically from both front quarters. After that milking, 250 mL of the collected samples were infused in the cows' respective rear quarters. No quarters were milked for the following 7 d (milk stasis period), and quarter milking was then resumed in all quarters for the last 7 d of the experiment (remilking period). Quarter milk samples were collected during the baseline period, before the milk stasis period, and during the remilking period. These samples were used for measuring milk components and the concentration of involution markers (SCC, BSA, and lactoferrin). Samples of mammary secretions were collected manually from the quarters during the milk stasis period for involution marker determination. We extracted RNA from samples collected from front quarters before the last milking before the milk stasis period for microRNA (miRNA) determination. As anticipated, the longer milk stasis period implemented for the right front quarter resulted in a more advanced involution than in the left front quarter, based on the concentration of involution markers in the mammary secretions, lower milk production recovery, and changes in milk composition during the remilking period. All 3 involution marker concentrations in the mammary secretions increased in both rear quarters, but were greater in the right quarter secretions than in the left quarter secretions. Resuming milking reinitiated milk production in all quarters, but milk production recovery in the right rear quarters was less robust than that in the left rear quarters (54.3 ± 1.4% vs. 61.6 ± 1.4%, respectively). Milk from the quarters infused with mammary secretions (right rear) had a lower lactose content, but a higher milk protein content and higher SCC than the quarters infused with milk. We detected a total of 359 miRNAs, 76 of which were differentially expressed in milk and mammary secretions. Expression of bta-miR-221 and bta-miR-223 was upregulated in mammary secretions 34- and 40-fold, respectively. The results of the present experiment support the contention that milk stasis leads to the accumulation of one or more factors that trigger involution. The results also indicate that milk stasis leads to changes in the miRNA profile of the milk, but whether such changes are a cause or a consequence of the involution process remains to be established.
Assuntos
Lactação , Glândulas Mamárias Animais , MicroRNAs , Leite , Animais , Feminino , Glândulas Mamárias Animais/metabolismo , Leite/química , Leite/metabolismo , BovinosRESUMO
Mammary gland secretory activity is modulated by systemic and local factors; however, the relationship between these factors is unknown. The aim of this study was to determine how a local factor, such as incomplete milking, affects mammary epithelial cell activity, number, and responsiveness to blood prolactin (PRL). Eight cows in mid-lactation were differentially milked (i.e., their right quarters were milked incompletely at approximately 70%, and their left quarters were milked completely, twice daily for 4 wk). Throughout the experiment, milk yield was measured at the quarter level. Milk samples were collected from each quarter once a week to assess the milk components, and epithelial cell concentrations, as well as to isolate milk fat globule RNA. In the weeks before and after the experiment, mammary gland functional capacity was evaluated by measuring the volume of milk harvested after complete filling of the gland. At the end of the last experimental week, mammary gland biopsies were performed on each rear quarter. The milk production of quarters milked completely remained stable during the treatment period, whereas, as expected, the milk production of quarters milked incompletely was only 53% of completely milked quarters at the end of the period. Accordingly, the expression of genes related to milk synthesis (CSN2, LALBA, and ACACA) in milk fat was lower in the quarters that were milked incompletely. Incomplete milking decreased the milk lactose content, indicating a loss of integrity of tight junctions. The total yield of epithelial cells in milk was not affected, but their concentration in milk, the BAX:BCL2 gene expression ratio, and the loss of mammary functional capacity were greater in the quarters milked incompletely, suggesting an acceleration of involution in those quarters. The expression of the short isoform of the PRL receptor gene (PRLR) tended to be lower, and the expression of STAT5A and STAT5B tended to decline in the quarters milked incompletely. In mammary gland biopsy samples, the number of both short and long isoforms of the PRLR were not affected, nor were the amount and activation of STAT3 and STAT5. However, the ratio of PRLR short isoform to PRLR long isoform was lower in the quarters milked incompletely. The decrease in milk yield induced by incomplete milking is rapid and associated with a decrease in mammary epithelial cell activity and a decrease in the number of secretory epithelial cells. The results of this experiment provide only limited support for the hypothesis that modulation of the mammary gland's responsiveness to PRL is part of the mechanism by which local factors, such as incomplete milking, modulate milk synthesis.
Assuntos
Lactação , Glândulas Mamárias Animais , Feminino , Bovinos , Animais , Glândulas Mamárias Animais/metabolismo , Lactação/fisiologia , Leite/metabolismo , Lactose/metabolismo , Células Epiteliais , Indústria de Laticínios/métodosRESUMO
INTRODUCTION: Neoadjuvant chemotherapy (NAC) is now recommended to treat muscle-invasive bladder cancer (MIBC) but is not always executed in real life. This study aims to evaluate the proportion of patients with MIBC who receive an optimal NAC, and to present the predictive factors of its achievement. METHODS: This monocenter retrospective study included all the patients who underwent radical cystectomy for≥pT2NxM0 MIBC between 2013, January and 2018, December. NAC consisted in 4-6 cycles of MVAC (methotrexate, vinblastine, adriamycin, and cisplatin) or 4 cycles of GC (gemcitabin, and carboplatin). Demographic (sex, age, ECOG-PS, glomerular filtration rate [GFR], and cN stage), surgical (urinary derivation, time of surgery, blood loss, and complications), and oncological characteristics were analyzed. Multivariate analysis are made to find predictors of administration of NAC. RESULTS: One hundred and twenty-seven patients were included. Thirty received CNA (24%). Patients who underwent CNA were younger, with better ECOG and better GFR. Multivariate analysis showed that cN+ stage and better GFR were significantly associated to administration of NAC. Eight patients (27%) couldn't receive an optimal treatment due to toxicity. Perioperative complication rates were similar, with or without NAC. Patients who underwent NAC had a worse GFR after treatment (-17 versus +5mL/min, P<0.01). CONCLUSION: Due to the risks of toxicity, NAC can only be proposed to selected population, which is not the current patients. Immunotherapy could allow to treat more patients because of better tolerance. LEVEL OF EVIDENCE: 3.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias da Bexiga Urinária/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Quimioterapia Adjuvante , Feminino , Humanos , Masculino , Terapia Neoadjuvante , Invasividade Neoplásica , Padrões de Prática Médica , Estudos Retrospectivos , Resultado do Tratamento , Neoplasias da Bexiga Urinária/patologia , UrologiaRESUMO
BACKGROUND: An elevated pre-treatment neutrophil to lymphocytes ratio (NLR) is associated with poor prognosis in various malignancies. Optimal cut-off is highly variable across studies and could not be determined individually for a patient to inform his prognosis. We hypothesize that NLR variations could be more useful than baseline NLR to predict progression-free survival (PFS) and overall survival (OS) in patients (pts) receiving anti-PD1 treatment. PATIENTS AND METHODS: All pts with metastatic renal cell carcinoma (mRCC) and metastatic non-small cell lung cancer (mNSCLC) who received anti-PD1 nivolumab monotherapy in second-line setting or later were included in this French multicentric retrospective study. NLR values were prospectively collected prior to each nivolumab administration. Clinical characteristics were recorded. Associations between baseline NLR, NLR variations and survival outcomes were determined using Kaplan-Meier's method and multivariable Cox regression models. RESULTS: 161 pts (86 mRCC and 75 mNSCLC) were included with a median follow-up of 18 months. On the whole cohort, any NLR increase at week 6 was significantly associated with worse outcomes compared to NLR decrease, with a median PFS of 11 months vs 3.7 months (p < 0.0001), and a median OS of 28.5 months vs. 18 months (p = 0.013), respectively. In multivariate analysis, NLR increase was significantly associated with worse PFS (HR 2.2; p = 6.10-5) and OS (HR 2.1; p = 0.005). Consistent results were observed in each cohort when analyzed separately. CONCLUSION: Any NLR increase at week 6 was associated with worse PFS and OS outcomes. NLR variation is an inexpensive and dynamic marker easily obtained to monitor anti-PD1 efficacy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , Linfócitos/imunologia , Neutrófilos/imunologia , Nivolumabe/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/sangue , Carcinoma Pulmonar de Células não Pequenas/imunologia , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma de Células Renais/imunologia , Carcinoma de Células Renais/mortalidade , Carcinoma de Células Renais/secundário , Estudos de Viabilidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Renais/imunologia , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Contagem de Leucócitos , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/mortalidade , Masculino , Pessoa de Meia-Idade , Prognóstico , Intervalo Livre de Progressão , Estudos Prospectivos , Estudos Retrospectivos , Medição de Risco/métodos , Adulto JovemRESUMO
Osteopontin (OPN) is now recognized as an important cytokine and extracellular integrin-binding protein at the crossroads of inflammation and homeostasis. In a previous study, we found that OPN gene (SPP1) polymorphisms are associated with milk performance traits and somatic cell score (SCS), a parameter used to estimate the genetic value of udder health in dairy cattle. In this study, we assessed whether the genetic variations had an impact on SPP1 promoter activity, immune response and the level of OPN secreted into milk. The influence of DNA polymorphisms on the promoter activity of SPP1 was confirmed in vitro. To measure the impact of the genetic variations on OPN secretion into milk, we measured OPN levels in both plasma and milk throughout lactation. Cows were grouped by the OPN haplotypes associated with a high (H2 × H3) or low (H1 × H4) SCS. For both H2 × H3 and H1 × H4, the OPN level in plasma remained low throughout lactation, although the concentration in the milk of H1 × H4 cows increased more in late lactation. Moreover, the macrophages of H1 × H4 cows expressed a lower SPP1 and proinflammatory IL6 in response to infection. Regarding the immune cell response, cows with the genetic potential to secrete higher OPN levels during late lactation had macrophages expressing fewer proinflammatory cytokines, a situation that might explain the genetic association with low somatic cells. Although OPN's favorable roles during late lactation remain to be elucidated, the tissue remodeling properties associated with OPN may be beneficial for reducing the incidence of infection during the transition period in lactating cows.
Assuntos
Variação Genética , Leite/química , Osteopontina/metabolismo , Regiões Promotoras Genéticas , Animais , Bovinos , Linhagem Celular , Feminino , Haplótipos , Lactação , Macrófagos/citologia , Dados de Sequência Molecular , Osteopontina/genéticaRESUMO
BACKGROUND: Patients with solid organ transplant (SOT) and solid tumors are usually excluded from clinical trials testing immune checkpoint blockers (ICB). As transplant rates are increasing, we aimed to evaluate ICB outcomes in this population, with a special focus on lung cancer. METHODS: We conducted a multicenter retrospective cohort study collecting real data of ICB use in patients with SOT and solid tumors. Clinical data and treatment outcomes were assessed by using retrospective medical chart reviews in every participating center. Study endpoints were: overall response rate (ORR), 6-month progression-free survival (PFS), and grade ≥3 immune-related adverse events. RESULTS: From August 2016 to October 2022, 31 patients with SOT (98% kidney) and solid tumors were identified (36.0% lung cancer, 19.4% melanoma, 13.0% genitourinary cancer, 6.5% gastrointestinal cancer). Programmed death-ligand 1 expression was positive in 29% of tumors. Median age was 61 years, 69% were males, and 71% received ICB as first-line treatment. In the whole cohort the ORR was 45.2%, with a 6-month PFS of 56.8%. In the lung cancer cohort, the ORR was 45.5%, with a 6-month PFS of 32.7%, and median overall survival of 4.6 months. The grade 3 immune-related adverse events rate leading to ICB discontinuation was 12.9%. Allograft rejection rate was 25.8%, and risk of rejection was similar regardless of the type of ICB strategy (monotherapy or combination, 28% versus 33%, P = 1.0) or response to ICB treatment. CONCLUSIONS: ICB could be considered a feasible option for SOT recipients with some advanced solid malignancies and no alternative therapeutic options. Due to the risk of allograft rejection, multidisciplinary teams should be involved before ICB therapy.
Assuntos
Inibidores de Checkpoint Imunológico , Transplante de Órgãos , Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Estudos Retrospectivos , Inibidores de Checkpoint Imunológico/uso terapêutico , Inibidores de Checkpoint Imunológico/farmacologia , Transplante de Órgãos/efeitos adversos , Transplante de Órgãos/métodos , Idoso , Neoplasias/tratamento farmacológico , Adulto , Transplantados , Estudos de CoortesRESUMO
The JAVELIN Bladder 100 phase III trial led to the incorporation of avelumab first-line (1L) maintenance treatment into international guidelines as a standard of care for patients with advanced urothelial carcinoma (UC) without progression after 1L platinum-based chemotherapy. JAVELIN Bladder 100 showed that avelumab 1L maintenance significantly prolonged overall survival (OS) and progression-free survival in this population compared with a 'watch-and-wait' approach. The aim of this manuscript is to review clinical studies of avelumab 1L maintenance in patients with advanced UC, including long-term efficacy and safety data from JAVELIN Bladder 100, subgroup analyses in clinically relevant subpopulations, and 'real-world' data obtained outside of clinical trials, providing a comprehensive resource to support patient management. Extended follow-up from JAVELIN Bladder 100 has shown that avelumab provides a long-term efficacy benefit, with a median OS of 23.8 months measured from start of maintenance treatment, and 29.7 months measured from start of 1L chemotherapy. Longer OS was observed across subgroups, including patients who received 1L cisplatin + gemcitabine, patients who received four or six cycles of 1L chemotherapy, and patients with complete response, partial response, or stable disease as best response to 1L induction chemotherapy. No new safety signals were seen in patients who received ≥1 year of avelumab treatment, and toxicity was similar in those who had received cisplatin or carboplatin with gemcitabine. Other clinical datasets, including noninterventional studies conducted in Europe, USA, and Asia, have confirmed the efficacy of avelumab 1L maintenance. Potential subsequent treatment options after avelumab maintenance include antibody-drug conjugates (enfortumab vedotin or sacituzumab govitecan), erdafitinib in biomarker-selected patients, platinum rechallenge in suitable patients, nonplatinum chemotherapy, and clinical trial participation; however, evidence to determine optimal treatment sequences is needed. Ongoing trials of avelumab-based combination regimens as maintenance treatment have the potential to evolve the treatment landscape for patients with advanced UC.
Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Cisplatino , Carcinoma de Células de Transição/tratamento farmacológico , Gencitabina , Neoplasias da Bexiga Urinária/tratamento farmacológico , DesoxicitidinaRESUMO
The CRISPR-Cas9 system has revolutionized our ability to precisely modify the genome and has led to gene editing in clinical applications. Comprehensive analysis of gene editing products at the targeted cut-site has revealed a complex spectrum of outcomes. ON-target genotoxicity is underestimated with standard PCR-based methods and necessitates appropriate and more sensitive detection methods. Here, we present two complementary Fluorescence-Assisted Megabase-scale Rearrangements Detection (FAMReD) systems that enable the detection, quantification, and cell sorting of edited cells with megabase-scale loss of heterozygosity (LOH). These tools reveal rare complex chromosomal rearrangements caused by Cas9-nuclease and show that LOH frequency depends on cell division rate during editing and p53 status. Cell cycle arrest during editing suppresses the occurrence of LOH without compromising editing. These data are confirmed in human stem/progenitor cells, suggesting that clinical trials should consider p53 status and cell proliferation rate during editing to limit this risk by designing safer protocols.
Assuntos
Sistemas CRISPR-Cas , Proteína Supressora de Tumor p53 , Humanos , Sistemas CRISPR-Cas/genética , Proteína Supressora de Tumor p53/genética , Pontos de Checagem do Ciclo Celular/genética , Divisão Celular , Separação Celular , RNARESUMO
Introduction: Urachal cancer (UrC) is a rare, non-urothelial malignancy. Its natural history and management are poorly understood. Although localized to the bladder dome, the most common histological subtype of UrC is adenocarcinoma. UrC develops from an embryonic remnant, and is frequently diagnosed in advanced stage with poor prognosis. The treatment is not standardized, and based only on case reports and small series. This large retrospective multicentric study was conducted by the French Genito-Urinary Tumor Group to gain a better understanding of UrC. Material and Methods: data has been collected retrospectively on 97 patients treated at 22 French Cancer Centers between 1996 and 2020. Results: The median follow-up was 59 months (range 44-96). The median age at diagnosis was 53 years (range 20-86), 45% were females and 23% had tobacco exposure. For patients with localized disease (Mayo I-II, n=46) and with lymph-node invasion (Mayo III, n=13) median progression-free-survival (mPFS) was 31 months (95% CI: 20-67) and 7 months (95% CI: 6-not reached (NR)), and median overall survival (mOS) was 73 months (95% CI: 57-NR) and 22 months (95% CI: 21-NR) respectively. For 45 patients with Mayo I-III had secondary metastatic progression, and 20 patients were metastatic at diagnosis. Metastatic localization was peritoneal for 54% of patients. Most patients with localized tumor were treated with partial cystectomy, with mPFS of 20 months (95% CI: 14-49), and only 12 patients received adjuvant therapy. Metastatic patients (Mayo IV) had a mOS of 23 months (95% CI: 19-33) and 69% received a platin-fluorouracil combination treatment. Conclusion: UrC is a rare tumor of the bladder where patients are younger with a higher number of females, and a lower tobacco exposure than in standard urothelial carcinoma. For localized tumor, partial cystectomy is recommended. The mOS and mPFS were low, notably for patients with lymph node invasion. For metastatic patients the prognosis is poor and standard therapy is not well-defined. Further clinical and biological knowledge are needed.
RESUMO
By providing contacts between hematopoietic cells and the bone marrow microenvironment, integrins are implicated in cell adhesion and thereby in control of cell fate of normal and leukemia cells. The ASB2 gene, initially identified as a retinoic acid responsive gene and a target of the promyelocytic leukemia retinoic acid receptor α oncoprotein in acute promyelocytic leukemia cells, encodes two isoforms, a hematopoietic-type (ASB2α) and a muscle-type (ASB2ß) that are involved in hematopoietic and myogenic differentiation, respectively. ASB2α is the specificity subunit of an E3 ubiquitin ligase complex that targets filamins to proteasomal degradation. To examine the relationship of the ASB2α structure to E3 ubiquitin ligase function, functional assays and molecular modeling were performed. We show that ASB2α, through filamin A degradation, enhances adhesion of hematopoietic cells to fibronectin, the main ligand of ß1 integrins. Furthermore, we demonstrate that a short N-terminal region specific to ASB2α, together with ankyrin repeats 1 to 10, is necessary for association of ASB2α with filamin A. Importantly, the ASB2α N-terminal region comprises a 9-residue segment with predicted structural homology to the filamin-binding motifs of migfilin and ß integrins. Together, these data provide new insights into the molecular mechanisms of ASB2α binding to filamin.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica , Células-Tronco Hematopoéticas/citologia , Integrinas/metabolismo , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Motivos de Aminoácidos , Animais , Adesão Celular , Fibronectinas/metabolismo , Células HeLa , Humanos , Camundongos , Músculos/metabolismo , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
The biofunctionalization of nanoelectromechanical systems (NEMS) is critical for the development of new classes of biosensors displaying improved performance and higher levels of integration. In this paper we propose a modified microcontact process (µCP) in order to biofunctionalize arrays of NEMS with a probe molecule on the active sensing areas together with an anti-fouling layer on the passive areas in a single, self-aligned step. We demonstrate the adequate functionalization/anti-fouling of arrays of freestanding nanocantilevers as dense as 10(5) nanostructures cm(-2) by using both fluorescence microscopy and dynamic measurements of the structures' resonant frequency. The proper bioactivity of an antibody deposited onto the cantilevers and the blocking property of a bovine serum albumin layer are both assessed by incubating specific and non-specific tagged secondary antibodies followed by fluorescence imaging. Furthermore, measurement of the resonant frequency of the nanocantilevers before and after functionalization and biological recognition demonstrate that using µCP for device functionalization does not damage the nanostructures and preserves the mechanical sensing capability of our NEMS.
Assuntos
Técnicas Biossensoriais/instrumentação , Imunoensaio/instrumentação , Sistemas Microeletromecânicos/instrumentação , Análise em Microsséries/instrumentação , Impressão Molecular/métodos , Nanopartículas/química , Nanotecnologia/instrumentação , Desenho de Equipamento , Análise de Falha de EquipamentoRESUMO
The aim of this study was to characterize the osteopontin (OPN) secreted in bovine milk and to determine whether the different forms are the product of spliced variants. Spliced variants of the human gene and secreted osteopontin isoforms have been reported in human tumor tissue. In bovine milk, we identified 2 major forms: one corresponding to the full-length coding transcript and a truncated version of this form. No alternative spliced transcripts were detected in the lactating mammary gland tissue, in milk somatic cells, or in peripheral blood immune cells. The 60-kDa bovine osteopontin (bOPN) and a truncated 40-kDa protein isoform were confirmed by mass spectrometry and further characterized by immunoblotting using a panel of 6 antibodies targeting different domains of the protein. Of the 3 human anti-OPN antibodies targeting the N-terminal segment of the protein, only one detected all forms on sodium dodecyl sulfate-PAGE; one human anti-OPN antibody failed to detect bOPN, whereas the other detected only the 60-kDa protein, albeit barely in its phosphorylated form. Detection was generally more sensitive when the 60-kDa protein was dephosphorylated. Two polyclonal antibodies raised against bOPN were tested: one targeting the milk-purified bOPN (bOPN-121) and one targeting a bovine epitope (synthetic peptide) corresponding to a carboxy-terminal domain of the protein (bOPN-117). The bOPN-121 antibody detected all forms irrespective of the phosphorylation status of bOPN. The bOPN-117 and the mouse anti-human OPN (hOPN-4) antibodies, which recognized different domains of the carboxy-terminal segment of the protein, also preferentially detected the dephosphorylated 60-kDa protein. Whereas phosphorylation had a major effect on detection for several antibodies, deglycosylation slightly decreased immunodetection for the tested antibodies. In particular, phosphorylation is the major posttranslational modification that influenced the weak detection capacity of several antibodies. This fact needs to be taken into account for immunodetection of milk content. In conclusion, the OPN forms secreted in bovine milk are not the product of alternative splicing. The 40-kDa protein appears to be a truncated hypophosphorylated variant of the full-length 60-kDa form, which is highly phosphorylated. Together, the proteomic and immunoblotting analyses used to characterize bovine milk OPN revealed the complex nature of the bovine milk OPN forms.
Assuntos
Osteopontina/genética , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Bovinos , Eletroforese em Gel de Poliacrilamida , Mapeamento de Epitopos/métodos , Epitopos/imunologia , Humanos , Proteínas do Leite/genética , Proteínas do Leite/imunologia , Dados de Sequência Molecular , Osteopontina/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Proteômica/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Cutaneous Central Follicular Lymphoma (CCFL) is a type B cutaneous lymphoma with a usually indolent course. Scalp localization of CCFL is extremely rare, we report a new case mimicking an epidural hematoma, and showing a rapid progression with aggressive infiltration of skin, calvaria, dura and brain parenchyma. CASE REPORT: A 58-year-old patient with an unlabeled polymalformative syndrome was admitted to the Emergency department following a head injury secondary to a self-resolving tonic-clonic epileptic seizure. The initial CT-scan was interpreted as a minor subcutaneous and epidural hematoma initially deemed for conservative management. Within 4 days, the patient showed a progressive neurological deterioration culminating into a stuporous status which prompted a constrast-enhanced brain MRI. The scan revealed a multilayered solid lesion, extending from the subgaleal compartment to the subdural space, threatening the integrity of overlying skin and causing infiltration of the brain parenchyma. Following emergency neurosurgical excision a definitive histology diagnosis of central follicular lymphoma was made. A focused chemotherapy with high-dose Methotrexate with R-CHOP protocol led to disease control until the latest follow up at 2 years. CONCLUSION: To our knowledge, this case represents the first CCFL invading the brain parenchyma and the second extending to the dura. Although such tumor is usually indolent the aggressive behavior herein reported extend the differential diagnosis to high-grade meningiomas, sarcomas, and metastases. Prognostication and appropriate adjuvant treatment require prompt surgical excision and histological confirmation.
Assuntos
Hematoma Epidural Craniano , Linfoma Folicular , Hematoma Epidural Craniano/diagnóstico , Hematoma Epidural Craniano/etiologia , Hematoma Epidural Craniano/cirurgia , Humanos , Linfoma Folicular/complicações , Metotrexato , Pessoa de Meia-Idade , Couro Cabeludo , Tomografia Computadorizada por Raios X/efeitos adversosRESUMO
Immobilization of live micro-organisms on solid substrates is an important prerequisite for atomic force microscopy (AFM) bio-experiments. The method employed must immobilize the cells firmly enough to enable them to withstand the lateral friction forces exerted by the tip during scanning but without denaturing the cell interface. In this work, a generic method for the assembly of living cells on specific areas of substrates is proposed. It consists in assembling the living cells within the patterns of microstructured, functionalized poly-dimethylsiloxane (PDMS) stamps using convective/capillary deposition. This versatile approach is validated by applying it to two systems of foremost importance in biotechnology and medicine: Saccharomyces cerevisiae yeasts and Aspergillus fumigatus fungal spores. We show that this method allows multiplexing AFM nanomechanical measurements by force spectroscopy on S. cerevisiae yeasts and high-resolution AFM imaging of germinated Aspergillus conidia in buffer medium. These two examples clearly demonstrate the immense potential of micro-organism assembly on functionalized, microstructured PDMS stamps by convective/capillary deposition for performing rigorous AFM bio-experiments on living cells.
Assuntos
Aspergillus fumigatus/ultraestrutura , Dimetilpolisiloxanos/química , Microscopia de Força Atômica/métodos , Saccharomyces cerevisiae/ultraestrutura , Esporos Fúngicos/ultraestrutura , Células Imobilizadas/ultraestruturaRESUMO
Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of leukemia inhibitory factor (LIF) cytokine. LIF starvation leads to cell commitment, and part of the ES-derived differentiated cells die by apoptosis together with caspase3-cleavage and p38alpha activation. Inhibition of p38 activity by chemical compounds (PD169316 and SB203580), along with LIF withdrawal, leads to different outcomes on cell apoptosis, giving the opportunity to study the influence of apoptosis on cell differentiation. By gene profiling studies on ES-derived differentiated cells treated or not with these inhibitors, we have characterized the common and specific set of genes modulated by each inhibitor. We have also identified key genes that might account for their different survival effects. In addition, we have demonstrated that some genes, similarly regulated by both inhibitors (upregulated as Bcl2, Id2, Cd24a or downregulated as Nodal), are bona fide p38alpha targets involved in neurogenesis and found a correlation with their expression profiles and the onset of neuronal differentiation triggered upon retinoic acid treatment. We also showed, in an embryoid body differentiation protocol, that overexpression of EGFP (enhanced green fluorescent protein)-BCL2 fusion protein and repression of p38alpha are essential to increase formation of TUJ1-positive neuronal cell networks along with an increase in Map2-expressing cells.
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Células-Tronco Embrionárias/metabolismo , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Neurônios/citologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Apoptose , Diferenciação Celular , Linhagem Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/enzimologia , Expressão Gênica/efeitos dos fármacos , Imidazóis/farmacologia , Camundongos , Neurônios/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Piridinas/farmacologia , Transcrição Gênica , Tretinoína/farmacologiaRESUMO
Branchio-oculo-facial syndrome (BOFS) is an autosomal-dominant condition characterized by three main features, respectively: branchial defects, ocular anomalies, and craniofacial defects including cleft lip and/or palate (CL/P). We report on one family with three affected, and two sporadic cases that have been found to carry missense mutations in the newly reported BOFS gene: TFAP2A. This report confirms the involvement of this transcription factor in this developmental syndrome with clinical variability. Moreover, we present CT scan temporal bone anomalies in the familial cases, related to branchial arch defects, highlighting the importance of radiological investigations for differential diagnosis.
Assuntos
Síndrome Brânquio-Otorrenal/genética , Osso Temporal/anormalidades , Fator de Transcrição AP-2/genética , Sequência de Aminoácidos , Sequência de Bases , Síndrome Brânquio-Otorrenal/complicações , Criança , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Dados de Sequência Molecular , Linhagem , Fator de Transcrição AP-2/fisiologiaRESUMO
The small intestine is a complex tissue with a crypt/villus architecture and high tissue polarity. Maintenance of tissue integrity and function is supported by a constant renewal of the epithelium, with proliferative cells located in the crypts and differentiated cells migrating upward to the top of villi. So far, most in vitro studies have been limited to 2D surfaces or 3D organoid cultures that do not fully recapitulate the tissue 3D architecture, microenvironment and cell compartmentalization found in vivo. Here, we report the development of a 3D model that reproduces more faithfully the architecture of the intestinal epithelium in vitro. We developed a new fabrication process combining a photopolymerizable hydrogel that supports the growth of intestinal cell lines with high-resolution stereolithography 3D printing. This approach offers the possibility to create artificial 3D scaffolds matching the dimensions and architecture of mouse intestinal crypts and villi. We demonstrate that these 3D culture models support the growth and differentiation of Caco-2â¯cells for 3 weeks. These models may constitute a complementary approach to organoid cultures to study intestinal homeostasis by allowing guided self-organization and controlled differentiation, as well as for in vitro drug screening and testing.
Assuntos
Hidrogéis/química , Mucosa Intestinal/citologia , Estereolitografia , Alicerces Teciduais/química , Fosfatase Alcalina/metabolismo , Células CACO-2 , Diferenciação Celular , Imunofluorescência , Humanos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Engenharia Tecidual/métodosRESUMO
Addiction develops from the gradual adaptation of the brain to chronic drug exposure, and involves genetic reprogramming of neuronal function. The central extended amygdala (EAc) is a network formed by the central amygdala and the bed nucleus of the stria terminalis. This key site controls drug craving and seeking behaviors, and has not been investigated at the gene regulation level. We used Affymetrix microarrays to analyze transcriptional activity in the murine EAc, with a focus on mu-opioid receptor-associated events because these receptors mediate drug reward and dependence. We identified 132 genes whose expression is regulated by a chronic escalating morphine regimen in the EAc from wild-type but not mu-opioid receptor knockout mice. These modifications are mostly EAc-specific. Gene ontology analysis reveals an overrepresentation of neurogenesis, cell growth and signaling protein categories. A separate quantitative PCR analysis of genes in the last of these groups confirms the dysregulation of both orphan (Gpr88) and known (DrD1A, Adora2A, Cnr1, Grm5, Gpr6) G protein-coupled receptors, scaffolding (PSD95, Homer1) and signaling (Sgk, Cap1) proteins, and neuropeptides (CCK, galanin). These transcriptional modifications do not occur following a single morphine injection, and hence result from long-term adaptation to excessive mu receptor activation. Proteins encoded by these genes are classically associated with spine modules function in other brain areas, and therefore our data suggest a remodeling of EAc circuits at sites where glutamatergic and monoaminergic afferences interact. Together, mu receptor-dependent genes identified in this study potentially contribute to drug-induced neural plasticity, and provide a unique molecular repertoire towards understanding drug craving and relapse.
Assuntos
Tonsila do Cerebelo/efeitos dos fármacos , Analgésicos Opioides/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Receptores Opioides mu/agonistas , Ativação Transcricional/efeitos dos fármacos , Tonsila do Cerebelo/metabolismo , Animais , Regulação da Expressão Gênica/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Morfina/farmacologia , Vias Neurais/efeitos dos fármacos , Vias Neurais/metabolismo , Plasticidade Neuronal/genética , Análise de Sequência com Séries de Oligonucleotídeos , Transtornos Relacionados ao Uso de Opioides/genética , Transtornos Relacionados ao Uso de Opioides/metabolismo , Transtornos Relacionados ao Uso de Opioides/fisiopatologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Opioides mu/metabolismo , Núcleos Septais/efeitos dos fármacos , Núcleos Septais/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Ativação Transcricional/genéticaRESUMO
The central extended amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the bed nucleus of the stria terminalis (BNST), the central nucleus of the amygdala (CeA) and the nucleus accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the mouse EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than twofold higher expression level in the EAc compared with whole brain. Among these, 43 genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to toward genetic manipulations within the EAc.
Assuntos
Tonsila do Cerebelo/fisiologia , Perfilação da Expressão Gênica/métodos , Expressão Gênica/fisiologia , Proteínas do Tecido Nervoso/genética , Tonsila do Cerebelo/anatomia & histologia , Animais , Mapeamento Encefálico , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The lateral hypothalamus (LH) is a brain structure that controls hedonic properties of both natural rewards and drugs of abuse. Mu opioid receptors are known to mediate drug reward, but whether overstimulation of these receptors impacts on LH function has not been studied. Here we have used a genome-wide microarray approach to identify LH responses to chronic mu opioid receptor activation at the transcriptional level. We have subjected wild-type and mu opioid receptor knockout mice to an escalating morphine regimen, which produces severe physical dependence in wild-type but not mutant animals. We have analyzed gene profiles in LH samples using the 430A.2 Affymetrix array and identified a set of 25 genes whose expression is altered by morphine in wild-type mice only. The regulation was confirmed for a subset of these genes using real-time quantitative PCR on samples from independent treatments. Altered expression of aquaporin 4, apolipoprotein D, and prostaglandin synthase is indicative of modified LH physiology. The regulation of two signaling genes (the serum glucocorticoid kinase and the regulator of G protein signaling 4) suggests that neurotransmission is altered in LH circuitry. Finally, the downregulation of apelin may indicate a potential role for this neuropeptide in opioid signaling and hedonic homeostasis. Altogether, our study shows that chronic mu opioid receptor stimulation induces gene expression plasticity in the LH and provides a unique collection of mu opioid receptor-dependent genes that potentially contribute to alter reward processes in addictive diseases.