Generation of human long-lived plasma cells by developmentally regulated epigenetic imprinting

Antibody secreting cells (ASCs) circulate after vaccination and infection and migrate to the BM where a subset known as long-lived plasma cells (LLPCs) persists and secrete antibodies for a lifetime. The mechanisms by which circulating ASCs become LLPCs are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared with BM LLPCs. Compared with blood ASCs, BM LLPCs have decreased nucleus/cytoplasm ratio but increased endoplasmic reticulum and numbers of mitochondria. LLPCs up-regulate pro-survival genes MCL1, BCL2, and BCL-XL while simultaneously down-regulating pro-apoptotic genes HRK1, CASP3, and CASP8. Consistent with reduced gene expression, the pro-apoptotic gene loci are less accessible in LLPCs. Of the pro-survival genes, only BCL2 is concordant in gene up-regulation and loci accessibility. Using a novel in vitro human BM mimetic, we show that blood ASCs undergo similar morphological and molecular changes that resemble ex vivo BM LLPCs. Overall, our study demonstrates that early-minted blood ASCs in the BM microniche must undergo morphological, transcriptional, and epigenetic changes to mature into apoptotic-resistant LLPCs.

Effect of glucose mediated oxidative stress on apoptotic gene expression in gingival mesenchymal stem cells

Background Diabetes is a common disease that causes gingival and periodontal problems. Stem cells isolated from dental sources are an emerging area of research with a potential to facilitate regenerative medicine. The stem cells retain the property of self-renewal and the ones isolated from dental sources are mainly multipotent mesenchymal stem cells that have the ability to self-renew as well as differentiation towards multiple lineages. Objectives We aimed to isolate and characterize gingival mesenchymal stem cells by pluripotency markers and investigated the effect of oxidative stress on growth kinetics and apoptotic gene expression of gingival cells exposed to glucose mediated oxidative stress. Methods In this study, we isolated gingival mesenchymal stem cells from gingiva. This was followed by morphologic analysis using inverted phase contrast microscopy and molecular profiling of these cells for the mRNA expression of specific genes. The isolated cells were cultured till passage 3 and then exposed to oxidative stress (high glucose concentration). We measured the apoptotic gene expression and compared their growth kinetics. Results The results showed that oxidative stress produced by glucose reduced growth kinetics and increased apoptotic gene expression in gingival mesenchymal stem cells. According to the genetic results, glucose activated TNF family to initiate apoptosis. Conclusion In conclusion, the present study demonstrated that high glucose obliterated cellular proliferation testified by evaluating growth kinetics and induced apoptotic gene expression in gingival mesenchymal stem cells. This initiated extrinsic apoptotic pathway mediated by TNF family. Therefore, in diabetes oral health condition is compromised and periodontal disease is common.

Global Reprogramming of Apoptosis-Related Genes during Brain Development

To enable long-term survival, mammalian adult neurons exhibit unique apoptosis competence. Questions remain as to whether and how neurons globally reprogram the expression of apoptotic genes during development. We systematically examined the in vivo expression of 1923 apoptosis-related genes and associated histone modifications at eight developmental ages of mouse brains. Most apoptotic genes displayed consistent temporal patterns across the forebrain, midbrain, and hindbrain, suggesting ubiquitous robust developmental reprogramming. Although both anti- and pro-apoptotic genes can be up- or downregulated, half the regulatory events in the classical apoptosis pathway are downregulation of pro-apoptotic genes. Reduced expression in initiator caspases, apoptosome, and pro-apoptotic Bcl-2 family members restrains effector caspase activation and attenuates neuronal apoptosis. The developmental downregulation of apoptotic genes is attributed to decreasing histone-3-lysine-4-trimethylation (H3K4me3) signals at promoters, where histone-3-lysine-27-trimethylation (H3K27me3) rarely changes. By contrast, repressive H3K27me3 marks are lost in the upregulated gene groups, for which developmental H3K4me3 changes are not predictive. Hence, developing brains remove epigenetic H3K4me3 and H3K27me3 marks on different apoptotic gene groups, contributing to their downregulation and upregulation, respectively. As such, neurons drastically alter global apoptotic gene expression during development to transform apoptosis controls. Research into neuronal cell death should consider maturation stages as a biological variable.

Alterations of miR-16, miR-let-7a and their target genes expression in human blastocysts following vitrification and re-vitrification

Embryo cryopreservation is a widely used technique in infertility management and today is an essential part of assisted reproductive technology (ART). In some cases, re-vitrification can be applied to good quality supernumerary warmed embryos that have not been transferred in the present cycle. However, there is no study about re-vitrification impact on microRNA and gene expression in human embryos. The purpose of this study is to evaluate miR-16, miR-let7a and target genes expression in in vitro produced human blastocysts following re-vitrification.Day3 embryos obtained from ICSI cycles of fertile couples referring for family balancing program were biopsied and cultured individually. On the fourth day (post-ICSI) male ones (choices of their parents) were transferred and the females (good quality embryos) were donated for research. Donated embryos were cultured to blastocyst stage and assigned to three groups: fresh, vitrified and re-vitrification. Embryos were vitrified on Cryotech carriers. Then blastocysts of three groups were individually assessed for expression of miR-16, miR-let7a and target genes.The results showed that re-vitrification of human blastocysts did not affect the ability to re-expand in culture. In addition, significant decrease was observed in miR-16 and miR-let7a expression in re-vitrified group compared to fresh (p < 0.05). A significant upregulation of the target genes ITGβ3 and BCL-2 in re-vitrified and vitrified embryos was observed compared to the fresh group (p < 0.05). The expression of BAX as a pro-apoptotic gene showed a significant decrease in re-vitrification group comparing with the fresh one (P < 0.05).The results of this research indicated that re-vitrification of embryos changes the expression of miR-16, miR-let-7a and their target genes. These alterations include increased expression of BCl-2 and ITGβ3 genes which play important roles in embryo survival and implantation, respectively. Clinical proof of these effects requires further research.

Apoptosis Induction with Combined Use of Cisplatin and Fisetin in Cisplatin- Resistant Ovarian Cancer Cells (A2780)

Background: Ovarian cancer is the leading cause of death caused by genital cancers. One of the most common treatments for this type of cancer is chemotherapy by cisplatin, which induces apoptosis in cancer cells. Apoptosis is a type of physiological cell death. Cisplatin chemotherapy usually has several side effects and cellular resistance to cisplatin is a common incidence. In order to overcome these problems, the use of combination therapies using natural substances has been considered. Fisetin is a flavonoid with anti-cancer activity which induces apoptosis. In this study, the apoptosis induced by cisplatin along with Fisetin in cisplatin-resistant ovarian cancer cell line (A2780) was investigated. Methods: In the present experimental study, the effect of combined use of Fisetin and cisplatin on ovarian cancer cell lines (A2780) was investigated by using MTT assay. Cell death was also determined by DAPI, acridine orange/propidium iodide, and Annexin/PI assay. Apoptotic gene expression of Bax, BCL-2, caspase 3, and caspase 9 was also assessed by real time PCR. Results: The results of MTT assay indicated that the combined treatment of Fisetin and cisplatin effectively inhibits proliferation of A2780 cells. The results of DAPI staining showed that fragmentation of chromatin in cells occurred in the combined treatment. Acridine orange-propidium iodide staining and Annexin/PI staining showed an increase in the rate of apoptotic cells in cells under combined treatment. The results of the study regarding changes in gene expression also indicated that Bax pro-apoptotic gene expression and BCL-2 anti-apoptotic gene expression increased in cells under treatment; moreover, gene expression of caspases 3 and 9 significantly increased as well. Conclusion: According to the findings of this study, the combined use of cisplatin and Fisetin increases the induction of apoptosis in cisplatin-resistant ovarian cancer cells (A2780); therefore, the combined use of cisplatin and Fisetin can be considered a promising strategy in the treatment of ovarian cancer.

Effects of Acute Diquat Poisoning on Liver Mitochondrial Apoptosis and Autophagy in Ducks

Diquat (DQ) is an effective herbicide and is widely used in agriculture. Due to persistent and frequent applications, it can enter into aquatic ecosystem and induce toxic effects to exposed aquatic animals. The residues of DQ via food chain accumulate in different tissues of exposed animals including humans and cause adverse toxic effects. Therefore, it is crucial and important to understand the mechanisms of toxic effects of DQ in exposed animals. We used ducks as test specimens to know the effects of acute DQ poisoning on mechanisms of apoptosis and autophagy in liver tissues. Results on comparison of various indexes of visceral organs including histopathological changes, apoptosis, autophagy-related genes, and protein expression indicated the adverse effects of DQ on the liver. The results of our experimental trial showed that DQ induces non-significant toxic effects on pro-apoptotic factors like BAX, BAK1, TNF-α, caspase series, and p53. The results revealed that anti-apoptotic gene Parkin was significantly upregulated, while an upward trend was also observed for Bcl2, suggesting that involvement of the anti-apoptotic factors in ducklings plays an important role in DQ poisoning. Results showed that DQ significantly increased the protein expression level of the autophagy factor Beclin 1 in the liver. Results on key autophagy factors like LC3A, LC3B, and p62 showed an upward trend at gene level, while the protein expression level of both LC3B and p62 reduced that might be associated with process of translation affected by the pro-apoptotic components such as apoptotic protease that inhibits the occurrence of autophagy while initiating cell apoptosis. The above results indicate that DQ can induce cell autophagy and apoptosis and the exposed organism may resist the toxic effects of DQ by increasing anti-apoptotic factors.

α-Lipoic Acid Induces Intrinsic Apoptosis in Human Oral Squamous Carcinoma Cells

Oral squamous cell carcinoma is one of the leading cancers in India and it is responsible for significant morbidity and mortality. α -lipoic acid, a co-factor for several metabolic enzymes, suppresses the tumor growth. In this study, we investigated the α-lipoic acid-induced cytotoxicity and apoptosis in human oral squamous carcinoma (SCC-25) cells. α-lipoic acid treatments were given to SCC-25 cells for 24 h and cell proliferation was evaluated by MTT assay. The reactive oxygen species expression was examined by dichloro-dihydro-fluorescein diacetate assay. Apoptosis-related morphological changes were detected by dual staining. Cytochrome c and RAS (H-Ras) expression was measured by dual staining and RT-PCR respectively. Intrinsic apoptosis-related markers are analyzed using qPCR.α-lipoic acid inhibited SCC-25 cell proliferation in a concentration-dependent manner. This treatment also increased intracellular reactive oxygen species expression and the percentage of apoptotic cells (up to 70% of the cell population). Dual staining further confirms cytochrome c cytosolic expression. The oncogene H-Ras protein and gene expression was also down-regulated upon α-lipoic acid treatment in SCC-25 cells. qPCR analysis further confirms α-lipoic acid-induced an upregulation of bax, Apaf-1, caspase 3 and − 9, pro-apoptotic gene expressions and downregulation of bcl-2, an anti-apoptotic gene expression. The present results suggest that α-lipoic acid has cytotoxic and pro-apoptotic potential and it also downregulates H-Ras oncogene expression in human oral squamous carcinoma cells. α-lipoic acid may have promising role in the treatment of human oral squamous carcinoma.

Study on the effects of the different polar group of EPA-enriched phospholipids on the proliferation and apoptosis in 95D cells

EPA-enriched phosphatidylcholine (EPA-PC) and EPA-enriched phosphatidylethanolamine (EPA-PE) are newly identified marine phospholipids. The polar group of phospholipids is known to influence EPA-phospholipid activity. However, the differences in anti-tumor effects between EPA-PC and EPA-PE have not been reported. In this study, we evaluated the effects of two forms of EPA on the proliferation and apoptosis in the lung-cancer cell line 95D as well as possible molecular mechanisms. Our results showed that EPA-PC effectively inhibited proliferative activity and promoted apoptosis of 95D cells in a dose-dependent manner, while EPA-PE had no effect on cell proliferation, although it slightly promoted apoptosis. Western blot results showed that EPA-PC and EPA-PE upregulated the expression of PPARγ, RXRα, and PTEN, and downregulated the PI3K/AKT signaling pathway. Furthermore, EPA-PC and EPA-PE induced the expression of the pro-apoptotic gene, Bax, and reduced the expression of the anti-apoptotic gene, Bcl-xl. Additionally, EPA-PC and EPA-PE promoted the release of cytochrome c and activated the apoptotic enzyme-cleaved caspase-3. These data suggest that the anti-tumor effect of EPA-phospholipids may be exerted via a PPARγ-related mechanism. EPA-PC was more efficacious as compared to EPA-PE, which might be due to the different polar groups of phospholipids.

Maturation of Human Long-lived Plasma Cells Results in Resistance to Apoptosis by Transcriptional and Epigenetic Regulation

Antibody secreting cells (ASC) circulate after vaccination and migrate to the bone marrow (BM) where a subset known as long-lived plasma cells (LLPC) persist and secrete antibodies for a lifetime. The mechanisms of how circulating ASC become LLPC are not well elucidated. Here, we show that human blood ASCs have distinct morphology, transcriptomes, and epigenetics compared to BM LLPC. LLPC acquire transcriptional and epigenetic changes in the apoptosis pathway to support their survival. Upregulation of pro-survival gene expression accompanies downregulation of pro-apoptotic gene expression in LLPC. While pro-apoptotic gene loci are less accessible, pro-survival gene loci are not always accompanied by accessibility changes. Importantly, we show similar LLPC morphological and transcriptional maturation of blood ASC in response to the novel in vitro BM mimetic. In all, our study demonstrates that blood ASC in the BM microniche must undergo morphological and molecular changes to mature into apoptotic-resistant LLPC.