Tamoxifen and its metabolites induce mitochondrial membrane depolarization and caspase-3 activation in equine neutrophils.

Neutrophils participate in innate immunity as the first line of host defence against microorganisms. However, persistent neutrophil activity and delayed apoptosis can be harmful to surrounding tissues; this problem occurs in diverse inflammatory diseases, including asthma-affected horses. Previous studies in horses with acute lung inflammation indicated that treatment with tamoxifen (TX), a selective oestrogen receptor modulator, produces a significant decrease in bronchoalveolar lavage fluid (BALF) neutrophil content. The aim of this study was to investigate the effect of tamoxifen and its metabolites (N-desmethyltamoxifen and endoxifen) on the mitochondrial membrane potential assay by flow cytometry, and the activation of effector caspase-3 through immunoblotting, in peripheral blood neutrophils obtained from healthy horses (n = 5). Results show that tamoxifen, N-desmethyltamoxifen and endoxifen depolarize the mitochondrial membrane and activate caspase-3 in healthy equine neutrophils in vitro. These findings suggest that tamoxifen and its metabolites may activate the intrinsic apoptotic pathway in equine neutrophils. However, more studies are necessary to further explore the signalling pathways of these drugs in the induction of apoptosis.

Immunoinflammatory response and bioactive potential of GuttaFlow bioseal and MTA Fillapex in the rat subcutaneous tissue.

To evaluate the effect of GuttaFlow bioseal (GFB) and MTA Fillapex (MTAF) in comparison with Endofill (EF) in the subcutaneous tissue. Polyethylene tubes with GFB, MTAF, EF or empty tubes (control group; CG) were implanted into subcutaneous of rats. After 7, 15, 30 and 60 days, the capsule thickness, inflammatory reaction, interleukin-6 (IL-6), vascular endothelial growth factor (VEGF), caspase-3, TUNEL-positive cells, von Kossa and ultrastructural features were evaluated. The data were statistically analyzed (p ≤ 0.05). At all periods, the number of IL-6- and VEGF-immunolabelled cells, and capsule thickness were lower in GFB than MTAF, which was lower than EF (p < 0.0001). At 60 days, the number of inflammatory cells was similar in GFB and MTAF (p = 0.58). Significant differences in the number of TUNEL- and caspase-3-positive cells were not observed among GFB, MTAF and CG whereas the highest values were found in EF specimens. The EF specimens exhibited several cells with condensed chromatin, typical of apoptosis. von Kossa-positive and birefringent structures were only observed in GFB and MTAF, suggesting the presence of calcite crystals. Taken together, these results show that cellular and structural damage induced by GFB and MTAF sealers were recovery over time. Moreover, these sealers express bioactive potential in subcutaneous tissue.

HIV-1 infection modulates IL-24 expression which contributes to cell apoptosis in vitro.

Although interleukin-24 (IL-24) has been extensively explored in the immunopathologies of autoimmune diseases, neoplasms, and infections, its role in HIV-1 infection has not been thoroughly elucidated to date. Therefore, the objective of this study was to evaluate the gene and protein expressions of IL-24 at the initial moments of HIV infection in PBMCs. Due to the pro-apoptotic role of IL-24, we evaluated the protein expression of caspase-3, as well as Annexin V/Propidium Iodide flow cytometry and phosphorylation of ERK, which may induce an apoptotic signal block when phosphorylated. The results of this study demonstrated that HIV-1 infection had an impact on the gene and protein expressions of IL-24 and ERK. Annexin V/Propidium Iodide assay demonstrated decrease in the mechanisms of apoptosis in infected cells after incubation of IL-24 neutralizing antibody. Studies on how HIV-1 regulates IL-24 expression may play a role in characterizing viral persistence mechanisms and designing antiretroviral strategies.

Therapeutic effects of vaccine derived from amastigote surface protein-2 (ASP-2) against Chagas disease in mouse liver.

This study investigated the efficacy of the vaccine in liver of mice infected with the Trypanosoma cruzi (T. cruzi) and immunized with AdASP-2. For this purpose, histopathological analysis and gene expression of COX-2, TNF-alpha, TNFR, iNOS, cytochrome C, caspase-3, TLR4, IL-6 and IL10 were evaluated. The following groups were used in this study: Group 1 - Control Group (CTRL) animals received AdßGal vehicle; Group 2 - Infected Group (TC) animals were infected with T. cruzi; Group 3 - Immunized Group (AdASP-2): animals were immunized by AdASP-2 vaccine; Group 4 - Immunized and Infected Group (AdASP-2+TC) animals were infected with T. cruzi and immunized by AdSP-2 vaccine. A significant decrease of amastigote nests was noticed in the group of animals that were immunized with AdASP-2 and infected on the same day. COX-2 and TNF-alpha gene expressions increased in TC group, whereas TNF-alpha decreased in the TC+AdASP-2 group. TNFR expression was high in AdASP-2+TC group. iNOS expression was high for all experimental groups whereas cytochrome C decreased for all experimental groups. Caspase 3 increased in TC and TC+AdASP-2 groups. The gene expression of TLR4 and IL-10 showed an increase in AdASP-2+TC group. Finally, hepatic fibrosis was noticed to TC and AdASP-2 + TC groups. Taken together, our results demonstrated that vaccination with AdASP-2 was effective against the acute phase of experimental Chagas disease as a result of a more powerful and rapid immune response closely related to expression of some inflammatory genes, such as iNOS, TNF-alpha, TLR 4, and IL-10.

Histologic evaluation of Ki-67 and cleaved caspase-3 expression in feline mammary carcinoma.

Objectives The purpose of this study was to evaluate histopathologic aspects of, and the expression of Ki-67 and cleaved caspase-3 in, feline mammary carcinoma (FMC). Methods Feline mammary tumors were surgically obtained by mastectomy from 30 female cats and were fixed with formalin and embedded in paraffin wax. Four-micron sections were stained with hematoxylin and eosin for histopathologic diagnosis. Ki-67 and cleaved caspase-3 were analyzed by immunohistochemistry. Results Samples were histologically confirmed as FMC. Positive immunostaining was observed in all cancer samples for both nuclear Ki-67 and cleaved caspase-3, with a mean positive staining percentage of 27.5% and 21.2%, respectively. No statistically significant correlations between Ki-67 and cleaved caspase-3 were observed within FMC. Conclusions and relevance A high proliferation index was found in feline mammary tumors. This is the first study evaluating cleaved caspase-3 expression in FMC.

Pro-inflammatory cytokines enhance ERAD and ATF6α pathway activity in salivary glands of Sjögren's syndrome patients.

Salivary gland (SG) acinar-cells are susceptible to endoplasmic reticulum (ER) stress related to their secretory activity and the complexity of synthesized secretory products. SGs of Sjögren's syndrome patients (SS)-patients show signs of inflammation and altered proteostasis, associated with low IRE1α/XBP-1 pathway activity without avert increases in apoptosis. Acinar-cells may avoid apoptosis by activation of the ATF6α pathway and ER-associated protein degradation (ERAD). The aim of this study was to evaluate the role of pro-inflammatory cytokines in ATF6α pathway/ERAD activation and cell viability in labial salivary glands (LSG) of SS-patients. In biopsies from SS-patients increased ATF6α signaling pathway activity, as evidenced by generation of the ATF6f cleavage fragment, and increased expression of ERAD machinery components, such as EDEM1, p97, SEL1L, gp78, UBE2J1, UBE2G2, HERP and DERLIN1, were observed compared to controls. Alternatively, for pro- (active-caspase-3) and anti-apoptotic (cIAP2) markers no significant difference between the two experimental groups was detected. Increased presence of ATF6f and ERAD molecules correlated significantly with increased expression of pro-inflammatory cytokines. These observations were corroborated in vitro in 3D-acini treated with TNF-α and/or IFN-γ, where an increase in the expression and activation of the ATF6α sensor and ERAD machinery components was detected under ER stress conditions, while changes in cell viability and caspase-3 activation were not observed. Cytokine stimulation protected cells from death when co-incubated with an ERAD machinery inhibitor. Alternatively, when cytokines were eliminated from the medium prior to ERAD inhibition, cell death increased, suggesting that the presence of pro-inflammatory cytokines in the medium is essential to maintain cell viability. In conclusion, the ATF6α pathway and the ERAD machinery are active in LSG of SS-patients. Both were also activated by TNF-α and IFN-γ in vitro in 3D-acini and aided in preventing apoptosis. IFN-γ levels were elevated in SS-patients and UPR responses triggered in vitro by this cytokine closely matched those observed in LSG from SS-patients, suggesting that cytokines may induce ER stress.

Effect of low-level laser therapy on irradiated parotid glands--study in mice.

The objective of this study was to evaluate the effect of low-level laser therapy (LLLT) on radiotherapy-induced morphological changes and caspase-3 immunodetection in parotids of mice. Forty-one Swiss mice were divided into control, radiotherapy, 2- and 4-J laser groups. The experimental groups were exposed to ionizing radiation in a single session of 10 Gy. In the laser groups, a GaAlAs laser (830 nm, 100 mW, 0.028 cm2, 3.57 W/cm2) was used on the region corresponding to the parotid glands, with 2-J energy (20 s, 71 J/cm2) or 4 J (40 s, 135 J/cm2) per point. LLLT was performed immediately before and 24 h after radiotherapy. One point was applied in each parotid gland. The animals were euthanized 48 h or 7 days after radiotherapy and parotid glands were dissected for morphological analysis and immunodetection of caspase-3. There was no significant difference between groups in the immunodetection of caspase-3, but the laser groups had a lower percentage compared to the radiotherapy group. LLLT promoted the preservation of acinar structure, reduced the occurrence of vacuolation, and stimulated parotid gland vascularization. Of the two LLLT protocols, the one using 4 J of energy showed better results.

Antiviral and myocyte protective effects of IL-28A in coxsackievirus B3-induced myocarditis.

OBJECTIVE: This study aimed to investigate whether interleukin-28A (IL-28A) plays a role in murine myocarditis induced by coxsackievirus B3 (CVB3), and to explore its possible mechanism involved. METHODS: Male BALB/c mice both infected and not infected by CVB3 were randomly divided into four groups (n=40), untreated or treated with different doses of IL-28A for 4 days, and then sacrificed on days 4 and 7 post-infection. The heart samples were collected for histopathologic examination. Cardiac viral load was determined by a plaque assay. Additionally, immunoblot analysis, TUNEL assay, and immunohistochemistry were performed to examine the expression of signal transducer, activator of transcription 1 and 2 (STAT1 and STAT2), CVB3-induced apoptosis and the expression of Bcl-2, BAX and Caspase-3. RESULTS: Compared to uninfected mice, the CVB3 infected mice exhibited higher mortality rate (p<0.001), apparent inflammation and myocardial lesion (p<0.01), and higher cardiac viral load (p<0.01). After CVB3 infection, IL-28A treated mice presented no death (p<0.001), reduced inflammation and myocardial lesion (p<0.01), and lower viral load (p<0.01) compared to untreated mice. Besides, treatment with IL-28A markedly increased the expressions of STAT1 and STAT2, and inhibited CVB3-induced apoptosis in myocardial cells with increased ratio of Bcl-2/BAX. CONCLUSION: The antiviral and myocyte protective effects of IL-28A in CVB3-induced myocarditis are regulated by STAT1 and STAT2.

ß-Actin-binding complementarity-determining region 2 of variable heavy chain from monoclonal antibody C7 induces apoptosis in several human tumor cells and is protective against metastatic melanoma.

Complementarity-determining regions (CDRs) from monoclonal antibodies tested as synthetic peptides display anti-infective and antitumor activities, independent of the specificity of the native antibody. Previously, we have shown that the synthetic peptide C7H2, based on the heavy chain CDR 2 from monoclonal antibody C7, a mAb directed to a mannoprotein of Candida albicans, significantly reduced B16F10 melanoma growth and lung colony formation by triggering tumor apoptosis. The mechanism, however, by which C7H2 induced apoptosis in tumor cells remained unknown. Here, we demonstrate that C7H2 interacts with components of the tumor cells cytoskeleton, being rapidly internalized after binding to the tumor cell surface. Mass spectrometry analysis and in vitro validation revealed that ß-actin is the receptor of C7H2 in the tumor cells. C7H2 induces ß-actin polymerization and F-actin stabilization, linked with abundant generation of superoxide anions and apoptosis. Major phenotypes following peptide binding were chromatin condensation, DNA fragmentation, annexin V binding, lamin disruption, caspase 8 and 3 activation, and organelle alterations. Finally, we evaluated the cytotoxic efficacy of C7H2 in a panel of human tumor cell lines. All tumor cell lines studied were equally susceptible to C7H2 in vitro. The C7H2 amide without further derivatization significantly reduced lung metastasis of mice endovenously challenged with B16F10-Nex2 melanoma cells. No significant cytotoxicity was observed toward nontumorigenic cell lines on short incubation in vitro or in naïve mice injected with a high dose of the peptide. We believe that C7H2 is a promising peptide to be developed as an anticancer drug.

Immunodetection of caspase-3 by Western blot using glutaraldehyde.

Cell death by apoptosis triggers the engagement of a conserved intracellular machinery of execution, involving mainly the activation of the caspase family of cysteine proteases. Caspase-3 is a common effector of most of the apoptotic pathways and is able to cleave several target proteins whose degradation will contribute to the execution phase of the cell demise program. Here we present a modification of the Western blot protocol to improve sensitivity of caspase-3 detection, providing a valuable tool to access its activation in biological specimens.

Tracheal cryopreservation: caspase-3 immunoreactivity in tracheal epithelium and in mixed glands.

Cryopreservation has an immunomodulating effect on tracheal tissue as a result of class II antigen depletion due to epithelium exfoliation. However, not all epithelium is detached. We evaluated the role of apoptosis in the remaining epithelium of 30 cryopreserved tracheal grafts. Caspase-3 immunoreactivity of tracheal epithelium was studied in canine tracheal segments cryopreserved with F12K medium, with or without subsequent storage in liquid nitrogen at -196 degrees C for 15 days. Loss of structural integrity of tracheal mixed glands was observed in all cryopreserved tracheal segments. Caspase-3 immunoreactivity in tracheal mucosa and in mixed glands was significantly decreased, in contrast to the control group and to cryopreserved tracheal segments in which it remained high, due to the effect of storage in liquid nitrogen (P < 0.05, ANOVA and Tukey test). We conclude that apoptosis can be triggered in epithelial cells during tracheal graft harvesting even prior to cryopreservation, and although the epithelial caspase-3 immunoreactivity is reduced in tracheal cryopreservation, this could be explained by increased cell death. Apoptosis cannot be stopped during tracheal cryopreservation.