Positive Apoptotic Cells Research Articles

Adrenalectomy promotes a permanent decrease of plasma corticoid levels and a transient increase of apoptosis and the expression of Transforming Growth Factor beta1 (TGF-beta1) in hippocampus: effect of a TGF-beta1 oligo-antisense.

BackgroundCorticosterone reduction produced by adrenalectomy (ADX) induces apoptosis in dentate gyrus (DG) of the hippocampus, an effect related to an increase in the expression of the pro-apoptotic gene bax. However it has been reported that there is also an increase of the anti-apoptotic gene bcl-2, suggesting the promotion of a neuroprotective phenomenon, perhaps related to the expression of transforming growth factor β1 (TGF-β1). Thus, we have investigated whether TGF-β1 levels are induced by ADX, and whether apoptosis is increased by blocking the expression of TGF-β1 with an antisense oligonucleotide (ASO) administered intracerebrally in corticosterone depleted rats.ResultsIt was observed an increase of apoptosis in DG, 2 and 5 days after ADX, in agreement with a reduction of corticosterone levels. However, the effect of ADX on the number of apoptotic positive cells in DG was decreased 5 days after the lesion. In CA1–CA3 regions, the effect was only observed 2 days after ADX. TGF-β1 mRNA levels were increased 2 days after ADX. The sustained intracerebro-ventricular administration of a TGF-β1 ASO via an osmotic mini pump increased apoptosis levels in CA and DG regions 5 days after ADX as well as sham-operated control animals. No significant effect was observed following a scrambled-oligodeoxynucleotide treatment.ConclusionThe changes in both the pattern and the magnitude of apoptotic-cell morphology observed 2 and 5 days after ADX suggest that, as a consequence of the reduction of corticosteroids, some trophic mechanisms restricting cell death to a particular time window are elicited. Sustained intracerebral administration of TGF-β1 ASO increased the apoptosis promoted by ADX, suggesting that TGF-β1 plays an anti-apoptotic role in vivo in hippocampus.

Protective effect of protein kinase C on heart function: an experiment with isolated rabbit hearts

To investigate the protective effect of protein kinase C on heart function. The hearts of 40 rabbits were isolated, underwent Langendorff perfusion, and randomly divided into 4 equal groups: Group I (control group, the heart underwent long ischemia or preservation for I hour, was re-warmed and re-infused, and then re-perfused with K-H fluid), Group II (ischemic preconditioning group, perfusion was stopped for 5 minutes before the long ischemia, then the aorta was re-infused), Group III (specific activator of PKC, phorbol myristate acetate was infused inversely via the aorta before the perfusion of K-H fluid), and Group IV (polymyxin B, a specific antagonist of PKC, was infused after the infusion of PKC). Before the preservation and by the end of reperfusion left ventricle end systolic pressure (LVESP), and left ventricle end diastolic pressure (LVDSP) were measured. At the end of experiment specimens of myocardium were collected from each heart to measure the water content, the levels of lactic dehydrogenase and MB isoenzyme of creatine kinase (CK-MB), superoxide dismutase (SOD), and malonyldialdehyde (MDA). TUNEL method was used to measure the number of apoptotic cardiomyocyte. All heart resumed beating. The LVESP of Group II and III were both significantly higher than those of Groups I and IV (all P < 0.05) and the LVESP of Group III was significantly higher than that of Group II too (P < 0.05). The LVEDP of Group II and III were both significantly lower than those of Groups I and IV (all P < 0.05) and the LVEDP of Group III was significantly lower than that of Group II too (P < 0.05). The levels of CK-MB, LDH, and SOD of Group II and III were all significantly higher than those of Groups I and IV (all P < 0.05) and the levels of CK-MB and LDH of Group III was significantly higher than that of Group II too (P < 0.05). The level of MDA of Group II and III were both significantly lower than those of Groups I and IV (all P < 0.05) and the level of MDA of Group III was significantly lower than that of Group II too (P < 0.05). The water contents of Group II and III were both significantly lower than those of Groups I and IV (all P < 0.05). The numbers of TUNEL positive cell and apoptotic cells of Group II and III were all significantly lower than those of Groups I and IV (all P < 0.05). PKC can be activated by transient ischemia and PMA. PKC protects the heart function effectively.

Close localization of DAP-kinase positive tumour-associated macrophages and apoptotic colorectal cancer cells

The death-associated protein kinase (DAP-kinase) is a cytoskeleton-associated protein crucially involved in the induction of early apoptotic pathways. Aberrant hypermethylation of the DAP-kinase promoter plays a major role in tumorigenesis. We aimed to investigate the inactivation of DAP-kinase and its association with apoptotic cell death in 94 colorectal carcinomas. DAP-kinase promoter hypermethylation and mRNA expression were investigated using methylation-specific PCR and real-time RT-PCR, respectively. The expression of DAP-kinase, Fas, and Fas-ligand (FasL) proteins was studied by immunohistochemistry and immunofluorescence. Apoptosis of tumour cells was investigated using the TUNEL assay. DAP-kinase was expressed in tumour cells and tumour-invading macrophages and was closely associated with high numbers of apoptotic tumour cells. DAP-kinase expression co-localized with FasL overexpression in tumour-associated macrophages, and aberrant promoter hypermethylation was verified in more than 50% of carcinomas. There was a tendency for proximal tumours to show DAP-kinase promoter methylation more frequently (p = 0.07). Promoter methylation resulted in a decrease or loss of DAP-kinase protein expression in tumour cells and tumour-associated macrophages. Simultaneously, a decreased apoptotic count and loss of Fas/FasL expression was observed in tumour cells. Our study is the first to demonstrate DAP-kinase expression in invading tumour-associated macrophages in colorectal cancer. The presence of similar expression levels of DAP-kinase in tumour cells and associated macrophages, and their dependence on the promoter methylation status of the tumour cells, suggests cross talk between these cell types during apoptotic cell death.

Expression of type 1 insulin-like growth factor receptor in marrow nucleated cells in malignant hematological disorders: correlation with apoptosis

To verify the expression of type 1 insulin-like growth factor receptor (IGF-IR) and its impact on hematopoietic cells apoptosis in myelodysplastic syndromes (MDS) and acute myeloid leukemias (AML), marrow samples from 16 patients with MDS and 16 patients with AML were examined along with 16 healthy donors as controls. Immunocytochemical methods (alkaline phosphatase anti-alkaline phosphatase) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (fluorescence) were used simultaneously on nucleated cell cytospins. The ratio of IGF-IR positive cells and apoptotic cells as well as the relationship between them were then analyzed separately. A quantitative real-time reverse transcriptase-polymerase chain reaction (PCR) was administrated for six MDS cases and two normal controls to validate IGF-IR expression detected by immunochemistry. In our assay, IGF-IR appeared to have higher to lower expression rate in turn from AML (86.8+/-13.8%) to MDS (56.8+/-14.3%) and then to normal controls (40.4+/-9.6%) (P<0.01 between each group). In MDS nucleated cells, IGF-IR showed stronger expression in refractory anemia with excess blasts (RAEB)/RAEB in transformation/chronic myelomonocytic leukemia subgroup when compared to RA/RA with ringed sideroblasts cases (64.1+/-3.2 vs 53.5+/-16.2%) (P>0.05). Nucleated cells from MDS marrow underwent more apoptosis (5.4+/-3.0%) than that in normal marrow (1.2+/-0.9%) (P<0.01) and AML marrow (0.3+/-0.4%) (also, P<0.01 between each compared group). For both AML and MDS cases, apoptotic signals presented mainly in individual IGF-IR negative cells (9.0+/-4.8%) and less so in IGF-IR positive cells (1.4+/-2.4%) (P<0.01). When analyzed by groups, cell number with IGF-IR expression showed a negative correlation to apoptotic cells amount (r=-0.852; P<0.01) but positive correlation to their blast count (r=0.677; P<0.01). Outcome from real-time quantitative PCR appeared to have a trend of enhanced IGF-IR expression in advanced MDS subtypes. In conclusion, overexpression of IGF-IR existed in hematopoietic cells in MDS and AML marrows, which appeared to be contributed to disease progress.

A mechanism of hyperthermia‐induced apoptosis in ras‐transformed lung cells

Lung cancer, the leading cause of cancer-related deaths in both men and women, is the consequence of disordered apoptosis, induction of which may have therapeutic utility. Hyperthermia has been identified as a stimulus for apoptosis. We investigated the mechanism of hyperthermia-induced cell death in ras-transformed lung cells. Effect of hyperthermia (43 degrees C for 180 min) was compared between two cell lines, an immortalized (sv-40) normal human bronchial epithelial (BEAS2-B) and its malignant transformed (H-ras transfected) counterpart (BZR-T33). Survival after hyperthermia: 7-d growth culture BEAS2-B, 1.03 +/- 0.007 and BZR-T33, 0.39 +/- 0.008 (P < 0.05); clonogenic assays BEAS2-B, 0.76 +/- 0.003 and BZR-T33, 0.41 +/- 0.004 (P < 0.05). Hoechst positive (apoptotic) cells: BEAS2-B, 11 +/- 3% and BZR-T33, 78 +/- 5% (P < 0.05). TUNEL, DNA fragmentation, and Annexin-V all corroborate this result. Western blot comparing the effect of hyperthermia in BZR-T33 cells to BEAS2-B cells revealed: TRAIL and FAS-L displayed significant increases (threefold and twofold, respectively); caspase-3 showed a decrease in uncleaved form and an increase in cleaved form, and a 50-fold increase in activity effectively blocked with the caspase-3 inhibitor DEVD-fmk; caspase-9 showed near depletion of uncleaved; poly (ADP-ribose) polymerase (PARP) degradation was clearly visible during heating. After hyperthermia, gene expression demonstrates a 5.7-fold increase in TRAIL and insignificant changes in tumor necrosis factor-alpha (TNF-alpha), FAS-L, and caspases 3, 8, 9 in transformed cells. Data demonstrated that hyperthermia induces apoptosis in transformed cells, and that apoptosis is mediated by caspase-3 as a result of activation of cell-death membrane receptors of the tumor-necrosis-factor family. In summary, these data suggest that hyperthermia could become an additional modality in the multidisciplinary approach to the treatment of lung cancer.

Role of p53 and reactive oxygen species in apoptotic response to copper and zinc in epithelial breast cancer cells

Previous studies revealed that cells may differ in their response to metal stress depending on their p53 status; however, the sequence of events leading to copper-induced apoptosis is still unclear. Exposure of copper (10 and 25 microM) and zinc (10 and 25 microM) caused activation of p53 in ER+/p53+ human epithelial breast cancer MCF7 cells and resulted in up-regulation of p21. Transactivation of p53 in MCF7 cells also led to increase in expression of Bax, proapototic Bcl-2 family member, triggering mitochondrial pore opening, and PIG3 (p53-induced gene 3 product), and also generation of intracellular reactive oxygen species (ROS). The treatment of MCF7 cells with either copper or zinc for 4 h also caused decrease in mitochondrial membrane potential (Delta psi(m)), accompanied by an elevation in the ROS production and redistribution of p53 into mitochondria. The loss of Delta psi(m) was correlated with accumulation of Annexin V positive apoptotic cells. However, the release of apoptosis inducing factor (AIF) and its translocation into nucleus was observed only in MCF7 cells treated with copper. In MDA-MB-231 (ER-/p53-) and MCF7-E6 (ER+/p53-) cells, both p53 and p21 protein levels were not altered in the presence of metals. These cells were resistant to metals, and there was no alteration in Delta psi(m). Copper treatment did not result in accumulation of ROS in these cell lines with an inactive p53 even after exposure to 50 microM of copper for 6 h, indicating a key role for p53 in the ROS generation. Pretreatment of MCF7 cells with p53 inhibitor, pifithrin-alpha, resulted in decrease of copper and zinc induced ROS production to the control level, suppression of both Bax expression and AIF release. Therefore, the activation of p53 seems to play a crucial role in copper and zinc induced generation of ROS in epithelial breast cancer cells, and expression of downstream targets of p53, such as PIG3 and Bax, responsible for increased generation of the intracellular ROS, as well as disruption of mitochondrial integrity. Our data suggest that copper induces apoptosis in MCF-7 cells with no caspases through the depolarization of mitochondrial membrane with release of AIF and its translocation into the nucleus. The results demonstrate that a functional p53 is required for the execution of apoptosis in epithelial cells.

Apoptosis and Proliferating Cell Nuclear Antigen in Lupus Nephritis (Class IV) and Membranoproliferative Glomerulonephritis

Background: The role of apoptosis in the pathogenesis of renal diseases has not been clearly established. Proliferating cell nuclear antigen (PCNA) is also a proliferation marker. In this study, we investigated the relationship between clinical course and PCNA apoptosis on baseline renal biopsy in patients with Lupus nephritis (LN) and membranoproliferative glomerulonephritis (MPGN). Methods: Thirty-nine patients with proliferative glomerulonephritis [lupus nephritis (LN)[21] and MPGN[18]] were included in this study. PCNA and apoptosis on renal biopsies were detected by immunohistochemical and terminal deoxynucleotidyl transferase mediated dUTP nick end labelling TUNEL methods, respectively. We calculated the ratios of intraglomerular apoptotic cells and PCNA positive cells per glomeruli, and total numbers of apoptotic tubular cells and PCNA positive tubular cells among the 100 tubular cells, and PCNA positive cell and apoptotic cell on two different tubulointerstitial areas (40 × 10). Results: In LN: Apoptotic indexes of glomerulus and tubulus were 1.08 ± 0.49 and 3.71 ± 1.38, respectively. PCNA positivities were found at 16.76 ± 11.34%, 46.57 ± 22.54%, and 40.28 ± 23.14% on glomerulus, tubulus, and interstitium, respectively. The activity index was 11.23 ± 3.41, and the chronicity index was 3.81 ± 1.99. In MPGN: Apoptotic indexes were found at 0.83 ± 0.25 and 3.55 ± 1.75 on glomerulus and tubulus, respectively. PCNA positivities were found at 21.33 ± 18.42%, 35.5 ± 25.99%, and 34.66 ± 26.84% on glomerulus, tubulus, and interstitium, respectively. In controls, apoptosis was not found. In LN: PCNA positivity on tubulus and interstitium were correlated with the activity index (r = 0.768, p < 0.001, r = 0.721, and p < 0.001, respectively). Glomerular PCNA and apoptosis on interstitium and glomerulus were not correlated with the activity index. The activity index also was not correlated with creatinine clearance and daily proteinuria (p = 0.35 for both). At the end of the first year, patients with recovered or stabilized renal function had higher interstitial and tubular PCNA than others in G1 and G2. Conclusion: It can be said that expression of PCNA on renal biopsy was correlated with activity indexes in LN. PCNA may be a prognostic indicator in MPGN and LN. However, apoptosis does not have a predictive value for MPGN and LN.

Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+and cell apoptosis in Caco-2 cells

To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 micromol/L) increased the FI of Caco-2 cells from 27.50+/-13.18 (control, n=150) to 35.71+/-13.99 (n=150, P<0.01), 72.19+/-35.40 (n=150, P<0.01) and 211.34+/-29.03 (n=150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 micromol/L). The FI value of Caco-2 cells treated by FAC was 185.85+/-33.77 (n=150, P<0.01), 122.73+/-58.47 (n=150, P<0.01), and 53.29+/-19.82 (n=150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 micromol/L) increased the FI of Caco-2 cells from 40.45+/-13.95 (control, n=150) to 45.19+/-21.95 (n=150, P<0.01), 89.87+/-43.29 (n=150, P<0.01) and 104.64+/-51.07 (n=150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Early testicular effects in rats perinatally exposed to DEHP in combination with DEHA—apoptosis assessment and immunohistochemical studies

This study aimed to characterize the effects of di(2-ethylhexyl) phthalate (DEHP) on the fetal rat testes and relate them to the effects seen in adults. Histopathological effects in fetal testes were examined with immunohistochemistry for anti-Müllerian hormone (AMH), 3β-hydroxysteroid dehydrogenase, smooth muscle actin (SMA), proliferating cell nuclear antigen (PCNA), histone H3 and vimentin. Additionally, testicular apoptosis levels were assessed in fetal, prepubertal and adult rats. As the plasticizer di(2-ethylhexyl) adipate (DEHA) has similarities with DEHP in chemical structure and metabolism, we investigated if the testicular effects of DEHP were modulated by co-administration with DEHA. Wistar rats were gavaged during gestation and lactation with vehicle, DEHP (300 or 750 mg/kg/day), or DEHP (750 mg/kg/day) in combination with DEHA (400 mg/kg/day), and male offspring were examined at gestation day (GD) 21, postnatal day (PND) 22, 26 and 190. In fetal testes, Leydig cells were found in large clusters containing AMH positive Sertoli cells. At GD 21, seminiferous chords appeared enlarged with an apparently increased number of gonocytes. However, proliferation of gonocytes did not appear increased. A few animals had a high number of TUNEL positive apoptotic cells in degenerating seminiferous tubules at PND 22 and 190, whereas most exposed animals had low levels of germ cell apoptosis at GD 21, PND 22 or PND 26, as evaluated by DNA laddering, TUNEL staining, Caspase-3 immunohistochemistry and Caspase-3 activity measurement. No differences between DEHP and DEHP + DEHA exposed groups were observed.

A Novel Mechanism for Imatinib Mesylate (STI571) Resistance in CML Cells: Contribution of TC-PTP to Modulating Signals Down-Stream from the BCR-ABL Fusion Protein.

Acquired resistance to imatinib mesylate (STI571) in chronic myelogenous leukemia (CML) patients has become a serious problem. To adress the novel molecular mechanism for imatinib-resistance in CML, we previously established imatinib-resistant sublines (designated KTR cells) from the CML cell line KT-1. We have analyzed p-glycoprotein expression, the number of bcr-abl fusion gene and the sequence of ATP binding site of ABL kinase domain. However, these were not responsible for imatinib-resistance in KTR cells. Interestingly, T-cell protein tyrosine phosphatase (TC-PTP) protein levels were markedly down-regulated in all KTR cells as compared to parental KT-1 cells. Therefore, we examined whether the suppression of TC-PTP expression might contribute to imatinib-resistance in KTR cells. We transduced the nuclear isoform of TC-PTP (TC45) and catalytically inactive TC45-D182A cDNA into KTR cells by retroviral gene transfer. Subsequently, we analyzed the sensitivity to imatinib by MTT proliferation assays. We also studied the signaling pathways in all transduced cells by Western blottings. KTR cells successfully expressed TC45 and TC45-D182A protein (designated KTR-TC45 and KTR-D182A cells, respectively). In MTT proliferation assays, the proliferation of KTR-TC45 cells restored their sensitivity to imatinib, but not in KTR-mock or KTR-D182A cells, indicating that transduced catalytically active TC45 restored the sensitivity to imatinib in KTR cells. In KTR2-mock cells, the percentage of annexin V positive apoptotic cells was 8% in the control and was increased to 25% upon imatinib treatment. In KTR-TC45 cells, the percentage of apoptotic cells was increased from 12% to 56% by the treatment with imatinib, suggesting that TC45 expression in KTR cells restored the susceptibility to apoptosis by imatinib mesylate. Taken together, these results indicate that the sensitivity to imatinib in KTR cells can be modulated by TC-PTP expression. In parental KT-1 cells, phosphorylation of STAT5 was abolished with the treatment of 0.5 μM imatinib for 1 hour. In contrast, STAT5 phosphorylation in KTR cells was stronger than that of KT-1 cells and only slightly suppressed upon exposure to 0.5 μM imatinib. In KTR-mock and KTR-D182A transduced cells, STAT5 phosphorylation was augmented compared to KTR-TC45 transduced cells. Upon treatment with 0.5 μM imatinib for 1 hour, phosphorylation of STAT5 was abolished in KTR2-TC45 cells whereas it remained elevated in KTR-mock and KTR-D182A cells. The expression of TC-PTP had no effect on the phosphorylation of the JAK2 or BCR-ABL in KTR cells. Besides, expression of TC-PTP did not alter protein kinase PKB/AKT or mitogen-activated protein kinase signaling in KTR cells. These results indicate that the loss of TC-PTP could enhance tyrosine phosphorylation of STAT5 and was involved in the acquired resistance to imatinib in KTR cells. In conclusion, we demonstrated that reconstitution of TC-PTP in imatinib-resistant KTR cells restored the sensitivity to imatinib. Although it will be necessary to ascertain the relevance of our studies in primary samples, we would like to propose that the loss of TC-PTP may represent a novel mechanism by which CML cells can acquire imatinib-resistance.

Fibronectin suppresses apoptosis and protects mice from endotoxic shock

Multiple-organ failure related to septicemia is a common cause of early mortality after liver transplantation. Endotoxemia following living donor hepatectomy may be a cause of postoperative death. Plasma fibronectin (Fn) exerts a broad range of biological effects on cellular adhesion, motility, differentiation, apoptosis, hemostasis, wound healing, reticuloendothelial system function, and ischemic injury. We studied the therapeutic effect of plasma Fn in mice after an intraperitoneal injection of lipopolysaccharide (LPS) and d-galactosamine (GalN). Female Balb/c mice received simultaneous intraperitoneal injection of LPS (50 μg/kg) and GalN (400 mg/kg). Thirty minutes prior to GalN/LPS administration, plasma Fn or bovine serum albumin was given intravenously. A single administration of plasma Fn (500 mg/kg) protected in dose-dependent fashion against lethal shock after GalN/LPS challenge. Plasma Fn significantly reduced the serum tumor necrosis factor-α, interferon-γ, and interleukin-6 levels and significantly increased the serum interleukin-10 levels after GalN/LPS administration. Furthermore, plasma Fn significantly inhibited liver necrosis at 9 hours after GalN/LPS injection. The fraction of apoptotic-positive cells in these plasma Fn-treated mice was significantly lower than in the control group. These results support the protective treatment of endotoxin-induced liver injury by plasma Fn.

Late onset of treatment with a chemokine receptor CCR1 antagonist prevents progression of lupus nephritis in MRL-Fas(lpr) mice.

Slowly progressive renal injury is the major cause for ESRD. The model of progressive immune complex glomerulonephritis in autoimmune MRL(lpr/lpr) mice was used to evaluate whether chemokine receptor CCR1 blockade late in the disease course can affect progression to renal failure. Mice were treated with subcutaneous injections of either vehicle or BX471, a nonpeptide CCR1 antagonist, three times a day from week 20 to 24 of age [corrected]. BX471 improved blood urea nitrogen levels (BX471, 35.1 +/- 5.3; vehicle, 73.1 +/- 39.6 mg/dl; P < 0.05) and reduced the amount of ERHR-3 macrophages, CD3 lymphocytes, Ki-67 positive proliferating cells, and ssDNA positive apoptotic cells in the interstitium but not in glomeruli. Cell transfer studies with fluorescence-labeled T cells that were pretreated with either vehicle or BX471 showed that BX471 blocks macrophage and T cell recruitment to the renal interstitium of MRL(lpr/lpr) mice. This was associated with reduced renal expression of CC chemokines CCL2, CCL3, CCL4, and CCL5 and the chemokine receptors CCR1, CCR2, and CCR5. Furthermore, BX471 reduced the extent of interstitial fibrosis as evaluated by interstitial smooth muscle actin expression and collagen I deposits, as well as mRNA expression for collagen I and TGF-beta. BX471 did not affect serum DNA autoantibodies, proteinuria, or markers of glomerular injury in MRL(lpr/lpr) mice. This is the first evidence that, in advanced chronic renal injury, blockade of CCR1 can halt disease progression and improve renal function by selective inhibition of interstitial leukocyte recruitment and fibrosis.

Persistent Proteinuria Up-Regulates Angiotensin II Type 2 Receptor and Induces Apoptosis in Proximal Tubular Cells

Apoptosis is implicated in the progressive cell loss and fibrosis both at glomerular and tubulointerstitial level. In this study, we examined the potential mechanisms by which persistent proteinuria (protein-overload model) could induce apoptosis. After uninephrectomy (UNX), Wistar rats received daily injections of 0.5 g of bovine serum albumin (BSA)/100 g body weight or saline. Both at day 8 and day 28, rats receiving BSA had proteinuria and renal lesions characterized by tubular atrophy and/or dilation and mononuclear cell infiltration. In relation to control-UNX rats, renal cortex of nephritic rats showed an increment in AT2 mRNA (reverse transcriptase-polymerase chain reaction) and protein (Western blot) expression. In both groups, AT2 receptor immunostaining was mainly localized in proximal tubular cells. Rats with persistent proteinuria showed a significantly increased number of terminal dUTP nick-end labeling positive apoptotic cells compared with UNX-controls, both in glomeruli and tubulointerstitium. Double staining for apoptosis and AT2 receptor showed that most terminal dUTP nick-end labeling positive cells were found in tubules expressing AT2 receptor. Using an antibody that recognizes the active form caspase-3, we observed an increment in caspase-3 activation in rats receiving BSA with respect to those receiving saline. Rats with persistent proteinuria showed a diminution in the phosphorylation of Bcl-2 with respect to UNX-controls both at day 8 and day 28. By contrast, no changes were observed either in the Bax or in the Bcl-2 protein levels. The administration of BSA to UNX rats induced a diminution in the phosphorylation of ERK with respect to UNX-control at all times studied. The changes observed in ERK activities took place without alterations of ERK1/2 protein levels. In summary, our data suggest that persistent proteinuria causes apoptosis in tubular cells through the activation of AT2 receptor, which can, in turn, inhibit MAP kinase (ERK1/2) activation and Bcl-2 phosphorylation.

Characteristics of Claudin Expression in Follicle-Associated Epithelium of Peyer's Patches: Preferential Localization of Claudin-4 at the Apex of the Dome Region

Gut-associated lymphoreticular tissues, such as Peyer's patches and cecal patches, are important inductive sites for mucosal immune responses. As such, gut-associated lymphoreticular tissues may have an epithelial barrier different from that of villous epithelium. In this study, we investigated the immunohistochemical distribution of the claudin family and occludin in the follicle-associated epithelium (FAE) of Peyer's patches and cecal patches of murine intestine. Unique profiles of claudin-2, -3, and -4 and occludin expression were noted in the tight junctions of the FAE: claudin-4 was preferentially expressed in the apex region; claudin-2 was only weakly expressed on the crypt side of the FAE compared with stronger expression on the crypt side of villous epithelial cells; and claudin-3 and occludin were found throughout the dome. These unique expression patterns were present also in cecal patch FAE. We also found that claudin-4 expression in the FAE of Peyer's patches and cecal patches correlated with the presence of TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling)-positive apoptotic cells, and Peyer's patch-deficient mice exhibited expression patterns of claudin and occludin in villous epithelia similar to those in wild-type mice. We conclude that claudin-4 expression was preferentially associated with the dome region of FAE, the mucosal inductive site of the murine intestine. In that location it might correlate with the cell life cycle, help maintain the apex configuration of the dome, or be a factor favoring the uptake of antigens by the FAE.

Apoptosis and increased expression of Fas ligand after uniocular anterior chamber (AC) inoculation of HSV-1

Purpose. To determine if apoptosis occurs in the eyes and brain following uniocular anterior chamber (AC) inoculation of HSV-1 and if expression of Fas ligand (FasL) or Fas is altered by virus infection. Methods. After inoculation of HSV-1 via the AC route, apoptotic cells in the eyes and brain were identified by TUNEL assay, and TUNEL results indicating DNA fragmentation in the brain were confirmed by DNA laddering assays. Expression of FasL and Fas in the eyes and brain was determined by immunohistochemistry and Western blotting and confirmed by RT-PCR, Southern blotting of RT-PCR products, and sequencing. Results. After AC inoculation of HSV-1, HSV-1 positive cells and apoptotic cells were observed in the eyes and brain. Immunofluorescent staining revealed that although both FasL and Fas were expressed in the cornea, retinal pigment epithelium cells (RPE), and photoreceptor cells of normal and virus infected mice, Fas and FasL were expressed at different locations in these cells. On day 8 p.i., more FasL DNA expression was observed in HSV-infected mice compared with uninfected mice. Conclusions. HSV-1 infection following AC inoculation of virus induced apoptosis in the eyes and brain. Although FasL expression was upregulated by HSV-1 infection, these results suggest there are likely to be factors in addition to FasL and Fas, such as Bcl-2 and caspases, that are involved in apoptosis observed in the eyes and brain of HSV-1- infected mice.

Apoptotic positive cells in Krabbe brain and induction of apoptosis in rat C6 glial cells by psychosine

Globoid cell leukodystrophy (GLD) is an autosomal recessive disorder of infants, caused by deficient activity of cerebroside-β-galactosidase resulting in loss of myelin accompanied by loss of oligodendrocytes. The loss of oligodendrocyte population is accompanied by accumulation of psychosine, which is considered as the molecule responsible for the observed pathophysiology of GLD. We were able to detect apoptotic cells by terminal dUTP nick-end labeling assay and nuclear localization of p53 in postmortem brain tissue of Krabbe's disease patients, which were not detected in the control brain. To study the role of psychosine in cell death, we investigated the effect of psychosine on C6 glial cell survival by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Similar to ceramide (43.8% loss) the galactopsychosine and glucopsychosine treatment killed up to 46.3 and 48.75% of cells, respectively. On the other hand, sphingosine had no effect. DNA laddering assay confirmed these results. Moreover, psychosine-induced detection of annexin-V positive cells supports a role for psychosine in C6 glial cell death via the apoptotic pathway. These results indicate that psychosine may play a role in apoptotic cell loss observed in GLD brain.

Bcl-2-linked apoptosis due to increase in NO synthase in brain of SAMP10

We examined the linkage of nitric oxide (NO)-induced apoptosis to acceleration of brain aging of senescence-accelerated mouse prone 10 (SAMP10). The expression of neuronal nitric oxide synthase (nNOS) increased in the cerebral cortex of the brain of SAMP10 in an age-dependent manner and significantly higher levels of neuronal nitric oxide synthase (nNOS) were observed in both young and old SAMP10 as compared to age-matched controls. Moreover, a lower level of anti-apoptotic protein Bcl-2 and a higher level of pro-apoptotic protein cytochrome c in cytosol were observed in SAMP10 compared to the control. However, there was no significant difference in the expression of pro-apoptotic protein p53 between SAMP10 and the control. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive apoptotic cells were more abundant in the cerebral cortex of aged SAMP10 than in the control. The present results suggest that an age-dependent increase of NO by up-regulation of nNOS promotes the Bcl-2-linked apoptosis in the cerebral cortex of SAMP10 and this may cause the acceleration of brain aging of SAMP10.

Therapeutic utility of aspirin in the ApcMin/+ murine model of colon carcinogenesis.

BackgroundIn recent years it has become evident that nonsteroidal anti-inflammatory drugs, in particular aspirin represent a potential class of cancer chemotherapeutic agents. Despite the wealth of knowledge gained from epidemiological, clinical and animal studies, the effectiveness of aspirin to treat established gastrointestinal cancer has not been determined. The present study examines the ability of aspirin to treat established polyposis in Min/+ mice.MethodsMin/+ mice with established polyposis were treated orally once daily from 12–16 weeks of age with either drug vehicle or aspirin (25 mg/kg). Upon completion of treatment, the number, location and size of intestinal tumours was determined. Additional variables examined were the number of apoptotic cells within tumours and COX activity.ResultsAdministration of aspirin for 4 weeks to Min/+ mice produce no effect on tumour number compared to vehicle-treated Min/+ mice (65 ± 8 vs. 63 ± 9, respectively). In addition, aspirin had no effect on tumour size or location. However, aspirin treatment produced a greater than 2-fold (p < 0.05) increase in the number of apoptotic positive cells within tumours and significantly decreased hepatic PGE2 content.ConclusionsAspirin was found to have no effect on tumour number and size when administered to Min/+ mice with established polyposis. The findings in the present study call in to question the utility of aspirin as a stand-alone treatment for established GI cancer. However, aspirin's ability to significantly promote apoptosis may render it suitable for use in combinatorial chemotherapy.

Pirfenidone suppresses tumor necrosis factor-α, enhances interleukin-10 and protects mice from endotoxic shock

A new experimental drug, pirfenidone (5-methyl-1-phenyl-1 H-pyridine-one; S-7701), has been reported to have beneficial effects for the treatment of certain fibrotic diseases. We investigated the anti-inflammatory properties in murine endotoxic shock to determine the pharmacological characteristics. The present study describes the prophylactic effect, cytokine regulatory profiles and therapeutic effect of pirfenidone in murine endotoxic shock, which was induced in mice using an intraperitoneal (i.p.) injection of lipopolysaccharide and d-galactosamine. First, we examined the prophylactic effect and cytokine regulatory profiles. A single oral administration of pirfenidone prior to lipopolysaccharide/ d-galactosamine challenge inhibited the production of circulating tumor necrosis factor-α (TNF-α), interleukin-12 and interferon-γ, markedly enhanced that of interleukin-10, and offered protection from subsequent lethal symptoms in a dose-dependent manner. Second, we examined the therapeutic effect. A single oral administration of pirfenidone 1, 2, 3, 4 and 5 h post lipopolysaccharide/ d-galactosamine challenge provided protection against lethal shock in a time-dependent manner. At the histopathological level, apoptotic positive cells were found to be suppressed in the liver. The transforming growth factor (TGF)-β1 level was markedly elevated in the liver of lipopolysaccharide/ d-galactosamine-challenged mice, suppressed in pirfenidone-treated mice. These findings may offer an alternative for both protective and therapeutical treatment of several human acute or chronic inflammatory diseases by pirfenidone.

Close association between Fas ligand (FasL; CD95L)-positive tumor-associated macrophages and apoptotic cancer cells along invasive margin of colorectal carcinoma: a proposal on tumor-host interactions.

Anti‐tumor immune responses are considered to be one of the key host reactions in human colorectal cancer, with T cells as important effector cells. For the induction of tumor‐specific immunity, processing of cancer cells and pruning of T cells by antigen‐presenting cells are important. The present study was designed to clarify the relationship between Fas ligand (FasL; CD95L) expression and apoptotic cancer cells. Immunohistochemistry using frozen sections taken from 58 patients with colorectal cancer revealed that stromal cells composed mainly of tumor‐associated macrophages expressed FasL, leaving cancer cells negative for FasL. These macrophages were abundantly distributed along the invasive margin. In situ hybridization revealed that these macrophages as well as cancer cells expressed FasL mRNA, whereas macrophages in the normal colon mucosa rarely expressed FasL. Apoptotic cancer cells recognized by monoclonal antibody M30 CytoDEATH were localized not only in cancer cell nests, but also in the stroma along the invasive margin showing a dissociated pattern, which was particularly evident in the areas of FasL+ macrophages. Furthermore, these macrophages, phenotypically similar to dendritic cells, occasionally contained M30+ apoptotic cancer cells in the cytoplasm. Clinicopathologic analyses in 123 cases revealed 1) a positive correlation between the degree of dissociated M30+ apoptotic cancer cells and the number of macrophages along the invasive margin and 2) an inverse association between the degree of dissociated M30+ apoptotic cancer cells and the occurrence of hematogenous metastasis after surgical resection of the primary tumor. In conclusion, the present study shows the impor‐ tance of FasL+ activated macrophages as one of the host defense mechanisms against cancer cell spread in human colorectal cancer.