Hallazgos ultraestructurales renales de pacientes con nefritis intersticial crónica de las comunidades agrícolas en El Salvador / Renal ultrastructural findings of patients with chronic interstitial nephritis from farming communities in El Salvador

Introducción: Desde hace 20 años se presenta en Centroamérica una enfermedad renal crónica que fundamentalmente afecta a hombres agricultores y no asociada a las causas tradicionales. Se caracteriza por presentar una nefritis intersticial crónica, en tanto las características ultraestructurales no se conocen con exactitud. En su origen se invoca el uso de agroquímicos y otros agentes nefrotóxicos, la deshidratación crónica, el consumo de medicamentos, entre otros factores. Objetivo: Describir las características ultraestructurales de la nefritis intersticial crónica en comunidades agrícolas. Método: Se realizó un estudio descriptivo de corte transversal. Se estudiaron muestras de biopsias renales de ocho pacientes con diagnóstico de nefritis intersticial crónica de las comunidades agrícolas. Resultados: De los ocho pacientes estudiados, dos (25 por ciento) trabajaban en labores agrícolas y cinco eran del sexo femenino (62,5 por ciento). Dos de los pacientes (25 por ciento) presentaban una enfermedad renal crónica estadio 2, y seis (75 por ciento) estadio 3. En cinco pacientes se hallaron fagolisosomas con presencia de componente lipídico entremezclado con material electrodenso en células del túbulo distal. En igual cantidad de pacientes se observaron cuerpos mieloides con zonas laminadas y núcleo central en células de túbulo proximal y de los vasos sanguíneos. Conclusiones: En pacientes de comunidades agrícolas que padecen nefritis intersticial crónica se evidencian fagolisosomas y estructuras mieloides en túbulos y vasos renales, cuyo contenido y origen se desconocen(AU)

Introduction: Chronic kidney disease mainly affecting male farmers and not associated to traditional causes has been present in Central America for twenty years. The condition is characterized by the presence of chronic interstitial nephritis, but its ultrastructural features are not fully known. Factors suggested as responsible for its occurrence include the use of agrochemicals and other nephrotoxic agents, chronic dehydration and medicine consumption. Objective: Describe the ultrastructural characteristics of chronic interstitial nephritis in farming communities. Method: A cross-sectional descriptive study was conducted of renal biopsy samples from eight patients diagnosed with chronic interstitial nephritis in farming communities. Results: Of the eight patients studied, two (25 percent) were farm workers and five (62.5percent) were female. Two of the patients (25 percent) had stage 2 and six (75 percent) stage 3 chronic kidney disease. In five patients evidence was found of phagolysosomes with lipid component mixed with electrodense material in distal tubule cells. An equal number of patients had myeloid bodies with laminated areas and central nucleus in proximal tubule and blood vessel cells. Conclusions: Evidence of phagolysosomes and myeloid structures of unknown content and origin was found in renal tubules and vessels of patients from farming communities diagnosed with chronic interstitial nephritis(AU)

Morphological analysis of autophagy / 药学学报

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional subcellular organelles and proteins in all living cells. The process of autophagy can be divided into three relatively independent steps: the initiation of phagophore, the formation of autophagosome and the maturation/degradation stage. Different morphological characteristics and molecular marker changes can be observed at these stages. Morphological approaches are useful to produce novel knowledge that would not be achieved through other experimental methods. Here we summarize the morphological methods in monitoring autophagy, the principles in data interpretation and the cautions that should be considered in the study of autophagy.

Inhibitory effect of red ginseng acidic polysaccharide from Korean red ginseng on phagocytic activity and intracellular replication of Brucella abortus in RAW 264.7 cells

Korean red ginseng (KRG) has long been used in traditional Korean and Oriental medicine. However, the anti-bacterial mechanism and therapeutic efficiency of KGR for intracellular Brucella infection are still unclear. In this study, the bactericidal activity of Korean red ginseng acidic polysaccharide (RGAP) on Brucella (B.) abortus and its cytotoxic effects on RAW 264.7 cells were evaluated. In addition, B. abortus internalization and intracellular replication in macrophages were investigated after RGAP treatment. RGAP-incubated cells displayed a marked reduction in the adherence, internalization and intracellular growth of B. abortus in macrophages. Furthermore, decreased F-actin fluorescence was observed relative to untreated B. abortus-infected cells. Western blot analysis of intracellular signaling proteins revealed reduced ERK, JNK and p38α phosphorylation levels in B. abortus-infected RGAP-treated cells compared to the control. Moreover, elevated co-localization of B. abortus-containing phagosomes with lysosome-associated membrane protein 1 (LAMP-1) were observed in RGAP-treated cells compared with the control. Overall, the results of this study suggest that RGAP can disrupt phagocytic activity of B. abortus via suppression of mitogen-activated protein kinases (MAPKs) signaling proteins ERK, JNK and p38 levels and inhibit intracellular replication of B. abortus by enhancing phagolysosome fusion, which may provide an alternative control of brucellosis.

Hepatitis B vaccination in adolescents living in Campinas, São Paulo, Brazil / Vacinação contra hepatite B em adolescentes residentes em Campinas, São Paulo, Brasil

Viral hepatitis is an important public health problem in Brazil and around the world.

As hepatites virais constituem importante problema de saúde pública no Brasil e em todo o mundo.

Phosphorylation of Atg31 is required for autophagy

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.

Genetic characterization of Toxoplasma gondii isolates from eared doves (Zenaida auriculata) in Brazil / Caracterização genética de isolados de Toxoplasma gondii de pombos (Zenaida auriculata) no Brasil

Eared doves (Zenaida auriculata), which are common in urban, rural and wild areas in many regions of Brazil, are frequently prey for domestic cats. Therefore Toxoplasma gondii isolates obtained from doves may reflect greater environmental diversity than those from other hosts. The aim of the present study was to evaluate T. gondii seroprevalence, isolate and genotype strains from Z. auriculata. Serum and tissue samples were collected from 206 doves for use in the modified agglutination test (MAT) and mouse bioassay. The prevalence of T. gondii antibodies in the doves was 22.3% (46/206), with titers ranging from 16 to 4096, and T. gondii strains were isolated from 12 of these doves. Five genotypes were detected by means of PCR-RFLP, including ToxoDB genotypes #1, #6, #17 and #65, and one genotype that had not previously been described (ToxoDB#182). This was the first report on isolation of T. gondii from Z. auriculata. This study confirmed the genetic diversity of T. gondii isolates and the existence of clonal type II (ToxoDB genotype #1) in Brazil.

Pombos silvestres (Zenaida auriculata), comuns em áreas urbanas, rurais e selvagens em muitas regiões do Brasil, são frequentemente predados por gatos domésticos. Sendo assim, os isolados de T. gondii obtidos de pombos podem refletir uma maior diversidade ambiental do que os outros hospedeiros. O objetivo do presente estudo foi avaliar a soroprevalência, isolar e genotipar T. gondii de Z. auriculata. Amostras de soro e tecido foram coletadas de 206 pombos para o teste de aglutinação modificado (MAT) e o bioensaio em camundongos. A prevalência de anticorpos contra T. gondii em pombos foi 22,3% (46/206), com títulos variando de 16 a 4096, e T. gondii foi isolado de 12 pombos. Cinco genótipos foram detectados por PCR-RFLP, incluindo os genótipos ToxoDB #1, #6, #17, #65 e um genótipo não descrito anteriormente (ToxoDB#182). Esse é o primeiro relato de isolamento de T. gondii de Z. auriculata. Este estudo também confirmou a diversidade dos isolados de T. gondii e a presença de tipo clonal II (ToxoDB #1) no Brasil.

Assessment of phagosomes infected with Mycobacterium tuberculosis as a vaccine candidate against tuberculosis.

The present study describes a novel and simple vaccination strategy that involve culturing of M. tuberculosis in the macrophage cells. Isolation of phagosome from macrophage (cell line J774) infected with M. tuberculosis (H37) and M. bovis (BCG) at early and late phase of infection was done ensuing the identification and characterization of these phagosome. In vitro study of apoptosis induced by phagosome infected with (H37) and (BCG) was performed. The vaccine candidate with H37 MOI- 1:10 at 3 h, MOI- 1:20 at 1, 1.5, 2.5 and 3 h and BCG MOI- 1:20 at 3.5 h showed percentage apoptosis as 38.64, 39.93, 34.66, 22.56,34.59 and 37.81% respectively. The results designates that macrophages provide cellular niche during infection and illustrate considerable immunogenic property. Novel antigens expressed or secreted by H37 in infected macrophages can provide evidence to be a successful vaccine candidate as it endures enhanced immune response than BCG.

Interferon-γ inhibits group B Streptococcus survival within human endothelial cells

Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS.

Effect of chaperone-mediated autophagy in MPP(+) -induced SH-SY5Y cells and interventional effect of puerarin / 中国中药杂志

<p><b>OBJECTIVE</b>To study the protective effect of puerarin on MPP(+) -induced SH-SY5Y cells by chaperone-mediated autophagy (CMA).</p><p><b>METHOD</b>The Parkinson's disease cell model was established by injuring SH-SY5Y cells with 1 mmol x L(-1) MPP+. The CCK-8 staining was adopted to detect the effect the puerarin of different concentrations on the survival rate of MPP(+)-induced SH-SYSY cells. The autophagosome formation was observed under transmission electron microscope. The AO staining showed the changes in the lysosome activity. RT-PCR was used to detect the changes in Lamp2a and Hsc70 mRNA expressions. The western blotting was adopted to test the expressions of Lamp2a, Hsc70 and alpha-synuclein protein in cells.</p><p><b>RESULT</b>Within the concentration range of 12. 5-50.0 micromol x L(-1), the pretreatment with puerain for 30 minutes could protect the injury of MPP+ in SH-SY5Y cells, and showed a certain dose-effect relationship. The AO staining and electron microscope showed the effect of puerain within the concentration range of 12.5-50.0 micromol x L(-1) on 1 mmol x L(-1) MPP(+)-induced SH-SY5Y cells; autophagosomes emerged in cells, and increased along with the rise in the puerarin dose. The results of the flow cytometry revealed that 50.0 micromol x L(-1) of puerarin could protect against the increase of the ROS level in 1 mmol x L(-1) MPP(+) -induced SH-SY5Y cells and prevent the oxidative injury. The results of RT-PCR and western blotting indicated that puerain within the concentration range of 12.5-50.0 micromol x L(-1) alleviated the MPP(+)-induced SH-SY5Y cell injury, and inhibited the accumulation of alpha-synuclein proteins in MPP(+) -induced SH-SY5Y cells by up-regulating Hsc70, Lamp2a mRNA and protein level.</p><p><b>CONCLUSION</b>Puerarin could protect against the MPP(+) -induced cell injury, whose protective mechanism may be related to the chaperone-mediated autophagy pathway of interventional molecules.</p>

Autophagy in ageing and ageing-related diseases / 药学学报

Autophagy is an important homeostatic cellular recycling mechanism responsible for degrading injured or dysfunctional cellular organelles and proteins in all living cells. Aging is a universal phenomenon characterized by progressive deterioration of cells and organs due to accumulation of macromolecular and organelle damage. Growing evidences indicate that the rate of autophagosome formation and maturation and the efficiency of autophagosome/lysosome fusion decline with age. Dysfunctional autophagy has also been observed in age-related diseases. Autophagy disruption resulted accumulation of mutated or misfolded proteins is the essential feature of neurodegenerative disorders. However, in cancers, fibroproliferative diseases or cardiovascular diseases, autophagy can play either a protective or destructive role in different types of disease, and even in different stages of the same disease. The review will discuss the cellular and molecular mechanisms of autophagy and its important role in the pathogenesis of aging and age-related diseases, and the ongoing drug discovery strategies for therapeutic intervention.

Coronavirus membrane-associated papain-like proteases induce autophagy through interacting with Beclin1 to negatively regulate antiviral innate immunity

Autophagy plays important roles in modulating viral replication and antiviral immune response. Coronavirus infection is associated with the autophagic process, however, little is known about the mechanisms of autophagy induction and its contribution to coronavirus regulation of host innate responses. Here, we show that the membrane-associated papain-like protease PLP2 (PLP2-TM) of coronaviruses acts as a novel autophagy-inducing protein. Intriguingly, PLP2-TM induces incomplete autophagy process by increasing the accumulation of autophagosomes but blocking the fusion of autophagosomes with lysosomes. Furthermore, PLP2-TM interacts with the key autophagy regulators, LC3 and Beclin1, and promotes Beclin1 interaction with STING, the key regulator for antiviral IFN signaling. Finally, knockdown of Beclin1 partially reverses PLP2-TM's inhibitory effect on innate immunity which resulting in decreased coronavirus replication. These results suggested that coronavirus papain-like protease induces incomplete autophagy by interacting with Beclin1, which in turn modulates coronavirus replication and antiviral innate immunity.

Atividade antimicrobiana de diferentes fármacos contra Mycobacterium abscessus organizada em biofilmes ou localizada em fagossomos / Antimicrobial activity of different drugs against Mycobacterium abscessus in biofilms organized or located in phagosomes

A Mycobacterium abscessus subspécie abscessus é um pesadelo quando envolvida em infecção pulmonar que são incuráveis, a despeito do uso de antimicrobianos com atividade in vitro, caso o tratamento não inclua a ressecção cirúrgica da área afetada. É a micobactéria patogência de crescimento rápido mais frequentemente isolada de culturas de sítios pulmonares. Há um número reduzido de opções terapêuticas para o tratamento dessas infecções, e é ainda mais reduzido o número de antimicrobianos que atingem concentrações terapêuticas no compartimento intracelular, em particular no fagossomo. O número limitado de antimicrobianos disponíveis para tratamento apontam a necessidade de determinação do perfil de susceptibilidade frente a antimicrobianos isolados e em combinação, nos compartimentos intra e extracelular. Os objetivos deste estudo foram avaliar: a sensibilidade de M. abscessus estruturadas em biofilmes e presentes no interior dos macrófagos; a ocorrência de sinergismo quando da associação entre fármacos, inibidores de betalactamase e o anti-inflamatório. As combinações entre os antimicrobianos foram apenas indiferente quanto ao FIC e a atividade dos fármacos em biofilme e em macrófagos é bacteriostático

Mycobacterium abscessus subspecies abscessus is a nightmare when involved in lung infection that is incurable, despite the use of antibiotics with in vitro activity, if the treatment does not include surgical resection of the affected area. It is a MCR - rapidly growing mycobacteria pathogenic most frequently isolated from cultures of lung sites. There are a small number of therapeutic options for the treatment of such infections is further reduced and the number of drugs that reach therapeutic concentrations in the intracellular compartment, particularly in the phagosome. The limited number of antimicrobials available for treatment indicate the need for determining the susceptibility profile against antimicrobials alone and in combination, in the intra and extracellular compartments. The objectives of this study were sensitivity of MCR structured biofilms and present in macrophages, the occurrence of synergism when the association between drugs, beta-lactamase inhibitors and anti-inflammatory. Combinations of antimicrobials were just indifferent and the activity of drugs on biofilms and macrophages was bacteriostatic

Autophagic failure promotes the exocytosis and intercellular transfer of alpha-synuclein

The accumulation of abnormal protein aggregates is a major characteristic of many neurodegenerative disorders, including Parkinson's disease (PD). The intracytoplasmic deposition of alpha-synuclein aggregates and Lewy bodies, often found in PD and other alpha-synucleinopathies, is thought to be linked to inefficient cellular clearance mechanisms, such as the proteasome and autophagy/lysosome pathways. The accumulation of alpha-synuclein aggregates in neuronal cytoplasm causes numerous autonomous changes in neurons. However, it can also affect the neighboring cells through transcellular transmission of the aggregates. Indeed, a progressive spreading of Lewy pathology among brain regions has been hypothesized from autopsy studies. We tested whether inhibition of the autophagy/lysosome pathway in alpha-synuclein-expressing cells would increase the secretion of alpha-synuclein, subsequently affecting the alpha-synuclein deposition in and viability of neighboring cells. Our results demonstrated that autophagic inhibition, via both pharmacological and genetic methods, led to increased exocytosis of alpha-synuclein. In a mixed culture of alpha-synuclein-expressing donor cells with recipient cells, autophagic inhibition resulted in elevated transcellular alpha-synuclein transmission. This increase in protein transmission coincided with elevated apoptotic cell death in the recipient cells. These results suggest that the inefficient clearance of alpha-synuclein aggregates, which can be caused by reduced autophagic activity, leads to elevated alpha-synuclein exocytosis, thereby promoting alpha-synuclein deposition and cell death in neighboring neurons. This finding provides a potential link between autophagic dysfunction and the progressive spread of Lewy pathology.

Identification of Atg8 Isoform in Encysting Acanthamoeba

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.

Suppression of low-dose hyper-radiosensitivity in human lung cancer cell line A549 by radiation-induced autophagy / 华中科技大学学报（医学）（英德文版）

This study explored the role of radiation-induced autophagy in low-dose hyperradiosensitivity (HRS) in the human lung cancer cell line A549. A549 cells, either treated with an autophagic inhibitor 3-methyladenine (3-MA), or with a vehicle control, were irradiated at different low doses (≤0.5 Gy). The generation of autophagy was examined by laser scanning confocal microscopy. Western blotting was used to detect the expression of microtubule-associated protein l light chain 3B II (LC3B-II). Flow cytometry (FCM) and clonogenic assays were used to measure the fraction of surviving cells at the low irradiation doses. Our results showed that there was a greater inhibition of autophagic activity, but a higher degree of low-dose HRS in A549 cells treated with 3-MA than in control group. Our data demonstrated that radiation-induced autophagy is correlated with HRS in A549 cells, and is probably one of the mechanisms underlying HRS.

The hydrophobic amino acids involved in the interdomain association of phospholipase D1 regulate the shuttling of phospholipase D1 from vesicular organelles into the nucleus

Phospholipase D (PLD) catalyzes the hydrolysis of phosphatidylcholine to generate the lipid second messenger, phosphatidic acid. PLD is localized in most cellular organelles, where it is likely to play different roles in signal transduction. PLD1 is primarily localized in vesicular structures such as endosomes, lysosomes and autophagosomes. However, the factors defining its localization are less clear. In this study, we found that four hydrophobic residues present in the N-terminal HKD catalytic motif of PLD1, which is involved in intramolecular association, are responsible for vesicular localization. Site-directed mutagenesis of the residues dramatically disrupted vesicular localization of PLD1. Interestingly, the hydrophobic residues of PLD1 are also involved in the interruption of its nuclear localization. Mutation of the residues increased the association of PLD1 with importin-beta, which is known to mediate nuclear importation, and induced the localization of PLD1 from vesicles into the nucleus. Taken together, these data suggest that the hydrophobic amino acids involved in the interdomain association of PLD1 are required for vesicular localization and disturbance of its nuclear localization.

Alteraciones en el reclutamiento y activación de proteínas Rab durante la infección micobacteriana: [revisão] / Alterations in recruitment and activation of Rab proteins during mycobacterial infection: [review]

En el fagosoma, Mycobacterium spp. altera la activación y reclutamiento de diferentes proteínas “del gen Ras de cerebro de rata”, comúnmente conocidas como Rab. En este manuscrito se revisa una serie de reportes que han demostrado que los fagosomas que contienen micobacterias tienen una expresión mayor y sostenida de Rab5, Rab11, Rab14 y Rab22a, y menor o ninguna expresión de Rab7, Rab9 y Rab6. Esto se correlaciona con aumento de la fusión de estos fagosomas con endosomas tempranos y de reciclaje, lo que les permite mantener ciertas características de compartimentos tempranos, permite que las bacterias obtengan acceso a nutrientes y previene la activación de mecanismos contra la micobacteria. La expresión de mutantes constitutivamente activos de las Rab de endosomas tempranos impide la maduración de fagosomas que contienen esferas de látex o micobacterias inactivadas por calor. Mientras que su silenciamiento, mediante ARN de interferencia o mediante dominantes negativos, induce la maduración de fagosomas micobacterianos. Los mecanismos exactos por los que las micobacterias alteran la dinámica de expresión de estas GTPasas, afectando la maduración fagolisosómica, no se han establecido. El problema podría explicarse por defectos en el reclutamiento de las proteínas que interactúan con Rab, como la cinasa-3 del fosfatidilinositol y el antígeno endosómico temprano 1. La identificación de los mecanismos empleados por Mycobacterium spp. para interrumpir el ciclo de activación de las Rab, será esencial para comprender la fisiopatología de la infección micobacteriana y útil como posibles blancos farmacológicos.

At the phagosome level, Mycobacterium spp. alters activation and recruitment of several “Ras gene from rat brain” proteins, commonly known as Rab. Mycobacterial phagosomes have a greater and sustained expression of Rab5, Rab11, Rab14 and Rab22a, and lowered or no expression of Rab7, Rab9 and Rab6. This correlates with increased fusion of the phagosomes with early and recycling endosomes acquiring some features of early phogosomes, allowing the bacteria to gain access to nutrients and preventing the activation of anti-mycobacterial mechanisms. The expression of constitutively active mutants of Rab from the early stage endosomes prevents the maturation of phagosomes containing latex beads or heat-inactivated mycobacteria. Silencing of these mutants by interference RNA or dominant negative forms induces the maturation of mycobacterial phagosomes. The mechanisms have not been established by which mycobacteria alter the expression of these GTPases and thereby shift the phagolysosomal maturation. The problem can be explained by alterations in the recruitment of proteins that interact with Rab, such as phosphoinositide 3-kinases and early endosomal antigen 1. Identifying the mechanisms used by Mycobacterium spp. to disrupt the cycle of Rab activation will be essential to understand the pathophysiology of mycobacterial infections and usefully to potential drug targets.

Autophagy is involved in 6-OHDA-induced dopaminergic cell death / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the role of autophagy in the death of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA).</p><p><b>METHODS</b>Rat models of Parkinson disease (PD) were established by stereotaxic administration of 6-OHDA (8 μg) into the unilateral substantia nigra par compact (SNpc). Autophagosomes in the SNpc were observed with transmission electron microscopy (TEM), and the expression of autophagy-related protein LC3 was determined with immunofluorescence (IF) assay.</p><p><b>RESULTS</b>Under TEM, the autophagosomes were found in the ipsilateral SNpc 6-24 h after 6-OHDA injection, which suggested the activation of autophagy. IF assay showed significantly increased LC3 expression in 6-OHDA-damaged TH-positive neurons as compared to the control group.</p><p><b>CONCLUSIONS</b>The increase of autophagosomes and activation of autophagy may play a role in dopaminergic neuron death induced by 6-OHDA.</p>

Discovery of Diagnostic Biomarkers for Legionnaires' Disease: Virulence Gene Expression Profiling of Legionella pneumophila serogroup 1 in A/J Mouse Model / 감염과화학요법

BACKGROUND: Legionella pneumophila is the causative agent of Legionnaires' disease, a severe form of pneumonia. After L. pneumophila is inhaled through contaminated aerosols, it is phagocytized by alveolar macrophages, multiplies in a specialized phagosome approximately 10 h postinfection, and eventually leads to the death of host cells. Currently available diagnostic tests for Legionella pneumonia have some limitations. This study was conducted to find diagnostic biomarkers for Legionella pneumonia using virulence gene expression profiling in a murine experimental model. MATERIALS AND METHODS: A/J mice were intranasally inoculated with L. pneumophila serogroup 1, and lungs were harvested 4, 8, 24, and 48 h postinfection. The strain grown in buffered yeast extract broth was used as reference samples. Cy-dye labeled cDNA samples were prepared with total RNA from lungs or broth culture, and hybridized on the oligo-microarray slide containing 2,895 genes of L. pneumophila serogroup 1. Virulence gene expression patterns were analyzed using a MIDAS software from TIGR (www.tigr.org). RESULTS: Among a total of 332 virulence genes examined, 17 genes including sidA, lepB, the genes related to flagella assembly (fliR and fliP), LPS lipid A biosynthesis, and the enhanced entry protein EnhA were up-regulated at all four time points. We further confirmed by quantitative real-time reverse transcription PCR that the expression of fliP gene was highly expressed in lung tissue as well as in bronchoalveolar lavage fluids from the mouse infected with L. pneumophila serogroup 1. CONCLUSIONS: Through gene expression analysis of L. pneumophila in a mouse model, several candidate biomarkers for diagnosing Legionnaires' disease could be identified.

CD40, autophagy and Toxoplasma gondii

Toxoplasmagondii represents a pathogen that survives within host cells by preventing the endosomal-lysosomal compartments from fusing with the parasitophorous vacuoles. The dogma had been that the non-fusogenic nature of these vacuoles is irreversible. Recent studies revealed that this dogma is not correct. Cell-mediated immunity through CD40 re-routes the parasitophorous vacuoles to the lysosomal compartment by a process called autophagy. Autophagosome formation around the parasitophorous vacuole results in killing of the T. gondii. CD40-induced autophagy likely contributes to resistance against T. gondii particularly in neural tissue.