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Is suppression of apoptosis a new therapeutic target in sepsis?

SUMMARY

Sepsis remains as a leading cause of death in critically ill patients. Unfortunately, there have been very few successful specific therapeutic agents that can significantly reduce the attributable mortality and morbidity of sepsis. Developing novel therapeutic strategies to improve outcomes of sepsis remains an important focus of ongoing research in the field of critical care medicine. Apoptosis has recently been identified as an important mechanism of cell death and evidence suggests that prevention of cell apoptosis can improve survival in animal models of sepsis and endotoxaemia. In this review article, we summarise the critical role of apoptosis of the immune cells in the pathophysiology of sepsis and propose that blocking cell-signaling pathways leading to apoptosis may present a promising specific therapy for sepsis. Various methods to inhibit apoptosis including the cell surface Fas receptor pathway inhibitors, caspase inhibitors, over-expression of anti-apoptotic genes and small interfering ribonucleic acid therapy are discussed.

Key Words: apoptosis, critical illness, endotoxaemia, lymphocyte, sepsis

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Sepsis is a natural systemic inflammatory response by the body to infectious pathogens that can lead to circulatory failure, acute lung injury and multiple organ failure. Sepsis is important because it is responsible for 50-60% of all deaths in intensive care units (1). The pathogenesis of sepsis involves an over-exuberant inflammatory response in which host's immune system induce significant cell and organ injury through the cytokines released during the infection (2). This hyper-inflammatory immune response is initiated by microbial products signalling through the toll-like receptors expressed on host immune cells (3). Indeed, some patients with sepsis may develop a prolonged immunosuppressive state or ineffective immune response, further worsening chances of recovery from sepsis (2,4).

Multiple pathways of cell death are typically involved in sepsis due to the interactions of pro-inflammatory, anti-inflammatory, coagulation and complement systems. By targeting a specific focus of the inflammatory response, numerous clinical trials of anti-cytokine or anti-inflammatory agents, such as anti-tumour necrosis factor (TNF) and interleukin-1 therapies, have failed to demonstrate any success in improving survival of patients with sepsis. As such, a reappraisal of the pathophysiology and molecular mechanisms involved in the development of sepsis and its associated mortality is required.

One of the major mechanisms of sepsis-induced immunosuppression is due to apoptosis of the cells of the adaptive immune system (5). Apoptosis of the immune cells, in particular lymphocytes, is increasingly considered an important step in inducing a state of 'immune paralysis' in sepsis and making the patient vulnerable to the invading pathogens (6). Manipulation of the apoptosis process has recently been suggested as a novel therapeutic approach for sepsis (4,7,8). Apoptosis-induced lymphocyte depletion in sepsis not only impairs the adaptive immune response, but it also compromises the innate immune system because of cross-talk between two systems (2). The loss of lymphocytes, dendritic cells and other immune effector cells may lead to an immunosuppressed state, which may contribute to the fatality of sepsis (5) (Figure 1).

Although apoptosis has been extensively studied in pathological conditions including cancer, neuro-degenerative diseases, autoimmune disorders and human immunodeficiency virus, its significance in sepsis and its inhibition as a therapeutic strategy in sepsis have only recently received attention. This review aims to provide a brief update on the critical role of apoptosis of the immune cells in the pathophysiology of sepsis and the potential of inhibiting apoptosis as a therapeutic strategy for sepsis. Various methods to inhibit apoptosis including the cell surface Fas receptor (FAS) pathway inhibitors (also known as CD95L pathway inhibitor), caspase inhibitors, overexpression of anti-apoptotic genes and small interfering ribonucleic acid (RNA) therapy are discussed.

MECHANISM OF APOPTOSIS

Apoptosis is a natural process of cell death that is genetically programmed and plays a vital role in normal physiology, such as tissue homeostasis regulation, and also in the pathophysiology of many diseases (9). Apoptosis is characterised morphologically by cell membrane damage, cell shrinkage, chromatin condensation and DNA fragmentation. Macrophages phagocytose the cells during apoptosis before cell membrane rupture, thereby reducing the inflammatory response that occurs due to release of cell contents (9-11).

Apoptosis is classically signalled by two major apoptotic pathways (12) (Figure 2): the extrinsic pathway (also called the death receptor caspase-8 mediated pathway) and the intrinsic pathway (also called mitochondria-initiated caspase-9 pathway). The extrinsic pathway involves the family of TNF receptor members and the intrinsic pathway involves interactions between apoptosis and anti-apoptotic members of the B cell lymphoma (Bcl)-2 family. Apoptosis may be initiated by many factors, including circulating glucocorticoid levels, pro-inflammatory cytokines such as TNF-[alpha], interleukin-1, interleukin-6, FAS ligand, heat shock proteins, oxygen free radicals, nitric oxide and cytotoxic T lymphocytes; all of these factors are activated in sepsis. It can be assumed that, even in early stages of sepsis, presence of these factors may shift the host's response towards an increase in pro-apoptotic events (13).

The extrinsic pathway is triggered when death ligands bind to their respective cell surface death receptors through recruitment of FAS-associated death domain (FADD) protein, procaspase-8 through the formation of a complex that induces cell death and activation of the caspases (caspase-8 and caspase-10). The intrinsic pathway is triggered by intracellular stress leading to an incease in mitochondrial outer membrane permeability and release of mitochondrial proteins, including cytochrome C. Cytochrome C combines with apoptosis activating factor 1 and the initiator caspase-9, to form apoptosome. Caspase-8 and caspase-9 then activate caspase-3, in the final common pathway of cell death (14,15).

Hotchkiss et al showed that both the extrinsic and intrinsic apoptotic pathways are important in the pathogenesis of sepsis (12) and that there may be communications and interactions between the two apoptotic pathways mediated by the truncated form of BH3, produced by caspase-8 mediated BID cleavage. The truncated form of BID acts to inhibit the Bcl-2-Bcl-xl pathway and to promote activation of Bcl-2-associated X protein and Bcl-2 homologous antagonist killer, thereby inducings cytochrome C release, leading to formation of the apoptosome (16). However, the cross-talk between both pathways appears to be ceil-type specific (e.g. hepatocytes) and may not be applicable for circulating lymphocytes (17).

APOPTOSIS IN SEPSIS

Inappropriate regulation of apoptosis of the immune cells may play a pivotal role in causing immune dysfunction and development of multiple organ failure in sepsis (18,19). Immune cells that may undergo altered apoptotic changes include neutrophils and macrophages, as well as various lymphocyte populations (7). The most common immune cells that are affected by dysregulated apoptosis appear to be the lymphocytes; relatively little overt apoptosis is observed in non-immune or non-lymphoid organs in experimental sepsis or clinical specimens from patients with sepsis (20).

Under normal circumstances, without sepsis, apoptosis of the lymphocytes will remove auto-reactive lymphocytes or resolve immune cell activation (7). The situation in sepsis is different because sepsis impairs the immune function by inducing defects in the innate immunity and widespread apoptosis of CD4+ T cell and B cell lymphocytes in the thymus (21), spleen (5,22) and lymph nodes, as well as in the circulating lymphocytes (23) both in animals and in human models of sepsis (22,23) Effron et al injected Escherichia coli into baboons and observed extensive apoptosis in the lymphoid tissue of the spleen and lymph nodes during fatal septic shock (24). The significance of lymphocytes to the survival of septic animals is supported by the markedly reduced capacity of lymphocyte-deficient mice to survive caecal ligation and puncture (CLP) (19). In a mouse model of pneumocytis pneumonia, although the infection was localised to the lungs, significant changes in apoptotic proteins in splenic lymphocytes were found, suggesting that an apparently localised pulmonary infection can also stimulate lymphocyte apoptosis at extra-pulmonary sites (25).

In contrast, neutrophils and macrophages are regulated differently during sepsis. Neutrophils are usually the first line of defence against invading pathogens. Activation of neutrophils may lead to deleterious inflammatory processes, including tissue injury. These cells are markedly down-regulated in sepsis, which is further linked to a reduction in caspase-3 and caspase-9 activity and maintenance of the mitochondrial membrane potential (26). Apoptotic death of neutrophils and their clearance is now recognised as a pivotal step in limiting injury to neighbouring cells or organs (7). Delay in neutrophil apoptosis has, in fact, been demonstrated to increase tissue damage and mortality in severse sepsis, acute lung injury and burn injury (7,27). Activation of anti-apoptotic factors seems to be the cause of delay in the death of neutrophils. It is still not clear which apoptotic pathway predominately regulates the cell death of recruited neutrophils in septic animals. Unlike lymphocytes, the severity of neutrophil apoptosis is associated with the severity of sepsis and sepsis-induced acute respiratory distress syndrome in critically ill patients in an inverse fashion. Further research is needed to explore the role of different specific receptors and intracellular signalling pathways in modulating neutrophil apoptosis in sepsis.

Similarly, apoptosis of other immune effector cells like monocytes (28), dendritic cells (29), bone marrow (13) and gut epithelial cells (30) are also accelerated in severe sepsis and further contribute to immunosuppression. In acute lung injury, apoptosis of alveolar epithelial cells and also respiratory endothelial cells has been observed in both animals and humans (31). In fact, neutrophils appear to play a vital role in inducing pulmonary epithelial cell apoptosis via the release of soluble FAS ligand; the administration of an inhibitory anti-FAS antibody could block the epithelial cell apoptosis (32). Apoptosis of endothelial cells, especially vascular endothelium cells, may be one of the mechanisms responsible for impaired perfusion, tissue hypoxia and subsequent organ dysfunction in sepsis. In polymicrobial sepsis mice model induced by CLP, endothelial cell apoptosis was extensively found in aortic tissues of mice (33).

Because it is difficult to distinguish apoptotic and dendritic cells from live cells that have ingested apoptotic cells in sepsis, few researchers have investigated the fate of macrophages and dendritic cells (32,34). Despite these technical difficulties, it has been reported that macrophages and selected subsets of dendritic cells undergo apoptosis in sepsis. Overexpression of anti-apoptotic proteins or deletion of pro-apoptotic proteins prevents loss of dendritic cells and macrophages, suggesting that these cell populations indeed undergo apoptosis in vivo in sepsis (42). Hence, it is suggested that the apoptotic destruction of lymphocytes and dendritic cells impairs the development of adaptive immune responses that are essential for the host's recovery, leading to an adverse effect on disease outcome (35).

Although we have enough evidence regarding the apoptosis of lymphoid cells during sepsis, the data on apoptosis of parenchymal cell in different solid organs in sepsis is much less convincing. Hotchkiss et al examined patients who had subsequently died from sepsis and found increased apoptosis of gut epithelial cells while parenchymal cells from the heart, lungs, kidney or liver did not show an increase in apoptosis (20). Hence, evidence suggests that organ failure in sepsis could be due to cellular apoptosis leading to tissue hypoperfusion and anoxic apoptosis (36).

It has been very well established in various human studies that the degree of lymphocyte apoptosis has a strong relationship with the severity of sepsis (6,23,37,38). Evidence suggests that apoptosis of circulating lymphocytes in severe sepsis is observed in the form of the 'up-regulation' of the pro-apoptotic genes Bcl2-associated X protein, Bcl-2 homologous antagonist killer and Bcl-2-interacting modulator of cell death, coupled with massive up-regulation of BID and FAS, and in the form of 'down-regulation' of anti-apoptotic genes Bcl-2 and Bcl-xl, respectively (24,38,39). The trend of up-regulation of BID and FAS suggests that they can represent robust biomarkers of apoptosis in routine sampling conditions (6,39) and the initial apoptosis percentage is more substantial in septic shock than in sepsis without circulatory failure shock. Multiple members of the mitochondrial pathway, both proapoptotic and anti-apoptotic, are up-regulated in sepsis and the ratio of apoptotic mediators appears to determine cell fate (38) (Table 1).

POTENTIAL ANTI-APOPTOTIC AGENTS IN SEPSIS

It has been observed that various anti-apoptotic therapies (Table 2) improve survival, not only by reducing apoptosis in thymus and spleen, but also through its uptake by hepatocytes and other organ cells (8,18,40,41). The blockade of apoptosis may also lead to inhibition of sepsis-induced gut epithelial apoptosis in both humans and animal models of sepsis, and reduces bacterial burden and translocation of bacteria from bowel into the circulation.

Caspases (cysteine aspartyl proteases) are upregulated in the lymphocytes of sepsis patients, which may hasten the death of lymphocytes (42). Many researchers have considered caspases to be important foci for any anti-apoptotic drugs. Inhibition of caspases was one of the earliest steps in sepsis research to ameliorate the effects of apoptosis.

Initial interventions in this regard started with the use of a broad-spectrum caspase inhibitor carboxyvalyl-alanyl-aspartyl-(o-methyl) that could improve survival by 40-45% in a CLP model of sepsis (43). The protective effects shown by caspase inhibitors are further supported by experiments using selective caspase-7 deficient mice that were found to be resistant to lipopolysaccharide-induced lymphocyte apoptosis as well as lipopolysaccharide-induced lethality, independent of the excessive production of serum cytokines (44). Like caspase-3, the executioner caspase-7 performs a critical role in the execution of apoptosis by cleaving a large set of substrates, ultimately leading to DNA fragmentation and other morphologic and biochemical hallmarks of apoptosis. The central role of caspase-7 in apoptosis suggests that inhibition of this caspase as a therapeutic strategy warrants further study.

Recently, Weber et al demonstrated that a broad caspase or pan caspase inhibitor (VX-166) substantially reduced the severity of lymphocyte apoptosis and mortality in two rodent models of sepsis endotoxin shock and CLP (45). VX-166 was administered by either repeat intravenous boluses or infusion through an implanted minibolus pump. VX-166 substantially reduce mortality in both models, even when they were administered up to eight hours after the initiation of sepsis. VX-166 has also been shown to reduced human primary endothelial cells death in vitro, but its effect on other cells remains uncertain and requires further research.

Overexpression of anti-apoptotic factors is an effective strategy to block the development of lymphocyte apoptosis (21,28,46). Moreover, apoptosis of macrophages and dendritic cells in a mouse CLP model is also inhibited by overexpression of antiapoptotic protein Bcl-2 (47) and reduced mortality was seen in transgenic mice that overexpress gut-specific Bcl-2 following CLP (48). In another study, Hotchkiss et al transferred T cells from mice with overexpression of Bcl-2 into wild mice and improved survival in sepsis (49). Similarly, BID knockout mice have been shown to have almost complete prevention of sepsis-induced lymphocyte apoptosis and a substantial survival benefit in polymicrobial sepsis in many experimental studies (50). BID, a pro-apoptotic protein, is necessary for hepatocyte apoptosis. Deletion of BID has effectively blocked FAS-mediated hepatocyte apoptosis and hence, liver damage; BID also has a role in lung apoptosis. Many studies have shown that BID deficiency ameliorates lung injury and provides early protection from mortality in a mouse model of endotoxaemia (51). Even apoptosis-inhibitory protein serine kinase Akt makes CLP-induced mice resistant to death from sepsis and there is less B and T lymphocyte apoptosis. The increased activation of the T cells that overexpress Akt is another potential mechanism for the protective effect of Akt (52).

Synthetic small interfering RNA or silencing RNA, another potential reversal inhibitor of active caspases, has been introduced as a recent therapeutic modality to ameliorate sepsis-induced immune dysfunction. Small interfering RNAs are basically double-stranded RNA molecules that consist of 20-25 base pairs that interfere with the expression of a specific gene (53, 54). RNA interference using synthetic small interfering RNA sequences has been shown to be very powerful in reading gene expression both in vitro and in vivo (55, 56). The therapeutic delivery of caspase-8 and caspase-3 gene small interfering RNA in vivo eliminated apoptosis in aortic endothelium and has been found to confer a dramatic survival advantage (more than seven days) to CLP mice (33). This survival benefit was observed despite administration of caspase-8/caspase-3 small interfering RNAs as late as ten hours after CLE The prevention of vascular endothelial cell apoptosis may be responsible for their beneficial effects in endotoxic shock. However, treatment with caspase-8/caspase-3 small interfering RNA has a significant effect on the survival of animals subjected to CLP, as compared to administration of either caspase-3 or caspase-8 alone. One previous study by Wesche-Soldato et al reported that treatment with a single dose of caspase-8 siRNA can significantly improve the survival of mice with polymicrobial endotoxic shock when the animals received this small interfering RNA at 30 minutes following the performance of CLP (41). Perl et al demonstrated that treatment with single intratracheal instillation of caspase-3 small interfering RNA in a rodent model of sepsis decreased lung tissue apoptosis/inflammation, as well as altering the sequelae of progression to acute lung injury, which in turn resulted in the ten-day survival benefit of septic mice (57). Caspase-3 may represent an important therapeutic target accessible by small interfering RNA treatment in vivo because of its location in the final common pathway of both extrinsic and intrinsic death pathways. Although these studies demonstrated that an anti-caspase strategy may be a promising novel treatment for sepsis, the potential risk of hyper-acute TNF-induced shock (58) and a lack of specificity of doses needed for such inhibition (49,59) remain the primary stumbling blocks for caspase inhibitors to be used clinically. Moreover, it is also difficult to get nearly complete inhibition of caspase-3.

Leukocytes, particularly neutrophils and monocytes, show increased expression of FAS in humans if treated with endotoxin (60). FAS is also expressed on a variety of immune (including lymphocytes, thymocytes and macrophages) and non-immune cells in different solid organs including the liver, lung and heart. FAS and FAS ligand expression on lymphocytes directly correlate with mortality (61). Hence, another approach to block lymphocyte apoptosis is to inhibit the FAS-mediated extrinsic apoptotic pathway using FAS-ligand deficient mice or administing FAS fusion protein. Both techniques have been shown to reduce mortality in experimental models of sepsis (18,62). Small interfering RNA therapy can also be used to inhibit synthesis of FAS and a 50% improvement in survival and reduction in apoptosis and organ damage in both the liver and the spleen were observed if small interfering RNA was given 30 minutes after CLP in experimental model of sepsis (40).

Similarly, the role of BIM small interfering RNA has been studied in a murine model of septic peritonitis to protect lymphocytes from sepsis-induced apoptosis (63). The degree of lymphocyte apoptosis in BIM small interfering RNA treated mice was markedly decreased and resulted in an overall survival of 90% as compared to just 50% in the control groups. It was observed that only a minute change in BIM protein expression is sufficient to have significant phenotypic alterations. The small interfering RNA therapy causes decrease in BIM expression and liberation of sufficient Bcl-2 which binds to and inhibits Bcl-2-associated X protein/Bcl2 homologous antagonist killer channel formation, resulting in blockade of apoptosis (64). Similarly, the rationale behind the use of small interfering RNAs Bcl-2 in protection against apoptosis is by binding and neutralising the pro-apoptotic Bcl-2 family members, for example, BIM, p53-upregulated modulator of Apoptosis and phobol-12-myristate-13-acetate-induced protein (64). However, Judge et al indicated that the potential concern with the use of small interfering RNAs, especially unmodified ones, is their ability to induce an inflammatory response through the stimulation of cell danger pathways by the uptake of small interfering RNAs via toll-like receptors (65). They also demonstrated that when uridine analog (2'-O-methyl uridine) or guanosine nucleosides are selectively incorporated into siRNA duplex strands, the immune-stimulatory effect of siRNAs can be abolished. In addition, using lower doses of siRNAs might ameliorate this potential adverse effect of small interfering RNAs.

Recently, systemic administration of small interfering RNAs targeting FADD, which attracts procaspase-8 into the death-inducing signalling complex, may have beneficial effect in septic acute lung injury and, thereafter, mortality. Survival benefit was seen despite administration of FADD small interfering RNA as late as ten hours after CLP-induced polymicrobial sepsis in mice (66). FADD small interfering RNA prevents apoptosis of pulmonary capillary endothelial cells, epithelial cells and spleen lymphocytes in sepsis. The effect of FADD small interfering RNA on septic mortality is more evident than that of FAS small interfering RNA, which was reportedly given 12 hours after CLP in experimental study (38). Similarly, the same authors demonstrated the effect of systemic delivery of FADD small interfering RNA on aortic endothelial cell apoptosis. FADD small interfering RNA prevented apoptosis of lymphocytes of the spleen and vascular endothelial cells in CLP mice (67). Abolition of the apoptosis-associated adaptor molecule FADD gene with the use of the specific small interfering RNA may represent a novel and efficacious therapeutic strategy for sepsis, although the results of animal studies may not be applicable to humans.

Despite all the advances, one of the major limitations of the clinical application of small interfering RNA is its method of delivery. Recently, Brahmamdam et al employed a cyclodextrin polymer-based, transferrin receptor-targeted delivery vehicle to co-administer BIM and PUMA small interfering RNA to mice immediately after CLR Anti-apoptotic small interfering RNA-based therapy targeting both BIM and p53-upregulated modulator of apoptosis (two most potent BH3-only proteins) markedly decreased apoptosis of CD4 T and B cells (68). Researchers have concluded that another limitation encountered during use of small interfering RNA therapy is the duration of response. Hence, it is essential to determine the optimal duration of the response after treatment with small interfering RNA before large scale clinical trials are conducted (68-70).

Another approach that needs further evaluation in sepsis is the use of human immunodeficiency virus protease inhibitors like nelfinavir and ritonavir. These drugs block the cleavage of human immunodeficiency virus propeptides and have been found to have direct anti-apoptotic effects as well. Weaver et al demonstrated that these drugs could prevent initiation of the intrinsic apoptotic pathway by stabilising the mitochondrial membrane potential. (71). Oral administration of nelfinavir and ritonavir to mice either before or four hours after CLP, could reduce lymphocyte apoptosis and mortality (71). Very recently, Iwata et al analysed the effects of two proteins of the Bcl-2 family (recombinant human Bcl-2 or Bcl-2A1 protein) with anti-apoptotic properties when applied intraperitoneally in a CLP-induced rodent model of sepsis. Total eight doses of 1 mcg recombinant proteins were administered to septic mice, including one intraperitoneal injection at 18 hours prior to CLP which could significantly decrease the apoptotic cells of the heart and intestine (72). The extracellular administration of recombinant human Bcl-2 or Bcl-2A1 protein may be useful as new strategies for the treatment of sepsis.

ROLE OF EXOGENOUS CYTOCHROME C IN SEPSIS

Recent research has concentrated on the role of exogenous cytochrome C as an anti-apoptotic agent in sepsis. Although cytochrome C released from mitochondria can activate the intrinsic apoptosis pathway, new evidence suggests that production of pro-apoptotic factors like cytochrome C offer protection in sepsis, especially with lung apoptosis. Impaired phosphorylation is also one of the proposed mechanisms to cause sepsis-induced organ dysfunction, but exactly how defective mitochondrial dysfunction causes morbidity and mortality in sepsis is not clear. A few researchers have recently tried to demonstrate a correlation between mitochondrial dysfunction and the pathophysiology of sepsis (73,74). Levy et al studied the role of exogenous cytochrome C on myocardial function in an animal model of sepsis 24 hours after CLP and found that exogenous cytochrome C restored myocardial cytochrome oxidase activity and improved cardiac function. In another study, Piel et al (74) evaluated that a single injection of exogenous cytochrome C, if administered at 24 hours, can sustain its effects on myocardial cytochrome oxidase activity and survival at several time points later. Only 15% of animals survived to 96 hours after CLP in the control group, while 50% of animals in the cytochrome C injection group survived to 96 hours after CLE The rationale behind the administration of cytochrome C is the inhibitation of activity of myocardial cytochrome oxidase in sepsis and limited availability of its substrate cytochrome C during the late phase of sepsis. Increased membrane permeability of mitochondria during sepsis helps in restoration of mitochondrial function as exogenous cytochrome C promptly gains access to the mitochondria of cardiomyocytes. In the aforementioned studies, focus is more on the potential beneficial effects of cytochrome C. Thus, the potential harms of exogenous cytochrome C on the mitochondrial apoptosis pathway or increase in oxidative stress needs to be evaluated in future studies.

Although evidence suggests that suppressing apoptosis can reduce cell deaths or improve survival in animal models of sepsis, there are still concerns regarding the efficacy and pharmacodynamics of these treatments and their possible clinical applications in humans. It is also possible that any survival benefit achieved by anti-apoptosis would not only be caused by inhibition of apoptosis of lymphocytes, but also by anti-apoptosis on endothelial cells and other immune cells. Discussion of various antiapoptotic agents in sepsis is incomplete without considering the limitations of their use. Since sepsis is a complex disease and involves triggering of multiple pathways with existence of cross-talk between different pathways, anti-apoptotic therapy has to overcome many issues, including the selectivity of their actions and risks of uncontrolled cell growth. Moreover, there are hardly any clinical trials related to the effects of antibiotic treatment or resuscitation on the expression of the pro-apoptotic or anti-apoptotic markers in the early stages of sepsis in humans. Few studies of the role of low-dose glucocorticoids on apoptosis have been done in sepsis patients and those that have, found their role to be non-significant (12, 61). Whether early aggressive management of sepsis, including early appropriate antibiotics and goal-directed therapy, may improve apoptosis of lymphocytes and other immune cells in early stages of sepsis remains uncertain, and this merits further evaluation.

CONCLUSION

Although clinical studies on specific mediator-directed therapies have proven to be unsuccessful in sepsis in the past two decades, the search for a therapeutic strategy that may improve outcomes of sepsis remains important. The 'one-size-fits-all' approach that has been used in many clinical trials may not be appropriate since sepsis involves triggering and interactions of multiple pro-inflammatory, anti-inflammatory, coagulation and control of cell death pathways. Recent animal studies suggest that a variety of strategies and agents could reduce apoptosis of many types of immune cells, especially lymphocytes, in the setting of sepsis. The harmful effects of these strategies in humans (and whether their beneficial effects can be modified by other early aggressive medical interventions) have not been thoroughly evaluated. Until clinical data on the safety of anti-apoptosis treatments are available, the potential benefits of anti-apoptosis therapy remain unknown for sepsis.

Caption: Figure 1: Progression of sepsis due to various cell deaths. IL=interleukin, TNF=tumour necrosis factor.

Caption: Figure 2: Extrinsic and intrinsic pathways of sepsis. FAS=cell surface fas receptor also known as CD95L, FADD=FAS-associated death domain, TNF=tumour necrosis factor, Bcl=B cell lymphoma, tBID=truncated form of BID, cFLIP=cellular flice like inhibitory protein, Smac=second mitochondria-derived activator of caspases, Diablo=homolog mitochondrial protein, IAPs=inhibitor of apoptosis proteins.

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M. HARJAI *, J. BOGRA ([dagger]), M. KOHLI ([double dagger]), A. B. PANT ([section])

Department of Anaesthesiology, Chhatrapati Shahuji Maharaj Medical University, Lucknow, Uttar Pradesh, India

* MB, BS, MD and PhD Fellow.

([dagger]) MD, Professor and Head of Department.

([double dagger])MD, PDCC, Professor and Trauma ICU Incharge.

([section]) PhD, Senior Scientist, In Vitro Division, Indian Institute of Toxicological Research, Lucknow.

Address for correspondence: Dr M. Harjai, MD, PhD Fellow, Department of Anaesthesiology, Chhatrapati Shahuji Maharaj Medical University, Lucknow, Uttar Pradesh, India 226 003. Email: mamtaharjaidoctor@yahoo. co.in

Accepted for publication on November 27,2012

Table 1

List of various pro-apoptotic and anti-apoptotic factors involved in
the sepsis

Pro-apoptotic factors               Anti-apoptotic factors

BAX                                 Bcl-2
Bcl-xs                              Bcl-xl, Bcl-Al
BIM, PUMA, NOXA                     Inhibitor of apoptosis proteins
FAS ligand                          Mcl-1
Tumour necrosis factor- a           Ras factor- a
BID, BAK
Apoptosis-inducing factor
p53

BAX=Bcl-2-associated X protein, Bcl=B cell lymphoma,
BIM=Bcl-2-interacting moderator of cell death, PUMA=p53-
upregulated modulator of apoptosis, NOXA=phorbol-12
myristate-l-acetate-induced protein, FAS=cell surface Fas
receptor also known as CD95L, Mcl=M cell lymphoma, Ras=rat
sarcoma, BID=BH3 interacting death domain, BAK=Bcl-2
homologous antagonist killer.

Table 2

Various treatments in experimental models of sepsis

Treatment                           Benefits

zVAD.fkm (43)                       Increases survival by 40-45%

VX-166 (45)                         Inhibits lymphocyte apoptosis and
                                    increases survival in rodent
                                    models

Caspase-8/caspase-3 gene small      Marked improvement in survival
interfering RNA (33)

Overexpression of anti-apoptotic    Inhibits T cell, macrophage and
protein Bcl-2 (46,47)               dendritic cell apoptosis and
                                    improves survival

FAS fusion protein in mice (62)     Marked survival benefit,
                                    decreases hepatic injury

FAS and caspase-8 small             Improves survival by 50% and
interfering RNA (38)                decreases apoptosis in liver and
                                    spleen

BIM small interfering RNA (63)      90% improvement in survival

FADD small interfering RNA (66)     Inhibits apoptosis of pulmonary
                                    capillary epithelial cells and
                                    splenic lymphocytes and increases
                                    survival

BIM and PUMA small interfering      Markedly decreases apoptosis of
RNA (68)                            CD4 T and B cells

Overexpression of Akt (52)          94% increase in survival and
                                    decrease in B and T cell
                                    apoptosis

HIV protease inhibitor (71)         Decreases lymphocyte apoptosis
                                    and improves survival

Extracellular administration of     Inhibits apoptosis in heart and
recombinant human Bcl-2 or Bcl-     intestine
2A1 (72)

zVAD.fkm = carboxy-valyl-alanyl-aspartyl-(o-methyl)-fluromethyl
ketone, VX-166=pan caspase inhibitor, RNA = ribonucleic acid, BcI = B
cell lymphoma, FAS = cell surface Fas receptor also known as CD95L,
BIM= Bcl-2-interacting moderator of cell death, FADD = FAS associated
death domain protein, PUMA = p53-upregulated modulator of apoptosis,
HIV = human immunodeficiency virus.
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Date:Mar 1, 2013
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