RESUMEN
Tobacco consumption increases the susceptibility to develop infectious diseases such as tuberculosis (TB). Nicotine (Nc) is the main component of cigarette smoke with immunomodulatory properties, however, its effect on Mycobacterium tuberculosis (Mtb) has been scarcely investigated. The present study evaluated the effect of nicotine on the growth of Mtb and on the induction of virulence-related genes. Mycobacteria were exposed to different concentrations of nicotine then Mtb growth was evaluated. Subsequently, the expression of the virulence-related genes lysX, pirG, fad26, fbpa, ompa, hbhA, esxA, esxB, hspx, katG, lpqh, and caeA was evaluated by RT-qPCR. The effect of nicotine on intracellular Mtb was also evaluated. The results showed that nicotine promotes the growth of Mtb both extracellularly and intracellularly and increases the expression of genes related to virulence. In summary, nicotine promotes the growth of Mtb and the expression of virulence-related genes that could be correlated with the increased the risk of smokers developing TB.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Humanos , Mycobacterium tuberculosis/genética , Virulencia/genética , Nicotina/farmacología , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Tobacco consumption is related to an increased risk to develop tuberculosis. Antimicrobial peptides are essential molecules in the response to Mycobacterium tuberculosis (Mtb) because of their direct antimicrobial activity. The aim of this study was to demonstrate that nicotine enters into Mtb infected epithelial cells and associates with the mycobacteria inducing genes related to antimicrobial peptides resistance. Epithelial cells were infected with virulent Mtb, afterwards cells were stimulated with nicotine. The internalization of nicotine was followed using electron and confocal microscopy. The lysX expression was evaluated isolating mycobacterial RNA and submitted to RT-PCR analysis. Our results indicated that nicotine promotes Mtb growth in a dose-dependent manner in infected cells. We also reported that nicotine induces lysX expression. In conclusion, nicotine associates to intracellular mycobacteria promoting intracellular survival.
Asunto(s)
Mycobacterium tuberculosis , Tuberculosis , Péptidos Antimicrobianos , Humanos , Macrófagos , Mycobacterium tuberculosis/genética , Nicotina/farmacologíaRESUMEN
The aim of the present work was to evaluate MTX treatment (0.1, 1 and 10 µg mL-1) in vitro in order to characterize its effects on cell proliferation alterations in cell cycle of HaCaT keratinocytes and wound healing in a Skh1 mice treated with MTX (low doses 30 mg kg-1, high doses 200 mg kg-1 and repeated doses at 1.5 mg kg-1). We analyzed the cytotoxic effect of methotrexate by a resazurin assay. The effects in the proliferation, cell cycle and apoptosis of HaCaT cells were analyzed by flow cytometry. The effects of MTX on wound healing in vivo were also analyzed. A trend toward reduction in the resazurin assay was found (p > 0.05). Reduced proliferation was also identified in a clonogenic assay and a CFSE assay (p < 0.05) due to the MTX treatment. A reduction in the G2/M and S phases was observed accompanied by apoptosis induction with increased sub G0 phase and annexin V FITC staining. Effect of MTX was evidenced in vivo on the wound closure process after day 10 (p < 0.05) with alterations in tissue architecture and remodeling. There is a marked effect of MTX on wound healing in vivo in Skh1 mice with implications for long-term therapy and surgical interventions.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Metotrexato/farmacología , Cicatrización de Heridas/efectos de los fármacos , Análisis de Varianza , Animales , Apoptosis/efectos de los fármacos , Modelos Animales de Enfermedad , Ratones , Estadísticas no ParamétricasRESUMEN
Aging is a major health issue due to the increased susceptibility of elderly people to infectious, autoimmune, and cardiovascular diseases. Innate immunity is an important mechanism to avoid primary infections; therefore, decreasing of its activity may lead to development of infections. Antimicrobial peptides (AMPs) are effector molecules of innate immunity that can eliminate microbial invaders. The role that cytokines play in the regulation of these innate immune mechanisms needs to be explored. Serum determinations of Th1, Th2, and Th17 cytokines were performed in order to evaluate their association with AMPs human beta-defensin (HBD)-2 and LL-37 in young adults, elder adults, and elder adults with recurrent infections. Our results showed differences in interleukin (IL)-10 and IL-6 among the different groups. Inverse correlations in serum cytokine levels and HBD-2 production were identified for IL-10, IL-2, IL-4, tumor necrosis factor-α, and IL-6. Also inverse correlations were identified for IL-10, IL-4, and cathelicidin (LL-37). Such results could impact the development of immunomodulators that promote AMP production to prevent and/or contain infectious diseases in this population.
Asunto(s)
Envejecimiento/inmunología , Péptidos Catiónicos Antimicrobianos/metabolismo , Infecciones/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , beta-Defensinas/sangre , Adulto , Anciano , Anciano de 80 o más Años , Péptidos Catiónicos Antimicrobianos/sangre , Células Cultivadas , Citocinas/metabolismo , Humanos , Inmunidad Innata , Persona de Mediana Edad , Recurrencia , Adulto Joven , CatelicidinasRESUMEN
Our objective was to identify transcriptional biomarkers in peripheral blood mononuclear cells (PBMC) that discriminate individuals with latent tuberculosis infection (LTBI) from those with pulmonary tuberculosis (PTB) in subjects with non-insulin-dependent diabetes mellitus (NIDDM) and in individuals without NIDDM. Using gene expression microarrays we identified differentially expressed genes from lungs of mice infected with Mycobacterium tuberculosis (Mtb) or a mutant (ΔsigH) representing a non-inflammatory model. Genes expressed in blood, with inflammatory related functions were evaluated in humans by RT-qPCR. NCF1 and ORM transcripts have the better discriminatory capacity to identify PTB subjects from LTBI and non-infected controls (NICs) independently of the presence of NIDDM. The sequential evaluation of the mRNA levels of NCF1 and ORM as multiple diagnostic tests showed 95% Sensitivity (Se) and 80% Specificity (Sp). In addition, FPR2 promises to be a good biomarker for the PTB detection in subjects with NIDDM (Se=100%; Sp=90%).
Asunto(s)
Biomarcadores , Diabetes Mellitus Tipo 2/complicaciones , Regulación de la Expresión Génica , Tuberculosis Pulmonar/complicaciones , Tuberculosis Pulmonar/diagnóstico , Adulto , Anciano , Animales , Análisis por Conglomerados , Femenino , Perfilación de la Expresión Génica , Humanos , Masculino , Ratones , Persona de Mediana Edad , Tuberculosis Pulmonar/fisiopatologíaRESUMEN
Elderly individuals are susceptible to develop infectious diseases; promoting innate immunity to prevent infections is a key issue. Human ß-defensin-2 (hBD-2) is an antimicrobial peptide with antimicrobial and immunomodulatory properties. L-isoleucine and vitamin D are important molecules that induce hBD-2. The Aim of this study was to determine the use L-isoleucine and Vitamin D to induce hBD-2 in cells from healthy elderly individuals and elderly individuals with recurrent infections. We explored three groups: young adults (n = 20) used as control group, elderly adults (n = 18) and elderly with recurrent infections (n = 11). PBMCs (peripheral blood mononuclear cells) were isolated from the different groups and then were treated with L-isoleucine or vitamin D3. hBD-2 concentration was assessed with a sandwich enzyme Immunosorbent assay by triplicate. Using the vehicle as a mock control. Our results showed that a percentage of the individuals responded to the treatments producing hBD-2 (p < 0.05). These results showed that both molecules induced hBD-2 in elderly individuals and can be potentially used as prophylactic therapy to decrease infection diseases rates in this vulnerable group.
RESUMEN
AIM: Explore the temporal expression of metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimental tuberculosis induced by virulent Mycobacterium tuberculosis strain H37Rv. METHODS: BALB/c mice were infected via endotracheal instillation with H37Rv. Groups of mice were euthanized at different time points during infection. RNA was isolated from the lungs, and the expression of MMP-3, 8, 9, 10, 12, 13 and TIMP-1-4 was determined by quantitative PCR. Immunohistochemical detection of MMP-3, MMP-9, and MMP-10 was done to determine the cell source. RESULTS: The infection with H37Rv-induced inflammation resulted in maximal up-regulation of MMP-3, 8, 9, 10, 12 and 13 at day 21 postinfection. Additionally, MMP-13 showed another expression peak during late disease at day 60. Airway epithelium and macrophages were the most common MMP-3 and MMP-9 immunopositive cells, while for MMP-10, macrophages and endothelial cells were the most common, particularly at days 14 and 21 in well-formed granulomas. During late disease, vacuolated macrophages in pneumonic areas and bronchial epithelium showed mild MMP immunostaining. CONCLUSIONS: MMP-3, 8, 9, 10, 12, and 13 are maximally expressed at the peak of granuloma formation in the mouse tuberculosis model, with no compensation in levels or timing of TIMP expression. This data opens the possibility of participation of these molecules in the granuloma process.
Asunto(s)
Metaloproteinasas de la Matriz/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Tuberculosis Pulmonar/enzimología , Animales , Modelos Animales de Enfermedad , Hidrolasas/inmunología , Masculino , Ratones Endogámicos BALB C , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tuberculosis Pulmonar/inmunologíaRESUMEN
A cohort of 123 adult contacts was followed for 18-24 months (86 completed the follow-up) to compare conversion and reversion rates based on two serial measures of QuantiFERON (QFT) and tuberculin skin test (TST) (PPD from TUBERSOL, Aventis Pasteur, Canada) for diagnosing latent tuberculosis (TB) in household contacts of TB patients using conventional (C) and borderline zone (BZ) definitions. Questionnaires were used to obtain information regarding TB exposure, TB risk factors and socio-demographic data. QFT (IU/mL) conversion was defined as <0.35 to ≥0.35 (C) or <0.35 to >0.70 (BZ) and reversion was defined as ≥0.35 to <0.35 (C) or ≥0.35 to <0.20 (BZ); TST (mm) conversion was defined as <5 to ≥5 (C) or <5 to >10 (BZ) and reversion was defined as ≥5 to <5 (C). The QFT conversion and reversion rates were 10.5% and 7% with C and 8.1% and 4.7% with the BZ definitions, respectively. The TST rates were higher compared with QFT, especially with the C definitions (conversion 23.3%, reversion 9.3%). The QFT conversion and reversion rates were higher for TST ≥5; for TST, both rates were lower for QFT <0.35. No risk factors were associated with the probability of converting or reverting. The inconsistency and apparent randomness of serial testing is confusing and adds to the limitations of these tests and definitions to follow-up close TB contacts.
Asunto(s)
Composición Familiar , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/transmisión , Prueba de Tuberculina/métodos , Adulto , Trazado de Contacto , Progresión de la Enfermedad , Exposición a Riesgos Ambientales , Estudios de Evaluación como Asunto , Femenino , Estudios de Seguimiento , Humanos , Tuberculosis Latente/clasificación , Tuberculosis Latente/epidemiología , Masculino , México/epidemiología , Persona de Mediana Edad , Factores de Riesgo , Factores Socioeconómicos , Encuestas y CuestionariosRESUMEN
Mycobacterium tuberculosis (Mtb) is one of the most important infectious agents worldwide and causes more than 1.5 million deaths annually. To make matters worse, the drug resistance among Mtb strains has risen substantially in the last few decades. Nowadays, it is not uncommon to find patients infected with Mtb strains that are virtually resistant to all antibiotics, which has led to the urgent search for new molecules and therapies. Over previous decades, several studies have demonstrated the efficiency of antimicrobial peptides to eliminate even multidrug-resistant bacteria, making them outstanding candidates to counterattack this growing health problem. Nevertheless, the complexity of the Mtb cell wall makes us wonder whether antimicrobial peptides can effectively kill this persistent Mycobacterium. In the present review, we explore the complexity of the Mtb cell wall and analyze the effectiveness of antimicrobial peptides to eliminate the bacilli.
Asunto(s)
Mycobacterium tuberculosis , Humanos , Antituberculosos/farmacología , Antituberculosos/uso terapéutico , Antituberculosos/química , Péptidos Antimicrobianos , Farmacorresistencia Bacteriana Múltiple , Pared Celular/químicaRESUMEN
Rosmarinus officinalis (Lamiaceae) possesses antioxidant activity and hepatoprotective effects, and so may provide a possible therapeutic alternative for chronic liver disease. The effect produced by a methanolic extract of Rosmarinus officinalis on CCl(4)-induced liver cirrhosis in rats was investigated using both prevention and reversion models. Over the course of the development of cirrhosis, the increased enzymatic activities of gamma-glutamyl transpeptidase and alanine aminotransferase, and the rise in bilirubin levels caused by CCl(4) administration, were prevented by Rosmarinus officinalis co-administration. When the cirrhosis by oxidative stress was evaluated as an increase on liver lipoperoxidation, total lipid peroxides, nitric oxide in serum, and loss of erythrocyte plasma membrane stability, R. officinalis was shown to prevent such alterations. On cirrhotic animals treated with CCl(4), histological studies showed massive necrosis, periportal inflammation and fibrosis which were modified by R. officinalis. These benefits on experimental cirrhosis suggest a potential therapeutic use for R. officinalis as an alternative for liver cirrhosis.
Asunto(s)
Cirrosis Hepática/tratamiento farmacológico , Fitoterapia , Extractos Vegetales/uso terapéutico , Rosmarinus , Animales , Tetracloruro de Carbono , Cromatografía Líquida de Alta Presión , Evaluación Preclínica de Medicamentos , Hígado/efectos de los fármacos , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/patología , Masculino , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
The development of a more efficient vaccine is needed to improve tuberculosis control. One of the current approaches is to identify immunogenic T-cell peptides that can elicit a protective and specific immune response. These peptides come from immunogenic proteins of the pathogen. The PE_PGRS33 protein of Mycobacterium tuberculosis has been proved immunogenic. However, little is known about immunogenic T-cell peptides of PE_PGRS33 and their interactions with MHC-II molecules. Therefore, we used the SYFPHEITHI database to determine the immunogenic PE_PGRS33 T-cell peptides. Next, we built homology models by using MOE v2018.1 software in order to obtain information about the specific interactions between the peptides and I-Ak. The AlgPred server was employed to look for allergenic sites in PE_PGRS33. We developed a sequence alignment between PE_PGRS33 and all the human proteins by using BLAST. Three peptides were commercially synthesized, and their activity was evaluated in vitro by the stimulation of PBMC from household contacts of TB patients. Our in silico results showed five immunogenic T-cell peptides. BLAST analysis showed low homology of PE_PGRS33 with human proteins and AlgPred did not reveal allergenic sites in PE_PGRS33. The three peptides triggered the activation of CD4+ T cells from the households contacts, showed by the production of IFN-γ. We identified three immunogenic peptides of PE_PGRS33 that demonstrated activity in vitro which allows to deepen into the immune response towards mycobacterial antigens, moving forward to the identification of new vaccine candidates.
Asunto(s)
Antígenos Bacterianos/inmunología , Linfocitos T CD4-Positivos/inmunología , Mycobacterium tuberculosis/inmunología , Vacunas contra la Tuberculosis/inmunología , Humanos , Activación de Linfocitos/inmunología , Péptidos/inmunología , Vacunas de Subunidad/inmunologíaRESUMEN
In the last few years, the great impact of antimicrobial peptides on infectious disease susceptibility and natural resistance has been reported. In some cases, susceptibility to diseases is related to antimicrobial peptide polymorphisms and gene copy numbers, but for the vast majority of infectious diseases, these phenomena need to be elucidated. This review is focused on the current knowledge about susceptibility and resistance conferred by genetic variations in antimicrobial peptide expression in infectious diseases.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/inmunología , Enfermedades Transmisibles/genética , Enfermedades Transmisibles/inmunología , Predisposición Genética a la Enfermedad , Animales , HumanosRESUMEN
Type 2 diabetes mellitus (DM2) is strongly associated with other comorbidities such as obesity, atherosclerosis, and hypertension. Obesity is associated with sustained low-grade inflammatory response due to the production of proinflammatory cytokines. This inflammatory process promotes the differentiation of some myeloid cells, including myeloid-derived suppressor cells (MDSCs). In this study, two groups of individuals were included: DM2 patients and non-DM2 individuals with similar characteristics. Immunolabeling of CD15+ CD14- and CD33+ HLA-DR-/low was performed from whole peripheral blood, and samples were analyzed by flow cytometry, and frequencies of MDSCs and the relationship of these with clinical variables, cytokine profile (measured by cytometric bead array), and anthropometric variables were analyzed. The frequency of CD33+ HLA-DR-/low MDSCs (that produce IL-10 and TGF-ß, according to an intracellular detection) is higher in patients with DM2 (P < 0.05), and there is a positive correlation between the frequency of CD15+ CD14- and CD33+ HLA-DR-/low MDSC phenotypes. DM2 patients have an increased concentration of serum IL-5 (P < 0.05). Also, a negative correlation between the frequency of CD15+ CD14- MDSCs and LDL cholesterol was found. Our group of DM2 patients have an increased frequency of mononuclear MDSC CD33+ HLA-DR-/low that produce TGF-ß and IL-10. These cytokines have been associated with immune modulation and reduced T cell responses. DM2 and non-DM2 subjects show a similar cytokine profile, but the DM2 patients have an increased concentration of IL-5.
Asunto(s)
Diabetes Mellitus Tipo 2/inmunología , Hipertensión/inmunología , Células Supresoras de Origen Mieloide/inmunología , Adulto , Femenino , Antígenos HLA-DR/análisis , Humanos , Interleucina-10/biosíntesis , Interleucina-5/sangre , Masculino , Persona de Mediana Edad , Lectina 3 Similar a Ig de Unión al Ácido Siálico/análisis , Factor de Crecimiento Transformador beta/biosíntesisRESUMEN
BACKGROUND: Pulmonary tuberculosis (PTB) is a public health problem with 10.4 million new cases reported in 2017 (1). According to the World Health Organization (WHO), accurate diagnostic tests based in serum biomarkers to detect new cases of tuberculosis are necessary. AIM OF THE STUDY: To evaluate antibodies against Mycobacterium. tuberculosis (Mtb) peptides (Ab-Mtb) and three soluble host biomarkers by ELISA serial multiple test in sera from non-infected controls (NIC, n = 31), latent tuberculosis (LTB, n = 37) and PTB (n = 28) patients in a diagnosis tuberculosis assay. MATERIALS AND METHODS: Levels of four Ab-Mtb peptides derived from Mtb and three host response molecules in serum from NIC, LTB and PTB were evaluated by ELISA as tuberculosis biomarkers. Multiple comparisons tests, determination of diagnostic values and ROC curves were performed. Serial and parallel multiple tests were performed with the biomarkers with the highest discriminatory capacity to improve diagnostic values of the test. RESULTS: We found significant differences between biomarkers levels in PTB comparing LTB and NIC to all candidate biomarkers; peptides P12033, P12037, and serum biomarkers such as sCD14 and chemokine CXCL9 showed the best sensitivity and specificity, the highest discriminatory power, and the best area under the curve (AUC) individually. In serial multiple tests, P12037 and sCD14 together have 92% of sensitivity and 91% of specificity, with positive and negative likelihood ratios greater than 10. CONCLUSIONS: Ab-Mtb peptide P12037 and sCD14 could be applied in a diagnostic test for suspected PTB to improve accuracy and time to diagnosis and could be implemented in a POCT device which can be affordable.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas Bacterianas/inmunología , Tuberculosis Latente/diagnóstico , Mycobacterium tuberculosis/inmunología , Tuberculosis Pulmonar/diagnóstico , Adulto , Área Bajo la Curva , Biomarcadores/sangre , Niño , Pruebas Diagnósticas de Rutina , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Tuberculosis Latente/microbiología , Masculino , Curva ROC , Sensibilidad y Especificidad , Tuberculosis Pulmonar/microbiologíaRESUMEN
BACKGROUND AND AIMS: Type 2 diabetes mellitus (DM2) confers a higher risk for active tuberculosis (TB). However, information on associated risk factors for latent tuberculosis infection (LTBI) inpatients with DM2 is limited. We conducted a cross-sectional study to elucidate the prevalence of LTBI and its associated factors on Mexican adults with DM2 receiving medical care at the Mexican Social Security Institute (IMSS). METHODS: Six hundred patients with DM2 without a prior history of TB from outpatient diabetes clinics were enrolled in the study. The tuberculin-skin-test (TST) was performed. The presence of LTBI was defined by a TST value of ≥ 5 mm. A standardized interview and physical examination were conducted to obtain clinical, demographic, and LTBI risk factor information; all subjects were laboratory tested to determine the presence of exclusion criteria. Microscopic examination of sputum samples and chest x-rays was performed to identify potential active TB. Subjects with any finding suggesting active TB or malignancy were excluded. A logistic regression model was used to identify variables associated with LTBI. RESULTS: LTBI prevalence among patients with DM2 was 51.3%. Risk factors for LTBI were living with a relative with TB, having been in prison, having hemoglobin values >14 g/dL, and glycosylated hemoglobin (HbA1c) values of > 7%. Blood pressure, economic income, or anthropometric measurements were not associated risk factors. CONCLUSIONS: Over one half of patients with DM harbor LTBI. Exposure to certain environmental conditions and poorly controlled DM2 (HbA1c > 7.0%) were risk factors for having LTBI in persons with DM2.
Asunto(s)
Diabetes Mellitus Tipo 2/epidemiología , Tuberculosis Latente/epidemiología , Estudios Transversales , Femenino , Humanos , Tuberculosis Latente/diagnóstico , Modelos Logísticos , Masculino , México/epidemiología , Persona de Mediana Edad , Prevalencia , Factores de Riesgo , Prueba de TuberculinaRESUMEN
Capacitative Ca(2+) entry is a process whereby the activation of Ca(2+) influx through the plasma membrane is triggered by depletion of intracellular Ca(2+) stores. Some transient receptor potential (TRPC) proteins have been proposed as candidates for capacitative Ca(2+) channels. Recent evidence indicates that capacitative Ca(2+) entry participates in the sperm acrosome reaction (AR), an exocytotic process necessary for fertilization. In addition, several TRPCs have been detected heterogeneously distributed in mouse sperm, suggesting that they may participate in other functions such as motility. Using reverse transcription-polymerase chain reaction (RT-PCR) analysis, RNA messengers for TRPC1, 3, 6 and 7 were found in human spermatogenic cells. Confocal indirect immunofluorescence revealed the presence of TRPC1, 3, 4 and 6 differentially localized in the human sperm, and immunogold transmission electron microscopy indicated that TRPC epitopes are mostly associated to the surface of the cells. Because all of them were detected in the flagellum, TRPC channel antagonists were tested in sperm motility using a computer-assisted assay. Our results provide what is to our knowledge the first evidence that these channels may influence human sperm motility.
Asunto(s)
Canales de Calcio/análisis , Canales de Calcio/fisiología , Motilidad Espermática , Espermatozoides/química , Espermatozoides/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Inmunohistoquímica , Masculino , ARN Mensajero/biosíntesis , Espermatozoides/ultraestructura , Canales Catiónicos TRPCRESUMEN
The induction of cytochrome P450 (CYP) 2E1 in testes and liver and the presence of trifluoroacetylated (TFA) adducts in spermatozoa, testes, liver and plasma were investigated in rats subchronically exposed by inhalation to halothane (15 ppm/4 h/day/5 days/week/9 weeks). After halothane exposure, p-nitrophenol hydroxylase (p-NPH) activity increased 3.2-fold and CYP2E1 apo-protein content 7-fold in testes, whereas in liver, p-NPH increased 2.3-fold and CYP2E1 apoprotein content 1.4-fold. These results suggest a differential inductive effect of halothane on CYP2E1 in these tissues. Moreover, TFA adducts were present in microsomes of testis and liver and in plasma of halothane-treated rats. The immunoblot analysis of testicular microsomes showed two intense TFA protein bands of 63 and 59 kDa, whereas in liver three intense bands of 100, 76 and 63 kDa were observed. Bands of similar molecular weights to those observed in liver were detected in the plasma of halothane-treated animals. In addition, TFA adducts were detected by immunofluorescence in spermatozoa, probably in the acrosome and/or perinuclear theca region, and in the distal tail of spermatozoa. The increase in CYP2E1 apoprotein and p-NPH activity observed in testis and liver microsomes suggests that halothane induces its own biotransformation both hepatically and extrahepatically and in addition, that the nature of the TFA adducts will depend on the proteins present in each tissue. Also, the presence of TFA adducts in spermatozoa may result from the activation of halothane in the reproductive tract. The detailed mechanism of TFA adduct formation and its consequences on the spermatozoa function remain to be fully clarified.
Asunto(s)
Anestésicos por Inhalación/toxicidad , Citocromo P-450 CYP2E1/biosíntesis , Halotano/toxicidad , Hígado/efectos de los fármacos , Espermatozoides/efectos de los fármacos , Testículo/efectos de los fármacos , Ácido Trifluoroacético/toxicidad , Administración por Inhalación , Animales , Biomarcadores , Peso Corporal/efectos de los fármacos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Cromatina/efectos de los fármacos , Citocromo P-450 CYP2E1/metabolismo , Electroforesis en Gel de Poliacrilamida , Inducción Enzimática/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Indicadores y Reactivos , Hígado/enzimología , Hígado/patología , Masculino , Microsomas Hepáticos/efectos de los fármacos , Microsomas Hepáticos/enzimología , Piridinas/farmacología , Ratas , Ratas Wistar , Espermatozoides/enzimología , Espermatozoides/patología , Testículo/enzimología , Testículo/patología , Ácido Trifluoroacético/sangreRESUMEN
Diabetic foot ulcers (DFUs) are chronic wounds with high matrix metalloproteinase (MMP) activity, and are a frequent complication on diabetics. This work studied the expression of selected MMP and tissue inhibitor of metalloproteinases (TIMP) gene family members in DFU and normal skin biopsies, and in vitamin D-treated keratinocytes cultured from those biopsies. We report for the first time the expression of some of these genes in healthy skin. Our results suggest that vitamin D may modulate the expression of some MMP gene family members in keratinocytes. Gene expression in DFU and in non-diabetic healthy skin (control) biopsies was evaluated by RT-qPCR for MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-19, TIMP-1 and TIMP-2, and also by immunohistochemistry for MMP-1 and MMP-9. Primary keratinocytes cultured from DFU and healthy skin biopsies were used for gene expression analyses of selected MMPs and TIMPs by RT-qPCR, both in the presence and absence of calcitriol. The expression of MMP-1, MMP-8, MMP-9, MMP-10, and TIMP-2 in healthy skin is reported here for the first time. DFUs showed increased MMP-1, MMP-9 and TIMP-1 expression, compared to healthy skin. Calcitriol down-regulated MMP-1 and MMP-10 expression in DFU-derived keratinocytes but not in those derived from healthy skin. Our data demonstrate the expression of certain MMPs that had not been previously described in healthy skin, and further support previous reports of MMP and TIMP up-regulation in DFUs. Our results point to calcitriol as a potential modulator for the expression of certain MMP members in DFUs.
Asunto(s)
Calcitriol/farmacología , Pie Diabético/enzimología , Queratinocitos/efectos de los fármacos , Queratinocitos/enzimología , Metaloproteinasas de la Matriz/metabolismo , Inhibidores Tisulares de Metaloproteinasas/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Biopsia , Estudios de Casos y Controles , Células Cultivadas , Pie Diabético/genética , Femenino , Regulación de la Expresión Génica , Humanos , Inmunohistoquímica , Masculino , Metaloproteinasas de la Matriz/genética , Persona de Mediana Edad , Inhibidores Tisulares de Metaloproteinasas/genética , Transcripción GenéticaRESUMEN
A cohort of 123 adult contacts was followed for 18‐24 months (86 completed the follow-up) to compare conversion and reversion rates based on two serial measures of QuantiFERON (QFT) and tuberculin skin test (TST) (PPD from TUBERSOL, Aventis Pasteur, Canada) for diagnosing latent tuberculosis (TB) in household contacts of TB patients using conventional (C) and borderline zone (BZ) definitions. Questionnaires were used to obtain information regarding TB exposure, TB risk factors and socio-demographic data. QFT (IU/mL) conversion was defined as <0.35 to ≥0.35 (C) or <0.35 to >0.70 (BZ) and reversion was defined as ≥0.35 to <0.35 (C) or ≥0.35 to <0.20 (BZ); TST (mm) conversion was defined as <5 to ≥5 (C) or <5 to >10 (BZ) and reversion was defined as ≥5 to <5 (C). The QFT conversion and reversion rates were 10.5% and 7% with C and 8.1% and 4.7% with the BZ definitions, respectively. The TST rates were higher compared with QFT, especially with the C definitions (conversion 23.3%, reversion 9.3%). The QFT conversion and reversion rates were higher for TST ≥5; for TST, both rates were lower for QFT <0.35. No risk factors were associated with the probability of converting or reverting. The inconsistency and apparent randomness of serial testing is confusing and adds to the limitations of these tests and definitions to follow-up close TB contacts.
Asunto(s)
Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Composición Familiar , Tuberculosis Latente/diagnóstico , Tuberculosis Latente/transmisión , Prueba de Tuberculina/métodos , Trazado de Contacto , Progresión de la Enfermedad , Exposición a Riesgos Ambientales , Estudios de Evaluación como Asunto , Estudios de Seguimiento , Tuberculosis Latente/clasificación , Tuberculosis Latente/epidemiología , México/epidemiología , Factores de Riesgo , Factores Socioeconómicos , Encuestas y CuestionariosRESUMEN
BACKGROUND: The transient receptor potential channel (TRP) family includes more than 30 proteins; they participate in various Ca(2+) dependent processes. TRPs are functionally diverse involving thermal, chemical and mechanical transducers which modulate the concentration of intracellular Ca(2+) ([Ca(2+)]i). Ca(2+) triggers and/or regulates principal sperm functions during fertilization such as motility, capacitation and the acrosome reaction. Nevertheless, the presence of the TRPM subfamily in sperm has not been explored. PRINCIPAL FINDINGS: Here we document with RT-PCR, western blot and immunocitochemistry analysis the presence of TRPM8 in human sperm. We also examined the participation of this channel in sperm function using specific agonists (menthol and temperature) and antagonists (BCTC and capsazepine). Computer-aided sperm analysis revealed that menthol did not significantly alter human sperm motility. In contrast, menthol induced the acrosome reaction in human sperm. This induction was inhibited about 70% by capsazepine (20 microM) and 80% by BCTC (1.6 microM). Activation of TRPM8 either by temperature or menthol induced [Ca(2+)]i increases in human sperm measured by fluorescence in populations or individual sperm cells, effect that was also inhibited by capsazepine (20 microM) and BCTC (1.6 microM). However, the progesterone and ZP3-induced acrosome reaction was not inhibited by capsazepine or BCTC, suggesting that TRPM8 activation triggers this process by a different signaling pathway. CONCLUSIONS: This is the first report dealing with the presence of a thermo sensitive channel (TRPM8) in human sperm. This channel could be involved in cell signaling events such as thermotaxis or chemotaxis.