Immunotherapy has shown promising survival out- come in patients with HCC. Immune checkpoint inhibitor (ICI) demonstrated encouraging ef ﬁ cacy in certain cancer types, particularly in melanoma and lung cancer. 12 Programmedcelldeathprotein-1 (PD-1), programmeddeath-ligand 1 (PD-L1), and cytotoxic T lymphocyte- associated protein-4 (CTLA-4) are the main targets of ICIs. 13 Following by the approval of ipilimumab for malignant melanoma in 2011, a number of ICIs, including three anti-PD-1 antibodies (nivolumab, pembrolizumab, and cemiplimab) and three anti-PD-L1 antibodies (atezo- lizumab, durvalumab, and avelumab), have been approved by the Food and Drug Administration (FDA) for different types of cancers. 14 In the present clinical trials, Nivolumab and pembrolizumab have shown promising ef ﬁ cacy and safety results in non-comparative, open-label Phase II studies of advanced HCC. 15,16 Based on these studies, the United States Food and Drug Administration (FDA) already granted accelerated conditional approval to both agents for sorafenib-experienced patients with HCC. Both nivolumab and pembrolizumab are currently being inves- tigated in ongoing Phase III trials.
In some C. elegans cells, programmedcelldeath is regulated by controlling transcription of egl-1. To begin to determine whether Hox proteins might promote survival or programmedcelldeath of cells by directly regulating transcription of egl-1, we constructed a reporter transgene in which the egl-1 open reading frame was precisely replaced by a histone:gfp fusion (see Materials and methods) and examined expression of this reporter in wild-type and Hox mutant backgrounds. In wild-type transgenic hermaphrodites, expression of the reporter in the posterior ventral nerve cord matches the wild-type pattern of programmedcell deaths, with expression of gfp in one nucleus each generated by the P9 and P10 lineages, two nuclei generated by the P11 lineage, and three nuclei generated by the P12 lineage (Fig. 2; data not shown). In mab-5 and ceh-20 mutants (see below) the pattern of fluorescent nuclei is changed to match the pattern of programmedcelldeath in the mutants (Fig. 2; data not shown); specifically, the reporter is not expressed in P(11,12).aaap. These results are consistent with transcriptional regulation of egl-1 to induce programmedcelldeath of these cells. The P(11,12).aaap cells do not undergo programmedcelldeath in egl-1 mutants (Table 1) consistent with the essential role of egl-1 in the programmedcelldeath of somatic cells (Conradt and Horvitz, 1998).
Studies imply that intestinal barrier dysfunction is a key contributor to morbid events associated with sepsis. Recently, the co-inhibitory molecule programmeddeath-ligand1 (PD-L1) has been shown to be involved in the regulation of intestinal immune tolerance and/or inflammation. Our previous studies showed that PD-L1 gene deficiency reduced sepsis-induced intestinal injury morphologically. However, it is not known how PD-L1 expression impacts intestinal barrier dysfunction during sepsis. Here we tested the hypothesis that PD-L1 expressed on intestinal epithelial cells (IECs) has a role in sepsis-induced intestinal barrier dysfunction. To address this, C57BL/6 or PD-L1 gene knockout mice were subjected to experimental sepsis and PD-L1 expression, intestinal permeability and tissue cytokine levels were assessed. Subsequently, septic or nonseptic colonic samples (assigned by pathology report) were immunohistochemically stained for PD-L1 in a blinded fashion. Finally, human Caco2 cells were used for in vitro studies. The results demonstrated that PD-L1 was constitutively expressed and sepsis significantly upregulates PD-L1 in IECs from C57BL/6 mice. Concurrently, we observed increased PD-L1 expression in colon tissue samples from septic patients. PD-L1 gene deficiency reduced ileal permeability and tissue levels of IL-6, TNF-α and MCP-1, and prevented ileal tight junction pro- tein loss compared with WT after sepsis. Comparatively, while Caco2 cell monolayers also responded to inflammatory cytokine stimulation with elevated PD-L1 expression, increased monolayer permeability and altered/decreased monolayer tight junction protein morphology/expression, these changes were reversed by PD-L1 blocking antibody. Together these data indicate that ligation of PD-L1 plays a novel role in mediating the pathophysiology of sepsis-induced intestinal barrier dysfunction.
In animals, cells may die either by a process called necrosis or by programmedcelldeath (PCD). PCD is an important process to control cell proliferation, generation of developmental patterns and the defense against pathogens and environmental conditions. The process removes superfluous, damaged or infected cells in an organized manner (Steller et al. 1995; Lawen 2003). The first step in the initiation of the celldeath program is the activation of caspase proteolytic cascades (Abraham et al. 2004). Caspases are the key executioners of apoptosis. They belong to a family of cysteine proteases, conserved through evolution (Kroemer & Martin 2005). Caspases are synthesized as proenzymes. The proenzymes are activated through cleavage at internal aspartate residues by other caspases or by autoactivation. Activated caspases cleave a variety of proteins after specific aspartate residues, ultimately leading to celldeath. The cleaved proteins are cytoskeletal proteins - such as lamins, α-fodrin and actin - proteins involved in DNA repair and cell-cycle regulation - such as poly(ADP-ribose) polymerase (PARP) and retinoblastoma protein - (Launay et al. 2005; Ruchaud et al. 2002; Zhivotovsky 2003). Caspase-3 is activated during most apoptotic processes. It is generally believed to be the main executioner caspase. Caspase-6 has been shown to cleave lamin and several nuclear proteins such as transcription factor activator protein-2α and SATB1 (special AT-rich sequence binding protein1) leading to the collapse of the nucleus (Nyormoi et al. 2001; Gotzmann et al. 2000). Caspase activation is strictly regulated by Bcl-2 family members. The Bcl-2 family controls the release of pro-apoptotic caspase activating factors - such as cytochrome c, APAF-1 and AIF - from the mitochondria into the cytosol (Broker et al. 2005). PCD is characterized by specific features such as cell shrinkage, blebbing of the plasma membrane, condensation and fragmentation of the nucleus, and internucleosomal cleavage of DNA (Nagata 2005; Zhivotovsky 2003).
vib-1 is required for the expression of the HET domain genes pin-c, tol, and het-6. Mutations in vib-1 suppress het-c/pin-c and mat HI, partially suppress het-6 HI, and increase the recovery of het-e- and het-8-incompatible partial diploid progeny (90). These data indicate that VIB-1 is a global mediator of fungal HI in Neurospora. A search of 500-bp promoter segments of the pin-c, het-6, and tol genes for common motifs identified a 12-bp consensus sequence (CTAC/GG/CA/CT/AC/GC/AC AC/T [E value ⫽ 3.10e ⫺06 ]). Interestingly, this consensus se- quence was enriched in the set of 55 predicted HET domain genes (P ⫽ 0.004 by the two-tail Fisher test). A number of these HET domain genes are polymorphic among isolates of Neurospora (N. L. Glass, unpublished results), which is a char- acteristic of cloned het loci (27, 70). The closest paralog of vib-1, NCU04729, also contained the 12-bp consensus se- quence, but NCU09915, the paralog with the highest similarity to NDT80, did not. These data suggest that NCU04729 might also be involved in HI and extracellular protease production. Five of the six molecularly characterized het interactions involve predicted proteins that share a common ⬃ 150-amino- acid HET domain (Pfam06985), including the proteins en- coded by N. crassa pin-c, tol, and het-6 and P. anserina het-D and het-E (22, 76). No function for these genes is known other than their role in HI. Our model for HI is that nonself recog- nition is mediated by a HET domain-containing protein inter- acting with another protein partner whose function can be diverse (e.g., transcription factors, such as those encoded by mat A-1 and mat a-1; plasma membrane proteins, such as that encoded by het-c; or ribonucleotide reductases, such as that encoded by un-24) (Fig. 9). Predicted HET domain genes are specific to and common among filamentous asco- mycete genomes (23). We hypothesize that proteins contain- ing this domain might be recruited to a celldeath signal transduction pathway and interact with downstream part- ners to trigger a common cellular response, mediated partly by VIB-1, to cause HI.
The human gene encoding PD-1, PDCD1, is localized on 2q37.3, which is a susceptibility locus for SLE . Allele T of a single-nucleotide polymorphism (SNP) corresponding to PD-1.5 C/T (dbSNP rs#cluster id rs2227981) was found to be associated with the development of RA (odds ratio (OR) 1.94, 95% confidence interval (CI) 1.25 to 3.01, p < 0.0025) but not SLE in Chinese patients living in Taiwan . So far more than 30 SNPs have been identified. Among the 30 SNPs, 7 (namely PD-1.1, PD-1.2, PD-1.3, PD-1.4, PD-1.5, PD-1.6 and PD-1.9) were examined in SLE . Two SNPs (PD-1.5 C/T (rs2227981) and PD-1.9 T/C/(rs2227982)) were selected because they occur in an exon, which affects protein synthesis. The change in PD-1.9 T/C causes a change in their synthesized amino acid from valine to alanine. Our study was therefore focused on determining whether PD-1.9 T/C SNP affects the development of AS.
In these processes, components of the extracellular matrix (ECM) (4) and in particular basement membrane act as sur- vival factors and suppress apoptosis. Proteolytic modification of matrix organization or disruption of cell–matrix contacts can result in initiation of PCD in epithelial cells, with induction of specific molecular effectors of apoptosis such as caspases (3, 4). Recently, it was shown that proteolytic processing of TNF- a and FAS ligand (FasL) from the surface of lymphoid cells by matrix metalloproteinase-like activity can also alter PCD (5, 6). We have recently examined the expression of ma- trix metalloproteinases (MMPs) and their endogenous inhibi- tors, the tissue inhibitors of metalloproteinases (TIMPs), in human normal and neoplastic lymphoid cell lines (7). In this study, TIMP-1 secretion was shown only by normal activated tonsillar B cells and by a subset of high-grade Burkitt’s lym- phomas but not by quiescent peripheral blood cells or follicu- lar lymphoma cell lines. MMP expression was variable and did not correlate with TIMP-1 production. These results are in agreement with previous reports from our laboratory and oth- ers that TIMP-1 expression correlates with clinical grade in non–Hodgkin’s lymphomas as well as in other malignancies but did not correlate with MMP expression (8–10). Therefore, these effects of TIMPs may be independent of their ability to inhibit MMPs (11, 12). In addition, TIMP-1 promotes survival of serum-dependent cell lines, including Burkitt’s lymphomas, in serum-free conditions (13). Together, these findings suggest that extracellular matrix proteases or their inhibitors (TIMPs) may directly influence malignancy and PCD in B cells as well as in other cell types. The present study was therefore under- taken to determine whether TIMP-1 regulates apoptosis in B cells. The results of our study demonstrate that TIMP-1 di- rectly controls apoptosis through a novel, non-MMP inhibitory pathway and suggest that this protein plays a pivotal role in maintenance of B cell homeostasis.
Identification of LR gene sequences that inhibit PCD. Three LR gene mutants were constructed to further characterize the sequences that were necessary to inhibit PCD (Fig. 3). LRT- stop contains three in-frame stop codons at the amino termi- nus of LR ORF2 and thus should prevent translation of any ORF encoded by a LR RNA. Insertion of stop codons at this position was previously shown to prevent expression of a 40- kDa protein that was recognized by a LR ORF2-specific anti- body P2 (21). Plasmid LRT⌬SmaI has a 258-bp SmaI deletion that spans the intron/exon borders of LR RNA (8, 21). In LRT⌬HX, 523 bp of the LR promoter was deleted. To test whether these constructs synthesized LR RNA, CV-1 cells were transfected, RNA was prepared 48 h after transfection, and RT-PCR was performed with LR-specific primers L3A and L3B (Fig. 3C). As expected, LRT wt synthesized RNA that
Schistosomiasis is a “neglected tropical disease” that affects over 200 million people in 78 countries and is considered a chronic and poverty-promoting disease . Schistosoma japonicum is the only human blood fluke that occurs in China. As of 2015, there were > 30, 000 villages (containing 68 million residents) endemic for schistosomiasis . As one of the countries routinely affected by epidemic diseases, China has made great strides in schistosomiasis. However, there are emerging challenges, including limitations in new drug develop- ment and the lack of an available vaccine [3–6]. Although some vaccines have been developed, the ideal immunity rate has not been achieved . One reason for this might be that growth and developmental mechanisms and its interactions with the host are poorly understood . To this end, the study of schistosomulum- specific molecules is not only important for identifying new functional genes as potential vaccine antigens or drug targets for human schistosomiasis but also helpful in revealing mechanisms of growth, development, and inter- actions with the host. Therefore, it is necessary to study the functions of the key genes involved in S. japonicum growth and development in more detail.
Abstract: The central nervous system (CNS) is regarded as an immune privileged environment; however, changes in the neuroimmunology paradigm have led to an increased interest in systematic immunotherapy in lung cancer therapy. The presence of the lymphatic system in the CNS as well as the physiological and biochemical changes in the blood – brain barrier in the tumor microenviron- ment suggests that immunocytes are fully capable of entering and exiting the CNS. Emerging clinical data suggest that inhibitors of programmeddeath receptor-1/programmeddeath ligand 1 (PD-1/PD-L1) can stimulate surrounding T cells and thus have antitumor effects in the CNS. For example, PD-1 antibody (pembrolizumab) monotherapy has displayed a 20 – 30% encephalic response rate in patients with brain metastases from malignant melanoma or non-small cell lung cancer. Combined application of nivolumab and ipilimumab anti-PD-1 and anti-cytotoxic T-lymphocyte-associated protein 4 showed an encephalic response rate of 55% in patients with brain metastases of melanoma. Further evidence is required to verify these response rates and identify the mechanisms of curative effects and drug tolerance. While regional treatments such as whole-brain radiosurgery, stereotactic radiosurgery, and brain surgery remain the mainstream, PD- 1/PD-L1 inhibitors display potential decreased neurotoxic effects. To date, ﬁ ve drugs have been approved for use in patients with encephalic metastases of lung carcinoma: the anti-PD-1 drugs, pembrolizumab and nivolumab, and the anti-PD-L1 agents, atezolizumab, durvalumab, and avelu- mab. In recent years, clinical trials of inhibitors in combination with other drugs to treat brain metastasis have also emerged. This review summarizes the biological principles of PD-1/PD-L1 immunotherapy for brain metastasis of lung cancer, as well as ongoing clinical trials to explore unmet needs.
The brain samples were fixed in 4% paraformaldehyde, embedded in paraffin, cut into 4-μm sections, and exam- ined by immunofluorescence staining. Then, the sections were stained with primary antibodies (all diluted 1:200; from Santa Cruz Biotechnology, Inc.) and appropriate secondary antibodies (1:500 dilution; Santa Cruz Bio- technology, Inc.) as described. Normal rabbit IgG was used as a negative control for immunofluorescence assay (data not shown). Finally, sections were observed by a fluorescence microscope (OLYMPUS BX50/BX-FLA/ DP70; Olympus Co., Japan). For fluorescence intensity assay, the relative fluorescence intensity was analyzed by use of ImageJ program by subtracting background. For positive cell counting, six microscopic fields in each tis- sue section and three sections per rat were examined and photographed in parallel for positive cell counting. Microscopy was performed by an experienced patholo- gist blind to the experimental condition. The number of positive cells was assessed in ≥ 100 cells.
protein 4, and indoleamine 2,3-dioxygenase, and infiltra- tion density of immune cells, including CD3(+), CD4(+), CD8(+), and PD-1(+) cells, in the tumor microenviron- ment . This discrepancy might be explained by the mechanisms of PD-L1 overexpression in tumor cells and by the difference in signatures of tumor immunity between Asian and non-Asian patients with gastric ade- nocarcinoma. On one hand, PD-L1 could be up-regulated by both intrinsic aberrant pathways involved in carcino- genesis and extrinsic cytokines produced by other stro- mal cells in the tumor microenvironment [38–40]. On the other hand, the density of tumor-infiltrating T cells was lower in Asian patients with gastric adenocarcinoma than non-Asian patients as reported in a previous study , revealing a large distinction in the immune status of tumor environment in patients from different geographic areas. Besides, different characteristics of gastric adeno- carcinoma between Asian and non-Asian populations might also be associated with the difference in median age of patients. The median age of patients in our study was 55 years old, which is consistent with the data reported by other studies in China [25, 42]. However, in the USA study, the median age of patients was 67 years old .
Recently, a pro-apoptotic Drosophila Bcl-2 family member was identified with the help of the database of genomic sequence; this gene is most closely related to mammalian Bok and potentially regulates apoptosis in the fly [21-24]. The Drosophila Bok homolog interacts with several anti- apoptotic, but not with several pro-apoptotic, Bcl-2 family members and, therefore, possibly functions by antagonizing pro-survival proteins. Ectopic expression of Bok protein pro- motes apoptosis in transgenic flies as well as in cultured cells. While one group reports that the caspase inhibitor p35 can block apoptosis induced by Drosophila Bok , another group did not see p35 inhibition . This differ- ence may be due to the slightly different constructs used in the experiments and the different assay systems. A third group shows that peptide caspase inhibitors can block Drosophila Bok-mediated apoptosis in cell culture, lending support to a model in which expression of Bok protein leads to caspase activation . Interestingly, ectopic expression of Drosophila Bok sensitizes the developing eye to celldeath induced by ultraviolet irradiation . Genetically, this Drosophila Bcl-2 family member appears to function upstream of, or in parallel to, the Drosophila ced-4/apaf-1 homolog and downstream of, or in parallel to, the Drosophila regulators of apoptosis, reaper, hid, and grim . The core components of the celldeath pathway are illustrated in Figure 1.
In the present study the protein kinase inhibitor STS (Omura et al., 1977; Tamaoki et al., 1986) was used as a potent and predictable inducer of PCD. Several vertebrate cells die by apoptosis in the presence of high concentrations of staurosporine, including thymocytes (Bertrand et al., 1993), lens epithelial cells (Ishizaki et al.,1993), chondrocytes (Ishizaki, 1994, hum an fibroblast cell lines (Jacobson et al., 1994) ohgodendrocytes and their precursors (Jacobson and Barres, unpublished observations). In the present study, we show th a t almost all of the cells in 6/6 organs can die by apoptosis: STS and CHX induce more than 90% pyknosis in postnatal explants of mouse heart, liver, lung, kidney, muscle, and pancreas, and rat gut, skin and cartilage (data not shown) within 18 hours. In no case, however, did 100% of the cells die. This may be due to technical reasons, since increasing the concentration of STS increased th a t pyknotic index in postnatal lung and pancreas. Furtherm ore, in dissociated-cell cultures 100% pyknosis is not seen until 48 hours after STS treatm ent, even though over 90% pyknosis is observed within 14 hours (Jacobson et al., 1993). A stochastic element in the timing of PCD in Rat-1 fibroblasts has been noted (Evan et al., 1992). It is also possible, but unlikely that a minority of cells in most organs cannot be
Monocytes play an essential role in the innate immune defense against microbial infection. Septic immunopara- lysis is first characterized by a monocytic deactivation of phagocytic function, proinflammatory cytokine release, and antigen-presenting capacity (probably due to a decreased expression of HLA-DR) [6,7]. Importantly, the persistence of immunoparalysis, is correlated with an increased risk of fatal outcomes . On the other hand, accumulating evidence points to the pivotal role of increased immune effector cell apoptosis in sepsis- induced immunosuppression [9,10]. Uptake of apoptotic cells by macrophages and dendritic cells (DCs) stimu- lates immune tolerance by inducing the release of anti- inflammatory cytokines, including IL-10 and transform- ing growth factor beta, and suppressing the release of proinflammatory cytokines. Inhibition of lymphocyte apoptosis can improve survival in animal models of sep- sis by using selective caspase inhibitors [11,12], by alter- ing proapoptotic/antiapoptotic protein expression [13,14], and by treatment with survival-promoting cyto- kines such as IL-7  and/or IL-15 .
the M4 sister cell (Hirose et al. 2010). The death of the M4 sister cell and the upregulation of egl-1 transcription in the M4 sister cell are also at least partially dependent on the speciﬁcity protein1 (SP1)-like transcription factor SPTF-3, which binds to another upstream cis-regulatory element of the egl-1 locus (Figure 3A) (Hirose and Horvitz 2013). In- terestingly, SPTF-3 is a direct transcriptional activator of both the egl-1 gene and the pig-1 gene, which encodes an AMPK-like protein kinase most similar to the mammalian kinase MELK and which, as discussed above (see The Q lineage), is a component of a genetic pathway that has been implicated in asymmetric cell division (by size and fate) in a number of cell lineages. Consistent with the notion that SPTF-3 activates pig-1 transcription in the M4 lineage to cause the death of the M4 sister cell; like the loss of sptf- 3, the loss of pig-1 blocks the death of 50% of the M4 sister cells (Hirose and Horvitz 2013). Based on these ﬁndings it was proposed that the death of the M4 sister cell is con- trolled by two parallel pathways that are both induced by sptf-3 function: the core apoptotic celldeath pathway that is activated by the sptf-3-, ceh-34-, and eya-1-dependent transcriptional upregulation of egl-1 (the sptf-3, ceh-34, eya-1, egl-1 pathway) and a pathway that is independent of the core apoptotic pathway and that is activated by sptf- 3-dependent transcriptional activation of pig-1 (the sptf-3, pig-1 pathway). Finally, the death of the M4 sister cell (as well as a number of other programmedcell deaths, includ- ing the death of the NSM sister cells) is also at least partially dependent on the yeast general control nondepressible ho- molog gcn-1 gene and the ABC transporter, class F 1 (abcf-1) gene, whose gene products physically interact and, based on sequence homologies, may function in the regulation of messenger RNA translation (Hirose and Horvitz 2014). This pathway (gcn-1, abcf-1 pathway) has been proposed to act in parallel to the two sptf-3-dependent pathways to contribute to the death of the M4 sister cell as well (Hirose and Horvitz 2014). However, based on the known function of pig-1 in asymmetric cell division and recent ﬁndings in the NSM lineage (Cordes et al. 2006; Chien et al. 2013; Chakraborty et al. 2015), it is also possible that, rather than acting in parallel to the sptf-3, ceh-34, eya-1, egl-1 pathway; the sptf-3, pig-1 pathway and the gcn-1, abcf-1 pathway may act at different time points in the M4 lineage to promote the asymmetric division of the M4 mother cell and the segre- gation of the apoptotic potential into the M4 sister cell (sptf-3, pig-1 pathway) as well as the synthesis of CED-3 protein in the M4 sister cell after cell division (gcn-1, abcf-1 pathway).
It is not known if the multiple causes of oocyte death reported above take different or the same PCD molecular pathways. In this regard, no progresses were obtained in the identification of the pro- apoptotic pathways activated following the withdrawal of the growth factors reported to protect mouse or human fetal oocytes from death (KL, LIF, IGF-1, NT4/5 and IL-1, see Klinger and De Felici, 2011). The results by Gawriluk and coll. (Gawriluk et al., 2011), reported above, indicate, however, that fetal oocytes are prone to activate autophagy. Actually, this could occur in the absence of adequate growth factor amount to preserve oocytes from death. Nevertheless, despite this, most oocytes undergo apoptosis likely because under prolonged starvation, autophagy turns into celldeath effector. This might happen both through typical autophagic lysosomal activity but also through apoptotic pathways. In fact, beclin 1 is able to bind BCL2, BCL-X or MCL1 (myeloid cell leukemia-1) and in this way favour the action of proapototic protein such as BAX. Actually, the balance between the anti-and proapoptotic proteins BCL-X and BAX was shown to be an important regulator of the oocyte death (for a review, see, Klinger and De Felici, 2011). A possible scenario is that, depending on different conditions, oocytes, can undergo PCD by autophagy or BCL-X/BAX rheostat. Under growth factor deprivation, in the absence of effective autophagy (Gawriluk et al., 2011), the BCL-X/BAX rheostat might lead to apoptosis, while in the absence of BAX, autophagy might induce oocyte to death (De Felici et al., 2008; Rodrigues et al., 2009) (Fig. 2).
Pyroptosis is an inflammatory caspase-dependent celldeath mechanism characterized by pore formation in the plasma membrane accompanied by relatively low intensity DNA-damage, and ADP-ribose polymerase activation. Caspase 1, 4, 5, and 11 constitute the family of inflammatory caspases that can be activated by inflammatory agents such as double-stranded DNA and bacterial toxins [25, 30]. In a canonical pyroptotic signaling pathway, the inflammatory stimuli lead to the direct activation of caspase. Caspase 1, in turn, cleaves the 54kDa protein known as gastermin D (GSDMD), following which the N-terminal fragment of gastermin-D forms oligomers that form pore in the plasma membrane and other pyroptotic events leading to celldeath . Caspase 1 has also been shown to cleave Caspase 3 and 7 independent of gastermin 2 cleavage . In the non-canonical pyroptotic signaling pathway, the inflammatory agents such as bacterial lipopolysaccharides activate caspase 4/5/11, which in turn cleaves GSDMD, releasing the pyroptotic N-terminal fragment of GSDMD, known as GSDMD-p30 [81, 82]. Although the precise mechanism remains to be elucidated, it has been shown that the GSDMD-p30, thus formed, associates with plasma membrane as well as mitochondrial membrane and induces the formation of pyroptotic pores . JNK- signaling appears to play a role in the non-canonical pyroptotic signaling pathway . It has been shown that the infection of bacterial pathogen in bone marrow derived macrophages leads to the increased production of ROS, ROS-induced activation of JNK1/2, and Subsequently, JNK-mediated increased expression of caspase 11 . As discussed above, increased in the levels of caspase 11 could trigger pyroptotic celldeath via GSDMD cleavage and the generation of GSDMD-p30. Such an increase in ROS following inflammatory stimuli has also been observed in lipopolysaccharide-induced caspase 1-dependent pyroptotic celldeath in human umbilical vein endothelial cells . Although the role of JNK in this cellular context was not assessed, it is possible that the increased ROS leads to the activation of JNK and JNK-mediated potentiation of non-canonical pyroptotic signaling via caspase 11 (Figure 4). Thus, JNKs appears to play a potentiating role in the regulation of pyroptosis.
During limb programmedcelldeath members of the different groups of apoptic regulators have been identified. As in other models of apoptosis, the final step of limb programmedcelldeath consists of the activation of caspases (Milligan et al., 1995; Jacobson et al., 1996; Mirkes et al., 2001). Associated with the pathway of caspases are the pro-apoptotic factors DIO-1 (Death Inducer-Obliterator-1; Garcia-Domingo et al., 1999), Gas1 and Gas2 (Growth Arrest Specific; Lee et al., 1999, Lee et al., 2001). The involvement of Apaf-1 in limb programmedcelldeath is supported by the occurrence of a reduced pattern of interdigital apoptosis and persistence of interdigital webs in mice mutant for this gene (Cecconi et al., 1998). Bax, a proapototic member of the Bcl-2 family, is expressed in the areas of celldeath (Dupe et al., 1999) and Bax (-/-)Bak (-/-) double knockout mice display persis- tence of interdigital webs (Lindsten et al., 2000). In addition, several antiapoptotic members of this family, including Bcl-2, Bcl- x and A1, are expressed in the digital rays but not in the interdigital spaces of the mice autopod (Novack and Korsmeyer, 1994; Carrio et al., 1996) while the interdigital regions prior to the onset of apoptosis express Bag-1 which encodes an antiapoptotic protein which binds to Bcl-2 (Crocoll et al., 2002). Another antiapoptotic factor, Dad-1 (Defender Against apoptotic cellDeath) has been implicated in the control of limb programmedcelldeath since heterozygous mutant mice for this gene display soft-tissue syndac- tyly (Nishii et al., 1999).