Microvascular in-vivo analysis of retrograde venous arterialization of ischemic skeletal muscle

@#<p style="text-align: justify;"><strong>OBJECTIVE:</strong> Nearly 20% of patients with critical limb ischemia will not be suitable for arterial bypass due to distal small vessel occlusion, and venous arterialization of the distal venous bed might be a valuable surgical option. This study demonstrates the in-vivo microcirculatory effects of this type of intervention.</p><p style="text-align: justify;"><strong>METHODS:</strong> Using intravital video microscopy, the authors studied the distal skeletal microcirculatory characteristics following venous arterialization of critical hindlimb ischemia in the rat. 25 Wistar rats underwent proximal ligation of the femoral arteries followed by venous arterialization carried out by anastomosing the saphenous vein to the femoral artery using microsurgery techniques. Microcirculatory hemodynamic conditions of the soleus muscle were observed under normal, ischemic, and arterialized conditions. Fluorescein-labeled red cells were used to measure red cell velocities (Vrbc) at the capillaries, and acridine orange injections used to stain endothelial cell nuclei to measure microcirculatory diameters, and leukocyte nuclei to measure leukocyte adhesion. Laser Doppler Perfusion (LDP) units at the distal limb were measured continuously throughout the procedure.</p><p style="text-align: justify;"><strong>RESULTS:</strong> Proximal femoral arterial ligation resulted in drastic reductions in LDP and Vrbc. Following distal venous arterialization, LDP returned to an average of 41% of baseline. Vrbc returned to near baseline values in 70% of the capillaries. Flow at the capillary and venular system showed frequent reversals and great variations in velocities. Venules and venu-venular anastomoses diameters increased by 50%. There was immediate macromolecular tracer leakage and leukocyte activation was significantly increased in both ischemic and arterialized groups (15 cells vs 156 and 178 cells respectively).</p><p style="text-align: justify;"><strong>CONCLUSION:</strong> Venous arterialization may provide an improvement in microcirculatory velocities but is accompanied by microcirculatory injury and dysfunction in the acute phase. These results suggest that mechanisms besides microcirculatory hemodynamics play a role in the overall picture of clinical effectivity of the procedure</p>

Zebrafish Larvae Model of Dilated Cardiomyopathy Induced by Terfenadine

BACKGROUND AND OBJECTIVES: Dilated cardiomyopathy can be the end-stage of severe cardiac disorders and directly affects the cardiac muscle, inducing cardiomegaly and heart failure (HF). Although a wide variety of animal models are available to study dilated cardiomyopathy, there is no model to assess dilated cardiomyopathy with non-invasive, simple, and large screening methods. METHODS: We developed a dilated cardiomyopathy model in zebrafish larvae using short duration terfenadine, a known cardiotoxic drug that induces ventricular size dilation. Fractional shortening of zebrafish hearts was calculated. RESULTS: We treated zebrafish with 5 to 10 µM terfenadine for 24 hours. In terfenadine-treated zebrafish, blood frequently pooled and clotted in the chamber, and circulation was remarkably reduced. Atria and ventricles were swollen, and fluid was deposited around the heart, mimicking edema. Cardiac contractility was significantly reduced, and ventricular area was significantly enlarged. Heart rate was markedly reduced even after terfenadine withdrawal. Acridine orange staining also showed that terfenadine increased cardiomyocyte apoptosis. A significant increase of natriuretic peptide B (NPPB) mRNA was found in terfenadine-treated zebrafish. A low dose of terfenadine (5–10 µM) did not show mortality in short-term treatment (24 hours). However, moderate dose (35–45 µM) terfenadine treatment reduced zebrafish survival within 1 hour. CONCLUSION: With advantages of rapid sample preparation procedure and transparent observation of the live heart, this model can potentially be applied to large-scale drug screening and toxicity assays for non-ischemic HF.

Reduced Autophagy in 5-Fluorouracil Resistant Colon Cancer Cells

We investigated the role of autophagy in SNUC5/5-FUR, 5-fluorouracil (5-FU) resistant SNUC5 colon cancer cells. SNUC5/5-FUR cells exhibited low level of autophagy, as determined by light microscopy, confocal microscopy, and flow cytometry following acridine orange staining, and the decreased level of GFP-LC3 puncta. In addition, expression of critical autophagic proteins such as Atg5, Beclin-1 and LC3-II and autophagic flux was diminished in SNUC5/5-FUR cells. Whereas production of reactive oxygen species (ROS) was significantly elevated in SNUC5/5-FUR cells, treatment with the ROS inhibitor N-acetyl cysteine further reduced the level of autophagy. Taken together, these results indicate that decreased autophagy is linked to 5-FU resistance in SNUC5 colon cancer cells.

Chracterization of THP-1 Cell Death Induced by Porphyromonas gingivalis Infection

BACKGROUND: Periodontitis is generally a chronic disorder characterized by the breakdown of tooth-supporting tissues. P. gingivalis, a Gram-negative anaerobic rod, is one of the major pathogens associated with periodontitis. Frequently, P. gingivalis infection leads to cell death. However, the correlation between P. gingivalis–induced cell death and periodontal inflammation remains to be elucidated. Among cell deaths, the death of immune cells appears to play a significant role in inflammatory response. Thus, the aim of this study was to examine P. gingivalis–induced cell death, focusing on autophagy and apoptosis in THP-1 cells. METHODS: Human acute monocytic leukemia cell line (THP-1) was used for all experiments. Autophagy induced by P. gingivalis in THP-1 cells was examined by Cyto ID staining. Intracellular autophagic vacuoles were observed by fluorescence microscopy using staining Acridine orange (AO); and 3-methyladenine (3-MA) was used to inhibit autophagy. Total cell death was measured by LDH assay. Cytokine production was measured by an ELISA method. RESULTS: P. gingivalis induced autophagy in an MOI-dependent manner in THP-1 cells, but 3-MA treatment decreased autophagy and increased the apoptotic blebs. P. gingivalis infection did not increase apoptosis compared to the control cells, whereas inhibition of autophagy by 3-MA significantly increased apoptosis in P. gingivalis-infected THP-1 cells. Inhibition of autophagy by 3-MA also increased total cell deaths and inflammatory cytokine production, including IL-1β and TNF-α. CONCLUSION: P. gingivalis induced autophagy in THP-1 cells, but the inhibition of autophagy by 3-MA stimulated apoptosis, leading to increased cell deaths and pro-inflammatory cytokines production. Hence, the modulation of cell deaths may provide a mechanism to fight against invading microorganisms in host cells and could be a promising way to control inflammation.

Dietary supplementation with astaxanthin may ameliorate sperm parameters and DNA integrity in streptozotocin-induced diabetic rats / 대한생식의학회지

OBJECTIVE: Diabetes mellitus (DM) is known to cause many systemic complications as well as male infertility. Astaxanthin (ASTX) is a powerful antioxidant that is involved in a variety of biologically active processes, including those with anti-diabetes effects. The present study investigates the effect of ASTX on the spermatozoa function in streptozotocin (STZ)-induced diabetic rats. METHODS: We divided 30 adult rats into three groups (10 rats per group), with a control group that received corn oil mixed with chow. DM was induced by intra-peritoneal injection of STZ. Eight weeks after the STZ injection, half of the diabetic animals were used as diabetic controls, and the rest were treated with ASTX for 56 days. Then the parameters and chromatin integrity of the epididymal sperm were analyzed using chromomycin A3, toluidine blue (TB), and acridine orange (AO) staining. RESULTS: The count, viability, and motility of the epididymal sperm were decreased significantly in the STZ group in comparison with the control group (count and viability, p<0.001; motility, p<0.001;0.01). ASTX increased normal morphology and viable spermatozoa compared to the STZ group (morphology, p=0.001; viability, p<0.001;0.05). The percentage of abnormal chromatins in TB and AO staining was higher in the STZ group compared to the control group (p<0.001;0.001). The mean percentage of TB and AO positive spermatozoa in STZ rats was significantly lower in the STZ+ASTX group (TB, p=0.001; AO, p<0.001;0.05). CONCLUSION: This study observed that in vivo ASTX treatment partially attenuates some detrimental effect of diabetes. Conversely, ASTX improved sperm viability, normal morphology, and DNA integrity.

Nitric Oxide-Induced Autophagy in MC3T3-E1 Cells is Associated with Cytoprotection via AMPK Activation

Nitric oxide (NO) is important in the regulation of bone remodeling, whereas high concentration of NO promotes cell death of osteoblast. However, it is not clear yet whether NO-induced autophagy is implicated in cell death or survival of osteoblast. The present study is aimed to examine the role of NO-induced autophagy in the MC3T3-E1 cells and their underlying molecular mechanism. The effect of sodium nitroprusside (SNP), an NO donor, on the cytotoxicity of the MC3T3-E1 cells was determined by MTT assay and expression of apoptosis or autophagy associated molecules was evaluated by western blot analysis. The morphological observation of autophagy and apoptosis by acridine orange stain and TUNEL assay were performed, respectively. Treatment of SNP decreased the cell viability of the MC3T3-E1 cells in dose- and time-dependent manner. SNP increased expression levels of p62, ATG7, Beclin-1 and LC3-II, as typical autophagic markers and augmented acidic autophagolysosomal vacuoles, detected by acridine orange staining. However, pretreatment with 3-methyladenine (3MA), the specific inhibitor for autophagy, decreased cell viability, whereas increased the cleavage of PARP and caspase-3 in the SNP-treated MC3T3-E1 cells. AMP-activated protein kinase (AMPK), a major autophagy regulatory kinase, was activated in SNP-treated MC3T3-E1 cells. In addition, pretreatment with compound C, an inhibitor of AMPK, decreased cell viability, whereas increased the number of apoptotic cells, cleaved PARP and caspase-3 levels compared to those of SNP-treated MC3T3-E1 cells. Taken together, it is speculated that NO-induced autophagy functions as a survival mechanism via AMPK activation against apoptosis in the MC3T3-E1 cells.

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (AO/EB) in vivo staining / Producto de excreción de shigella dysenteriae (pesdy) induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (NA/BE)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, were investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5, v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental models for the evaluation of the toxic action of new molecules and new products with therapeutic potential.

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 101, 10-2, 10-3, 10-4 y 10-5, v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del SdyEP, se expresó como dependiente de la concentracion y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10%, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una granpoblación de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental de pez cebra, para la evaluación de la acción tóxica de nuevas moléculas y nuevos productos de excreción bacterial con potencial terapéutico.

NaF-induced Autophagy on SCC25 Human Tongue Squamous Cell Carcinoma Cells

Fluoride has been accepted as an important material for oral health and is widely used to prevent dental caries in dentistry. However, its safety is still questioned by some. Autophagy has been implicated in cancer cell survival and death, and may play an important role in oral cancer. This study was undertaken to examine whether sodium fluoride (NaF) modulates autophagy in SCC25 human tongue squamous cell carcinoma cells. NaF demonstrated anticancer activity via autophagic and apoptotic cell death. Autophagic vacuoles were detectable using observed to form by monodansylcadaverine (MDC) and acridine orange (AO). Analysis of NaF-treated SCC25 cells for the presence of biochemical markers revealed direct effects on the conversion of LC-3II, degradation of p62/SQSTM1, cleavage formation of ATG5 and Beclin-1, and caspase activation. NaF-induced cell death was suppressed by the autophagy inhibitor 3-methyladenine (3-MA). NaF-induced autophagy was confirmed as a pro-death signal in SCC25 cells. These results implicate NaF as a novel anticancer compound for oral cancer therapy.

Excretion product of shigella dysenteriae (sdyep) induced cell death in early larval stage of zebrafish (danio rerio): acridine orange and ethidium bromide (ao/eb) in vivo staining / El producto de excreción de la shigella dysenteriae (pesdy) Induce muerte celular en larvas de pez cebra (danio rerio): marcaje in vivo con naranja de acridina y bromuro de etidio (na/be)

In the field of studies of acute toxicity induced by bacterial agents, Shiga toxins have been relevant due to the severity of the extra-intestinal diseases they cause. Numerous studies have shown that Shiga toxin induced apoptosis in different cell types; however, this important process has been little studied in vivo experimental models. In this study, the effects of excretion products of Shigella dysenteriae, in which Shiga toxin is present, we investigated on early larval stages of Zebrafish, an animal model with many advantages over other in vivo experimental models traditionally used. Both the collection of eggs and larvae of Zebrafish, and the product from excretion from Shigella dysenteriae (SdyEP) were performed according to laboratory standards. Also, toxicity bioassay, larvae treatment with pure and diluted solution, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v SdyEP and cell death in vivo using Acridine Orange (AO) and Ethidium Bromide (EB) were applied. The excretion product of Shigella dysenteriae (SdyEP) effect was expressed in terms of larval mortality and dependent dilution rather than incubation time. The larval population surviving treatment with Shigella excretion product presents severe morphological effects. The larval population generally presents notable severe morphological damage, the necrosis state is represented by the opacity of the larvae after being treated for 24 h (b) compared to control. Other changes associated with larval anatomy were also observed; particularly the caudal end curvature was significant into 10%. The use of AO/EB revealed a distribution pattern from fluorescence into green and orange in surviving larvae SdyEP poisoning, there was a large population of dead cells around the anal and caudal region as evidenced by the presence of orange nuclei in greater numbers as controls in the larvae. The results support the application of coloring AO/EB in Zebrafish experimental...

En el campo de los estudios de toxicidad aguda inducida por agentes bacterianos, las toxinas Shiga resultan relevantes debido a la severidad de las enfermedades extra-intestinales que causan. Numerosos estudios han demostrado que la toxina Shiga induce la apoptosis en diferentes tipos de células, sin embargo, este importante proceso ha sido poco estudiado en modelos experimentales in vivo. En este estudio, fueron evaluados los efectos del productos de excreción de Shigella dysenteriae (PESdy), sobre estadios larvarios de pez cebra (Danio rerio), un modelo animal con muchas ventajas sobre otros modelos experimentales in vivo utilizados tradicionalmente. Tanto la recolección de los huevos y larvas de pez cebra, así como la obtención del producto de la excreción, se realizaron de acuerdo a los estándares de laboratorio. Poblaciones larvarias, fueron tratadas con distintas soluciones; pura y diluidas, 10-1, 10-2, 10-3 , 10-4 and 10-5 v/v de PESdy. La muerte celular in vivo, usando naranja de acridina (NA) y bromuro de etidio (BE) fue evaluada. El efecto del PESdy, se expresó como dependiente de la concentración y del tiempo de exposición. La población de larvas sobrevivientes, presentaron curvatura troncal en un 10 por ciento, en relación a los controles. La necrosis se puso en evidencia a través de la opacidad de las larvas después de 24 h. El uso de NA/BE reveló un patrón de distribución de la fluorescencia en verde y naranja en larvas sobrevivientes al tratamiento. Una gran población de células muertas, alrededor de la región anal y caudal, se ponen en evidencia por la presencia de núcleos de naranja en mayor número que en los controles. Los resultados apoyan la aplicación de la coloración NA/BE en modelo experimental...

Evaluation of micronucleus frequency by acridine orange fluorescent staining in bucccal epithelial cells of oral submucosus fibrosis [OSMF] patients

Oral submucosus fibrosis [OSMF] is a collagen-related disorder seen in habitual betel quids and smokers. This is a high risk precancerous condition in which the connective tissue fibers of the lamina propria and deeper parts of the mucosa becomes stiff with restricted mouth opening. Patients with severe cases have symptoms like difficulties in chewing, swallowing and speaking. In the present study 25 individuals were gutkha chewers and 25 were OSMF patients [chewing gutkha along with smoking] and 25 individuals were taken as controls. A significant increase in the frequency of micronuclei was observed in OSMF patients [34.4 +/- 1.79] as compared to gutkha chewers [14.4 +/- 0.73] and controls [4.36 +/- 0.27]. The number of micronucleated cells in OSMF, gutkha chewers and control groups were 19.84 +/- 0.69, 12.6 +/- 0.51 and 4.20 +/- 0.27, respectively and are significantly different at p < 0.05. Acridine orange is used due its fluorescence nature and easier visibility of the micronucleus present in the buccal epithelial cells. It is concluded that chewing gutkha along with smoking is more dangerous for human health as it hastens the incidence of OSMF

Penetration of erythromycin through Staphylococcus epidermidis biofilm / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The catheter related infection caused by Staphylococcus epidermidis biofilm is increasing and difficult to treat by antimicrobial chemotherapy. The properties of biofilms that give rise to antibiotic resistance are only partially understood. This study aimed to elucidate the penetration of erythromycin through Staphylococcus epidermidis biofilm.</p><p><b>METHODS</b>The penetration ratio of erythromycin through Staphylococcus epidermidis biofilms of 1457, 1457-msrA, and wild isolate S68 was detected by biofilm penetration model at different time points according to the standard regression curve. The RNA/DNA ratio and the cell density within the biofilms were observed by confocal laser microscope and transmission electromicroscope, respectively.</p><p><b>RESULTS</b>The penetration ratios of erythromycin through the biofilms of 1457, 1457-msrA, and S68 after cultivation for 36 hours were 0.93, 0.55 and 0.4, respectively. The erythromycin penetration ratio through 1457 biofilm (0.58 after 8 hours) was higher than that through the other two (0.499 and 0.31 after 24 hours). Lower growth rate of the cells in biofilm was shown, with reduction of RNA/DNA proportion observed by confocal laser microscope through acridine orange stain. Compared with the control group observed by transmission electrmicroscope, the cell density of biofilm air face was lower than that of agar face, with more cell debris.</p><p><b>CONCLUSIONS</b>Erythromycin could penetrate to the Staphylococcus epidermidis biofilm, but could not kill the cells thoroughly. The lower growth rate of the cells within biofilm could help decreasing the erythromycin susceptibility.</p>

Acridine orange stain of Mycobacterium leprae / 나학회지

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the diagnosis of leprosy. However, the fluorescent stain performs better and allows the detection of more positive smears. The limitation for its widespread use has been the high cost for fluorescent microscopes. Novel light-emitting diodes (LED) are inexpensive solutions for fluorescent microscopes, and thus fluorescent stain may be a cost-effective step to improve the diagnosis of leprosy in resource-poor countries. And the comparison of auramine and acridine orange for staining of acid-fast bacteria was showed significantly more acid-fast rods after using acridine orange and the number of "false positive" results was somewhat higher on auramine staining. So acridine orange offers a good alternative to auramine which is considered carcinogenic. This study evaluated the comparison of the Ziehl-Neelson's AFB stain and the acridine orange stain in the skin smear based on PCR. As PCR results were taken as gold standard, results of the study revealed that the sensitivity of Ziehl-Neelson's AFB stain was 50% and that of acridine orange stain was 92.2%. This study confirmed that the fluorescence stain method is more sensitive than the Ziehl-Neelsen's staining method. It is suggested that the training of laboratory technicians on fluorescence microscopy should be scaled up for increased disease control.

The Role of HS-1200 Induced Autophagy in Oral Cancer Cells

Bile acids and synthetic bile acid derivatives induce apoptosis in various kinds of cancer cells and thus have anticancer properties. Recently, it has been suggested that autophagy may play an important role in cancer therapy. However, few data are available regarding the role of autophagy in oral cancers and there have been no reports of autophagic cell death in OSCCs (oral squamous cell carcinoma cells) induced by HS-1200, a synthetic bile acid derivative. We thus examine whether HS-1200 modulates autophagy in OSCCs. Our findings indicate that HS-1200 has anticancer effects in OSCCs, and we observed in these cells that autophagic vacuoles were visible by monodansylcadaverine (MDC)and acridine orange staining. When we analyzed HS-1200-treated OSCC cells for the presence of biochemical markers, we observed that this treatment directly affects the conversion of LC-3II, degradation of p62/SQSTM1 and full-length beclin-1, cleavage of ATG5-12 and the activation of caspase. An autophagy inhibitor suppressed HS-1200-induced cell death in OSCCs, confirming that autophagy acts as a pro-death signal in these cells. Furthermore, HS-1200 shows anticancer activity against OSCCs via both autophagy and apoptosis. Our current findings suggest that HS-1200 may potentially contribute to oral cancer treatment and thus provide useful information for the future development of a new therapeutic agent.

Evaluation of flow cytometry as a diagnostic method for detection of Giardia lambila in comparison to IFAT and other conventional staining techniques in fecal samples

Giardia lamblia is one of the most common diarrhea-related parasites in humans, where infection ranges from asymptomatic to acute or chronic disease. Because of the non-characteristic symptoms of giardiasis, as well as significant prevalence of cyst carriers, it is necessary to upgrade parasitological techniques for examination of feces, in order to avoid false negative results. Accurate diagnosis is important to exclude other parasitic causes of diarrhea. The present study is designed to assess the efficacy of flow cytometry [FC] as a sensitive method for detection of G. lamblia cysts in stool samples in comparison with other standard conventional diagnostic methods. 70 patients [30 males and 40 females] ranging in age from 5 to 60 years were included in this study. Stool samples were taken from each patient on three successive days to detect Giardia cysts and evaluate the intensity of infection. Different methods were used that included concentration methods, permanent stained slides, immunofluorescent antibody test [IFAT] and FC. Giardia lamblia cysts were detected in 24 samples by IFAT with detection rate of 34.3%. Using trichrome stain 20 samples [28.6%] were positive, modified Ziehl- Neelsen Stain [MZN] detected 11samples [15.7%], while Acridine Orange [AO] detected 14 positive samples [20%]. Flow cytometry detected 18 samples with detection rate of 25.7%. Sensitivity and specificity of FC were 90% and 100%, respectively and IFAT sensitivity and specificity were100% and 92%, respectively. Results of the present study clearly demonstrated that incorporation of IFAT and FC can improve sensitivity of detection of Giardia cysts in stool samples. Although FC is more expensive than the other staining methods and IFAT, it is rapid, simple and accurate in estimating the quantity of parasites in each sample. Thus, FC can be recommended for detection of protozoa in stool

Dual Inhibition of PI3K/Akt/mTOR Pathway and Role of Autophagy in Non-Small Cell Lung Cancer Cells / 결핵및호흡기질환

BACKGROUND: The phosphoinositide 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit this pathway are currently under development for lung cancer treatment. In the present study, we have tested whether dual inhibition of PI3K/Akt/mTOR signaling can lead to enahnced antitumor effects. We have also examined the role of autophagy during this process. METHODS: We analyzed the combination effect of the mTOR inhibitor, temsirolimus, and the Akt inhibitor, GSK690693, on the survival of NCI-H460 and A549 non-small cell lung cancer cells. Cell proliferation was determined by MTT assay and apoptosis induction was evaluated by flow cytometry and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Autophagy induction was also evaluated by acridine orange staining. Changes of apoptosis or autophagy-related proteins were evaluated by western blot analysis. RESULTS: Combination treatment with temsirolimus and GSK690693 caused synergistically increased cell death in NCI-H460 and A549 cells. This was attributable to increased induction of apoptosis. Caspase 3 activation and poly(ADP-ribose) polymerase cleavage accompanied these findings. Autophagy also increased and inhibition of autophagy resulted in increased cell death, suggesting its cytoprotective role during this process. CONCLUSION: Taken together, our results suggest that the combination of temsirolimus and GSK690693 could be a novel strategy for lung cancer therapy. Inhibition of autophagy could also be a promising method of enhancing the combination effect of these drugs.

Comparative analysis of Fluorescence stain and Ziehl-Neelsen's AFB stain for Mycobacterium leprae (pilot study) / 나학회지

The Ziehl-Neelson's AFB staining method was mainly used for the AFB observation of the mycobacteria. However, this method has several issues of false negative results, and hence a comparative experiment of the Ziehl-Neelson's AFB staining and the fluorescence staining method was done to remedy this problem. As the fluorescence staining method brightly highlights the AFB in a dark field, and also as it is observed with the lower power objective, it is a method that can better the observation and shorten the time of observation as well. The fluorescence staining method that was used in this experiment did a comparative analysis of the Auramine O-Rhodamine B and the Acridine Orange. The results showed that although the Auramine O-Rhodamine B allows easier observation of the AFB with a high fluorescence expression rate for the multibacillary leprosy sample, the darkness on the periphery makes it hard to observe anything else, while also making it hard to observe the cell changes and paucibacillary leprosy of the AFB. However, the Acridine Orange staining method highlights the cells in dark green and changes the color of the AFB from bright red to orange making it easier to observe bacilli. The results of the study show that the Acridine Orange method is superior to the Auramine O-Rhodamine B method in detecting acid fast bacilli in specimen.

Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons / 소아과

PURPOSE: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). METHODS: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 microM, 10 microM, and 100 microM) on OGD-induced neurotoxicity. RESULTS: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 microM and 100 microM of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 microM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). CONCLUSION: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

Reliability of acridine orange fluorescence microscopy in oral cytodiagnosis.

Context and Aims: The oral cavity is the most predominant location in the head and neck region for primary malignant epithelial tumors. Oral cancer is estimated to be the sixth most common malignancy. Early recognition is imperative for successful treatment and good prognosis. Exfoliative cytology is a simple and reasonably effective technique for rapid initial evaluation of a suspicious oral lesion. The present study was conducted to determine the reliability of acridine orange fluorescence microscopy for cytodiagnosis as a more rapid and easier method for the final evaluation of the cytological specimen. Materials and Methods: Smears were collected from 20 individuals with oral lesions suspicious of malignancy, oral lesions not suggestive of malignancy and normal buccal mucosa. One smear was stained with Papanicolaou stain and another one with acridine orange stain. The differences in the study group and control group were compared by means of the χ2 (Chi-square) test. The results were considered statistically significant whenever P was <0.05. Results: The acridine orange fluorescence stain reliably demonstrated malignant cells based on the differential fluorescence - a cytochemical criterion. The efficacy of the stain was higher than the conventional Papanicolaou stain in screening of oral lesions suspicious of malignancy. However, the acridine orange fluorescence stain did not differentiate effectively between malignant cells and rapidly proliferating cells, as the technique is based on the nucleic acid content. Conclusion: The fluorescent acridine orange method can be used reliably for the screening of carcinomas and it is especially helpful in the follow-up detection of recurrent carcinoma in previously treated cases.

Autophagy of Human Tenon's Capsule Fibroblasts Induced by Mitomycin-C

PURPOSE: The present study investigated whether an autophagic process is involved in the apoptotic death of human tenon's capsule fibroblasts (HTCFs) caused by mitomycin-C. METHODS: An autophagic phenotype was tested using fluorescence microscopy and flow cytometry with specific biological staining dyes including monodansylcadaverine and acridine orange and microtubule-associated protein 1 light chain 3 (LC3). RESULTS: Treatment with mitomycin-C (0.4 mg/ml) increased the acidic vesicular organelles of tenon's capsule fibroblasts in a time dependent manner. Mitomycin-C induced both LC3-II cleavage and beclin-1 expression. 3-MA, a pharmacological inhibitor of autophagy, inhibited the mitomycin-C induced increase of acidic vesicular organelleS. CONCLUSIONS: Autophagy was induced with 0.4 mg/ml mitomycin-C in tenon's capsule fibroblasts. And, autophagic mechanisms may be involved in the early stage of apoptosis of fibroblasts.

Induction of Autophagy by Low Dose of Cisplatin in H460 Lung Cancer Cells / 결핵및호흡기질환

BACKGROUND: Most lung cancer patients receive systemic chemotherapy at an advanced stage disease. Cisplatin-based chemotherapy is the main regimen for treating advanced lung cancer. Recently, autophagy has become an important mechanism of cellular adaptation under starvation or cell oxidative stress. The purpose of this study was to determine whether or not autophagy can occurred in cisplatin-treated lung cancer cells. METHODS: H460 cells were incubated with RPMI 1640 and treated in 5 micrometer or 20 micrometer cisplatin concentrations at specific time intervals. Cells surviving cisplatin treatment were measured and compared using an MTT cell viability assay to cells that underwent apoptosis with autophagy by nuclear staining, apoptotic or autophagic related proteins, and autophagic vacuoles. The development of acidic vascular organelles was using acridine orange staining and fluorescent expression of GFP-LC3 protein in its transfected cells was observed to evaluate autophagy. RESULTS: Lung cancer cells treated with 5 micrometer cisplatin-treated were less sensitive to cell death than 20 micrometer cisplatin-treated cells in a time-dependent manner. Nuclear fragmentation at 5 micrometer was not detected, even though it was discovered at 20 micrometer. Poly (ADP-ribose) polymerase cleavages were not detected in 5 micrometer within 24 hours. Massive vacuolization in the cytoplasm of 5 micrometer treated cells were observed. Acridine orange stain-positive cells was increased according in time-dependence manner. The autophagosome-incorporated LC3 II protein expression was increased in 5 micrometer treated cells, but was not detected in 20 micrometer treated cells. The expression of GFP-LC3 were increased in 5 micrometer treated cells in a time-dependent manner. CONCLUSION: The induction of autophagy occurred in 5 micrometer dose of cisplatin-treated lung cancer cells.