Presence Of Apoptotic Cells Research Articles

Inhibition of tumor growth by recombinant adenovirus containing human lactoferrin through inducing tumor cell apoptosis in mice bearing EMT6 breast cancer

Human lactoferrin (hLTF), an 80-kDa iron-binding glycoprotein, has antitumor activity. In this study, a recombinant adenovirus containing the human lactoferrin cDNA (ad-rhLTF) was constructed and its effect on tumor growth was investigated in mice bearing EMT6 breast cancer. Ad-rhLTF was injected seven times within 14 days into the tumor site at two concentrations (10(8) and 5 × 10(8) pfu/mL) in mice bearing EMT6 breast cancer. Injected ad-rhLTF had considerable cytotoxicity on mice breast cancer, and significantly reducing the weight of tumor produced and increasing the tumor inhibition rate up to 52.64%. The presence of apoptotic cells was confirmed using TUNEL staining and flow cytometry assays. At the same time, RTPCR and Western blot analyses demonstrated that ad-rhLTF also decreased expression of Bcl-2 and increased Bax and caspase 3 expressions. Therefore, we conclude that ad-rhLTF inhibits tumor growth by inducing tumor cell apoptosis in mice with breast cancer by triggering the mitochondrial-dependent pathway and activation of caspase 3. The results indicate that ad-rhLTF might be a promising drug for breast cancer gene therapy.

Antiendothelial Cell Antibodies Induce Apoptosis of Bone Marrow Endothelial Progenitors in Systemic Sclerosis

Patients with systemic sclerosis (SSc) have significantly fewer and functionally impaired endothelial progenitor cells (EPC) in peripheral blood and bone marrow; further, endothelial apoptosis seems to play a primary role in the pathogenesis of vascular damage. We investigated whether the failure of bone marrow EPC is related to their apoptotic phenotype and analyzed the possible mechanisms inducing apoptosis. The presence of apoptotic cells was investigated in bone marrow aspirates taken from patients with SSc; microvessel density (MVD) and the immunohistochemical expression of vascular endothelial growth factor (VEGF) were also measured in bone marrow biopsies. A correlation between EPC apoptosis and the presence of antiendothelial cell antibodies (AECA) was also investigated. We confirmed the presence of bone marrow EPC dysfunction in SSc, while hematopoiesis was not impaired. Bone marrow studies showed a high percentage of apoptotic progenitors, no signs of fibrosis or an altered MVD, and an increased VEGF index. The patients' bone marrow plasma showed significant titers of AECA, and their presence correlated with that of apoptotic progenitors. These findings were further confirmed by an in vitro assay in which the apoptosis of normal progenitors was induced by the addition of AECA+ purified IgG. Our results showed that apoptosis in patients with SSc involves the source compartment of endothelial progenitors and correlates with AECA activity. These findings support the hypothesis that AECA may play a pathogenetic role by affecting the bone marrow EPC machinery that should repair the peripheral vascular lesions.

Cone loss is delayed relative to rod loss during induced retinal degeneration in the diurnal cone-rich rodent Arvicanthis ansorgei

Cone photoreceptor breakdown underlies functional vision loss in many blinding diseases. Cone loss is often secondary to that of rods, but little experimental data are available on the relationship between the two populations. Because of its high cone numbers, we used the diurnal rodent Arvicanthis ansorgei to explore changes in rod and cone survival and function during chemically-induced retinal degeneration. Adult animals received intraperitoneal injections of N-methyl-N-nitrosourea (MNU), and changes in retinal fundus appearance, histology, phenotype, apoptosis (TUNEL staining) and functionality (scotopic and photopic electroretinography) were monitored as a function of post-treatment time and retinal topography. Relative to control animals injected with vehicle only, MNU-injected animals showed time-, region- and population-specific changes as measured by morphological and immunochemical criteria. Histological (gradual thinning of photoreceptor layer) and phenotypical (reduced immunostaining of rhodopsin and rod transducin, and mid wavelength cone opsin and cone arrestin) modifications were first observed in superior central retina at 11 days post-injection. These degenerative changes spread into the superior peripheral and inferior hemisphere during the following 10 days. Rod loss preceded that of cones as visualized by differential immunolabelling and presence of apoptotic cells in rod but not cone cells. By 3 months post-injection, degeneration of the photoreceptor layer was complete in the superior hemisphere, but only partial in the inferior hemisphere. Despite the persistence of cone photoreceptors, scotopic and photopic electroretinography performed at 90 days post-treatment showed that both rod and cone function were severely compromised. In conclusion, MNU-induced retinal degeneration in Arvicanthis follows a predictable spatial and temporal pattern allowing clear separation of rod- and cone-specific pathogenic mechanisms. Compared to other rodents in which MNU has been used, Arvicanthis ansorgei demonstrates pronounced resistance to photoreceptor cell loss.

Effect of fluoride exposure on different immune parameters in humans

Context: T regulatory (Treg) cells play an important role in the modulation of the immune response, and are implicated in the pathogenesis of autoimmune diseases. Many people is exposed to fluoride (F), mainly through drinking water. Objective: The aim of this work was to assess the possible effect of F exposure on different immune parameters, mainly Treg cells.Materials and methods: We studied 61 subjects from a community of the state of Durango, Mexico, where the population is exposed to F levels over 2.0 ppm in drinking water. Peripheral blood mononuclear cells (PBMC) were isolated and the level and function of Treg cells was analyzed by flow cytometry and cell proliferation assays. In addition, we detected the presence of apoptotic cells, the expression of TLR/CD14, and the in vitro synthesis of TNF-α by monocytes.Results: We found a negative correlation between urinary F and percentage of CD4+CD25+ Treg cells (r = −0.55, P < 0.001). Accordingly, a defective function of these cells was detected in 30% of individuals exposed to F. In contrast, a positive association between levels of CD4+TGF-β+ or CD4+IL-10+ Treg lymphocytes and F urine concentrations was detected. In addition, a negative correlation was detected between the F urinary levels and the proportion of apoptotic cells, in PBMC or T cells or monocytes (P < 0.05 in all cases). Finally, no apparent association between F exposure and TLR4/CD14 expression or the synthesis of TNF-α was detected.Conclusion: Our data suggest that F exposure exerts a complex and relevant effect on Treg cells in humans.

Cyanoacrylate Surgical Glue as an Alternative to Suture Threads for Mesh Fixation in Hernia Repair

In recent years, the use of synthetic glues has become an established practice in several areas of surgical treatment. For example, they are used in open and laparoscopic surgery and in digestive tract endoscopy, interventional radiology, and vascular neuroradiology. The experiments in this study were aimed at elucidating that suture-based permanent mesh fixation can be replaced by fixation with N-butyl 2-cyanoacrylate glue (Glubran2) for surgical repair of abdominal wall hernias. In 25 Wistar rats, two hernia defects (1.5 cm in diameter) per animal were created bilaterally in the midline of the abdominal wall. The peritoneum was spared. The lesions were left untreated for 10 d to achieve a chronic condition. Then the defects were covered with TiMESH extralight (2 × 2 cm) and fixed by 30 μL of Glubran2 or traditional suture. The time points of sacrifice were 17 and 28 d, 3, 4, and 5mo. At autopsy, histology and immunohistochemistry were performed to evaluate the inflammatory response and the presence of apoptotic cells respectively. Mesh fixation was excellent in all samples at each time point. At application sites, the inflammatory reaction was mild with a small number of macrophages and vascularized connective tissue presence around glue and mesh threads. Glue residues were observed in histologic sections at each time point. No presence of apoptotic cells was found. This study demonstrated that Glubran2 can effectively replace traditional suture in mesh fixation without affecting tissue healing and determining a physiological inflammatory reaction at the abdominal wall site.

Sonodynamically Induced Apoptosis by Protoporphyrin IX on Hepatoma-22 cells In Vitro

The synergistic effect of ultrasound and certain chemicals on cells is known as sonodynamic therapy (SDT). It has been reported that the direct sonochemical and subsequent redox reactions induced by SDT treatment can lead to apoptotic cell death. However, the detailed biologic mechanism about it is not well understood until now. In this study the effect of low-intensity ultrasound on Hepatoma-22 cells (H22) in the presence of the sonosensitizing drug protoporphyrin IX (PpIX) was evaluated at different incubation times after sonication. Trypan blue exclusion was used to detect cell viability. The presence of apoptotic cells was identified by 4'-6-diamidino-2-phenylindole (DAPI) nuclear staining and transmission electric microscope (TEM) observation. An inverted confocal laser scanning microscope was used to detect the release of mitochondrial protein cytochrome c (Cyt c) and the redistribution of Bcl-2 family proteins Bid and Bax. Additionally, the generation of intracellular reactive oxygen species (ROS) and the loss of mitochondria membrane potential (MMP) were quantificationally measured by a fluorescence microplate reader. The results indicated that the synergistic cytotoxicity of PpIX and ultrasound increased in a time-dependent manner and the mitochondria damage may be the main factor for sonodynamically induced apoptosis by PpIX in H22 cells. (E-mail: lshaof@snnu.edu.cn)

The effect of apoptotic cells on virus-specific immune responses detected using IFN-gamma ELISPOT

Interferon (IFN)-γ ELISPOT can be used to monitor the magnitude of virus-specific cellular immune responses in vaccine trials. Often, IFN-γ ELISPOT is performed with cryopreserved peripheral blood mononuclear cells (PBMC). However, it has not been well defined yet to what extent diminished cell viability of PBMC following cryopreservation affects IFN-γ responses in ELISPOT. Therefore, we assessed the influence of apoptotic cells on the number of spot-forming cells (SFC) in IFN-γ ELISPOT using a gradient of UV-irradiated apoptotic PBMC and viral antigens derived from varicella zoster virus (VZV) and cytomegalovirus (CMV). No SFC were observed when UV-irradiated apoptotic cells were stimulated with VZV or CMV antigens. Moreover, presence of apoptotic cells among viable T cells hampered the detection of SFC following stimulation with VZV or CMV cell lysates, but not with CMVpp65 peptide pool. Statistical analysis showed that mainly late apoptotic cells, staining both Annexin V and 7-amino-actinomycin D (7-AAD), were associated with a decreased number of SFC. In conclusion, it is recommended to use highly viable thawed PBMC for the detection of virus-specific cellular immune responses by IFN-γ ELISPOT, since the detection of CMV- and VZV-specific T cell responses stimulated by cell lysates was significantly impeded by the presence of apoptotic cells.

Review: Endothelial cell dysfunction, medial arterial calcification and osteoprotegerin in diabetes

Macrovascular complications such as cardiovascular disease and peripheral vascular disease are the leading cause of increased mortality and morbidity, respectively, in patients with diabetes mellitus. The aetiopathogenesis of macrovasculopathy in diabetes is multifactorial and differs in types 1 and 2 diabetes. Endothelial cell dysfunction is an early feature of diabetic vasculopathy and is associated with poor glycaemic control. Chronic hyperglycaemia may promote an adverse vascular milieu leading to early endothelial cell apoptosis, in the long run. The presence of apoptotic cells in the vascular lumen may trigger a cascade of reactions between the promoters and inhibitors of arterial calcification. Medial arterial calcification, a characteristic feature of diabetes, is an important predictor of cardiovascular disease and occurs independently of atherosclerosis. Medial arterial calcification may occur in the presence of normal serum calcium and phosphate levels. Osteoprotegerin is an important modulator of mineral metabolism and manifests its effects in the bone and arteries. It is hypothesised that osteoprotegerin is a key inhibitor of arterial calcification which is released by endothelial cells as a protective measure for survival in adverse conditions. It is a potential risk marker for early identification and monitoring of disturbed mineral metabolism and vasculopathy in diabetes.

Phagocytosis of Apoptotic Cells by Neutrophil Granulocytes: Diminished Proinflammatory Neutrophil Functions in the Presence of Apoptotic Cells

Neutrophil granulocytes are rapidly recruited from the bloodstream to the site of acute inflammation where they die in large numbers. Because release of toxic substances from dead neutrophils can propagate the inflammatory response leading to tissue destruction, clearance of dying inflammatory neutrophils has a critical function in the resolution of the inflammatory response. Apoptotic neutrophils are phagocytosed primarily by macrophages, provided these cells are present in adequate numbers. However, macrophages are rare at sites of acute inflammation, whereas the number of neutrophils can be extremely high. In the current study, in vitro experiments with human neutrophils were carried out to investigate whether neutrophils can ingest apoptotic neutrophils. We show that naïve granulocytes isolated from venous blood have a limited capacity to phagocytose apoptotic cells. However, exposure to activating stimuli such as LPS, GM-CSF and/or IFN-gamma results in enhanced phagocytosis of apoptotic cells. The efficient uptake of apoptotic cells by neutrophils was found to depend on the presence of heat labile serum factors. Importantly, the contact to or uptake of apoptotic cells inhibited neutrophil functions such as respiratory burst and the release of the proinflammatory cytokines TNF-alpha and interferon-inducible protein-10. Contact to apoptotic cells, however, induced the secretion of IL-8 and growth-related oncogene-alpha, which was independent of NF-kappaB and p38 MAPK but involved C5a and the ERK1/2 pathway. The data suggest that activated neutrophils participate in the clearance of apoptotic cells. In addition, because apoptotic cells inhibit proinflammatory functions of neutrophils, uptake of apoptotic cells by neutrophils contributes to the resolution of inflammation.

The Role of Toll‐Like Receptor 2 in the Recognition of Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans (previously Actinobacillus actinomycetemcomitans) is a Gram-negative bacterium present in the oral cavity and is usually associated with localized aggressive periodontitis. Isolated antigens from A. actinomycetemcomitans can activate innate immune cells through Toll-like receptors (TLRs), which are molecules that recognize structural components conserved among microorganisms. In this study, we evaluate the role of TLR2 in the recognition of A. actinomycetemcomitans. Macrophages and neutrophils from knockout mice with targeted disruption of TLR2 (TLR2(-/-) mice) and wild-type mice were collected and used for the subsequent assays. The production of cytokines and chemokines was evaluated by enzyme-linked immunosorbent assay (ELISA), and the presence of apoptotic cells was determined by flow cytometry. In addition, the mechanisms that modulate the outcome of A. actinomycetemcomitans-induced periodontal disease in TLR2(-/-) mice were examined. The results show that TLR2-deficient mice developed more severe periodontitis after A. actinomycetemcomitans infection, characterized by significantly higher bone loss and inflammatory cell migration to periodontal tissues. The inflammatory cell influx into the peritoneal cavities of TLR2(-/-) mice was three-fold lower than that observed for the littermate controls. A significantly diminished production of the cytokines tumor necrosis factor-alpha and interleukin-1beta as well as the chemokine CC-ligand-5 in the peritoneal cavities of TLR2(-/-) mice was observed. In addition, a high frequency of apoptotic cells in the inflammatory exudates from TLR2(-/-) mice was observed. Phagocytosis and nitric oxide production was diminished in cells from TLR2(-/-) mice, facilitating the dissemination of the pathogen to the spleen. The results of this study highlight the involvement of TLR2 in recognizing A. actinomycetemcomitans and its essential role in controlling A. actinomycetemcomitans infection.

Inhibitory effects of persistent apoptotic cells on monoclonal antibody production in vitro

Cells undergoing apoptosis in vivo are rapidly detected and cleared by phagocytes. Swiftrecognition and removal of apoptotic cells is important for normal tissue homeostasisand failure in the underlying clearance mechanisms has pathological consequencesassociated with inflammatory and auto-immune diseases. Cell cultures in vitro usuallylack the capacity for removal of non-viable cells because of the absence of phagocytesand, as such, fail to emulate the healthy in vivo micro-environment from which dead cellsare absent. While a key objective in cell culture is to maintain viability at maximal levels,cell death is unavoidable and non-viable cells frequently contaminate cultures insignificant numbers. Here we show that the presence of apoptotic cells in monoclonalantibody-producing hybridoma cultures has markedly detrimental effects on antibodyproductivity. Removal of apoptotic hybridoma cells by macrophages at the time ofseeding resulted in 100% improved antibody productivity that was, surprisingly to us,most pronounced late on in the cultures. Furthermore, we were able to recapitulate thiseffect using novel super-paramagnetic Dead-Cert™Nanoparticles to remove non-viablecells simply and effectively at culture seeding. These results (1) provide direct evidencethat apoptotic cells have a profound influence on their non-phagocytic neighbours inculture and (2) demonstrate the effectiveness of a simple dead-cell removal strategy forimproving antibody manufacture in vitro.

Bioeffects of Low-Energy Continuous Ultrasound on Isolated Sarcoma 180 Cells

Background: The aim of this study was to investigate the mechanism underlying bioeffects of low-intensity continuous ultrasound on isolated sarcoma 180 (S180) cells and cellular responses to these effects. Methods: After sonication, several structural and functional parameters were examined to elucidate ultrasound-induced cell damage. Results: Instant disruption of the cell membrane might be caused by acoustic cavitation, producing mechanical and chemical effects that acted simultaneously on S180 cells; this could be reflected by immediate (morphological) changes such as membrane permeability, membrane fluidity, lipid peroxidation and the generation of hydroxyl radicals in culture medium. Our results of the delayed effects also indicated S180 cells were sensitive to ultrasound-induced apoptosis, and the rate of apoptosis rose gradually with a prolonged incubation time. The presence of apoptotic cells was identified by a distinct morphological form characterized by membrane blebbing, cell shrinkage, chromatin condensation and DNA fragmentation. Moreover, delayed cytotoxicity was accompanied by an increase in intracellular reactive oxygen species (ROS) and a decrease in the mitochondrial membrane potential, and the two events presented obviously a negative correlation. Conclusion: ROS secondarily generated from damaged mitochondria may play a role in the induction of apoptosis.

Propolis Induced Apoptosis in MC Coy-Plovdiv Cells

ABSTRACTPropolis is a natural product with different biological activities. Current research is focused on its ability to suppress tumor growth by induction of apoptosis. However, its selective influence on normal and transformed cells is not fully studied yet.Our previous investigations determined the McCoy-Plovdiv cell line as a model system for in vitro examination of propolis. In the present study we analysed ethanol extracts of propolis from the region of East Rodopi mountains in final concentrations 0,001; 0,1; 1,0; 10 μg/ml.The proliferative activity was examined with MTT and NR cell vitality tests. The type of cell dead was proven via TUNEL test and by fluorescent detection of phosphatidylserine (PS) externalization. Cytotoxicity tests showed decreased cell vitality from 100 to 0% increasing with propolis concentration. The TUNEL test proved the presence of apoptotic cells. Propolis in concentrations 0,001-1,0 μg/ml induced apoptosis in a large number of cells. The results from PS externalization confirmed the same observation. Increasing propolis concentrations augment the intensity of the fluorescence signal and the number of apoptotic cells.Our results evidence that Bulgarian propolis induces apoptosis in McCoy-Plovdiv cells.

Acid-Sensing Ion Channel 3 in Retinal Function and Survival

Changes in extracellular pH occur in the retina and directly affect retinal activity and phototransduction. The authors analyzed the expression in rodent retina of ASIC3, a sensor of extracellular acidosis, and used ASIC3 knockout mice to explore its role in retinal function and survival. The expression and the role of ASIC3 were examined by immunolocalization and by comparing retinas from wild-type and knockout mice at different ages through electroretinography, retinal histology (light and electron microscopy), expression of glial fibrillary acidic protein (GFAP), analysis of cell apoptosis (TUNEL assay), and patch-clamp recordings in primary cultures of retinal ganglion cells (RGCs). ASIC3 is present in the rod inner segment of photoreceptors and in horizontal and some amacrine cells. ASIC3 is also detected in RGCs but does not significantly contribute to ASIC currents recorded in cultured RGCs. At 2 to 3 months, knockout mice experience a 19% enhancement of scotopic electroretinogram a-wave amplitude and a concomitant increase of b-wave amplitude without alteration of retinal structure. Older (8-month-old) knockout mice have 69% and 64% reductions in scotopic a- and b-waves, respectively, and reductions in oscillatory potential amplitudes associated with complete disorganization of the retina and degenerating rod inner segments. GFAP and TUNEL staining performed at 8 and 12 months of age revealed an upregulation of GFAP expression in Müller cells and the presence of apoptotic cells in the inner and outer retina. Inactivation of ASIC3 enhances visual transduction at 2 to 3 months but induces late-onset rod photoreceptor death, suggesting an important role for ASIC3 in maintaining retinal integrity.

Increased apoptosis in circulating lymphocyte cultures of anti-RNA polymerase III positive patients with systemic sclerosis

The sera of 39 patients (38 women and 1 man), 16 with limited and 23 with diffuse clinical form of systemic sclerosis (SSc), were tested for anti-centromere (ACA), anti-topoisomerase I (ATA) and anti-RNA polymerase III (ARA) antibodies. The presence of apoptotic cells in cultures of circulating lymphocytes was investigated using the TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling) technique. ACAs were present in 16 (41%), ATA in 15 (38%) and ARA in 8 (21%) cases. The mean frequency of apoptotic lymphocytes was statistically higher in the ARA positive patients with respect to that in the control population (P < 0.001), in ACA (P < 0.001) and in the ATA (P < 0.001) groups. Moreover, apoptosis was distributed homogenously in ACA and ATA positive subjects, but not in the ARA patients. Our results show that there is an increase in apoptosis in the lymphocytes of ARA positive SSc patients.

Side effects of oxysterols: cytotoxicity, oxidation, inflammation, and phospholipidosis

Oxysterols are 27-carbon atom molecules resulting from autoxidation or enzymatic oxidation of cholesterol. They are present in numerous foodstuffs and have been demonstrated to be present at increased levels in the plasma of patients with cardiovascular diseases and in atherosclerotic lesions. Thus, their role in lipid disorders is widely suspected, and they might also be involved in important degenerative diseases such as Alzheimer's disease, osteoporosis, and age-related macular degeneration. Since atherosclerosis is associated with the presence of apoptotic cells and with oxidative and inflammatory processes, the ability of some oxysterols, especially 7-ketocholesterol and 7beta-hydroxycholesterol, to trigger cell death, activate inflammation, and modulate lipid homeostasis is being extensively studied, especially in vitro. Thus, since there are a number of essential considerations regarding the physiological/pathophysiological functions and activities of the different oxysterols, it is important to determine their biological activities and identify their signaling pathways, when they are used either alone or as mixtures. Oxysterols may have cytotoxic, oxidative, and/or inflammatory effects, or none whatsoever. Moreover, a substantial accumulation of polar lipids in cytoplasmic multilamellar structures has been observed with cytotoxic oxysterols, suggesting that cytotoxic oxysterols are potent inducers of phospholipidosis. This basic knowledge about oxysterols contributes to a better understanding of the associated pathologies and may lead to new treatments and new drugs. Since oxysterols have a number of biological activities, and as oxysterol-induced cell death is assumed to take part in degenerative pathologies, the present review will focus on the cytotoxic activities of these compounds, the corresponding cell death signaling pathways, and associated events (oxidation, inflammation, and phospholipidosis).

The hypothalamic nonapeptides oxytocin and arginine vasopressin promote cardiac myocyte survival in rats with heart failure

The current study examines the hypothesis that the hypothalamic nonapeptides oxytocin (OT) and arginine vasopressin (AVP) are involved in morphogenic modulation of LV remodeling during development of heart failure in rats. Experimental heart failure (HF) has been induced in male Wistar rats (180–230g) by i.m. injections of 0.1 ml 1% mesatonum (phenylephrine) solution and subsequent swimming until exhaustion for 10 days. The small pieces of LV myocardium from the hearts of control and HF rats were explanted for 3 days in medium 199 supplemented with 20 % FBS. The explants were treated with OT (0.5·10−5 IU) or AVP (0.5·10−5 IU) for 72 hrs. The LV myocardium and the hypothalamic‐hypophyseal neurosecretory system (HHNS) were analyzed using light and electron microscopy. The presence of apoptotic cells were detected using TUNEL technique and p53 immunohistochemistry. We found that OT and AVP similarly reduced the number of necrotic and apoptotic cardiac myocytes compared to untreated explants (~4% and ~2% vs. ~15% and ~10%, respectively, P&lt;0.05). However, although neurosecretory cells of the paraventricular and supraoptic nuclei of the hypothalamus were activated in rats with HF, there were signs of inhibition of release of OT and AVP from neurohypophysial terminals. Thus, we suggest that OT and AVP demonstrate a cardioprotective potential by preventing the cardiac myocyte death and that the inhibition of their release from the HHNS during development of heart failure might play a modulatory role in progression of LV remodeling.

Identification and quantification of apoptosis in the kidney using morphology, biochemical and molecular markers

Renal cell apoptosis is important in both physiological conditions such as normal renal development and pathological processes affecting the glomerular, vascular or tubulointerstitial compartments. Apoptosis may result in the detrimental loss of cells following many renal diseases or damaging changes, with significant loss of function. In contrast, apoptosis may control and limit inflammatory processes in both the acute and chronic phases of renal disease. Investigators interested in the presence of apoptotic cells in different forms of renal disease and development need methods to accurately determine the level of apoptosis within the kidney. Apoptosis is a gene-driven mode of cell death that may be identified by distinct morphological features, endonuclease-initiated DNA degradation, and by the involvement of specific apoptosis-regulating proteins. Many research papers that analyse the presence of apoptosis use the in situ terminal deoxyribonucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) assay that detects DNA strand breaks in situ in tissue sections. Localization of activated caspase-3 is now seen as an alternative to TUNEL. This review will discuss some methods of identifying apoptosis in the kidney, using both morphological and biochemical or molecular characteristics, and also discuss some of the pitfalls of entire reliance on biochemical means of apoptotic cell identification without some morphological checks and balances. Although there are some caveats to the methods for identifying apoptotic cells in renal disease, those investigators who take the time to undertake such analysis often gain insightful data that provide explanations for the disease or condition being studied.

Disappearance of Vimentin in Sertoli Cells: A Mono(2-ethylhexyl) Phthalate Effect

The effects of mono(2-ethylhexyl) phthalate (MEHP) on 21-day-old C57Bl/6N mice and their Sertoli cell cultures were studied. Mice were given a single dose of 800 mg/kg MEHP by oral gavage and sacrificed 24 h later. At the same time, testes were harvested from another batch of mice for Sertoli cell cultures. Cultures were subsequently exposed to 0, 1, and 100 nmol/ml MEHP for 0, 3, 6, 12, and 24 h. An antivimentin antibody was used to detect intermediate filament changes in Sertoli cells. Meanwhile, detection of preapoptotic signals and presence of apoptotic cells were done using annexin V-FITC (fluorescein isothiocyanate) and TUNEL (deoxynucleotidyltransferase-mediated dUTP nick end labeling) analyses, respectively. In vivo results showed a correlation between the increase in TUNEL-positive cells and the vimentin disruption in treated mice. Toluidine blue staining of the Sertoli cell cultures showed the increased number and size of vacuoles in Sertoli cell cytoplasm. Vimentin immunohistochemistry showed gradual disappearance of vimentin in Sertoli cell cultures as time and dose increased. Some Sertoli cells were found to be annexin V-FITC positive, but no TUNEL-positive cells were found. Taken together, these results show that the appearance of vacuoles and the vimentin disappearance caused by MEHP in the Sertoli cells are related with each other and can be observed in relation to time. This can be used as an indicator of the loss of mechanical support for spermatogenic cells, which in the end causes apoptosis of spermatogenic cells.

GM-CSF-secreting cancer immunotherapies: preclinical analysis of the mechanism of action.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-secreting tumor cell immunotherapies have demonstrated long-lasting, and specific anti-tumor immune responses in animal models. The studies reported here specifically evaluate two aspects of the immune response generated by such immunotherapies: the persistence of irradiated tumor cells at the immunization site, and the breadth of the immune response elicited to tumor associated antigens (TAA) derived from the immunotherapy. To further define the mechanism of GM-CSF-secreting cancer immunotherapies, immunohistochemistry studies were performed using the B16F10 melanoma tumor model. In contrast to previous reports, our data revealed that the irradiated tumor cells persisted and secreted high levels of GM-CSF at the injection site for more than 21 days. Furthermore, dense infiltrates of dendritic cells were observed only in mice treated with GM-CSF-secreting B16F10 cells, and not in mice treated with unmodified B16F10 cells with or without concurrent injection of rGM-CSF. In addition, histological studies also revealed enhanced neutrophil and CD4+ T cell infiltration, as well as the presence of apoptotic cells, at the injection site of mice treated with GM-CSF-secreting tumor cells. To evaluate the scope of the immune response generated by GM-CSF-secreting cancer immunotherapies, several related B16 melanoma tumor cell subclones that exist as a result of genetic drift in the original cell line were used to challenge mice previously immunized with GM-CSF-secreting B16F10 cells. These studies revealed that GM-CSF-secreting cancer immunotherapies elicit T cell responses that effectively control growth of related but antigenically distinct tumors. Taken together, these studies provide important new insights into the mechanism of action of this promising novel cancer immunotherapy.