Interferons (IFNs) are proteins of the family of cytokines. Their antiproliferative function has been taken into account for several clinical therapies against malignant diseases. In this family, IFNs α and γ have demonstrated the highest antitumor effects. HerberPAG® is a new co-formulation with IFNs, α2b and γ. It has been obtained to increase the antiproliferative effect of individual IFNs and decrease their associated toxicity. Glioblastoma multiforme (GBM) is the most common primary brain tumor and one of the most deadly forms of cancer. The objective of the present work is to obtain insights into the regulation of Interferon-STAT-pathways and apoptosis in U87MG, at the transcriptional level. As a pharmacogenomic strategy we quantified mRNAs levels in vitro by quantitative PCR, using the cell line U87MG as a model. Some of the genes involved in the first steps of IFNs signaling pathways (stat1 and stat3) and apoptosis events (tp53, bax, bcl-2, bad, caspase3 (casp3), caspase8 (casp8) and caspase9 (casp9)) were studied. The detected mRNAs expression pattern for stat1and stat3 indicates a higher tumor suppressor activity of HerberPAG® compared to individuals IFNs. The up-regulation of tp53, bax, bad, casp3, casp8 and casp9 genes and the down regulation of bcl-2 gen, after the treatment with HerberPAG® show a pro-apoptotic function. HerberPAG® gene-induced profile shows an advantage in relation to IFN α2b and γ with a higher stat1 expression and a downregulation of bcl-2 which increases bax:bcl-2 ratio. The regulation of genes involved in IFN-STAT-pathways and apoptosis may be the first evidences to explain the increased antiproliferative properties of this co-formulation.

Colorectal cancer (CRC) is one of the most common types of cancer seen in the world. 5-Fluorouracil (5-Fu) plus Oxaliplatin (1-OHP) remains the backbone of CRC chemotherapeutics, but with limited success. Phenoxodiol (Pxd) is an isoflavone analog with antitumor activity against various types of cancers, and sensitizes chemoresistant cancer cells to chemotherapeutics including platinum and taxanes. This study was, therefore, undertaken to examine whether Pxd pre-treatment with conventional chemotherapeutic agent(s) 5-Fu and 1-OHP co-administration be a therapeutic strategy for CRC. Cell viability and cytotoxicity were evaluated using dimethyl-thiazolyl diphenyl tetrazolium bromide (MTT) and lactate dehydrogenase assays. The percentage of apoptotic and necrotic cells were determined by fluorescence microscopy analysis. Besides, active Caspase-3 levels by ELISA and relative mRNA levels of Caspase 3 (CASP3), CASP8 and CASP9 genes were determined by quantitative real-time PCR (qPCR) analysis. The pre-treatment of Pxd followed by 5-Fu and 1-OHP co-administration was more effective at inhibiting cell viability than either chemotherapeutic agents treatment alone. When compared to 5-Fu with 1-OHP alone treatment, Pxd pre-treatment overwhelmingly increased apoptotic Caspase-3 activity levels in CRC cells. Moreover, qPCR analyses showed that CASP3 and CASP9 mRNA levels significantly increased after pre-treatment with Pxd followed by 5-Fu and 1-OHP treatments, compared to 5-Fu with 1-OHP alone. Our results suggested that Pxd enhanced the in vitro antitumor activity of 5-Fu and 1-OHP. Our study also suggested that Pxd may be a potential candidate agent in advanced CRC and inclusion of Pxd to the conventional chemotherapeutic agent(s) could be an effective therapeutic strategy for CRC.

Keywords: 5-Fluorouracil; apoptosis; colorectal cancer; oxaliplatin; phenoxodiol.

Nobiletin as a nontoxic dietary citrus flavonoid has anticancer effects in cancer. Toll-like receptor three has a role in prostate cancer progression. However the relationship among NOB and TLR3 signaling in PCa has not been elucidated, yet. Therefore, we aimed to evaluate the effects of NOB on the activation of TLR3 signaling pathways in PCa In Vitro. PC-3, LNCaP and HUVEC cells were used for comparison of NOB-mediated TLR3 signaling pathways. After treatment with NOB and Poly I:C alone and NOB + Poly I:C, RT-PCR, western blotting and ELISA assay were performed to evaluate changes in gene and protein expression level, as well as CASP8. NOB potentially induced TLR3/IRF3 signaling pathway and the activation of TLR3/IRF3 signaling pathway by both NOB and Poly I:C was more profound in LNCaP than PC-3 cells. However, the level of TRIF protein and CASP8 decreased after both NOB and Poly I:C incubation. NOB could mediate TLR3 signaling pathways. NOB + Poly I:C could improve the activation of TLR3/IRF3 signaling pathway. However, the activation of TRIF/RIPK1/FADD signaling pathway reduced. Therefore, the elucidation of molecular mechanisms of TLR3 signaling pathways and the combination effects of NOB + Poly I:C on apoptotic cell death are further studied.

Triple-negative breast cancer (TNBC) is notoriously difficult to treat due to the lack of biological targets and poor sensitivity to conventional therapies. Chemotherapy is the main clinical therapy, but the effective screening strategy for chemotherapy drugs is poorly investigated. Drug repositioning has been the center of attention in recent years attracting numerous studies. Here, we firstly found multiple common features between leukemia and TNBC by analyzing the global transcriptome profiles based on the transformed comparison data from NCI60. Therefore, we investigated the role of the classic leukemia drug thioguanine (6-TG) in TNBC cancer cells. Our results indicated that 6-TG inhibited cell proliferation and tumor cell progression by suppressing PI3K-AKT pathway via downregulating the DNA methylation level of PTEN. Moreover, apoptosis was induced via the activation of PI3K-AKT downstream TSC1 and the downregulation of methylation levels of DAXX, TNF, FADD and CASP8 etc. These findings indicated 6-TG exerts its anti-tumor effects in vitro and in vivo through regulating the DNA methylation levels of genes involved in PI3K-AKT and apoptosis pathway. Meanwhile, our study suggested that transcriptome-based drug screening has potential implications for breast cancer therapy and drug selection.

Keywords: PI3K–AKT; methylation; thioguanine; transcriptome; triple-negative breast cancer.

Theaflavin (TF) is a major active pigment and polyphenol of tea, possessing anti-cancer activities. However, little is known about its activity and mechanism on melanoma cells. To fill this gap, we conducted in vitro experiments (cell viability assay, morphology observation, DAPI staining, and flow cytometry) and in vivo experiment by using a xenograft model of larval zebrafishes. Real-time PCR (qPCR) and Western blot (WB) analyses were conducted to explore the mechanism of TF. The in vitro data showed that TF exerted significant anti-proliferative and pro-apoptotic effects on A375 cells in a concentration-dependent manner. In vivo, TF significantly inhibited A375 tumor growth in larval zebrafishes at 0.67 and 2.0 μg/ml (1.3 to 3.9 μM). qPCR and WB data showed that TF significantly activated the P53 pathway-related proteins (ATM, CHK1/2, P53, and CASP8/3) and the JNK pathway-related proteins (ASK1, JNK, and C-JUN) through phosphorylation and cleavage, followed by activation of pro-apoptotic molecules (PARP, BAX, BIM, PUMA, and P53). In sum, TF possessed cytotoxic pro-apoptotic and tumor-inhibitory effects on A375 cells through activations of P53 and JNK pathways. This is the first report on TF regarding its effects and mechanism on A375 cells, making it a promising candidate of natural products for clinical treatment of melanoma.

Keywords: JNK; P53; green tea; melanoma; theaflavin; zebrafish.

BACKGROUND

Caspase-8 and caspase-9 (encoded by CASP8 and CASP9) are executive caspases of programmed cell death (apoptosis). Dysregulation of apoptosis plays an important role in cancer development, progression, and resistance to anticancer therapy. The goal of this work was to evaluate potential associations between polymorphisms in CASP8 and CASP9, previously linked to breast cancer risk, and the transcript levels of these genes (including their alternative anti-apoptotic variants) in tumor tissues and the clinical characteristics of the patients.

METHODS

Sanger sequencing, high resolution melting (HRM) analysis, and allelic discrimination were used to identify polymorphisms in DNA samples isolated from tumor tissues and peripheral blood lymphocytes of 60 breast carcinoma patients. Total transcript levels of CASP8 and CASP9, and levels of alternative splicing variants CASP8L and CASP9B, were quantified by real-time PCR in tumor tissues. Clinically interesting associations were validated in DNA from lymphocytes of 615 breast carcinoma patients.

RESULTS

A haplotype in CASP9 composed of three polymorphisms rs4645978-rs2020903-rs4646034 was significantly associated with CASP9 expression in tumors, with the expression of the progesterone receptor and ERBB2, and with the TNBC subtype of breast carcinoma in the validation study. The associations between the rs3834129 polymorphism in CASP8 and stage of disease, rs6435074 with grade, expression of estrogen receptor and ERBB2, and rs6723097 with ERBB2 expression have not yet been validated. However, rs6723097 was associated with disease-free survival in patients treated with hormonal therapy.

CONCLUSIONS

This study reveals a previously unknown and presumably functional (in silico) association between a haplotype in CASP9 and molecular and clinical phenotypes of breast carcinoma. The potential clinical utility of this association for prognostication of breast carcinoma should be evaluated by independent studies.Key words: breast carcinoma - caspases - polymorphisms - functional - clinical - importanceThis work was supported by grant of the CU Grant Agency No. 1444313, and grant of the Internal Grant Agency of the Czech Ministry of Health No. 15-25618A.The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study.The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 3. 3. 2016Accepted: 26. 10. 2016.

Objective: This study is aimed at understanding the molecular mechanisms and exploring potential therapeutic targets for atrial fibrillation (AF) by multiomics analysis.

Methods: Transcriptomics and methylation data of AF patients were retrieved from the Gene Expression Omnibus (GEO). Differentially expressed genes (DEGs) and differentially methylated sites between AF and normal samples were screened. Then, highly expressed and hypomethylated and lowly expressed and hypermethylated genes were identified for AF. Weighted gene coexpression network analysis (WGCNA) was presented to construct AF-related coexpression networks. 52 AF blood samples were used for whole exome sequence. The mutation was visualized by the maftools package in R. Key genes were validated in AF using independent datasets.

Results: DEGs were identified between AF and controls, which were enriched in neutrophil activation and regulation of actin cytoskeleton. RHOA, CCR2, CASP8, and SYNPO2L exhibited abnormal expression and methylation, which have been confirmed to be related to AF. PCDHA family genes had high methylation and low expression in AF. We constructed two AF-related coexpression modules. Single-nucleotide polymorphism (SNP) was the most common mutation type in AF, especially T > C. MUC4 was the most frequent mutation gene, followed by PHLDA1, AHNAK2, and MAML3. There was no statistical difference in expression of AHNAK2 and MAML3, for AF. PHLDA1 and MUC4 were confirmed to be abnormally expressed in AF.

Conclusion: Our findings identified DEGs related to DNA methylation and mutation for AF, which may offer possible therapeutic targets and a new insight into the pathogenesis of AF from a multiomics perspective.

Lymphoproliferative disease (LPD) is a comprehensive concept covering diseases ranging from transient lymphadenopathy to lymphoma. LPD is frequently associated with Epstein-Barr virus (EBV) infections and tends to occur in patients with inborn errors of immunity (IEI) and in patients after organ transplantation. Most patients with severe combined immunodeficiency or X-linked lymphoproliferative disease develop LPD. Autoimmune lymphoproliferative syndrome (ALPS), a typical LPD disease, is caused by germline mutations in FAS, FASL, CASP10, CASP8 and FADD, which are involved in the apoptosis pathway. ALPS patients develop autoimmune diseases and LPDs such as hepatosplenomegaly and lymphadenopathy. On the other hand, RAS-associated ALPS-like syndrome and CTLA4 haploinsufficiency also belong to ALPS-associated diseases. EBV-associated LPD is a clinical condition that should be noted in patients with IEI. Patients with genetic defects in SH2D1A, XIAP, CD27, CD70, CD137, ITK, CTPS, RASGRP1, and MAGT1 are prone to EBV-associated LPD.

Keywords: Epstein-Barr virus; Immunodeficiency-associated lymphoproliferative disorders; Inborn errors of immunity; Lymphoma.

Introduction: The impaired balance between cell proliferation and cell death, followed the inability to receive the death signals, cells push towards the neoplasia pathway. Fibulin 1 (FBLN1) plays a role as a co-factor in the mechanism of action of a protease such as a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS-1), which has important roles in angiogenesis, can also act as both tumor suppressor gene (TSG) and an oncogene in the main constituent of the extra-cellular matrix. This preliminary study has investigated the effects of silencing FBLN1 with siRNA on autophagy, proliferation, apoptosis pathways in the MSM cell line.

Material and methods: It was transfected siRNA specific to FBLN1 incubated MSM SPC212 cells, and compared with negative control siRNAs by a real-time polymerase chain reaction. It was determined apoptosis, proliferation, autophagy-related genes in mRNA levels.

Results: It was observed that increased anti-apoptosis genes, such as CASP2, CASP7, DDFA, and BCL2, anti-apoptotic gene, reduced APAF1, CASP8. Proliferation induced through while increased ADAMTS1, CDH1, CDH6, CLDN7, CSF3, MMP7, MMP13 genes. Autophagy increased via increasing MAP1LC3B, ATG-16L1 genes while decreased via suppressed ULK1, and ATG7 genes by silencing FBLN1 with siRNAs (p < 0.05).

Conclusions: Proliferation can be induction with silencing of FBLN1 with siRNA in processing mechanism MSM. It was concluded that FBLN1 could be act as pleiotropic on autophagy, and apoptosis pathways in proliferation processing for MSM. Therefore we think that FBLN1 acts like a TSG. FBLN1 can be considered as a targeted treatment option in advanced stage MSM.

Keywords: apoptosis; autophagy; fibulin 1; mesothelioma; proliferation.

Objective: Hua-Feng-Dan (HFD) is a Chinese medicine for stroke. This study is to predict and verify potential molecular targets and pathways of HFD against stroke using network pharmacology.

Methods: The TCMSP database and TCMID were used to search for the active ingredients of HFD, and GeneCards and DrugBank databases were used to search for stroke-related target genes to construct the "component-target-disease" by Cytoscape 3.7.1, which was further filtered by MCODE to build a core network. The STRING database was used to obtain interrelationships by topology and to construct a protein-protein interaction network. GO and KEGG were carried out through DAVID Bioinformatics. Autodock 4.2 was used for molecular docking. BaseSpace was used to correlate target genes with the GEO database.

Results: Based on OB ≥ 30% and DL ≥ 0.18, 42 active ingredients were extracted from HFD, and 107 associated targets were obtained. PPI network and Cytoscape analysis identified 22 key targets. GO analysis suggested 51 cellular biological processes, and KEGG suggested that 60 pathways were related to the antistroke mechanism of HFD, with p53, PI3K-Akt, and apoptosis signaling pathways being most important for HFD effects. Molecular docking verified interactions between the core target (CASP8, CASP9, MDM2, CYCS, RELA, and CCND1) and the active ingredients (beta-sitosterol, luteolin, baicalein, and wogonin). The identified gene targets were highly correlated with the GEO biosets, and the stroke-protection effects of Xuesaitong in the database were verified by identified targets.

Conclusion: HFD could regulate the symptoms of stroke through signaling pathways with core targets. This work provided a bioinformatic method to clarify the antistroke mechanism of HFD, and the identified core targets could be valuable to evaluate the antistroke effects of traditional Chinese medicines.

Platinum-based adjuvant chemotherapy is very common for gastric cancer (GC) patients, but the chemotherapy sensitivity is very heterogeneous. The genomic variants and the gene-gene interactions involved in Fas-mediated apoptosis pathway including Fas (FAS 1377 G > A and 670 A > G), FasL (FASL 844 C > T) and caspase-8 (CASP8 -652 6N ins > del or I > D), may paly vital roles in the response to platinum-based treatment. In our investigation, 662 stage II-III postoperative GC patients were enrolled between 1998 and 2006. 261 patients accepted platinum-based regimens and the remaining 401 were not. The log rank tests, Kaplan Meier plots, Pearson chi-square tests, Student t-tests and Cox regression analyses were performed. For the chemotherapy cohort, FAS 1377 G > A or FAS 670 A > G variants alone was related with inferior survival, and a greater than additive effect was identified when patients simultaneously carrying FAS 1377 GA and FAS 670 GA genotypes. But the poor response was neutralized when patients simultaneously carrying FASL 844 C > T or CASP8 -652 6N ins > del mutations. Our study suggested that FAS 1377 G > A and FAS 670 A > G variants may serve as potential biomarkers to predict the response to platinum-based adjuvant chemotherapy, and the gene-gene interactions involved in Fas-mediated apoptosis pathway may enhance or neutralize the chemosensitivity.

Keywords: Fas-mediated apoptosis; chemotherapy; gastric cancer (GC); genetic variants.

We analyzed the peculiarities of the copy number variation of genes that regulate apoptosis, DNA repair, cell proliferation, metabolism, and estrogen reception in tumor and normal cells of high-grade and low-grade serous adenocarcinoma of the ovaries. Using real-time qPCR method, the relative copy number of 34 genes (BAX, BCL2, TP53, MDM2, CASP9, CASP3, CASP7, CASP8, PRKCI, SOX2, OCT4, PIK3, PTEN, C-MYC, SOX18, AKT1, NOTCH1, BRCA1, BRCA2, EXO1, SCNN1A, KRAS, EGFR, BRAF, CYP1A1, CYP1A2, CYP1B1, CYP19A, ESR1, ESR2, GPER, STS, SULT1A, and SULT1E1) was determined in normal and tumor cells of the ovaries extracted by contactless capture laser microsection from FFPE-blocks of 200 patients. The most typical molecular markers of ovarian serous adenocarcinoma cells were identified: copy number of PIK3CA, BCL2, BAX, CASP3, and CASP8 genes. Based on the differences in the gene copy number variation, two molecular subtypes of serous adenocarcinoma were identified, corresponding to two histological subtypes: high-grade (MDM2, SOX2, ESR1, CYP1B1, SULT1E1, TP53, BRCA2) and low-grade (PIK3CA, PTEN, BCL2, BAX, and CASP3). Each of these subtypes was also characterized by molecular heterogeneity and can be subdivided into several subgroups: 3 subgroups for high-grade and 4 subgroups for low-grade serous adenocarcinoma. These findings extend our understanding of the molecular mechanisms of carcinogenesis in the ovarian tissue, confirm molecular difference between the two histological subtypes of serous adenocarcinoma probably underlying their different clinical course.

Keywords: apoptosis; cell proliferation; gene copy number variation; laser microdissection; serous ovarian adenocarcinoma.

Majoranolide, a butanolide isolated from the nonpolar fraction of an ethanol extract of Mezilaurus crassiramea (Lauraceae) fruits, is being reported for the first time in this genus and the third time in plants. Structurally identified from 1D and 2D NMR and HRESIMS data, majoranolide proved cytotoxic against cancer cells-MCF-7 and MDA-MB-231 (breast), HT-29 (colon), PC-3 (prostate), 786-0 (renal), and HL-60 (leukemia)-inhibiting growth in HL-60 cells (GI₅₀ = 0.21 μM) and exhibiting higher selectivity for this line than for nonneoplastic NIH/3T3 murine fibroblasts. Effects on the cell cycle, caspase-3 activation, and plasma membrane integrity were evaluated by flow cytometry. Expression of genes related to apoptotic pathways (BAX, BCL2, BIRC5, and CASP8) was investigated using RT-qPCR. At 50 μM, majoranolide induced cell cycle arrest at G1 in 24 h increased the sub-G1 population in 48 h and increased caspase-3 activation in a time-dependent manner. The compound upregulated BAX and CASP8 transcription (proapoptotic genes) and downregulated BIRC5 (antiapoptotic). Loss of plasma membrane integrity in 30% of cells occurred at 48 h, but not at 24 h, characterizing gradual, programmed death. The results suggest that majoranolide cytotoxicity involves apoptosis induction in HL-60 cells, although other mechanisms may contribute to this cell death.

Prostate cancer remains the most common non-cutaneous malignancy among men in the United States. To discover potential serum-based biomarkers for high-risk prostate cancer, we performed a high-multiplex immunoassay utilizing patient-matched pre-operative and post-operative serum samples from ten men with high-grade and high-volume prostate cancer. Our study identified six (CASP8, MSLN, FGFBP1, ICOSLG, TIE2 and S100A4) out of 174 proteins that were significantly decreased after radical prostatectomy. High levels of CASP8 were detected in pre-operative serum samples when compared to post-operative serum samples and serum samples from patients with benign prostate hyperplasia (BPH). By immunohistochemistry, CASP8 protein was expressed at higher levels in prostate cancer tissues compared to non-cancerous and BPH tissues. Likewise, CASP8 mRNA expression was significantly upregulated in prostate cancer when compared to benign prostate tissues in four independent clinical datasets. In addition, mRNA levels of CASP8 were higher in patients with recurrent prostate cancer when compared to patients with non-recurrent prostate cancer and high expression of CASP8 was associated with worse disease-free survival and overall survival in renal cancer. Together, our results suggest that CASP8 may potentially serve as a biomarker for high-risk prostate cancer and possibly renal cancer.

Objective: In Jordan, breast cancer (BC) affects a substantial proportion of Jordanian women, highlighting the need for studies to be carried out regarding the genetic component of the disease. The aim of the present study was to investigate the interaction between BC risk and prognosis and polymorphisms in genes (ATM, CASP8, FGFR2, FN1, IGF1, LSP1, MAP3K, MMP7, and RHOC) that were chosen for this study previously reported as having a role in the disease.

Materials and methods: Blood samples were collected from 242 BC patients and 231 disease-free volunteers recruited from the Jordanian population. DNA was extracted from blood and each sample was sent to the Australian Genome Research Facility for genotyping.

Results: The rs1219648 SNP of the FGFR2 gene was the only investigated variant to show any direct association with BC in Jordanian women (p-value = 0.04). However, the CASP8rs6760993 SNP was found to be significantly associated with BC (p-value = 0.04) when using the dominant model. Other gene polymorphisms showed varying levels of association between some investigated SNPs and different BC risk and prognostic factors.

Conclusion: Despite reports to the contrary in other populations, most of the investigated genes and their respective SNPs did not show any significant association with BC in Jordanian women. Our results underline the need for independent BC research to be carried out in the Jordanian population to decipher the genetic basis of the disease.<br />.

Keywords: Genetic susceptibility; SNPs; breast cancer.

This corrects the article DOI: 10.1038/nm.4290.

Background: This observational clinical study evaluated the expression levels and predictive values of some apoptosis-related genes in granulosa cells (GCs) and follicular fluid (FF) of women undergoing in vitro fertilization (IVF).

Methods: GCs and FF were obtained at oocyte retrieval from 31 consecutive patients with heterogeneous infertility diagnosis (age: 34.3 ± 5.8 years, body mass index: 24.02 ± 3.12 kg/m2, duration of infertility: 4.2 ± 2.1 years). mRNA expression of pro-apoptotic (BAX, CASP3, CASP8) and anti-apoptotic (BCL2, AMH, AMHR, FSHR, LHR, CYP19A1) factors was determined by quantitative RT-PCR using ROCHE LightCycler 480.

Results: No significant difference in GC or FF mRNA expression of pro- and anti-apoptotic factors could be demonstrated between IVF patients with (9 patients) or without (22 patients) clinical pregnancy. Each transcript investigated was detected in FF, but their levels were markedly reduced and independent of those in GCs. The number of retrieved oocytes was positively associated with GC AMHR (r = 0.393, p = 0.029), but the day of embryo transfer was negatively associated with GC LHR (r = - 0.414, p = 0.020) and GC FSHR transcripts (r = - 0.535, p = 0.002). When pregnancy positive group was analysed separately the impact of apoptosis- related gene expressions on some selected measures of IVF success could be observed. Strong positive relationship was found between gene expression levels of pro- and anti-apoptotic factors in GCs.

Conclusion: Our study provides only marginal evidences for the apoptosis dependence of IVF outcome and suggests that the apoptosis process induces adaptive increases of the anti-apoptotic gene expression to attenuate apoptosis and to protect cell survival.

Keywords: Apoptosis; Follicular fluid; Granulosa cell; In vitro fertilization; mRNA expression.

The present study describes a 19-year-old woman with systemic herpes simplex virus (HSV)-1 infection postpartum, and a fatal course of neonatal herpesvirus infection. The mother experienced an unusual disease course with hemophagocytic lymphohistiocytosis (HLH) and persistence of HSV-1 DNA for 15 weeks. Functional investigation of cells from the mother demonstrated significantly impaired induction of antiviral interferons and cytokines in response to viruses and various ligands in the context of normal activation of the transcription factors NF-κB and IRF3. Whole exome sequencing did not reveal any functionally validated genetic variants. We suggest that the functionally impaired antiviral responses, potentially caused by a mutation in CASP8 or other variants in non-coding regions of the genome, contributed to the unusually severe disease course in two generations with disseminated HSV-1 infection evolving into HLH in the mother, and a fatal neonatal HSV-1 infection.

Keywords: Herpes simplex virus; hemophagocytic lymphohistiocytosis; innate immunity; interferon; neonatal herpes.

NLRP3 inflammasome is involved in several chronic inflammatory diseases. The inflammatory effect of the NLRP3 inflammasome is executed through IL-1β and IL-18. Therefore, IL-1β is one of the primary targets in chronic inflammatory conditions. However, current treatment regimens are dependent on anti- IL-1β biologicals. The therapies targeting IL-1β through inhibition of NLRP3 inflammasome are thus being actively explored. We identified safranal, a small molecule responsible for the essence of saffron as a potential inhibitor of the NLRP3 inflammasome. Safranal significantly suppressed the release of IL-1β from ATP stimulated J774A.1 and bone marrow-derived macrophages (BMDMs) by regulating CASP1 and CASP8 dependent cleavage of pro-IL-1β. Safranal markedly suppressed the expression of NLRP3 and its ATPase activity. Safranal treatment enhanced the expression of NRF2, whereas, si-RNA mediated silencing of Nrf2 abrogated the anti-NLRP3 effect of safranal. Furthermore, safranal inhibited ASC oligomerization and formation of ASC specks. Safranal also displayed anti-NLRP3 activity in multiple mice models. Treatment of animals with safranal reduced the production of IL-1β in ATP elicited peritoneal inflammation, MSU induced air pouch inflammation, and MSU injected foot paw edema in mice. Thus, our data projects safranal as a potential preclinical drug candidate against NLRP3 inflammasome triggered chronic inflammation.

Keywords: ASC; Caspase-1; IL-1β; Macrophages; NLRP3 inflammasome; Saffron; Safranal.