Therapeutic evaluation of immunomodulators in reducing surgical wound infection.

Despite many advances in infection control practices, including prophylactic antibiotics, surgical site infections (SSIs) remain a significant cause of morbidity, prolonged hospitalization, and death worldwide. Our innate immune system possesses a multitude of powerful antimicrobial strategies which make it highly effective in combating bacterial, fungal, and viral infections. However, pathogens use various stealth mechanisms to avoid the innate immune system, which in turn buy them time to colonize wounds and damage tissues at surgical sites. We hypothesized that immunomodulators that can jumpstart and activate innate immune responses at surgical sites, would likely reduce infection at surgical sites. We used three immunomodulators; fMLP (formyl-Methionine-Lysine-Proline), CCL3 (MIP-1α), and LPS (Lipopolysaccharide), based on their documented ability to elicit strong inflammatory responses; in a surgical wound infection model with Pseudomonas aeruginosa to evaluate our hypothesis. Our data indicate that one-time topical treatment with these immunomodulators at low doses significantly increased proinflammatory responses in infected and uninfected surgical wounds and were as effective, (or even better), than a potent prophylactic antibiotic (Tobramycin) in reducing P. aeruginosa infection in wounds. Our data further show that immunomodulators did not have adverse effects on tissue repair and wound healing processes. Rather, they enhanced healing in both infected and uninfected wounds. Collectively, our data demonstrate that harnessing the power of the innate immune system by immunomodulators can significantly boost infection control and potentially stimulate healing. We propose that topical treatment with these immunomodulators at the time of surgery may have therapeutic potential in combating SSI, alone or in combination with prophylactic antibiotics.

Pseudomonas aeruginosa ExoT induces G1 cell cycle arrest in melanoma cells.

Recently, we demonstrated that Pseudomonas aeruginosa Exotoxin T (ExoT) employs two distinct mechanisms to induce potent apoptotic cytotoxicity in a variety of cancer cell lines. We further demonstrated that it can significantly reduce tumour growth in an animal model for melanoma. During these studies, we observed that melanoma cells that were transfected with ExoT failed to undergo mitosis, regardless of whether they eventually succumbed to ExoT-induced apoptosis or survived in ExoT's presence. In this report, we sought to investigate ExoT's antiproliferative activity in melanoma. We delivered ExoT into B16 melanoma cells by bacteria (to show necessity) and by transfection (to show sufficiency). Our data indicate that ExoT exerts a potent antiproliferative function in melanoma cells. We show that ExoT causes cell cycle arrest in G1 interphase in melanoma cells by dampening the G1/S checkpoint proteins. Our data demonstrate that both domains of ExoT; (the ADP-ribosyltransferase (ADPRT) domain and the GTPase activating protein (GAP) domain); contribute to ExoT-induced G1 cell cycle arrest in melanoma. Finally, we show that the ADPRT-induced G1 cell cycle arrest in melanoma cells likely involves the Crk adaptor protein. Our data reveal a novel virulence function for ExoT and further highlight the therapeutic potential of ExoT against cancer.

Impact of preconditioned diabetic stem cells and photobiomodulation on quantity and degranulation of mast cells in a delayed healing wound simulation in type one diabetic rats.

Herein, we report the influence of administering different protocols of preconditioned diabetic adipose-derived mesenchymal stem cells (ADSs) with photobiomodulation in vitro, and photobiomodulation in vivo on the number of mast cells (MCs), their degranulation, and wound strength in the maturation step of a Methicillin-resistant Staphylococcus aureus (MRSA)-infectious wound model in rats with type one diabetes. An MRSA-infectious wound model was generated on diabetic animals, and they were arbitrarily assigned into five groups (G). G1 were control rats. In G2, diabetic ADS were engrafted into the wounds. In G3, diabetic ADS were engrafted into the wound, and the wound was exposed to photobiomodulation (890 nm, 890 ± 10 nm, 80 Hz, 0.2 J/cm2) in vivo. In G4, preconditioned diabetic ADS with photobiomodulation (630 and 810 nm; each 3 times with 1.2 J/cm2) in vitro were engrafted into the wound. In G5, preconditioned diabetic ADS with photobiomodulation were engrafted into the wound, and the wound was exposed to photobiomodulation in vivo. The results showed that, the maximum force in all treatment groups was remarkably greater compared to the control group (all, p = 0.000). Maximum force in G4 and G5 were superior than that other treated groups (both p = 0.000). Moreover, G3, G4, and G5 showed remarkable decreases in completely released MC granules and total numbers of MC compared to G1 and G2 (all, p = 0.000). We concluded that diabetic rats in group 5 showed significantly better results in terms of accelerated wound healing and MC count of an ischemic infected delayed healing wound model.

Pseudomonas aeruginosa ExoT Induces Atypical Anoikis Apoptosis in Target Host Cells by Transforming Crk Adaptor Protein into a Cytotoxin.

Previously, we demonstrated that Pseudomonas aeruginosa ExoT induces potent apoptosis in host epithelial cells in a manner that primarily depends on its ADP-ribosyltransferase domain (ADPRT) activity. However, the mechanism underlying ExoT/ADPRT-induced apoptosis remains undetermined. We now report that ExoT/ADPRT disrupts focal adhesion sites, activates p38ß and JNK, and interferes with integrin-mediated survival signaling; causing atypical anoikis. We show that ExoT/ADPRT-induced anoikis is mediated by the Crk adaptor protein. We found that Crk-/- knockout cells are significantly more resistant to ExoT-induced apoptosis, while Crk-/- cells complemented with Crk are rendered sensitive to ExoT-induced apoptosis. Moreover, a dominant negative (DN) mutant form of Crk phenocopies ExoT-induced apoptosis both kinetically and mechanistically. Crk is generally believed to be a component of focal adhesion (FA) and its role in cellular survival remains controversial in that it has been found to be either pro-survival or pro-apoptosis. Our data demonstrate that although Crk is recruited to FA sites, its function is likely not required for FA assembly or for survival per se. However, when modified by ExoT or by mutagenesis, it can be transformed into a cytotoxin that induces anoikis by disrupting FA sites and interfering with integrin survival signaling. To our knowledge, this is the first example whereby a bacterial toxin exerts its cytotoxicity by subverting the function of an innocuous host cellular protein and turning it against the host cell.

Pseudomonas aeruginosa ExoT Induces Mitochondrial Apoptosis in Target Host Cells in a Manner That Depends on Its GTPase-activating Protein (GAP) Domain Activity.

Pseudomonas aeruginosa is the most common cause of hospital-acquired pneumonia and a killer of immunocompromised patients. We and others have demonstrated that the type III secretion system (T3SS) effector protein ExoT plays a pivotal role in facilitating P. aeruginosa pathogenesis. ExoT possesses an N-terminal GTPase-activating protein (GAP) domain and a C-terminal ADP-ribosyltransferase (ADPRT) domain. Because it targets multiple non-overlapping cellular targets, ExoT performs several distinct virulence functions for P. aeruginosa, including induction of apoptosis in a variety of target host cells. Both the ADPRT and the GAP domain activities contribute to ExoT-induced apoptosis. The ADPRT domain of ExoT induces atypical anoikis by transforming an innocuous cellular protein, Crk, into a cytotoxin, which interferes with integrin survival signaling. However, the mechanism underlying the GAP-induced apoptosis remains unknown. In this study, we demonstrate that the GAP domain activity is both necessary and sufficient to induce mitochondrial (intrinsic) apoptosis. We show that intoxication with GAP domain results in: (i) JNK1/2 activation; (ii) substantial increases in the mitochondrial levels of activated pro-apoptotic proteins Bax and Bid, and to a lesser extent Bim; (iii) loss of mitochondrial membrane potential and cytochrome c release; and (iv) activation of initiator caspase-9 and executioner caspase-3. Further, GAP-induced apoptosis is partially mediated by JNK1/2, but it is completely dependent on caspase-9 activity. Together, the ADPRT and the GAP domains make ExoT into a highly versatile and potent cytotoxin, capable of inducing multiple forms of apoptosis in target host cells.

Pseudomonas aeruginosa uses T3SS to inhibit diabetic wound healing.

Diabetic foot ulcers are responsible for more hospitalizations than any other complication of diabetes. Bacterial infection is recognized as an important factor associated with impaired healing in diabetic ulcers. Pseudomonas aeruginosa is the most frequently detected Gram-negative pathogen in diabetic ulcers. P. aeruginosa infection has been shown to impair healing in diabetic wounds in a manner that correlates with its ability to form biofilm. While the majority of infections in diabetic ulcers are biofilm associated, 33% of infections are nonbiofilm in nature. P. aeruginosa is the most prevalent Gram-negative pathogen in all diabetic wound types, which suggests that the deleterious impact of P. aeruginosa on healing in diabetic wounds goes beyond its ability to form biofilm and likely involves other factors. The Type III Secretion System (T3SS) virulence structure is required for the pathogenesis of all P. aeruginosa clinical isolates, suggesting that it may also play a role in the inhibition of wound repair in diabetic skin ulcers. We evaluated the role of T3SS in mediating P. aeruginosa-induced tissue damage in the wounds of diabetic mice. Our data demonstrate that P. aeruginosa establishes a robust and persistent infection in diabetic wounds independent of its ability to form biofilm and causes severe wound damage in a manner that primarily depends on its T3SS.

Short-term glycemic control is effective in reducing surgical site infection in diabetic rats.

BACKGROUND: Patients and animals with diabetes exhibit enhanced vulnerability to bacterial surgical infections. Despite multiple retrospective studies demonstrating the benefits associated with glycemic control in reducing bacterial infection after cardiac surgery, there are fewer guidelines on the use of glycemic control for noncardiac surgeries. In the current study, we investigated whether long-term (begun 2 weeks before surgery) or immediate (just before surgery) glycemic controls, continued postoperatively, can reduce surgical site infection in type 1 diabetic-induced rats. METHODS: Rats were injected with streptozotocin to induce type 1 diabetes. Four groups of animals underwent surgery and thigh muscle Staphylococcus aureus bacteria challenge (1 × 10 colony forming units) at the time of surgery. Group 1 diabetic rats received insulin treatment just before surgery and continued until the end of study (short-term glycemic control group). Group 2 diabetic rats received insulin treatment 2 weeks before surgery and continued until the end of study (long-term glycemic control). Group 3 diabetic rats received no insulin treatment (no glycemic control group). Group 4 nondiabetic rats served as a healthy control group. Rats were euthanized at 3 or 6 days after surgery. Blood glucose and muscle bacterial burden were measured at 3 or 6 days after surgery. RESULTS: Glycemic control was achieved in both long- and short-term insulin-treated diabetic rats. Compared with untreated diabetic rats, the bacterial burden in muscle was significantly lower in both groups of glycemic controlled diabetic rats at 3 (all P < 0.003) and 6 (all P < 0.0001) days after surgery. CONCLUSIONS: A short-term glycemic control regimen, initiated just before surgery and bacterial exposure, was as effective in reducing surgical site infection as a long-term glycemic control in type 1 diabetic rats. These data suggest that immediately implementing glycemic control in type 1 diabetic surgical patients before undergoing noncardiac surgery may decrease the risk of infection.

Macrophage and Neutrophil Dysregulation in Diabetic Wounds.

SIGNIFICANCE: The incidence of diabetes continues to rise throughout the world in an alarming rate. Diabetic patients often develop diabetic foot ulcers (DFUs), many of which do not heal. Non-healing DFUs are a major cause of hospitalization, amputation, and increased morbidity. Understanding the underlying mechanisms of impaired healing in DFU is crucial for its management. RECENT ADVANCES: This review focuses on the recent advancements in macrophage and neutrophils in diabetic wounds and DFUs. In particular, we will discuss diabetes-induced dysregulations and dysfunctions of macrophage and neutrophil functions. CRITICAL ISSUES: It is well established that diabetic wounds are characterized by stalled inflammation that results in impaired healing. Recent findings in the field suggest that dysregulation of macrophages and neutrophils play a critical role in impaired healing in DFUs. The delineation of mechanisms that restore macrophage and neutrophil function in diabetic wound healing is the focus of intense investigation. FUTURE DIRECTIONS: The breadth of recently generated knowledge on the activity of macrophages and neutrophils in diabetic wound healing is impressive. Experimental models have delineated pathways that hold promise for the treatment of diabetic wounds and DFUs. These pathways may be useful targets for further clinical investigation.

Animal models for type 1 and type 2 diabetes: advantages and limitations.

Diabetes mellitus, commonly referred to as diabetes, is a group of metabolic disorders characterized by chronic elevation in blood glucose levels, resulting from inadequate insulin production, defective cellular response to extracellular insulin, and/or impaired glucose metabolism. The two main types that account for most diabetics are type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM), each with their own pathophysiological features. T1D is an autoimmune condition where the body's immune system attacks and destroys the insulin-producing beta cells in the pancreas. This leads to lack of insulin, a vital hormone for regulating blood sugar levels and cellular glucose uptake. As a result, those with T1D depend on lifelong insulin therapy to control their blood glucose level. In contrast, T2DM is characterized by insulin resistance, where the body's cells do not respond effectively to insulin, coupled with a relative insulin deficiency. This form of diabetes is often associated with obesity, sedentary lifestyle, and/or genetic factors, and it is managed with lifestyle changes and oral medications. Animal models play a crucial role in diabetes research. However, given the distinct differences between T1DM and T2DM, it is imperative for researchers to employ specific animal models tailored to each condition for a better understanding of the impaired mechanisms underlying each condition, and for assessing the efficacy of new therapeutics. In this review, we discuss the distinct animal models used in type 1 and type 2 diabetes mellitus research and discuss their strengths and limitations.

Reduced Bioactive Microbial Products (Pathogen-Associated Molecular Patterns) Contribute to Dysregulated Immune Responses and Impaired Healing in Infected Wounds in Mice with Diabetes.

Diabetic chronic ulcers are plagued with persistent nonresolving inflammation. However, diabetic wound environment early after injury suffers from inadequate inflammatory responses due to reductions in proinflammatory cytokines levels. Diabetic neutrophils have known impairments in bactericidal functions. We hypothesized that reduced bacterial killing by diabetic neutrophils, due to their bactericidal functional impairments, results in reduced bioactive bacterial products, known as pathogen-associated molecular patterns, which in turn contribute to reduced signaling through toll-like receptors, leading to inadequate production of proinflammatory cytokines in infected diabetic wound early after injury. We tested our hypothesis in db/db type 2 obese diabetic mouse wound infection model with Pseudomonas aeruginosa. Our data indicate that despite substantially higher levels of infection, toll-like receptor 4-mediated signaling is reduced in diabetic wounds early after injury owing to reduced bioactive levels of lipopolysaccharide. We further demonstrate that topical treatment with lipopolysaccharide enhances toll-like receptor 4 signaling, increases proinflammatory cytokine production, restores leukocyte trafficking, reduces infection burden, and stimulates healing in diabetic wounds. We posit that lipopolysaccharide may be a viable therapeutic option for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process, which is intended to reset chronic ulcers into acute fresh wounds.

Pseudomonas aeruginosa: Infections, Animal Modeling, and Therapeutics.

Pseudomonas aeruginosa is an important Gram-negative opportunistic pathogen which causes many severe acute and chronic infections with high morbidity, and mortality rates as high as 40%. What makes P. aeruginosa a particularly challenging pathogen is its high intrinsic and acquired resistance to many of the available antibiotics. In this review, we review the important acute and chronic infections caused by this pathogen. We next discuss various animal models which have been developed to evaluate P. aeruginosa pathogenesis and assess therapeutics against this pathogen. Next, we review current treatments (antibiotics and vaccines) and provide an overview of their efficacies and their limitations. Finally, we highlight exciting literature on novel antibiotic-free strategies to control P. aeruginosa infections.

Pseudomonas aeruginosa Cytotoxins: Mechanisms of Cytotoxicity and Impact on Inflammatory Responses.

Pseudomonas aeruginosa is one of the most virulent opportunistic Gram-negative bacterial pathogens in humans. It causes many acute and chronic infections with morbidity and mortality rates as high as 40%. P. aeruginosa owes its pathogenic versatility to a large arsenal of cell-associated and secreted virulence factors which enable this pathogen to colonize various niches within hosts and protect it from host innate immune defenses. Induction of cytotoxicity in target host cells is a major virulence strategy for P. aeruginosa during the course of infection. P. aeruginosa has invested heavily in this strategy, as manifested by a plethora of cytotoxins that can induce various forms of cell death in target host cells. In this review, we provide an in-depth review of P. aeruginosa cytotoxins based on their mechanisms of cytotoxicity and the possible consequences of their cytotoxicity on host immune responses.

An Updated Review of Hypertrophic Scarring.

Hypertrophic scarring (HTS) is an aberrant form of wound healing that is associated with excessive deposition of extracellular matrix and connective tissue at the site of injury. In this review article, we provide an overview of normal (acute) wound healing phases (hemostasis, inflammation, proliferation, and remodeling). We next discuss the dysregulated and/or impaired mechanisms in wound healing phases that are associated with HTS development. We next discuss the animal models of HTS and their limitations, and review the current and emerging treatments of HTS.

IL-10 Dysregulation Underlies Chemokine Insufficiency, Delayed Macrophage Response, and Impaired Healing in Diabetic Wounds.

Persistent inflammation is a major contributor to healing impairment in diabetic chronic wounds. Paradoxically, diabetic wound environment during the acute phase of healing is completely different because it exhibits a reduced macrophage response owing to inadequate expression of CCL2 proinflammatory cytokine. What causes a reduction in CCL2 expression in diabetic wounds early after injury remains unknown. In this study, we report that in contrast to prolonged exposure to high glucose, which makes monocytes proinflammatory, short-term exposure to high glucose causes a rapid monocyte reprogramming, manifested by increased expression and secretion of IL-10, which in an autocrine/paracrine fashion reduces glucose uptake and transforms monocytes into an anti-inflammatory phenotype by dampening signaling through toll-like receptors. We show that IL-10 expression is significantly increased in diabetic wounds during the acute phase of healing, causing significant reductions in toll-like receptor signaling and proinflammatory cytokine production, delaying macrophage and leukocyte responses, and underlying healing impairment in diabetic wounds. Importantly, blocking IL-10 signaling during the acute phase of healing improves toll-like receptor signaling, increases proinflammatory cytokine production, enhances macrophage and leukocyte responses, and stimulates healing in diabetic wounds. We posit that anti-IL-10 strategies have therapeutic potential if added topically after surgical debridement, which resets chronic wounds into acute fresh wounds.

CrkII/Abl phosphorylation cascade is critical for NLRC4 inflammasome activity and is blocked by Pseudomonas aeruginosa ExoT.

Type 3 Secretion System (T3SS) is a highly conserved virulence structure that plays an essential role in the pathogenesis of many Gram-negative pathogenic bacteria, including Pseudomonas aeruginosa. Exotoxin T (ExoT) is the only T3SS effector protein that is expressed in all T3SS-expressing P. aeruginosa strains. Here we show that T3SS recognition leads to a rapid phosphorylation cascade involving Abl / PKCδ / NLRC4, which results in NLRC4 inflammasome activation, culminating in inflammatory responses that limit P. aeruginosa infection in wounds. We further show that ExoT functions as the main anti-inflammatory agent for P. aeruginosa in that it blocks the phosphorylation cascade through Abl / PKCδ / NLRC4 by targeting CrkII, which we further demonstrate to be important for Abl transactivation and NLRC4 inflammasome activation in response to T3SS and P. aeruginosa infection.

Presence of CrkI-containing microvesicles in squamous cell carcinomas could have ramifications on tumor biology and cancer therapeutics.

Recently, we described a phenomenon whereby apoptotic cells generate and release CrkI-containing microvesicles, which stimulate proliferation in surrounding cells upon contact to compensate for their own demise. We termed these microvesicles "ACPSVs" for Apoptotic Compensatory Proliferation Signaling microvesicles. As immune cells and a majority of current cancer therapeutics destroy tumor cells primarily by apoptosis, we conducted a small pilot study to assess the possibility that ACPSVs may also be generated in squamous cell carcinomas. We first evaluated a primary and a metastatic squamous cell carcinoma cancer cell lines for their ability to produce ACPSVs under normal and apoptotic conditions. We next conducted a pilot study to assess the occurrence of ACPSVs in solid tumors extracted from 20 cancer patients with squamous cell carcinomas. Both cancer cell lines produced copious amounts of ACPSVs under apoptotic conditions. Interestingly, the metastatic squamous cell carcinoma cancer cell line also produced high levels of ACPSVs under healthy condition, suggesting that the ability to generate ACPSVs may be hijacked by these cells. Importantly, ACPSVs were also abundant in the solid tumors of all squamous cell carcinoma cancer patients. Detection of ACPSVs in cancer has potentially important ramifications in tumor biology and cancer therapeutics which warrants further investigation.

Overriding impaired FPR chemotaxis signaling in diabetic neutrophil stimulates infection control in murine diabetic wound.

Infection is a major co-morbidity that contributes to impaired healing in diabetic wounds. Although impairments in diabetic neutrophils have been blamed for this co-morbidity, what causes these impairments and whether they can be overcome, remain largely unclear. Diabetic neutrophils, isolated from diabetic individuals, exhibit chemotaxis impairment but this peculiar functional impairment has been largely ignored because it appears to contradict the clinical findings which blame excessive neutrophil influx as a major impediment to healing in chronic diabetic ulcers. Here, we report that exposure to glucose in diabetic range results in impaired chemotaxis signaling through the formyl peptide receptor (FPR) in neutrophils, culminating in reduced chemotaxis and delayed neutrophil trafficking in the wound of Leprdb (db/db) type two diabetic mice, rendering diabetic wound vulnerable to infection. We further show that at least some auxiliary receptors remain functional under diabetic conditions and their engagement by the pro-inflammatory cytokine CCL3, overrides the requirement for FPR signaling and substantially improves infection control by jumpstarting the neutrophil trafficking toward infection, and stimulates healing in diabetic wound. We posit that CCL3 may have therapeutic potential for the treatment of diabetic foot ulcers if it is applied topically after the surgical debridement process which is intended to reset chronic ulcers into acute fresh wounds.

The Pseudomonas aeruginosa type III secreted toxin ExoT is necessary and sufficient to induce apoptosis in epithelial cells.

Type III secreted (T3SS) effectors are important virulence factors in acute infections caused by Pseudomonas aeruginosa. PA103, a well-studied human lung isolate, encodes and secretes two effectors, ExoU and ExoT. ExoU is a potent cytotoxin that causes necrotic cell death. In addition, PA103 can induce cell death in macrophages in an ExoU-independent but T3SS-dependent manner. We now demonstrate that ExoT is both necessary and sufficient to cause apoptosis in HeLa cells and that it activates the mitochondrial/cytochrome c-dependent apoptotic pathway. We further show that ExoT induction of cell death is primarily dependent on its ADP ribosyltransferase domain activity. Our data also indicate that the T3SS apparatus can cause necrotic cell death, which is effectively blocked by ExoT, suggesting that P. aeruginosa may have evolved strategies to prevent T3SS-induced necrosis.

Pseudomonas aeruginosa ExoS Induces Intrinsic Apoptosis in Target Host Cells in a Manner That is Dependent on its GAP Domain Activity.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes serious infections in immunocompromised individuals and cystic fibrosis patients. ExoS and ExoT are two homologous bifunctional Type III Secretion System (T3SS) virulence factors that induce apoptosis in target host cells. They possess a GTPase Activating Protein (GAP) domain at their N-termini, which share ~76% homology, and an ADP-ribosyltransferase (ADPRT) domain at their C-termini, which target non-overlapping substrates. Both the GAP and the ADPRT domains contribute to ExoT's cytotoxicity in target epithelial cells, whereas, ExoS-induced apoptosis is reported to be primarily due to its ADPRT domain. In this report, we demonstrate that ExoS/GAP domain is both necessary and sufficient to induce mitochondrial apoptosis. Our data demonstrate that intoxication with ExoS/GAP domain leads to enrichment of Bax and Bim into the mitochondrial outer-membrane, disruption of mitochondrial membrane and release of and cytochrome c into the cytosol, which activates initiator caspase-9 and effector caspase-3, that executes cellular death. We posit that the contribution of the GAP domain in ExoS-induced apoptosis was overlooked in prior studies due to its slower kinetics of cytotoxicity as compared to ADPRT. Our data clarify the field and reveal a novel virulence function for ExoS/GAP as an inducer of apoptosis.

Apoptosis and Compensatory Proliferation Signaling Are Coupled by CrkI-Containing Microvesicles.

Apoptosis has been implicated in compensatory proliferation signaling (CPS), whereby dying cells induce proliferation in neighboring cells as a means to restore homeostasis. The nature of signaling between apoptotic cells and their neighboring cells remains largely unknown. Here we show that a fraction of apoptotic cells produce and release CrkI-containing microvesicles (distinct from exosomes and apoptotic bodies), which induce proliferation in neighboring cells upon contact. We provide visual evidence of CPS by videomicroscopy. We show that purified vesicles in vitro and in vivo are sufficient to stimulate proliferation in other cells. Our data demonstrate that CrkI inactivation by ExoT bacterial toxin or by mutagenesis blocks vesicle formation in apoptotic cells and inhibits CPS, thus uncoupling apoptosis from CPS. We further show that c-Jun amino-terminal kinase (JNK) plays a pivotal role in mediating vesicle-induced CPS in recipient cells. CPS could have important ramifications in diseases that involve apoptotic cell death.