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The obligate intracellular bacterium, Chlamydia trachomatis, has evolved to depend on its human host for many metabolites, including most amino acids and three of the four nucleotides. Given this, it is not surprising that depletion of a single amino acid in the host cell growth medium blocks chlamydial replication. Paradoxically, supra-normal levels of some amino acids also block productive replication of Chlamydia. Here, we have determined how elevated serine levels, generated by exogenous supplementation, impede chlamydial inclusion development and reduce the generation of infectious progeny. Our findings reveal that human serine racemase, which is broadly expressed in multiple tissues, potentiates the anti-chlamydial effect of elevated serine concentrations. In addition to reversibly converting l-serine to d-serine, serine racemase also deaminates serine via ß-elimination. We have determined that d-serine does not directly impact Chlamydia; rather, ammonia generated by serine deamination limits the productive chlamydial replication. Our findings imply that ammonia produced within host cells can traverse the chlamydial inclusion membrane. Further, this property of serine deaminase can be exploited to sensitize Chlamydia to concentrations of doxycycline that are otherwise not bactericidal. Because exogenously elevated levels of serine can be tolerated over extended periods, the broad expression pattern of serine racemase indicates it to be a host enzyme whose activity can be directed against multiple intracellular bacterial pathogens. From a therapeutic perspective, demonstrating host metabolism can be skewed to generate an anti-bacterial metabolite that synergizes with antibiotics, we believe our results provide a new approach to target intracellular pathogens.
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Antibacterianos , Chlamydia trachomatis , Serina , Humanos , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/efeitos dos fármacos , Serina/metabolismo , Antibacterianos/farmacologia , Células HeLa , Racemases e Epimerases/metabolismo , Desaminação , Infecções por Chlamydia/metabolismo , Infecções por Chlamydia/tratamento farmacológico , Infecções por Chlamydia/microbiologiaRESUMO
PURPOSE: To our knowledge, there are very few studies evaluating if the levels of folate modify the risk of cervical intraepithelial neoplasia grade 2 and higher (CIN2+ and CIN3+) associated with the levels of HPV genome methylation, two cofactors related to single carbon metabolism and independently associated with cervical cancer in previous studies. We conducted a case-control study nested in a three-arm randomized clinical pragmatic trial (ASCUS-COL trial) to evaluate the risk of CIN3+ associated with methylation levels according to serum folate concentrations. METHODS: Cases (n = 155) were women with histologically confirmed CIN2+ (113 CIN2, 38 CIN3, and 4 SCC) and controls were age and follow-up time at diagnosis-matched women with histologically confirmed ≤ CIN1 (n = 155), selected from the 1122 hrHPV + women of this trial. The concentrations of serum folate were determined by the radioimmunoassay SimulTRAC-SNB-VitaminB12/Folate-RIAKit and the methylation levels by the S5 classifier. Stepwise logistic regression models were used to estimate the association between folate or methylation levels and CIN2+ or CIN3+. The joint effect of folate levels and methylation on the risk of CIN3+ was estimated using combinations of categorical stratifications. RESULTS: Folate levels were significantly lower in women with CIN3+ than in other diagnostic groups (p = 0.019). The risk of CIN3+ was eight times higher (OR 8.9, 95% CI 3.4-24.9) in women with folate deficiency and high methylation levels than in women with normal folate and high methylation levels (OR 1.4, 95% CI 0.4-4.6). CONCLUSION: High methylation and deficient folate independently increased the risk of CIN3+ while deficient folate combined with high methylation was associated with a substantially elevated risk of CIN3+.
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Células Escamosas Atípicas do Colo do Útero , Deficiência de Ácido Fólico , Displasia do Colo do Útero , Feminino , Humanos , Estudos de Casos e Controles , Metilação de DNA , Ácido Fólico , Deficiência de Ácido Fólico/genética , Displasia do Colo do Útero/diagnóstico , Displasia do Colo do Útero/genética , Displasia do Colo do Útero/patologiaRESUMO
Diversifying the biomedical research workforce is crucial for eliminating cancer health disparities. To address this need, Moffitt Cancer Center and Louisiana State University Health Sciences formed the Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD). A key component of SPIRIT-CHD is the Cancer Research Education Program (CREP), designed to train underrepresented undergraduate and medical students in biomedical science research. The CREP featured an 8-week summer internship with a web-based curriculum, community outreach, and mentored research experiences. Three cohorts (n = 39) completed the CREP. Students were evaluated before and after the internship using the Goal Attainment Scale (GAS), Science Teaching Efficacy Belief Instrument (STEBI), and Research Appraisal Inventory (RAI), modified to assess CREP outcomes. These scales measured students' intentions to pursue cancer research careers, self-efficacy in communicating scientific information, and perceived research abilities. Paired test results showed significant increases (p < 0.001) in scores across the scales (GAS, STEBI, RAI) pre- and post-training. Trainees reported heightened intentions to pursue cancer research careers (GAS; mean increase of 5.3, p < 0.001) and greater self-efficacy in relaying scientific information (STEBI; mean increase of 9.2, p < 0.001). They also showed increased self-confidence in conducting research (RAI; mean increase of 58.2, p < 0.001). These findings demonstrate the program's success in fostering interest in cancer research careers and enhancing research confidence. Results support the development of programs like CREP to positively impact the academic and professional trajectories of underrepresented students, ultimately creating a more diverse and inclusive biomedical research workforce equipped to address health disparities.
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Since 2018, we have evaluated the effectiveness of various teaching technologies for training young investigators on translational research in cancer health disparities. The Southeast Partnership for Improving Research and Training in Cancer Health Disparities (SPIRIT-CHD) unites Moffitt Cancer Center and the Louisiana State University Health Sciences Center. One of the main components of the SPIRIT-CHD is the Cancer Research Education Program (CREP) for training undergraduate and medical students from underrepresented backgrounds. The CREP utilizes a web-based didactic curriculum to engage students at both institutions in biobanking, precision medicine, and cancer health disparities topics. We report experiences from our cross-institutional cancer education program, specifically evaluating the cohorts' satisfaction and learning gains using various communication technologies and instructional approaches. Trainees completed a survey with questions evaluating the curriculum and technology. Trainees reported satisfaction with the flipped classroom model (FCM) content and overall program (mean score = 3.2, SD = 0.79), and would recommend the program to peers. Yet, despite improved program delivery, trainees felt interaction between the two sites (mean score = 1.5, SD = 0.85) and engagement with faculty (mean score = 2.80, SD = 1.14) could be improved. The technology with the highest reported use was e-mail, with a mean score of 4.6 (SD = 0.52). LinkedIn and Twitter had the lowest frequency of use with mean scores at 1.90 (SD = 0.99) and 1.30 (SD = 1.34). Our study highlights the successes and challenges of remote learning using technology to increase interaction and engagement among trainees and faculty in a multi-site cancer research training program.
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Educação de Graduação em Medicina , Estudantes de Medicina , Bancos de Espécimes Biológicos , Currículo , Humanos , AprendizagemRESUMO
BACKGROUND: Triple-negative breast cancer (TNBC) subtypes are clinically aggressive and cannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derived xenograft methods. METHODS: We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. RESULTS: Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole and intact breast tumor, specific cell populations within the tumor, and the extracellular matrix. CONCLUSIONS: Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.
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Proliferação de Células/genética , Claudinas/genética , Recidiva Local de Neoplasia/genética , Neoplasias de Mama Triplo Negativas/genética , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Camundongos , Recidiva Local de Neoplasia/patologia , Neoplasias de Mama Triplo Negativas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Human Immunodeficiency Virus (HIV)-infected individuals are at increased risk for developing neurocognitive disorders and depression. These conditions collectively affect more than 50% of people living with HIV/AIDS and adversely impact adherence to HIV therapy. Thus, identification of early markers of neurocognitive impairment could lead to interventions that improve psychosocial functioning and slow or reverse disease progression through improved treatment adherence. Evidence has accumulated for the role and function of microRNAs in normal and pathological conditions. We have optimized a protocol to profile microRNAs in body fluids. Using this methodology, we have profiled plasma microRNA expression for 30 age-matched, HIV-infected (HIV(+) ) patients and identified highly sensitive and specific microRNA signatures distinguishing HIV(+) patients with cognitive impairment from those without cognitive impairment. These results justify follow-on studies to determine whether plasma microRNA signatures can be used as a screening or prognostic tool for HIV(+) patients with neurocognitive impairment. J. Cell. Physiol. 231: 829-836, 2016. © 2015 Wiley Periodicals, Inc.
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Disfunção Cognitiva/sangue , Disfunção Cognitiva/complicações , Infecções por HIV/sangue , Infecções por HIV/complicações , MicroRNAs/sangue , Adulto , Disfunção Cognitiva/genética , Demografia , Infecções por HIV/genética , Humanos , MicroRNAs/genética , Pessoa de Meia-Idade , Curva ROC , Reação em Cadeia da Polimerase em Tempo Real , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Our laboratory established a role for poly(ADP-ribose)polymerase (PARP) in asthma. To increase the clinical significance of our studies, it is imperative to demonstrate that PARP is actually activated in human asthma, to examine whether a PARP inhibitor approved for human testing such as olaparib blocks already-established chronic asthma traits in response to house dust mite (HDM), a true human allergen, in mice and to examine whether the drug modulates human cluster of differentiation type 4 (CD4(+)) T-cell function. To conduct the study, human lung specimens and peripheral blood mononuclear cells (PBMCs) and a HDM-based mouse asthma model were used. Our results show that PARP is activated in PBMCs and lung tissues of asthmatics. PARP inhibition by olaparib or gene knockout blocked established asthma-like traits in mice chronically exposed to HDM including airway eosinophilia and hyper-responsiveness. These effects were linked to a marked reduction in T helper 2 (Th2) cytokine production without a prominent effect on interferon (IFN)-γ or interleukin (IL)-10. PARP inhibition prevented HDM-induced increase in overall cellularity, weight and CD4(+) T-cell population in spleens of treated mice whereas it increased the T-regulatory cell population. In CD3/CD28-stimulated human CD4 (+)T-cells, olaparib treatment reduced Th2 cytokine production potentially by modulating GATA binding protein-3 (gata-3)/IL-4 expression while moderately affecting T-cell proliferation. PARP inhibition inconsistently increased IL-17 in HDM-exposed mice and CD3/CD28-stimulated CD4(+) T cells without a concomitant increase in factors that can be influenced by IL-17. In the present study, we provide evidence for the first time that PARP-1 is activated in human asthma and that its inhibition is effective in blocking established asthma in mice.
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Antialérgicos/farmacologia , Antiasmáticos/farmacologia , Antígenos de Dermatophagoides , Asma/prevenção & controle , Pulmão/efeitos dos fármacos , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Asma/enzimologia , Asma/imunologia , Asma/fisiopatologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Humanos , Mediadores da Inflamação/metabolismo , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/fisiopatologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Poli(ADP-Ribose) Polimerase-1 , Poli(ADP-Ribose) Polimerases/deficiência , Poli(ADP-Ribose) Polimerases/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/enzimologia , Linfócitos T Reguladores/imunologia , Células Th2/efeitos dos fármacos , Células Th2/enzimologia , Células Th2/imunologiaRESUMO
INTRODUCTION: Cervical cancer is characterized by an immunosuppressive microenvironment and a Th2-type cytokine profile. Expression of arginase (ASE), the enzyme that converts L-arginine into L-ornithine and urea, is stimulated by Th2-type cytokines. OBJECTIVE: To assess the association of ASE activity and L-Arg metabolism products with cervical cancer. METHODS: Sera of 87 and 41 women with histologically confirmed by colposcopy-directed biopsy SCC and CIN3 respectively and 79 with normal cytology or Low-Grade Squamous Intraepithelial Lesion (LSIL), were evaluated. Cytokines were measured using Milliplex Human cytokine/chemokine kit. Arginase (ASE) activity was determined using an enzymatic assay. Levels of L-arginine, L-ornithine, putrescine and spermine were determined by HPLC. RESULTS: Significantly higher levels of ASE activity were observed in women with CIN3 (age-adjusted OR: 24.3; 95%CI: 3.82-155) and SCC (AOR: 9.8; 95%CI: 2.34-40.8). As expected, possibly due to high levels of ASE activity, higher levels of l-Arg were negatively associated with CIN3 (AOR: 0.03; 95%CI: 0.004-0.19) and SSC (AOR: 0.06; 95%CI: 0.02-0.24). Consistent with the role of ASE in the conversion of L-arginine to L-ornithine and polyamine production therefrom, women with cervical cancer had higher levels of spermine and putrescine. A correlation analysis revealed a significant albeit weak relationship between high levels of IL-10 and high levels of ASE (Pearson r=0.32, p-value=0.003) in women with cervical cancer. CONCLUSION: This study indicates that ASE activity and L-Arg degradation mechanisms of immunosuppression are present in cervical cancer. The results foster research in the design of possible strategies to inhibit ASE activity for therapy of cervical cancer.
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Arginase/metabolismo , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/imunologia , Displasia do Colo do Útero/enzimologia , Displasia do Colo do Útero/imunologia , Neoplasias do Colo do Útero/enzimologia , Neoplasias do Colo do Útero/imunologia , Adulto , Idoso , Arginina/metabolismo , Carcinoma de Células Escamosas/sangue , Feminino , Humanos , Tolerância Imunológica , Pessoa de Meia-Idade , Neoplasias do Colo do Útero/sangue , Adulto Jovem , Displasia do Colo do Útero/sangueRESUMO
Polycyclic aromatic hydrocarbons (PAHs) are the products of incomplete combustion of organic materials, which are present in cigarette smoke, deep-fried food, and in natural crude oil. Since PAH-metabolites form DNA adducts and cause oxidative DNA damage, we asked if these environmental carcinogens could affect transforming potential of the human Polyomavirus JC oncoprotein, T-antigen (JCV T-antigen). We extracted DMSO soluble PAHs from Deepwater Horizon oil spill in the Gulf of Mexico (oil-PAHs), and detected several carcinogenic PAHs. The oil-PAHs were tested in exponentially growing cultures of normal mouse fibroblasts (R508), and in R508 stably expressing JCV T-antigen (R508/T). The oil-PAHs were cytotoxic only at relatively high doses (1:50-1:100 dilution), and at 1:500 dilution the growth and cell survival rates were practically unaffected. This non-toxic dose triggered however, a significant accumulation of reactive oxygen species (ROS), caused oxidative DNA damage and the formation of DNA double strand breaks (DSBs). Although oil-PAHs induced similar levels of DNA damage in R508 and R508/T cells, only T-antigen expressing cells demonstrated inhibition of high fidelity DNA repair by homologous recombination (HRR). In contrast, low-fidelity repair by non-homologous end joining (NHEJ) was unaffected. This potential mutagenic shift between DNA repair mechanisms was accompanied by a significant increase in clonal growth of R508/T cells chronically exposed to low doses of the oil-PAHs. Our results indicate for the first time carcinogenic synergy in which oil-PAHs trigger oxidative DNA damage and JCV T-antigen compromises DNA repair fidelity.
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Antígenos Virais de Tumores/genética , Vírus JC/genética , Mutagênese/efeitos dos fármacos , Hidrocarbonetos Policíclicos Aromáticos/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Animais , Morte Celular/efeitos dos fármacos , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Dimetil Sulfóxido/química , Histonas/metabolismo , Humanos , Camundongos , Estresse Oxidativo/efeitos dos fármacos , PetróleoRESUMO
Cytokine-driven hyper inflammation has been identified as a critical factor behind poor outcomes in patients severely infected with SARS-CoV-2 virus. Notably, protein ISGylation, a protein conjugated form of Type 1 IFN-inducible ubiquitin-like protein ISG15 (Interferon-Stimulated Gene 15), induces cytokine storm (CS) and augments colonic inflammation in colitis-associated colon cancers in mouse models. However, whether ISGylation is increased and causally responsible for CS and hyper inflammation in symptomatic COVID-19 patients is unknown. Here, we measured ISGylation levels in peripheral blood mononuclear cells (PBMCs) from 10 symptomatic (SARS-CoV-2-positive with symptoms) and asymptomatic (SARS-CoV-2-positive with no symptoms) COVID-19 patients, and 4 uninfected individuals (SARS-CoV-2-negative), using WesTm assay. Strikingly, we note significant increases in protein ISGylation and MX-1 (myxovirus-resistance protein-1) protein levels, both induced by type-I IFN, in symptomatic but not in asymptomatic patients and uninfected individuals. Knowing that ISGylation augments CS and intestinal inflammation in colon cancers, we propose that increased ISGylation may be an underlying cause of CS and inflammation in symptomatic patients.
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COVID-19 , Ubiquitinas , Animais , Citocinas/metabolismo , Humanos , Inflamação , Leucócitos Mononucleares/metabolismo , Camundongos , SARS-CoV-2 , Ubiquitinas/metabolismoRESUMO
Background: Videoconferencing platforms are being used for the purposes of interviewing in academic medicine because of the coronavirus disease 2019 pandemic. We present considerations applicable to interviewers and interviewees in the virtual space, with a focus on medical school and residency applicants. Methods: We reviewed the literature regarding the virtual interview process for medical school and residency by searching PubMed using the following keywords and terms: "interview," "academic medicine," "medical school application," "residency application," "virtual interviews," and "videoconferencing." Our search identified 701 results, from which we selected 36 articles for review. Results: The garnered information focuses on strategies for optimizing the virtual interview process from the standpoint of both the interviewer and the interviewee. We discuss the advantages and disadvantages of the virtual interview process and present recommendations. Conclusion: While the future of the interview process for medical school and residency is uncertain, virtual interviewing is a common and growing practice that will continue to be at least part of the medical interview process for years to come. Interviewers and interviewees should prepare to adapt to the evolving changes in the process.
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BACKGROUND: Previous studies have shown that different alcoholic beverage types impact prostate cancer (PCa) clinical outcomes differently. However, intake patterns of specific alcoholic beverages for PCa status are understudied. The study's objective is to evaluate intake patterns of total alcohol and the three types of beverage (beer, wine, and spirits) by the PCa risk and aggressiveness status. METHOD: This is a cross-sectional study using 10,029 men (4676 non-PCa men and 5353 PCa patients) with European ancestry from the PCa consortium. Associations between PCa status and alcohol intake patterns (infrequent, light/moderate, and heavy) were tested using multinomial logistic regressions. RESULTS: Intake frequency patterns of total alcohol were similar for non-PCa men and PCa patients after adjusting for demographic and other factors. However, PCa patients were more likely to drink wine (light/moderate, OR = 1.11, p = 0.018) and spirits (light/moderate, OR = 1.14, p = 0.003; and heavy, OR = 1.34, p = 0.04) than non-PCa men. Patients with aggressive PCa drank more beer than patients with non-aggressive PCa (heavy, OR = 1.48, p = 0.013). Interestingly, heavy wine intake was inversely associated with PCa aggressiveness (OR = 0.56, p = 0.009). CONCLUSIONS: The intake patterns of some alcoholic beverage types differed by PCa status. Our findings can provide valuable information for developing custom alcohol interventions for PCa patients.
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Gamma interferon (IFN-γ) induces expression of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO1) in human epithelial cells, the permissive cells for the obligate intracellular bacterium Chlamydia trachomatis. IDO1 depletes tryptophan by catabolizing it to kynurenine with consequences for C. trachomatis, which is a tryptophan auxotroph. In vitro studies reveal that tryptophan depletion can result in the formation of persistent (viable but noncultivable) chlamydial forms. Here, we tested the effects of the IDO1 inhibitor, levo-1-methyl-tryptophan (L-1MT), on IFN-γ-induced C. trachomatis persistence. We found that addition of 0.2 mM L-1MT to IFN-γ-exposed infected HeLa cell cultures restricted IDO1 activity at the mid-stage (20 h postinfection [hpi]) of the chlamydial developmental cycle. This delayed tryptophan depletion until the late stage (38 hpi) of the cycle. Parallel morphological and gene expression studies indicated a consequence of the delay was a block in the induction of C. trachomatis persistence by IFN-γ. Furthermore, L-1MT addition allowed C. trachomatis to undergo secondary differentiation, albeit with limited productive multiplication of the bacterium. IFN-γ-induced persistent infections in epithelial cells have been previously reported to be more resistant to doxycycline than normal productive infections in vitro. Pertinent to this observation, we found that L-1MT significantly improved the efficacy of doxycycline in clearing persistent C. trachomatis forms. It has been postulated that persistent forms of C. trachomatis may contribute to chronic chlamydial disease. Our findings suggest that IDO1 inhibitors such as L-1MT might provide a novel means to investigate, and potentially target, persistent chlamydial forms, particularly in conjunction with conventional therapeutics.
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Chlamydia trachomatis/efeitos dos fármacos , Células Epiteliais/microbiologia , Indolamina-Pirrol 2,3,-Dioxigenase/antagonistas & inibidores , Interferon gama/farmacologia , Triptofano/análogos & derivados , Antibacterianos/farmacologia , Chlamydia trachomatis/fisiologia , Relação Dose-Resposta a Droga , Doxiciclina/farmacologia , Células HeLa , Humanos , Indolamina-Pirrol 2,3,-Dioxigenase/metabolismo , Cinurenina/análise , Fatores de Tempo , Triptofano/análise , Triptofano/farmacologiaRESUMO
BACKGROUND: Systemic lupus erythematosus (SLE) is characterized by a wide spectrum of clinical and immunological abnormalities. New data have emerged about the role of inflammasomes in autoimmune diseases. We aimed to investigate whether basal inflammasome activation occurs in SLE patients, and whether a relationship between inflammasome-related-cytokines and disease activity exists. METHODS: Fourteen (14) consecutive SLE patients and 13 healthy individuals, matched by sex, age and ethnicity, were included. Demographics, laboratory and clinical data were recorded. Peripheral blood mononuclear cells (PBMCs) from patients and controls were obtained and monocytes were isolated by negative selection. Purified monocytes were stimulated with LPS in the presence or absence of Caspase-1 inhibitor. CD14 and Caspase-1 expression were analyzed by flow cytometry. Cytokine levels were determined in plasma and culture supernatants by ELISA. Student's t test and Mann-Whitney tests were used for statistical analysis. RESULTS: The percentage of CD14+/Caspase-1+ was significantly higher in monocytes from SLE patients compared to normal controls (p<0.01). These findings paralleled with higher plasma levels of IL-1ß (p<0.05) and IL-18 (p<0.01) in those patients. Purified monocytes from SLE patients displayed a robust inflammatory response after LPS stimulation where Caspase-1, IL-1ß and IL-18 were highly expressed. Plasma levels of IL-18 were also significantly higher in SLE patients with active disease (p<0.05). In addition, the production of IL-18 was reduced by 3 fold when Caspase-1 inhibitor was added to the cultures. CONCLUSIONS: Monocytes from SLE patients exhibited increased inflammasome activation, characterized by high expression of Caspase-1, IL-1ß and IL-18. Caspase-1 specific inhibitor decreased inflammasome activation (in vitro) by suppressing the production of IL-18.
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Epstein-Barr nuclear antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B cells. EBNA1 transactivates viral promoters for genes that are necessary for immortalization when it is bound to a cluster of 20 cognate binding sites, termed the family of repeats. A region of EBNA1 from amino acids (aa) 40 to 89, termed linking region 1 (LR1), has been identified previously as being sufficient for transactivation. LR1 contains two domains that are conserved in the EBNA1 orthologs of other gamma herpesviruses. The first of these, termed unique region 1 (UR1), corresponds to aa 65 to 89 of EBNA1. UR1 is necessary for transactivation and contains a conserved recognition site for cyclic AMP-dependent protein kinase (PKA), corresponding to serine 78 of EBNA1. We have pharmacologically modulated PKA activity to determine if PKA controls EBNA1's ability to transactivate. Our results indicate that PKA activators and inhibitors do not affect transactivation by EBNA1. In addition, site-directed mutagenesis demonstrates that transactivation is not influenced by the phosphorylation status of serine 78 in the UR1 domain. The second conserved domain within LR1 is a glycine-arginine repeat, corresponding to aa 40 to 54 of EBNA1. This domain, termed ATH1, functions as an AT-hook, a DNA-binding motif found in architectural transcription factors such as HMGA1a. We demonstrate that deletion of the ATH1 domain decreases EBNA1 transactivation ability, which is consistent with a transcriptional role for ATH1. Furthermore, transactivation is restored when ATH1 is replaced by equivalent AT-hook motifs from HMGA1a. Our data strongly indicate a role for AT-hooks in EBNA1's ability to transactivate, a function necessary for EBV to immortalize naïve B-cells.
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Antígenos Nucleares do Vírus Epstein-Barr/metabolismo , Herpesvirus Humano 4/metabolismo , Ativação Transcricional/genética , Motivos de Aminoácidos , Animais , Linhagem Celular , Sequência Conservada , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Antígenos Nucleares do Vírus Epstein-Barr/química , Antígenos Nucleares do Vírus Epstein-Barr/genética , Herpesvirus Humano 4/química , Herpesvirus Humano 4/genética , Dados de Sequência Molecular , Monoéster Fosfórico Hidrolases/metabolismo , Alinhamento de Sequência , Serina/genética , Serina/metabolismoRESUMO
BACKGROUND: L-arginine (L-Arg) is deficient in sickle cell disease (SSD) during vasoocclusion. We investigated possible causal relationship between L-Arg deficiency and immune dysfunction in SSD in steady-state. PROCEDURE: Fifteen patients with SSD in steady-state and 13 controls were studied. Plasma L-Arg levels were measured using liquid chromatography. T cell subsets and CD3zeta (CD3zeta) chain expression were analyzed using flow cytometry. Lymphocyte proliferative response to phytohemagglutinin (PHA) and production of IL-6 and interferon-gamma (IFN-gamma) were evaluated with and without L-Arg. RESULTS: SSD patients had significantly lower L-Arg levels than controls. CD3 and CD19 cell populations were comparable for both groups, but SSD patients had above normal numbers of natural killer cells (P = 0.06). Patients and controls exhibited significantly increased lymphocyte blastogenesis to PHA after introduction of L-Arg to cultures; response of patients was significantly greater than values for control individuals. Proliferative response to candida in SSD patients was significantly lower than in controls; L-Arg supplementation did not increase this response. L-Arg had no effect on blastogenic response to PPD and candida albicans. No effect was likewise seen in production of IL-6 and IFN-gamma after addition of L-Arg. CD3zeta chain expression increased after addition of L-Arg in both groups; differences were insignificant. CONCLUSION: L-Arg levels in steady-state SSD are significantly lower than in controls. L-Arg supplementation enhanced lymphocyte blastogenesis to PHA for both controls and patients, but not in response to antigen. There were no significant differences in CD3zeta chain expression although upregulation of expression occurred after L-Arg supplementation for both groups.
Assuntos
Anemia Falciforme/imunologia , Arginina/farmacologia , Imunidade/efeitos dos fármacos , Subpopulações de Linfócitos T/patologia , Adolescente , Arginina/sangue , Arginina/deficiência , Complexo CD3/biossíntese , Estudos de Casos e Controles , Células Cultivadas , Criança , Feminino , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Fito-Hemaglutininas/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Regulação para Cima , Adulto JovemRESUMO
PURPOSE: Immune dysfunction reported in renal cell carcinoma (RCC) patients may contribute to tumor progression. Myeloid-derived suppressor cells (MDSC) represent one mechanism by which tumors induce T-cell suppression. Several factors pivotal to the accumulation of MDSC are targeted by the tyrosine kinase inhibitor, sunitinib. The effect of sunitinib on MDSC-mediated immunosuppression in RCC patients has been investigated. EXPERIMENTAL DESIGN: Patient peripheral blood levels of MDSC and regulatory T-cell (Treg) and T-cell production of IFN-gamma were evaluated before and after sunitinib treatment. Correlations between MDSC and Treg normalization as well as T-cell production of IFN-gamma were examined. The in vitro effect of sunitinib on patient MDSC was evaluated. RESULTS: Metastatic RCC patients had elevated levels of CD33(+)HLA-DR(-) and CD15(+)CD14(-) MDSC, and these were partially overlapping populations. Treatment with sunitinib resulted in significant reduction in MDSC measured by several criteria. Sunitinib-mediated reduction in MDSC was correlated with reversal of type 1 T-cell suppression, an effect that could be reproduced by the depletion of MDSC in vitro. MDSC reduction in response to sunitinib correlated with a reversal of CD3(+)CD4(+)CD25(hi)Foxp3(+) Treg cell elevation. No correlation existed between a change in tumor burden and a change in MDSC, Treg, or T-cell production of IFN-gamma. In vitro addition of sunitinib reduced MDSC viability and suppressive effect when used at >/=1.0 microg/mL. Sunitinib did not induce MDSC maturation in vitro. CONCLUSIONS: Sunitinib-based therapy has the potential to modulate antitumor immunity by reversing MDSC-mediated tumor-induced immunosuppression.
Assuntos
Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Células Mieloides/imunologia , Pirróis/farmacologia , Fatores Supressores Imunológicos/efeitos dos fármacos , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma de Células Renais/imunologia , Feminino , Humanos , Indóis/uso terapêutico , Interferon gama/biossíntese , Neoplasias Renais/imunologia , Masculino , Pessoa de Meia-Idade , Pirróis/uso terapêutico , Sunitinibe , Fatores Supressores Imunológicos/fisiologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/fisiologiaRESUMO
Despite a decline in overall incidence rates for cancer in the past decade, due in part to impressive advancements in both diagnosis and treatment, breast cancer (BC) remains the leading cause of cancer-related deaths in women. BC alone accounts for â¼30% of all new cancer diagnoses in women worldwide. Triple-negative BC (TNBC), defined as having no expression of the estrogen or progesterone receptors and no amplification of the HER2 receptor, is a subtype of BC that does not benefit from the use of estrogen receptor-targeting or HER2-targeting therapies. Differences in socioeconomic factors and cell intrinsic and extrinsic characteristics have been demonstrated in Black and White TNBC patient tumors. The emergence of patient-derived xenograft (PDX) models as a surrogate, translational, and functional representation of the patient with TNBC has led to the advances in drug discovery and testing of novel targeted approaches and combination therapies. However, current established TNBC PDX models fail to represent the diverse patient population and, most importantly, the specific ethnic patient populations that have higher rates of incidence and mortality. The primary aim of this review is to emphasize the importance of using clinically relevant translatable tumor models that reflect TNBC human tumor biology and heterogeneity in high-risk patient populations. The focus is to highlight the complexity of BC as it specifically relates to the management of TNBC in Black women. We discuss the importance of utilizing PDX models to study the extracellular matrix (ECM), and the distinct differences in ECM composition and biophysical properties in Black and White women. Finally, we demonstrate the crucial importance of PDX models toward novel drug discovery in this patient population.
Assuntos
COVID-19 , Regulação para Baixo , Proteína S , SARS-CoV-2 , Trombose , Humanos , COVID-19/complicações , COVID-19/sangue , COVID-19/virologia , Proteína S/metabolismo , Masculino , Feminino , Trombose/sangue , Pessoa de Meia-Idade , IdosoRESUMO
Tumor-induced tolerance is a well-established phenomenon in cancer patients that can severely impair the therapeutic efficacy of immunotherapy. One mechanism leading to T-cell tolerance is the generation of myeloid-derived suppressor cells (MDSC) by soluble factors produced by the tumor. MDSC express CD11b(+) as a common marker but may vary in their stage of maturation, depending on the tumor factors being produced. Arginase production by MDSC depletes arginine from the tumor microenvironment and impairs T-cell signal transduction and function. We studied whether an increase in MDSC could explain the molecular alterations and dysfunction found in T cells of patients with renal cell carcinoma (RCC). Arginase activity in the peripheral blood mononuclear cells of 117 RCC patients was increased between 6- to 8-fold compared with normal controls. The increased arginase activity was limited to the CD11b(+)CD14(-) myeloid cells and resulted in significantly decreased serum levels of arginine and increased ornithine in patients. Depletion of MDSC restored IFN-gamma production and T-cell proliferation. Preliminary data suggest that prostaglandin E(2) produced by the tumor induces arginase I expression in MDSC. Therefore, blocking MDSC activity may enhance the therapeutic efficacy of immunotherapy in RCC.