Inactivated whole-virion vaccine BBV152/Covaxin elicits robust cellular immune memory to SARS-CoV-2 and variants of concern.

BBV152 is a whole-virion inactivated vaccine based on the Asp614Gly variant. BBV152 is the first alum-imidazoquinolin-adjuvanted vaccine authorized for use in large populations. Here we characterized the magnitude, quality and persistence of cellular and humoral memory responses up to 6 months post vaccination. We report that the magnitude of vaccine-induced spike and nucleoprotein antibodies was comparable with that produced after infection. Receptor binding domain-specific antibodies declined against variants in the order of Alpha (B.1.1.7; 3-fold), Delta (B.1.617.2; 7-fold) and Beta (B.1.351; 10-fold). However, pseudovirus neutralizing antibodies declined up to 2-fold against the Delta followed by the Beta variant (1.7-fold). Vaccine-induced memory B cells were also affected by the Delta and Beta variants. The SARS-CoV-2-specific multicytokine-expressing CD4+ T cells were found in ~85% of vaccinated individuals. Only a ~1.3-fold reduction in efficacy was observed in CD4+ T cells against the Beta variant. We found that antigen-specific CD4+ T cells were present in the central memory compartment and persisted for at least up to 6 months post vaccination. Vaccine-induced CD8+ T cells were detected in ~50% of individuals. Importantly, the vaccine was capable of inducing follicular T helper cells that exhibited B-cell help potential. These findings show that inactivated vaccine BBV152 induces robust immune memory to SARS-CoV-2 and variants of concern that persists for at least 6 months after vaccination.

An efficient immunoassay for the B cell help function of SARS-CoV-2-specific memory CD4+ T cells.

The B cell "help" function of CD4+ T cells is an important mechanism of adaptive immunity. Here, we describe improved antigen-specific T-B cocultures for quantitative measurement of T cell-dependent B cell responses, with as few as ∼90 T cells. Utilizing M. tuberculosis (Mtb), we show that early priming and activation of CD4+ T cells is important for productive interaction between T and B cells and that similar effects are achieved by supplementing cocultures with monocytes. We find that monocytes promote survivability of B cells via BAFF and stem cell growth factor (SCGF)/C-type lectin domain family 11 member A (CLEC11A), but this alone does not fully recapitulate the effects of monocyte supplementation. Importantly, we demonstrate improved activation and immunological output of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific memory CD4+ T-B cell cocultures with the inclusion of monocytes. This method may therefore provide a more sensitive assay to evaluate the B cell help quality of memory CD4+ T cells, for example, after vaccination or natural infection.

Effectiveness of ChAdOx1 nCoV-19 vaccine against SARS-CoV-2 infection during the delta (B.1.617.2) variant surge in India: a test-negative, case-control study and a mechanistic study of post-vaccination immune responses.

BACKGROUND: SARS-CoV-2 variants of concern (VOCs) have threatened COVID-19 vaccine effectiveness. We aimed to assess the effectiveness of the ChAdOx1 nCoV-19 vaccine, predominantly against the delta (B.1.617.2) variant, in addition to the cellular immune response to vaccination. METHODS: We did a test-negative, case-control study at two medical research centres in Faridabad, India. All individuals who had a positive RT-PCR test for SARS-CoV-2 infection between April 1, 2021, and May 31, 2021, were included as cases and individuals who had a negative RT-PCR test were included as controls after matching with cases on calendar week of RT-PCR test. The primary outcome was effectiveness of complete vaccination with the ChAdOx1 nCoV-19 vaccine against laboratory-confirmed SARS-CoV-2 infection. The secondary outcomes were effectiveness of a single dose against SARS-CoV-2 infection and effectiveness of a single dose and complete vaccination against moderate-to-severe disease among infected individuals. Additionally, we tested in-vitro live-virus neutralisation and T-cell immune responses to the spike protein of the wild-type SARS-CoV-2 and VOCs among healthy (anti-nucleocapsid antibody negative) recipients of the ChAdOx1 nCoV-19 vaccine. FINDINGS: Of 2379 cases of confirmed SARS-CoV-2 infection, 85 (3·6%) were fully vaccinated compared with 168 (8·5%) of 1981 controls (adjusted OR [aOR] 0·37 [95% CI 0·28-0·48]), giving a vaccine effectiveness against SARS-CoV-2 infection of 63·1% (95% CI 51·5-72·1). 157 (6·4%) of 2451 of cases and 181 (9·1%) of 1994) controls had received a single dose of the ChAdOx1 nCoV-19 vaccine (aOR 0·54 [95% CI 0·42-0·68]), thus vaccine effectiveness of a single dose against SARS-CoV-2 infection was 46·2% (95% CI 31·6-57·7). One of 84 cases with moderate-to-severe COVID-19 was fully vaccinated compared with 84 of 2295 cases with mild COVID-19 (aOR 0·19 [95% CI 0·01-0·90]), giving a vaccine effectiveness of complete vaccination against moderate-to-severe disease of 81·5% (95% CI 9·9-99·0). The effectiveness of a single dose against moderate-to-severe disease was 79·2% (95% CI 46·1-94·0); four of 87 individuals with moderate-to-severe COVID-19 had received a single dose compared with 153 of 2364 participants with mild disease (aOR 0·20 [95% CI 0·06-0·54]). Among 49 healthy, fully vaccinated individuals, neutralising antibody responses were lower against the alpha (B.1.1.7; geometric mean titre 244·7 [95% CI 151·8-394·4]), beta (B.1.351; 97·6 [61·2-155·8]), kappa (B.1.617.1; 112·8 [72·7-175·0]), and delta (88·4 [61·2-127·8]) variants than against wild-type SARS-CoV-2 (599·4 [376·9-953·2]). However, the antigen-specific CD4 and CD8 T-cell responses were conserved against both the delta variant and wild-type SARS-CoV-2. INTERPRETATION: The ChAdOx1 nCoV-19 vaccine remained effective against moderate-to-severe COVID-19, even during a surge that was dominated by the highly transmissible delta variant of SARS-CoV-2. Spike-specific T-cell responses were maintained against the delta variant. Such cellular immune protection might compensate for waning humoral immunity. FUNDING: Department of Biotechnology India, Council of Scientific and Industrial Research India, and Fondation Botnar.

Japanese Encephalitis Virus Infected Human Monocyte-Derived Dendritic Cells Activate a Transcriptional Network Leading to an Antiviral Inflammatory Response.

A comprehensive understanding of the human immune response to virus infection is imperative for developing effective therapies, antivirals, and vaccines. Dendritic cells (DCs) are among the first cells to encounter the virus and are also key antigen-presenting cells that link the innate and adaptive immune system. In this study, we focus on the human immune response to the mosquito-borne Japanese encephalitis virus (JEV), which is the leading cause of virus-induced encephalitis in south-east Asia and has the potential to become a global pathogen. We describe the gene regulatory circuit of JEV infection in human monocyte-derived DCs (moDCs) along with its functional validation. We observe that JEV can productively infect human moDCs leading to robust transcriptional activation of the interferon and NF-κB-mediated antiviral and inflammatory pathways. This is accompanied with DC maturation and release of pro-inflammatory cytokines and chemokines TNFα, IL-6, IL-8, IL-12, MCP-1. and RANTES. JEV-infected moDCs activated T-regulatory cells (Tregs) in allogenic mixed lymphocyte reactions (MLR) as seen by upregulated FOXP3 mRNA expression, suggestive of a host response to reduce virus-induced immunopathology. The virus also downregulated transcripts involved in Peroxisome Proliferator Activated Receptor (PPAR) signalling and fatty acid metabolism pathways suggesting that changes in cellular metabolism play a crucial role in driving the DC maturation and antiviral responses. Collectively, our data describe and corroborate the human DC transcriptional network that is engaged upon JEV sensing.

Immune Memory in Mild COVID-19 Patients and Unexposed Donors Reveals Persistent T Cell Responses After SARS-CoV-2 Infection.

Understanding the causes of the diverse outcome of COVID-19 pandemic in different geographical locations is important for the worldwide vaccine implementation and pandemic control responses. We analyzed 42 unexposed healthy donors and 28 mild COVID-19 subjects up to 5 months from the recovery for SARS-CoV-2 specific immunological memory. Using HLA class II predicted peptide megapools, we identified SARS-CoV-2 cross-reactive CD4+ T cells in around 66% of the unexposed individuals. Moreover, we found detectable immune memory in mild COVID-19 patients several months after recovery in the crucial arms of protective adaptive immunity; CD4+ T cells and B cells, with a minimal contribution from CD8+ T cells. Interestingly, the persistent immune memory in COVID-19 patients is predominantly targeted towards the Spike glycoprotein of the SARS-CoV-2. This study provides the evidence of both high magnitude pre-existing and persistent immune memory in Indian population. By providing the knowledge on cellular immune responses to SARS-CoV-2, our work has implication for the development and implementation of vaccines against COVID-19.

An immune epigenetic insight to COVID-19 infection.

Severe acute respiratory syndrome coronavirus-2 is a positive-sense RNA virus, a causal agent of ongoing COVID-19 pandemic. ACE2R methylation across three CpG sites (cg04013915, cg08559914, cg03536816) determines the host cell's entry. It regulates ACE2 expression by controlling the SIRT1 and KDM5B activity. Further, it regulates Type I and III IFN response by modulating H3K27me3 and H3K4me3 histone mark. SARS-CoV-2 protein with bromodomain and protein E mimics bromodomain histones and evades from host immune response. The 2'-O MTases mimics the host's cap1 structure and plays a vital role in immune evasion through Hsp90-mediated epigenetic process to hijack the infected cells. Although the current review highlighted the critical epigenetic events associated with SARS-CoV-2 immune evasion, the detailed mechanism is yet to be elucidated.

Immune memory in mild COVID-19 patients and unexposed donors from India reveals persistent T cell responses after SARS-CoV-2 infection.

Understanding the causes of the diverse outcome of COVID-19 pandemic in different geographical locations is important for the worldwide vaccine implementation and pandemic control responses. We analyzed 42 unexposed healthy donors and 28 mild COVID-19 subjects up to 5 months from the recovery for SARS-CoV-2 specific immunological memory. Using HLA class II predicted peptide megapools, we identified SARS-CoV-2 cross-reactive CD4+ T cells in around 66% of the unexposed individuals. Moreover, we found detectable immune memory in mild COVID-19 patients several months after recovery in the crucial arms of protective adaptive immunity; CD4+ T cells and B cells, with a minimal contribution from CD8+ T cells. Interestingly, the persistent immune memory in COVID-19 patients is predominantly targeted towards the Spike glycoprotein of the SARS-CoV-2. This study provides the evidence of both high magnitude pre-existing and persistent immune memory in Indian population. By providing the knowledge on cellular immune responses to SARS-CoV-2, our work has implication for the development and implementation of vaccines against COVID-19.

A haemagglutination test for rapid detection of antibodies to SARS-CoV-2.

Serological detection of antibodies to SARS-CoV-2 is essential for establishing rates of seroconversion in populations, and for seeking evidence for a level of antibody that may be protective against COVID-19 disease. Several high-performance commercial tests have been described, but these require centralised laboratory facilities that are comparatively expensive, and therefore not available universally. Red cell agglutination tests do not require special equipment, are read by eye, have short development times, low cost and can be applied at the Point of Care. Here we describe a quantitative Haemagglutination test (HAT) for the detection of antibodies to the receptor binding domain of the SARS-CoV-2 spike protein. The HAT has a sensitivity of 90% and specificity of 99% for detection of antibodies after a PCR diagnosed infection. We will supply aliquots of the test reagent sufficient for ten thousand test wells free of charge to qualified research groups anywhere in the world.

CD8+ T cells are crucial for humoral immunity establishment by SA14-14-2 live attenuated Japanese encephalitis vaccine in mice.

The live attenuated SA14-14-2 Japanese encephalitis (JE) vaccine is a historical vaccine that protects against JE. Despite its extensive use, the mechanism of protective immunity conferred by the SA14-14-2 vaccine is not well established. Here, we used mouse models to understand the mechanism of the development of humoral immunity against the vaccine. The vaccine induces robust GC responses within a week postimmunization. In lethal virus challenge, we show that CD4+ T cells alone, but not CD8+ T cells, are sufficient to confer vaccine-mediated protection. However, the CD4-mediated protection was potentiated in the presence of vaccine-primed CD8+ T cells. Employing CD8-deficient mice, we show that both the protective traits of CD4+ T cells and the quality of antibody response to the vaccine are impaired in absence of CD8+ T cells. We further demonstrate that the poor protective immune response induced by the vaccine in absence of CD8+ T cells is mainly due to the impaired differentiation and function of follicular Th cells, leading to suboptimal GC reaction. Our study highlights an unprecedented role of CD8+ T cells in the establishment of humoral responses to the vaccine. By elucidating underlying cellular determinants of vaccine-induced protective immunity, our work has implications for rational design of vaccines against JE virus and related flaviviruses.

Immunogenicity of long-lasting recombinant factor VIII products.

Replacement therapy for patients with hemophilia A using plasma-derived or recombinant factor VIII (FVIII) is complicated by the short half-life of the FVIII products and by the occurrence of neutralizing antibodies in a substantial number of patients. In the recent years, enormous efforts have been invested to develop new generations of coagulation factors with extended half-lives. Presumably, the use of long-lasting FVIII products should reduce the frequency of administration to the patients and drastically improve their quality of life. The question of their immunogenicity remains however unanswered as yet. The present review proposes a summary of the different strategies developed to enhance the half-life of FVIII, including fusion of FVIII to the Fc fragment of the human IgG1 or to human serum albumin, or attachment of polyethylene glycol. Based on the available literature, we hypothesize on the potential benefits or risks associated with each of the latter strategies in terms of immunogenicity of the newly derived hemostatic drugs.

Neutralization of Japanese Encephalitis Virus by heme-induced broadly reactive human monoclonal antibody.

Geographical expansion and re-emerging new genotypes of the Japanese encephalitis virus (JEV) require the development of novel therapeutic approaches. Here, we studied a non-conventional approach for antibody therapy and show that, upon exposure to heme, a fraction of natural human immunoglobulins acquires high-affinity reactivity with the antigenic domain-III of JEV E glycoprotein. These JEV-reactive antibodies exhibited neutralizing activity against recently dominant JEV genotypes. This study opens new therapeutic options for Japanese encephalitis.

Materno-Fetal Transfer of Preproinsulin Through the Neonatal Fc Receptor Prevents Autoimmune Diabetes.

The first signs of autoimmune activation leading to ß-cell destruction in type 1 diabetes (T1D) appear during the first months of life. Thus, the perinatal period offers a suitable time window for disease prevention. Moreover, thymic selection of autoreactive T cells is most active during this period, providing a therapeutic opportunity not exploited to date. We therefore devised a strategy by which the T1D-triggering antigen preproinsulin fused with the immunoglobulin (Ig)G Fc fragment (PPI-Fc) is delivered to fetuses through the neonatal Fc receptor (FcRn) pathway, which physiologically transfers maternal IgGs through the placenta. PPI-Fc administered to pregnant PPIB15-23 T-cell receptor-transgenic mice efficiently accumulated in fetuses through the placental FcRn and protected them from subsequent diabetes development. Protection relied on ferrying of PPI-Fc to the thymus by migratory dendritic cells and resulted in a rise in thymic-derived CD4(+) regulatory T cells expressing transforming growth factor-ß and in increased effector CD8(+) T cells displaying impaired cytotoxicity. Moreover, polyclonal splenocytes from nonobese diabetic (NOD) mice transplacentally treated with PPI-Fc were less diabetogenic upon transfer into NOD.scid recipients. Transplacental antigen vaccination provides a novel strategy for early T1D prevention and, further, is applicable to other immune-mediated conditions.

Regulation of immune responses to protein therapeutics by transplacental induction of T cell tolerance.

Central tolerance plays a key role in modulating immune responses to self and exogenous antigens. The absence of self-antigen expression, as in patients with genetic deficiencies, prevents the development of antigen-specific immune tolerance. Hence, a substantial number of patients develop neutralizing antibodies to the corresponding protein therapeutics after replacement treatment. In this context, the administration of missing antigens during fetal development, a key period for self-tolerance establishment, should confer early and long-lasting antigen-specific tolerance. To this end, we exploited the physiological pathway of the neonatal Fc receptor (FcRn) through which maternal immunoglobulins are transplacentally transferred to fetuses. We demonstrate that Fc-fused antigens administered to pregnant mice reach fetal lymphoid organs in an FcRn-dependent manner, accumulate in antigen-presenting cells of myeloid origin, and promote the generation of both thymic and peripheral antigen-specific regulatory T cells. This strategy was successfully pursued in a mouse model of hemophilia A, where maternofetal transfer of the Fc-fused immunodominant domains of coagulation factor VIII conferred antigen-specific tolerance. Transplacental tolerance induction with Fc-fused proteins may thus prove valuable to prevent alloimmunization after replacement protein therapy for congenital deficiencies.

Japanese encephalitis virus expands regulatory T cells by increasing the expression of PD-L1 on dendritic cells.

The mechanisms underlying Japanese encephalitis virus (JEV) pathogenesis need to be thoroughly explored to delineate therapeutic approaches. It is believed that JEV manipulates the innate and adaptive compartments of the host's immune system to evade immune response and cross the blood-brain barrier. The present study was thus designed to investigate the functional modulation of DCs after exposure to JEV and to assess the consequences on CD4(+) T-lymphocyte functions. Human monocyte-derived DCs were either infected with 1 MOI of live virus, UV-inactivated virus, or were mock-infected. Replication-competent JEV induced a significant increase in the expression of maturation markers 48 h postinfection, along with that of programmed cell death 1 ligand 1 (PD-L1; also called B7-H1 and CD274). JEV-infected DCs expanded the Treg cells in allogenic mixed lymphocyte reactions. The expansion of Treg cells by JEV-infected DCs was significantly reduced upon blocking PD-L1 using an antagonist. In addition, JEV-infected DCs significantly altered the proliferation and reduced the polarization of Th cells toward the Th1-cell phenotype. The results, for the first time, suggest that JEV evades the host's immune system by modulating the crosstalk between DCs and T lymphocytes via the PD-L1 axis.

Affinity-purified respiratory syncytial virus antibodies from intravenous immunoglobulin exert potent antibody-dependent cellular cytotoxicity.

Mixed infections are one of the major therapeutic challenges, as the current strategies have had limited success. One of the most common and widespread conditions of mixed infection is respiratory syncytial virus-mediated pathology of the respiratory tract in children. There is a dire need for the development of novel therapeutic approaches during mixed infections. Therapeutic intravenous immunoglobulin preparations, obtained from plasma pools of healthy donors have been used in immune deficiencies. This study was thus designed to characterize the functional efficacy of RSV-specific antibodies in IVIg. To explore the functional ability of these affinity-purified RSV-specific antibodies, the antibody-dependent and complement dependent cytotoxicity was determined using peripheral cells of healthy donors. This study demonstrates the existence of highly potent RSV-specific antibodies in IVIg preparations and provides the basis for the use of IVIg as broad-spectrum protective shield to RSV-infected children during mixed infections.

Transcriptomic profile of host response in mouse brain after exposure to plant toxin abrin.

Abrin toxin is a plant glycoprotein, which is similar in structure and properties to ricin and is obtained from the seeds of Abrus precatorius (jequirity bean). Abrin is highly toxic, with an estimated human fatal dose of 0.1-1 µg/kg, and has caused death after accidental and intentional poisoning. Abrin is a potent biological toxin warfare agent. There are no chemical antidotes available against the toxin. Neurological symptoms like delirium, hallucinations, reduced consciousness and generalized seizures were reported in human poisoning cases. Death of a patient with symptoms of acute demyelinating encephalopathy with gastrointestinal bleeding due to ingestion of abrin seeds was reported in India. The aim of this study was to examine both dose and time-dependent transcriptional responses induced by abrin in the adult mouse brain. Mice (n=6) were exposed to 1 and 2 LD50 (2.83 and 5.66 µg/kg respectively) dose of abrin by intraperitoneal route and observed over 3 days. A subset of animals (n=3) were sacrificed at 1 and 2 day intervals for microarray and histopathology analysis. None of the 2 LD50 exposed animals survived till 3 days. The histopathological analysis showed the severe damage in brain and the infiltration of inflammatory cells in a dose and time dependent manner. The abrin exposure resulted in the induction of rapid immune and inflammatory response in brain. Clinical biochemistry parameters like lactate dehydrogenase, aspartate aminotransferase, urea and creatinine showed significant increase at 2-day 2 LD50 exposure. The whole genome microarray data revealed the significant regulation of various pathways like MAPK pathway, cytokine-cytokine receptor interaction, calcium signaling pathway, Jak-STAT signaling pathway and natural killer cell mediated toxicity. The comparison of differential gene expression at both the doses showed dose dependent effects of abrin toxicity. The real-time qRT-PCR analysis of selected genes supported the microarray data. This is the first report on host-gene response using whole genome microarray in an animal model after abrin exposure. The data generated provides leads for developing suitable medical counter measures against abrin poisoning.

Transcriptomic profile of host response in Japanese encephalitis virus infection.

BACKGROUND: Japanese encephalitis (JE) is one of the leading causes of acute encephalopathy with the highest mortality rate of 30-50%. The purpose of this study was to understand complex biological processes of host response during the progression of the disease. Virus was subcutaneously administered in mice and brain was used for whole genome expression profiling by cDNA microarray. RESULTS: The comparison between viral replication efficiency and disease progression confirms the active role of host response in immunopathology and disease severity. The histopathological analysis confirms the severe damage in the brain in a time dependent manner. Interestingly, the transcription profile reveals significant and differential expression of various pattern recognition receptors, chemotactic genes and the activation of inflammasome. The increased leukocyte infiltration and aggravated CNS inflammation may be the cause of disease severity. CONCLUSION: This is the first report that provides a detailed picture of the host transcriptional response in a natural route of exposure and opens up new avenues for potential therapeutic and prophylactic strategies against Japanese encephalitis virus.

Transcriptional regulation and activation of the mitogen-activated protein kinase pathway after Japanese encephalitis virus infection in neuroblastoma cells.

Japanese encephalitis virus (JEV), the most frequent and the single most important cause of encephalitis worldwide, has spread throughout most of Asia. For the development of appropriate and effective therapy, there is an immediate requirement to understand the role of host factors in JEV-induced neuropathogenesis. In the present study, we investigated the role of mitogen-activated protein kinases (MAPKs) in JEV infection of mouse neuroblastoma (N2A) cells. The MAPK pathway was studied at the transcriptional level to access the gene expression profile at different time points after JEV infection. The effector MAPK genes were also analyzed for protein expression and activation. Gene expression analysis showed a significant regulation of extracellular signal-regulated kinases (ERK)1, ERK2 and c-Jun N-terminal kinase (JNK)3 genes along with their downstream transcription factors such as Mef2c, c-Jun and Sfn. Experiments with the JNK inhibitor, SP600125, and the ERK inhibitor, PD98059, showed the involvement of JNK in JEV-induced caspase-3 activation and apoptosis, but ERK1/2 had no effect. Overall, our results show the transcriptional regulation of the MAPK pathway and the essential role of JNK in JEV-induced apoptosis in neuroblastoma cells. These findings provide a new insight into the role of the mitogen- and stress-activated kinases in JEV pathogenesis and opens up new avenues of therapeutics.

Modulation of ROS/MAPK signaling pathways by okadaic acid leads to cell death via, mitochondrial mediated caspase-dependent mechanism.

Okadaic acid (OA) is a specific and potent protein phosphatase inhibitor and tumor promoter. The present study establishes the role of reactive oxygen species (ROS) and mitogen activated protein kinases in cell death induced by okadaic acid. The study showed that okadaic acid is cytotoxic at 10 nM with an IC50 of 100 nM in U-937 cells. The CVDE assay and mitochondrial dehydrogenase assay showed a time dependent cytotoxicity. The phase contrast visualization of the OA treated cells showed the apoptotic morphology and was confirmed with esterase staining for plasma membrane integrity. OA activated caspases-7, 9 and 3, PARP cleavage and induced nuclear damage in a time and dose dependent manner. Compromised mitochondrial membrane potential, release of cytochrome-c and apoptosis inducing factor confirms the involvement of mitochondria. A time dependent decrease in glutathione levels and a dose dependent increase in ROS with maximum at 30 min were observed. ROS scavenger-N-acetyl cysteine, mitochondrial stabilizer-cyclosporin-A, and broad spectrum caspase inhibitor Z-VAD-FMK inhibited the OA induced caspase-3 activation, DNA damage and cell death but caspase-8 inhibitor had no effect. OA activated p38 MAPK and JNK in a time dependent manner, but not ERK½. MAP kinase inhibitors SB203580, SP600125 and PD98059 confirm the role of p38 MAPK and JNK in OA induced caspase-3 activation and cell death. Over all, our results indicate that OA induces cell death by generation of ROS, and activation of p38 MAPK and JNK, and executed through mitochondrial mediated caspase pathway.