ABSTRACT
SAMHD1 restricts HIV-1 infection of myeloid-lineage and resting CD4+ T-cells. Most likely this occurs through deoxynucleoside triphosphate triphosphohydrolase activity that reduces cellular dNTP to a level where reverse transcriptase cannot function, although alternative mechanisms have been proposed recently. Here, we present combined structural and virological data demonstrating that in addition to allosteric activation and triphosphohydrolase activity, restriction correlates with the capacity of SAMHD1 to form "long-lived" enzymatically competent tetramers. Tetramer disruption invariably abolishes restriction but has varied effects on in vitro triphosphohydrolase activity. SAMHD1 phosphorylation also ablates restriction and tetramer formation but without affecting triphosphohydrolase steady-state kinetics. However phospho-SAMHD1 is unable to catalyse dNTP turnover under conditions of nucleotide depletion. Based on our findings we propose a model for phosphorylation-dependent regulation of SAMHD1 activity where dephosphorylation switches housekeeping SAMHD1 found in cycling cells to a high-activity stable tetrameric form that depletes and maintains low levels of dNTPs in differentiated cells.
Subject(s)
Biocatalysis , HIV-1/pathogenicity , Monomeric GTP-Binding Proteins/metabolism , Cell Line , Chromatography, Gel , Chromatography, High Pressure Liquid , Crystallography, X-Ray , Flow Cytometry , Humans , Monomeric GTP-Binding Proteins/chemistry , Phosphorylation , Protein Conformation , Reverse Transcriptase Polymerase Chain Reaction , SAM Domain and HD Domain-Containing Protein 1 , Spectrophotometry, AtomicABSTRACT
OBJECTIVE: To present the baseline clinical and demographic characteristics of CRSwNP patients over the age of 18 enrolled in a Patient Support Program (PSP) prior to biologic treatment. METHODS: Descriptive, cross-sectional study performed in a Colombian CRSwNP asthma PSP sponsored by Sanofi from Aug-2021 to Jul-2022. Data was collected from CRSwNP patients, prior to the start of Dupilumab treatment, who consented to the use of their data. The following information was reported: Age, reporting city, treating medical specialty, comorbidities, and persistence of treatment. RESULTS: 339 patients were included, 171 (50,4%) were women and 168 (49,6%) were men. The mean age at Dupilumab treatment initiation was 52,4 years. 62,8% began treatment during adulthood (26-59y), while 34.1% started at elderly (+60y) and 3.1% were young adults (18-25y). Most cases (29,7%) were included in Bogotá, followed by Antioquia (19%), Valle del Cauca (7,3%) and the remaining 44% nationwide. Comorbidities were present in 67,1% of the patients, with diagnosis of allergic rhinitis, atopic dermatitis, asthma, and other non-type 2 inflammatory diseases. Nasal surgical history was present in 89,6% of the patients, most of them with one to three previous surgeries. Continuous treatment was observed in 70,3% of patients for 6 to 12 months, in 21,3% for more than 12 months and in 8,4% for less than six months. The most frequently treating medical specialty was otorhinolaryngology (79,6%), followed by allergology (16%) and other medical professionals (4,4%). CONCLUSIONS: There is concordance with the literature on a higher presentation of the disease in women than in men. There is a large proportion of patients with nasal surgical history and type 2 inflammatory comorbidities by the moment of biologic treatment initiation. The care and identification of CRSwNP colombian patients is mainly provided by otorhinolaryngologists, followed by allergologists.
OBJETIVO: Presentar las características clínicas y demográficas iniciales de los pacientes con RSCcPN, mayores de 18 años, inscritos en un Programa de Soporte al Paciente (PSP), antes del inicio de tratamiento biológico. MÉTODOS: Estudio descriptivo y transversal realizado en un PSP para RSCcPN en Colombia, entre agosto de 2021 y julio de 2022, patrocinado por Sanofi. Los datos se recopilaron de pacientes con RSCcPN, antes de comenzar el tratamiento con Dupilumab, quienes dieron su consentimiento para el uso de sus datos. Se reportó la siguiente información: edad, ciudad de origen, especialidad médica tratante, comorbilidades y persistencia del tratamiento. RESULTADOS: Se incluyeron 339 pacientes, 171 mujeres (50,4%), y 168 hombres (49,6%). La edad promedio al inicio del tratamiento con Dupilumab, fue de 52,4 años. El 62,8% inició tratamiento durante la edad adulta (entre 26 y 59 años), mientras que el 34,1% comenzó en la vejez (+60 años), y el 3,1% entre los 18 y 25 años. La mayoría de los casos (29,7%) se incluyeron en Bogotá, seguidos por Antioquia (19%), Valle del Cauca (7,3%) y el 44% restante en todo el país. Las comorbilidades estuvieron presentes en el 67,1% de los pacientes, con diagnóstico de rinitis alérgica, dermatitis atópica, asma y otras enfermedades no inflamatorias tipo 2. El 89,6% de los pacientes tenía antecedentes de cirugía nasal, la mayoría de ellos con entre una y tres cirugías previas. Se observó tratamiento continuo en el 70,3% de los pacientes durante 6 y 12 meses, en el 21,3%, durante más de 12 meses, y en el 8,4% durante menos de 6 meses. La especialidad médica que trató a los pacientes con más frecuencia fue la otorrinolaringología (79,6%), seguida por la alergología (16%) y otros profesionales médicos (4,4%). CONCLUSIONES: Existe concordancia con la literatura con una mayor presentación de la enfermedad en mujeres que en hombres. Hay una gran proporción de pacientes con antecedentes de cirugía nasal y comorbilidades inflamatorias tipo 2, al inicio del tratamiento biológico. La atención e identificación de los pacientes colombianos con RSCcPN es proporcionada principalmente por otorrinolaringólogos, seguidos por alergólogos.
Subject(s)
Nasal Polyps , Rhinosinusitis , Adolescent , Adult , Aged , Female , Humans , Male , Middle Aged , Young Adult , Antibodies, Monoclonal, Humanized/therapeutic use , Chronic Disease , Cohort Studies , Colombia/epidemiology , Comorbidity , Cross-Sectional Studies , Nasal Polyps/epidemiology , Nasal Polyps/complications , Rhinosinusitis/epidemiologyABSTRACT
The cornea contains a reservoir of self-regenerating epithelial cells that are essential for maintaining its transparency and good vision. The study of stem cells in this functionally important organ has grown over the past four decades, partly due to the ease with which this tissue is visualized, its accessibility with minimally invasive instruments, and the fact that its stem cells are segregated within a transitional zone between two functionally diverse epithelia. While human, animal, and ex vivo models have been instrumental in progressing the corneal stem cell field, there is still much to be discovered about this exquisitely sensitive window for sight. This review will provide an overview of the human cornea, where its stem cells reside and how components of the microenvironment including extracellular matrix proteins and their integrin receptors are thought to govern corneal stem cell homeostasis.
Subject(s)
Epithelial Cells/cytology , Limbus Corneae/cytology , Stem Cell Niche , Stem Cells/physiology , Animals , Antigens, Differentiation/metabolism , Cell Shape , Extracellular Matrix/metabolism , Humans , Integrins/metabolism , Stem Cells/metabolismABSTRACT
Nonnucleoside reverse transcriptase (RT) inhibitors (NNRTIs) are important components of current combination therapies for human immunodeficiency virus type 1 (HIV-1) infection. In screening of chemical libraries, we found 6-azido-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AzBBU) and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl)uracil (AmBBU) to be highly active and selective inhibitors of HIV-1 replication in vitro. To determine the resistance profiles of these compounds, we conducted a long-term culture of HIV-1-infected MT-4 cells with escalating concentrations of each compound. After serial passages of the infected cells, escape viruses were obtained, and they were more than 500-fold resistant to the uracil derivatives compared to the wild type. Sequence analysis was conducted for RT of the escape viruses at passages 12 and 24. The amino acid mutation Y181C in the polymerase domain of RT was detected for all escape viruses. Docking studies using the crystal structure of RT showed that AmBBU requires the amino acid residues Leu100, Val106, Tyr181, and Trp229 for exerting its inhibitory effect on HIV-1. Four additional amino acid changes (K451R, R461K, T468P, and D471N) were identified in the RNase H domain of RT; however, their precise role in the acquisition of resistance is still unclear. In conclusion, the initial mutation Y181C seems sufficient for the acquisition of resistance to the uracil derivatives AzBBU and AmBBU. Further studies are required to determine the precise role of each mutation in the acquisition of HIV-1 resistance.
Subject(s)
Anti-HIV Agents/pharmacology , HIV Reverse Transcriptase/antagonists & inhibitors , HIV-1/drug effects , Reverse Transcriptase Inhibitors/pharmacology , Uracil/analogs & derivatives , Amino Acid Sequence , Amino Acid Substitution , Anti-HIV Agents/chemistry , Anti-HIV Agents/metabolism , Binding Sites , Cell Line , Computer Simulation , Drug Resistance, Viral/drug effects , Drug Resistance, Viral/genetics , HIV Reverse Transcriptase/chemistry , HIV Reverse Transcriptase/metabolism , HIV-1/enzymology , HIV-1/genetics , Humans , Models, Molecular , Molecular Sequence Data , Mutation , Protein Structure, Tertiary , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/metabolism , Sequence Analysis , Small Molecule Libraries , T-Lymphocytes/drug effects , T-Lymphocytes/virology , Uracil/chemistry , Uracil/metabolism , Uracil/pharmacologyABSTRACT
Sterile α-motif/histidine-aspartate domain-containing protein 1 (SAMHD1) inhibits replication of HIV-1 in quiescent myeloid cells. U937 cells are widely used as a convenient cell system for analyzing SAMHD1 activity due to a low level of SAMHD1 RNA expression, leading to undetectable endogenous protein expression. Based on similar assays developed in the Stoye laboratory to characterize other retroviral restriction factors, the Bishop lab developed a two-color restriction assay to analyze SAMHD1 in U937 cells. Murine Leukaemia Virus-like particles expressing SAMHD1, alongside YFP expressed from an IRES, are used to transduce U937 cells. Cells are then treated with phorbol myristate acetate to induce differentiation to a quiescent phenotype. Following differentiation, cells are infected with HIV-1 virus-like particles expressing a fluorescent reporter. After 48 h, cells are harvested and analyzed by flow cytometry. The proportion of HIV-infected cells in the SAMHD1-expressing population is compared to that in internal control cells lacking SAMHD1. This comparison reveals a restriction ratio. SAMHD1 expression leads to a five-fold reduction in HIV infection, corresponding to a restriction ratio of 0.2. Our recent substitution of RFP for the original GFP as the reporter gene for HIV infection has facilitated flow cytometry analysis. This assay has been successfully used to characterize the effect of amino acid substitutions on SAMHD1 restriction by transducing with viruses encoding altered SAMHD1 proteins, derived from site-directed mutagenesis of the expression vector. For example, the catalytic site substitutions HD206-7AA show a restriction phenotype of 1, indicating a loss of restriction activity. Equally, the susceptibility of different tester viruses can be determined. The assay can be further adapted to incorporate the effect of differentiation status, metabolic status, and SAMHD1 modifiers to better understand the relationship between SAMHD1, cell metabolic state, and viral restriction.
Subject(s)
HIV Infections , Monomeric GTP-Binding Proteins , Animals , Flow Cytometry , Humans , Mice , Monomeric GTP-Binding Proteins/genetics , Monomeric GTP-Binding Proteins/metabolism , SAM Domain and HD Domain-Containing Protein 1/genetics , U937 Cells , Virus ReplicationABSTRACT
RESUMEN Objetivo: Identificar aspectos críticos de la Cadena de Frío en el Perú Metodología: Estudio descriptivo. Se analizaron datos del Ministerio de Economía y Finanzas (MEF) y del Ministerio de Salud (MINSA) de los años 2020-2021, así como los registros administrativos de las Estrategias Sanitarias Regionales de inmunizaciones del MINSA en 2020. Se consideraron aspectos técnicos de la Norma Técnica de Manejo de cadena de frío, como es obsolescencia, dotación, funcionalidad y capacidad de almacenamiento. Resultados: En el año 2020, en términos de obsolescencia el 61.8% de los equipos de cadena de frío a nivel nacional presentaban obsolescencia, siendo regiones claves como Lima Metropolitana (capital del país) la más afectada con un 88%. En cuanto a la dotación de equipos, el 9% de los establecimientos de salud del primer nivel carecen de equipos de refrigeración, siendo Loreto la región con mayor déficit 46%, seguida de Huancavelica con un 21% de brecha. En términos de funcionamiento, se registra que el 84% de los equipos a nivel nacional funcionan, y el 16% reportan fallas técnicas, lo cual representa alto riesgo en la seguridad y potencia inmunogénica de las vacunas a prever. En términos de capacidad, al considerar el almacenamiento trimestral o mensual para las vacunas contra la COVID-19 u otras emergencias sanitarias se identificaron brechas significativas. Conclusiones: Existen riesgos en la operatividad, suministro y capacidad de almacenamiento de las vacunas del esquema nacional de inmunizaciones de Perú incluso ante emergencias sanitarias.
ABSTRACT Objective: The aim of this study was to identify critical aspects of the Cold Chain in the immunization process in Peru. Methodology: A descriptive study was conducted, analyzing data from the Ministry of Economy and Finance (MEF) and the Ministry of Health (MINSA) for the years 2020-2021, as well as administrative records from the MINSA's Regional Health Strategies for immunizations in 2020. Technical aspects established in the Health Technical Standard for Cold Chain Management, such as obsolescence, allocation, functionality, and storage capacity were taken into account. Results: In the year 2020, at the national level, 61.8% of the cold chain equipment showed obsolescence, with some regions exceeding 75%, with Lima's metropolitan region being the most affected at 88%. Concerning equipment allocation, 9% of the first-level health facilities lacked refrigeration equipment, with Loreto having the highest deficit (46%), followed by Huancavelica with a 21% gap. The overall equipment functionality nationwide was 84%, meaning that 16% of health facilities experienced technical failures, affecting vaccine's storage capacity and posing risks to their safety and immunogenicity. Significant gaps were identified when considering quarterly or monthly storage for COVID-19 vaccines or other health emergencies. Conclusions: This study highlights potential risks in the operability and storage capacity of the national immunization program's vaccines in Peru during contingencies such as the COVID-19 pandemic or other health emergencies.
ABSTRACT
SAMHD1 is an intracellular enzyme that specifically degrades deoxynucleoside triphosphates into component nucleoside and inorganic triphosphate. In myeloid-derived dendritic cells and macrophages as well as resting T-cells, SAMHD1 blocks HIV-1 infection through this dNTP triphosphohydrolase activity by reducing the cellular dNTP pool to a level that cannot support productive reverse transcription. We now show that, in addition to this direct effect on virus replication, manipulating cellular SAMHD1 activity can significantly enhance or decrease the anti-HIV-1 efficacy of nucleotide analogue reverse transcription inhibitors presumably as a result of modulating dNTP pools that compete for recruitment by viral polymerases. Further, a variety of other nucleotide-based analogues, not normally considered antiretrovirals, such as the anti-herpes drugs Aciclovir and Ganciclovir and the anti-cancer drug Clofarabine are now revealed as potent anti-HIV-1 agents, under conditions of low dNTPs. This in turn suggests novel uses for nucleotide analogues to inhibit HIV-1 in differentiated cells low in dNTPs.
Subject(s)
HIV-1/physiology , Nucleotides/chemistry , SAM Domain and HD Domain-Containing Protein 1/metabolism , Acyclovir/pharmacology , Adenine Nucleotides/pharmacology , Allosteric Regulation , Arabinonucleosides/pharmacology , Cell Line , Clofarabine , Ganciclovir/pharmacology , Humans , Myeloid Cells/virology , Nucleotides/metabolism , Nucleotides/pharmacology , Reverse Transcriptase Inhibitors/chemistry , Reverse Transcriptase Inhibitors/pharmacology , Virus Replication/drug effectsABSTRACT
Epstein-Barr virus (EBV) infection in tumor cells is usually restricted to the latent form, indicating that the induction of viral lytic infection may present a novel approach for the treatment of EBVassociated tumors. By contrast, EBV lytic replication is inhibited by highlevels of nuclear factor (NF)κB, which suggests that NFκB inhibitors may activate lytic replication from the latent form. In the current study, the addition of NFκB inhibitors (Bay117082, ZLLFCHO and aspirin) was observed to induce the EBV lytic genes BZLF1, BRLF1 and BMRF1 in EBVpositive gastric cancer (GC) cells. Both EBVpositive and negative GC cells were treated with different concentrations of antiherpes agents and the cytotoxic effects were measured at different time points following induction of EBV lytic replication. A marginal dose and timedependent reduction in cell viability was observed for EBVpositive andnegative GC cells. The cytotoxic effects of NFκB inhibitors on EBVpositive GC cells were enhanced by the addition of the antiherpes agents, ganciclovir, acyclovir, foscarnet and brivudine (P<0.05). However, there was no significant synergistic effect on EBVnegative GC cells. The combination of 5 mM aspirin and ganciclovir exhibited the highest cytotoxic effect in EBVpositive GC cells (CC50=7.2 µg/ml).
Subject(s)
Antineoplastic Agents/pharmacology , Antiviral Agents/pharmacology , Epstein-Barr Virus Infections/complications , Herpesvirus 4, Human/drug effects , NF-kappa B/antagonists & inhibitors , Stomach Neoplasms/etiology , Stomach Neoplasms/metabolism , Cell Line, Tumor , Cells, Cultured , Dose-Response Relationship, Drug , Drug Synergism , Epstein-Barr Virus Infections/virology , Gene Expression , Humans , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Transcription Factor RelB/antagonists & inhibitors , Transcription Factor RelB/genetics , Transcription Factor RelB/metabolism , Virus Activation/drug effects , Virus Replication/drug effectsABSTRACT
Objetivo. Optimizar una técnica PCR que permita evaluar la presencia de C. trachomatis en hisopados anorrectales provenientes de HSH. En Colombia se notifican anualmente más de 70.000 casos nuevos de ITS, de los cuales se estima que aproximadamente el 9.3% corresponde a uretritis entre las que se encuentran las causadas por C. trachomatis. Métodos. Uno de los problemas en el método de detección de C. trachomatis por PCR en muestras de hisopado anorrectal es la extracción de ADN, el uso de equipos automatizados dispuestos en el mercado resulta costoso y en muchos de los casos no están disponibles en el laboratorio clínico de rutina. En este estudio se realizó una PCR para detección de C. trachomatis, estableciendo un protocolo para la toma de muestra y extracción de ADN a partir de hisopos anorrectales. Resultados. Se procesaron 27 muestras correspondientes a HSH voluntarios pertenecientes al Grupo de apoyo y estudio de la Diversidad Sexual (GAEDS) de la Universidad Nacional de Colombia. Se encontraron 5 muestras positivas para C. trachomatis en hombres sintomáticos y asintomáticos relacionado con el riesgo de adquirir infección por sus prácticas sexuales.
Objective. optimize a PCR technique to evaluate the presence of C. trachomatis in anorectal swabs from MSM. In Colombia there are reported each year more than 70,000 new cases of STIs, of which it is estimated that approximately 9.3% is urethritis among which are those caused by C. trachomatis. Methods. DNA extraction is one of the problems in the method of detecting C. trachomatis by PCR anorectal swab samples. Besides, the use of automated equipment arranged on the market is expensive and in many cases the samples are not available in the clinical laboratory routine. In this study it was performed PCR for detection of C. trachomatis protocol establishing the sampling and DNA extraction from anorectal swabs. Results. 27 samples were processed corresponding HSH volunteers belonging to the Support group and study of Sexual Diversity (GAEDS) of the National University of Colombia. 5 samples positive for C. trachomatis associated with both symptomatic and asymptomatic men at high risk of acquiring infection because of their sexual practices were found.
Subject(s)
Humans , Chlamydia trachomatis , Sexual Behavior , Homosexuality , Sexually Transmitted DiseasesABSTRACT
BACKGROUND: Nine novel uracil analogues were synthesized and evaluated as inhibitors of HIV-1. METHODS: Key structural modifications included replacement of the 6-chloro group of 1-benzyl-6-chloro-3-(3,5-dimethylbenzyl)uracil by other functional groups or N(1)-alkylation of 3-(3,5-dimethylbenzyl)-5-fluorouracil. RESULTS: These compounds showed only micromolar potency against HIV-1 in MT-4, though two of them; 6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil were highly potent (half maximal effective concentration =0.067 and 0.069 µM) and selective (selectivity index =685 and 661), respectively. Structure-activity relationships among the newly synthesized uracil analogues suggest the importance of the H-bond formed between 6-amino group of 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil and amide group of HIV-1 reverse transcriptase. CONCLUSIONS: We discovered two 6-substituted 1-benzyl-3-(3,5-dimethylbenzyl) uracils, (6-azido-1-benzyl-3-(3,5-dimethylbenzyl) uracil and 6-amino-1-benzyl-3-(3,5-dimethylbenzyl) uracil) as novel anti-HIV agents. These compounds should be further pursued for their toxicity and pharmacokinetics in vivo as well as antiviral activity against non-nucleoside reverse transcriptase inhibitor-resistant strains.
Subject(s)
Anti-HIV Agents/chemical synthesis , Anti-HIV Agents/pharmacology , Uracil/analogs & derivatives , Anti-HIV Agents/chemistry , Cell Line, Tumor , Drug Evaluation, Preclinical/methods , HIV-1/drug effects , Humans , Hydrogen Bonding , Structure-Activity Relationship , Uracil/chemistryABSTRACT
AIM: To clarify the reason for the low frequency of Epstein-Barr virus-associated gastric carcinoma (EBVaGC) in Peru, despite the high frequency reported in neighboring countries, the distribution of the distinctive EBV (type i/XhoI+) strain in EBVaGC and a healthy population was examined. MATERIALS AND METHODS: EBV polymorphisms in BamHI W1/I1 and XhoI restriction site of the latent membrane protein 1 gene (LMP1) were examined among 11 EBVaGCs and 172 healthy controls from Peru, and these frequencies were compared with those in a previous study of Chile and Colombia (n=303). RESULTS: The frequency of the distinctive EBV strain in EBVaGCs (55%) was significantly higher than that in controls (7%). Furthermore, the frequency of this EBV type in Peruvian controls was significantly lower than that in controls from Chile and Colombia (27%, p<0.001). CONCLUSION: The low frequency of the distinctive EBV strain among the Peruvian population might be a reason for the lower incidence of EBVaGC in Peru, as compared with neighboring countries.