Validation of a clinical trial composite endpoint for patients with necrotizing soft tissue infections.

OBJECTIVE: Our objective was to develop and validate a composite endpoint for patients with necrotizing soft tissue infections that incorporates: local tissue injury, systemic organ dysfunction, and mortality. METHODS: The Necrotizing Infection Clinical Composite Endpoint (NICCE) was defined as follows:(i) alive at day 28, (ii) three or less debridements before day 14, (iii) no amputation beyond first debridement, (iv) modified sequential organ failure assessment score score (mSOFA) at day 14 ≤ 1. To be considered a success, all individual criteria must be met. Several data sets were used to assess validity: (i) a retrospective data set of 198 patients treated during 2013 at 12 US trauma centers; (ii) a subset with high disease acuity, admission mSOFA score of 3 or higher (n = 69); and (iii) 40 patients from a multicenter, phase 2 randomized trial of a CD28 immunomodulator (AB103). Clinical success based on each parameter and the composite score was assessed. RESULTS: Using the retrospective data set for all patients and those with high disease severity (respectively), survival rates were 92% and 84%; day 14 mSOFA 1 or lower score was 69% and 51%; three or less debridements was 84% and 77%; and no subsequent amputations were 96% and 94%. Overall, the percent meeting all success criteria for NICCE was 58% (all patients) and 33% (mSOFA > 3). NICCE success was also associated with reduced utilization of health care resources, intensive care unit-free days were median (interquartile range) of 25.3 (21.9-28) and 19.6 (4.3-25.1) days (one-sided Wilcoxon p < 0.001) and ventilator-free days were 28 (26-28) versus 25 (14-28) (p < 0.001) for NICCE success versus failure, respectively. Using the phase 2 data set, the treated group (0.5 mg/kg, n = 15) demonstrated a NICCE success rate of 73.3% versus 40% for placebo (n = 10). CONCLUSION: These data demonstrate internal consistency of the components and face and criterion validity of the NICCE endpoint. NICCE offers an opportunity to demonstrate a clinically relevant treatment effect for patients enrolled in clinical trials for necrotizing soft tissue infection. LEVEL OF EVIDENCE: Prognostic/Epidemiological, level III; Therapeutic, level IV.

Impact and Progression of Organ Dysfunction in Patients with Necrotizing Soft Tissue Infections: A Multicenter Study.

BACKGROUND: Necrotizing soft tissue infections (NSTI) represent a rare but devastating disease for which the systemic manifestations have been poorly characterized. In an effort to define an optimal endpoint for clinical trials in this condition, the objective of this study was to establish the pattern of organ dysfunction over time and determine the correlation between organ dysfunction and clinical outcome in patients with NSTI. METHODS: We conducted a multicenter, retrospective clinical study of patients with NSTI presenting to 12 academic medical centers in the U.S. during 2013. Patients with a diagnosis of NSTI confirmed by surgical findings were included. Organ dysfunction was assessed using a modified Sequential Organ Failure Assessment (SOFA) score (mSOFA: excluding liver) on admission and on hospital days 1, 2, 3, 7, 10, and 14. The presence of organ dysfunction on admission and resolution of organ dysfunction were correlated with clinical parameters, including intensive care unit (ICU)-free days (of 28 d), ventilator-free days, number of debridements, and mortality rate. The incidence of acute kidney injury (AKI) and recovery also were assessed. RESULTS: There were 198 patients enrolled, of whom 62% were male, the mean age was 51 years, and 40% had monomicrobial infections. The mean mSOFA score on admission was 2.4 ± 3.0, with 49% of the patients having a score ≥2 and 35% a score of ≥3. Patients typically demonstrated worsening of the mSOFA score over the first 24 h followed by gradual resolution. An mSOFA ≥3 at admission was associated with a significant decrease in ventilator-free days (mean 20.1 vs. 25.6 days; p < 0.001); ICU-free days (15.2 vs. 23.1, p < 0.001); more debridements (mean 2.3 vs. 2.0; p = 0.11); a higher mortality rate (15.9% vs. 3.1%; p = 0.003); and a higher rate of AKI (59.4 vs. 35.9%; p < 0.001). The persistence of organ dysfunction (mSOFA >1) among survivors at day 14 was associated with fewer ICU-free days (17.8 vs. 23.6; p < 0.001) and ventilator-free days (23.6 vs. 27; p = 0.001) and a lower recovery rate from AKI (38.7% vs. 81.3%; p < 0.001). CONCLUSION: Early development of systemic organ dysfunction in patients with NSTI is associated with higher morbidity and mortality rates. Failure of the resolution of organ dysfunction by day 14 forecasts a poor outcome. The mSOFA score may be a useful marker for patient selection for inclusion in interventional trials, and the resolution of organ dysfunction by day 14 may be an important clinical endpoint.

CD28 homodimer interface mimetic peptide acts as a preventive and therapeutic agent in models of severe bacterial sepsis and gram-negative bacterial peritonitis.

BACKGROUND: Severe gram-negative bacterial infections and sepsis are major causes of morbidity and mortality. Dysregulated, excessive proinflammatory cytokine expression contributes to the pathogenesis of sepsis. A CD28 mimetic peptide (AB103; previously known as p2TA) that attenuates CD28 signaling and T-helper type 1 cytokine responses was tested for its ability to increase survival in models of polymicrobial infection and gram-negative sepsis. METHODS: Mice received AB103, followed by an injection of Escherichia coli 0111:B4 lipopolysaccharide (LPS); underwent induction E. coli 018:K1 peritonitis induction, followed by treatment with AB103; or underwent cecal ligation and puncture (CLP), followed by treatment with AB103. The effects of AB103 on factors associated with and the lethality of challenge infections were analyzed. RESULTS: AB103 strongly attenuated induction of tumor necrosis factor α and interleukin 6 (IL-6) by LPS in human peripheral blood mononuclear cells. Receipt of AB103 following intraperitoneal injection of LPS resulted in survival among 73% of CD1 mice (11 of 15), compared with 20% of controls (3 of 15). Suboptimal doses of antibiotic alone protected 20% of mice (1 of 5) from E. coli peritonitis, whereas 100% (15 of 15) survived when AB103 was added 4 hours following infection. Survival among mice treated with AB103 12 hours after CLP was 100% (8 of 8), compared with 17% among untreated mice (1 of 6). In addition, receipt of AB103 12 hours after CLP attenuated inflammatory cytokine responses and neutrophil influx into tissues and promoted bacterial clearance. Receipt of AB103 24 hours after CLP still protected 63% of mice (5 of 8). CONCLUSIONS: Single-dose AB103 reduces mortality in experimental models of polymicrobial and gram-negative bacterial infection and sepsis, warranting further studies of this agent in clinical trials.

Single administration of p2TA (AB103), a CD28 antagonist peptide, prevents inflammatory and thrombotic reactions and protects against gastrointestinal injury in total-body irradiated mice.

The goal of this study was to elucidate the action of the CD28 mimetic peptide p2TA (AB103) that attenuates an excessive inflammatory response in mitigating radiation-induced inflammatory injuries. BALB/c and A/J mice were divided into four groups: Control (C), Peptide (P; 5 mg/kg of p2TA peptide), Radiation (R; total body irradiation with 8 Gy γ-rays), and Radiation + Peptide (RP; irradiation followed by p2TA peptide 24 h later). Gastrointestinal tissue damage was evaluated by analysis of jejunum histopathology and immunohistochemistry for cell proliferation (Cyclin D1) and inflammation (COX-2) markers, as well as the presence of macrophages (F4/80). Pro-inflammatory cytokines IL-6 and KC as well as fibrinogen were quantified in plasma samples obtained from the same mice. Our results demonstrated that administration of p2TA peptide significantly reduced the irradiation-induced increase of IL-6 and fibrinogen in plasma 7 days after exposure. Seven days after total body irradiation with 8 Gy of gamma rays numbers of intestinal crypt cells were reduced and villi were shorter in irradiated animals compared to the controls. The p2TA peptide delivery 24 h after irradiation led to improved morphology of villi and crypts, increased Cyclin D1 expression, decreased COX-2 staining and decreased numbers of macrophages in small intestine of irradiated mice. Our study suggests that attenuation of CD28 signaling is a promising therapeutic approach for mitigation of radiation-induced tissue injury.

A Novel Drug for Treatment of Necrotizing Soft-Tissue Infections: A Randomized Clinical Trial.

IMPORTANCE: Necrotizing soft-tissue infections (NSTI) have high morbidity and mortality rates despite aggressive surgical debridement and antibiotic therapy. AB103 is a peptide mimetic of the T-lymphocyte receptor, CD28. We hypothesized that AB103 will limit inflammatory responses to bacterial toxins and decrease the incidence of organ failure. OBJECTIVES: To establish the safety of AB103 in patients with NSTI and evaluate the potential effects on clinically meaningful parameters related to the disease. DESIGN, SETTING, AND PARTICIPANTS: A prospective, randomized, placebo-controlled, double-blinded study was performed in 6 academic medical centers in the United States. Participants included adults with NSTI. Of 345 patients screened, 43 were enrolled for the intent-to-treat analysis, and 40 met criteria for the modified intent-to-treat analysis; 15 patients each were included in the high-dose and low-dose treatment arms, and 10 in the placebo arm. INTERVENTION: Single intravenous dose of AB103 (0.5 or 0.25 mg/kg) within 6 hours after diagnosis of NSTI. MAIN OUTCOMES AND MEASURES: Change in the Sequential Organ Failure Assessment score within 28 days, intensive care unit-free and ventilator-free days, number and timing of debridements, plasma and tissue cytokine levels at 0 to 72 hours, and adverse events. RESULTS: Baseline characteristics were comparable in the treatment groups. The Sequential Organ Failure Assessment score improved from baseline in both treatment groups compared with the placebo group at 14 days (change from baseline score, -2.8 in the high-dose, -2 in the low-dose, and +1.3 in the placebo groups; P = .04). AB103-treated patients had a similar number of debridements (mean [SD], 2.2 [1.1] for the high-dose, 2.3 [1.2] for the low-dose, and 2.8 [2.1] for the placebo groups; P = .56). There were no statistically significant differences in intensive care unit-free and ventilator-free days or in plasma and tissue cytokine levels. No drug-related adverse events were detected. CONCLUSIONS AND RELEVANCE: AB103 is a safe, promising new agent for modulation of inflammation after NSTI. Further study is warranted to establish efficacy. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT01417780.

A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes.

Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable ß chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

18F-ML-10, a PET tracer for apoptosis: first human study.

UNLABELLED: Clinical PET of apoptosis may have substantial value in advancing patient care. We report here the first-in-humans study with (18)F-labeled 2-(5-fluoropentyl)-2-methyl malonic acid ((18)F-ML-10), a small-molecule PET tracer for apoptosis. Presented are the dosimetry, biodistribution, stability, and safety profiles of this PET tracer in healthy human volunteers. Also reported is tracer binding to targeted apoptotic cells in testicular tissue, where a relative abundance of apoptotic cells is normally observed. METHODS: (18)F-ML-10 (233 ± 90 MBq) was intravenously administered to 8 healthy subjects, followed by whole-body PET/CT for 220 min. Serial blood and urine samples were collected for radioactivity measurement, and plasma tracer stability was assessed by high-performance liquid chromatography. Dosimetry calculations were performed using OLINDA/EXM software. RESULTS: (18)F-ML-10 manifested high stability in vivo and rapid distribution followed by fast clearance, with an elimination half-life of 1.3 ± 0.1 and 1.1 ± 0.2 h from the blood and from all other organs, respectively, and excretion through the urine. Dosimetry showed an average effective whole-body dose of 15.4 ± 3.7 µSv/MBq, with the urinary bladder being the dose-limiting organ. Selective accumulation and retention of the tracer in the testes was observed in all male subjects, a finding also demonstrated in mice using both small-animal PET and histopathology, confirming binding to apoptotic cells. Administration of (18)F-ML-10 was safe, without adverse effects. CONCLUSION: (18)F-ML-10 administered to healthy humans demonstrated a favorable dosimetry, biodistribution, stability, and safety profile. Binding to apoptotic sites was also demonstrated. These data support further development of this small-molecule probe for clinical PET of apoptosis.

Small-molecule biomarkers for clinical PET imaging of apoptosis.

Apoptosis is a fundamental biologic process. Molecular imaging of apoptosis in vivo may have important implications for clinical practice, assisting in early detection of disease, monitoring of disease course, assessment of treatment efficacy, or development of new therapies. Although a PET probe for clinical imaging of apoptosis would be highly desirable, this is yet an unachieved goal, mainly because of the required challenging integration of various features, including sensitive and selective detection of the apoptotic cells, clinical aspects such as favorable biodistribution and safety profiles, and compatibility with the radiochemistry and imaging routines of clinical PET centers. Several approaches are being developed to address this challenge, all based on novel small-molecule structures targeting various steps of the apoptotic cascade. This novel concept of small-molecule PET probes for apoptosis is the focus of this review.

Novel fluorescence molecular imaging of chemotherapy-induced intestinal apoptosis.

Chemotherapy-induced enteropathy (CIE) is one of the most serious complications of anticancer therapy, and tools for its early detection and monitoring are highly needed. We report on a novel fluorescence method for detection of CIE, based on molecular imaging of the related apoptotic process. The method comprises systemic intravenous administration of the ApoSense fluorescent biomarker (N,N(')-didansyl-L-cystine DDC) in vivo and subsequent fluorescence imaging of the intestinal mucosa. In the reported proof-of-concept studies, mice were treated with either taxol+cyclophosphamide or doxil. DDC was administered in vivo at various time points after drug administration, and tracer uptake by ileum tissue was subsequently evaluated by ex vivo fluorescent microscopy. Chemotherapy caused marked and selective uptake of DDC in ileal epithelial cells, in correlation with other hallmarks of apoptosis (i.e., DNA fragmentation and Annexin-V binding). Induction of DDC uptake occurred early after chemotherapy, and its temporal profile was parallel to that of the apoptotic process, as assessed histologically. DDC may therefore serve as a useful tool for detection of CIE. Future potential integration of this method with fluorescent endoscopic techniques, or development of radio-labeled derivatives of DDC for emission tomography, may advance early diagnosis and monitoring of this severe adverse effect of chemotherapy.

Molecular imaging of neurodegeneration by a novel cross-disease biomarker.

Current pre-mortem diagnosis of neurodegenerative disorders such as Alzheimer's disease (AD) or amyotrophic lateral sclerosis (ALS) is based on clinical assessment of neurological deficits. However, symptoms and signs emerge only late in the disease course, thus indicating an urgent need for novel tools for detection of the underlying neuropathology. NST-729 (MW=310) is a novel molecular imaging probe, which is a member of the ApoSense family of small molecule detectors of apoptosis. We now report on the ability of NST-729, upon its systemic administration in vivo, to detect characteristic neuropathology in pre-clinical models of AD (Tg2576 transgenic mice) and ALS (transgenic SOD-1 G93A mutation mice). In the AD model, NST-729 clearly and selectively bound and imaged amyloid plaques, in excellent correlation with a typical amyloid ex vivo staining (Congo red). In the ALS model, NST-729 distinctly and selectively imaged multiple degenerating neurons in the motor nuclei in the pons, medulla and spinal cord, manifesting numerous multifocal irregularities and disruptions of neuritic projections, typical of axonal apoptosis. Study results therefore support the potential utility of NST-729 as a cross-disease biomarker for neurodegeneration, and also its potential role as the first molecular probe for ALS. Future radio-labeled NST-729 analogues may assist in the early diagnosis of disease, and in the development of neuroprotective therapies for these severe neurological disorders.

From the Gla domain to a novel small-molecule detector of apoptosis.

Apoptosis plays a pivotal role in the etiology or pathogenesis of numerous medical disorders, and thus, targeting of apoptotic cells may substantially advance patient care. In our quest for novel low-molecular-weight probes for apoptosis, we focused on the uncommon amino acid gamma-carboxyglutamic acid (Gla), which plays a vital role in the binding of clotting factors to negatively charged phospholipid surfaces. Based on the alkyl-malonic acid motif of Gla, we have developed and now present ML-10 (2-(5-fluoro-pentyl)-2-methyl-malonic acid, MW=206 Da), the prototypical member of a novel family of small-molecule detectors of apoptosis. ML-10 was found to perform selective uptake and accumulation in apoptotic cells, while being excluded from either viable or necrotic cells. ML-10 uptake correlates with the apoptotic hallmarks of caspase activation, Annexin-V binding and disruption of mitochondrial membrane potential. The malonate moiety was found to be crucial for ML-10 function in apoptosis detection. ML-10 responds to a unique complex of features of the cell in early apoptosis, comprising irreversible loss of membrane potential, permanent acidification of cell membrane and cytoplasm, and preservation of membrane integrity. ML-10 is therefore the most compact apoptosis probe known to date. Due to its fluorine atom, ML-10 is amenable to radio-labeling with the (18)F isotope, towards its potential future use for clinical positron emission tomography imaging of apoptosis.

Monitoring of tumor response to chemotherapy in vivo by a novel small-molecule detector of apoptosis.

Utilization of molecular imaging of apoptosis for clinical monitoring of tumor response to anti-cancer treatments in vivo is highly desirable. To address this need, we now present ML-9 (butyl-2-methyl-malonic acid; MW = 173), a rationally designed small-molecule detector of apoptosis, based on a novel alkyl-malonate motif. In proof-of-concept studies, induction of apoptosis in tumor cells by various triggers both in vitro and in vivo was associated with marked uptake of (3)H-ML-9 administered in vivo, in correlation with the apoptotic hallmarks of DNA fragmentation, caspase-3 activation and membrane phospholipid scrambling, and with correlative tumor regression. ML-9 uptake following chemotherapy was tumor-specific, with rapid clearance of the tracer from the blood and other non-target organs. Excess of non-labeled "cold" compound competitively blocked ML-9 tumor uptake, thus demonstrating the specificity of ML-9 binding. ML-9 may therefore serve as a platform for a novel class of small-molecule imaging agents for apoptosis, useful for assessment of tumor responsiveness to treatment.

Molecular imaging of neurovascular cell death in experimental cerebral stroke by PET.

UNLABELLED: Clinical molecular imaging of apoptosis is a highly desirable yet unmet challenge. Here we provide the first report on (18)F-labeled 5-fluoropentyl-2-methyl-malonic acid ((18)F-ML-10), a small-molecule, (18)F-labeled PET tracer for the imaging of apoptosis in vivo; this report includes descriptions of the synthesis, radiolabeling, and biodistribution of this novel apoptosis marker. We also describe the use of (18)F-ML-10 for small-animal PET of neurovascular cell death in experimental cerebral stroke in mice. METHODS: (18)F-ML-10 was synthesized by nucleophilic substitution from the respective mesylate precursor, and its biodistribution was assessed in healthy rats. Permanent occlusion of the middle cerebral artery (MCA) was induced in mice, and small-animal PET was performed 24 h later. RESULTS: Efficient radiolabeling of ML-10 with (18)F was achieved. Biodistribution studies with (18)F-ML-10 revealed rapid clearance from blood (half-life of 23 min), a lack of binding to healthy tissues, and rapid elimination through the kidneys. No significant tracer metabolism in vivo was observed. Clear images of distinct regions of increased uptake, selectively in the ischemic MCA territory, were obtained in the in vivo small-animal PET studies. Uptake measurements ex vivo revealed 2-fold-higher uptake in the affected hemisphere and 6- to 10-fold-higher uptake in the region of interest of the infarct. The cerebral uptake of (18)F-ML-10 was well correlated with histologic evidence of cell death. The tracer was retained in the stroke area but was cleared from blood and from intact brain areas. CONCLUSION: (18)F-ML-10 is useful for noninvasive PET of neurovascular histopathology in ischemic cerebral stroke in vivo. Such an assessment may assist in characterization of the extent of stroke-related cerebral damage and in the monitoring of disease course and effect of treatment.

Targeting cell death in vivo in experimental traumatic brain injury by a novel molecular probe.

Traumatic brain injury (TBI) remains a frequent and major challenge in neurological and neurosurgical practice. Apoptosis may play a role in cerebral tissue damage induced by the traumatic insult, and thus its detection and inhibition may advance patient care. DDC (N,N'-didansyl-L-cystine) is a novel fluorescent probe for detection of apoptotic cells. We now report on the performance of DDC in experimental TBI. Closed head injury was induced in mice by weight-drop. DDC was administered intravenously in vivo. Two hours later, animals were sacrificed, and brain tissue was subjected to fluorescent microcopy, for assessment of DDC uptake, in correlation with histopathological assessment of apoptosis by TUNEL and caspase substrates, and also in correlation with the neurological deficits, as assessed by Neurological Severity Score (NSS). Selective uptake of DDC was observed at the primary site of injury, and also at remote sites. Uptake was at the cellular level, with accumulation of DDC in the cytoplasm. Cells manifesting DDC uptake were confirmed as apoptotic cells by detection of the characteristic apoptotic DNA fragmentation (positive TUNEL staining) and detection of activated caspases. The damaged region stained by DDC fluorescence correlated with the severity of neuronal deficits. Our study confirms the role of apoptosis in TBI, and proposes DDC as a useful tool for its selective targeting and detection in vivo. Such imaging of apoptosis, following future radiolabeling of DDC, may advance care for patients with head injury, by allowing real-time evaluation of the extent of tissue damage, assessment of novel therapeutic strategies, and optimization of treatment for the individual patient.

Monitoring of chemotherapy-induced cell death in melanoma tumors by N,N'-Didansyl-L-cystine.

Early assessment of the efficacy of anticancer agents is a highly desirable and an unmet need in clinical oncology. Clinical imaging of cell-death may be useful in addressing this need, as induction of tumor cell-death is the primary mechanism of action of most anticancer drugs. In this study, we examined the performance of N,N'-Didansyl-L-cystine (DDC), a member of the ApoSense family of novel small molecule detectors of cell-death, as a potential tool for monitoring cell-death in cancer models. Detection of cell-death by DDC was examined in fluorescent studies on B16 melanoma cells both in vitro and ex vivo following its in vivo administration. In vitro, DDC manifested selective uptake and accumulation within apoptotic cells that was highly correlated with Annexin-V binding, changes in mitochondrial membrane potential, and caspase activation. Uptake was not ATP-dependent, and was inducible by calcium mobilization. In vivo, DDC selectively targeted cells undergoing cell-death in melanoma tumors, while not binding to viable tumor cells. Chemotherapy caused marked tumor cell-death, evidenced by increased DDC uptake, which occurred before a detectable change in tumor size and was associated with increased animal survival. These data confirm the usefulness of imaging of cell-death by DDC as a tool for early monitoring of tumor response to anti-cancer therapy.

Novel molecular imaging of cell death in experimental cerebral stroke.

Cell death is the basic neuropathological substrate in cerebral ischemia, and its non-invasive imaging may improve diagnosis and treatment for stroke patients. ApoSense is a novel family of low-molecular weight compounds for detection and imaging of cell death in vivo. We now report on imaging of cell death and monitoring of efficacy of neuroprotective treatment in vivo by intravenous administration of the ApoSense compound DDC (didansylcystine), in experimental stroke in rodents. Rats and mice were subjected to a short-term (2 h) or permanent occlusion of the middle cerebral artery (MCA) and injected with DDC or 3H-labeled DDC. Fluorescent and autoradiographic studies, respectively, were performed ex vivo, comprising assessment of DDC uptake in the infarct region, in correlation with tissue histopathology. Neuroprotection was induced by a caspase inhibitor (Q-VD-OPH), and its effect was monitored by DDC. Following its intravenous administration, DDC accumulated selectively in injured neurons within the region of infarct. Caspase inhibition exerted a 45% reduction in infarct volume, which was well reported by DDC. This is the first report on a small molecule probe for detection in vivo of cell death in cerebral stroke. DDC may potentially assist in addressing the current "neuroimaging/neurohistology gap", for molecular assessment of the extent of stroke-related cell death.

Molecular imaging of cell death in vivo by a novel small molecule probe.

Apoptosis has a role in many medical disorders, therefore assessment of apoptosis in vivo can be highly useful for diagnosis, follow-up and evaluation of treatment efficacy. ApoSense is a novel technology, comprising low molecular-weight probes, specifically designed for imaging of cell death in vivo. In the current study we present targeting and imaging of cell death both in vitro and in vivo, utilizing NST-732, a member of the ApoSense family, comprising a fluorophore and a fluorine atom, for both fluorescent and future positron emission tomography (PET) studies using an (18)F label, respectively. In vitro, NST-732 manifested selective and rapid accumulation within various cell types undergoing apoptosis. Its uptake was blocked by caspase inhibition, and occurred from the early stages of the apoptotic process, in parallel to binding of Annexin-V, caspase activation and alterations in mitochondrial membrane potential. In vivo, NST-732 manifested selective uptake into cells undergoing cell-death in several clinically-relevant models in rodents: (i) Cell-death induced in lymphoma by irradiation; (ii) Renal ischemia/reperfusion; (iii) Cerebral stroke. Uptake of NST-732 was well-correlated with histopathological assessment of cell-death. NST-732 therefore represents a novel class of small-molecule detectors of apoptosis, with potential useful applications in imaging of the cell death process both in vitro and in vivo.

Examination of cellular and molecular events associated with optic nerve axotomy.

PURPOSE: Analyzing cellular behavior during scar formation and determining the expression of growth inhibiting molecules in the optic nerve and retina following acute optic nerve injury. METHODS: A rat model of complete transection of the optic nerve that spares the vascular supply and the neural scaffold was used. The response of the optic nerve and retinas to axotomy was studied by immunological and biochemical approaches. RESULTS: Optic nerve axotomy led to massive cell invasion at the site of injury that spread along both sides of the nerve. The cells were microglia, oligodendrocytes, and to a lesser extent astrocytes. A marked induction of semaphorin 3A was evident, especially in the area of the scar, and persisted up to the 28th day of the experiment. Expression of neuropilin-1, a component of the semaphorin 3A receptor, increased following injury. The molecular events associated with axotomy were studied by measuring the levels of semaphorin 3A, p38 MAPK, and ERK1/2 in the retina. Semaphorin 3A levels and the activated form of p38 were elevated 3 days post-axotomy and then declined; ERK1/2 activation levels reached their peak 14 days post axotomy. Acute nerve injury led to morphological alterations in oligodendrocytes, astrocytes, and the extracellular matrix, disrupting the delicate internal organization of the optic nerve. CONCLUSIONS: We suggest that cell invasion, semaphorin 3A and neuropilin-1 induction, and disruption of the internal organization of the optic nerve contribute to axotomy-induced degenerative processes.

Immunosuppressive role of semaphorin-3A on T cell proliferation is mediated by inhibition of actin cytoskeleton reorganization.

Timely negative regulation of the immune system is critical to allow it to perform its duty while maintaining it under tight control to avoid overactivation. We previously reported that the neuronal receptor neuropilin-1 (NP-1) is expressed in human lymph nodes. However, the role of NP-1 interaction with its physiological ligand semaphorin-3A (Sema-3A) on immune cells remains elusive. Here we show that Sema-3A is expressed by activated DC and T cells, and that its secretion in DC/T cell cocultures is delayed. Sema-3A/NP-1 interaction down-modulated T cell activation since addition of Sema-3A in DC/T cell cocultures dramatically inhibited allogeneic T cell proliferation. More importantly, neutralization by blocking antibodies or by antagonist peptide of endogenous Sema-3A produced by DC/T cell cocultures resulted in a 130% increase in T cell proliferation. Sema-3A acted directly on T cells, since it could block anti-CD3/CD28-stimulated proliferation of T cells. Finally, immunomodulatory functions of Sema-3A relied on the blockage of actin cytoskeleton reorganization, affecting TCR polarization and interfering with early TCR signal transduction events such as ZAP-70 or focal adhesion kinase phosphorylation. Therefore, we propose that Sema-3A secretion and the resulting NP-1/Sema-3A interaction are involved in a late negative feedback loop controlling DC-induced T cell proliferation.

ApoSense: a novel technology for functional molecular imaging of cell death in models of acute renal tubular necrosis.

PURPOSE: Acute renal tubular necrosis (ATN), a common cause of acute renal failure, is a dynamic, rapidly evolving clinical condition associated with apoptotic and necrotic tubular cell death. Its early identification is critical, but current detection methods relying upon clinical assessment, such as kidney biopsy and functional assays, are insufficient. We have developed a family of small molecule compounds, ApoSense, that is capable, upon systemic administration, of selectively targeting and accumulating within apoptotic/necrotic cells and is suitable for attachment of different markers for clinical imaging. The purpose of this study was to test the applicability of these molecules as a diagnostic imaging agent for the detection of renal tubular cell injury following renal ischemia. METHODS: Using both fluorescent and radiolabeled derivatives of one of the ApoSense compounds, didansyl cystine, we evaluated cell death in three experimental, clinically relevant animal models of ATN: renal ischemia/reperfusion, radiocontrast-induced distal tubular necrosis, and cecal ligature and perforation-induced sepsis. RESULTS: ApoSense showed high sensitivity and specificity in targeting injured renal tubular epithelial cells in vivo in all three models used. Uptake of ApoSense in the ischemic kidney was higher than in the non-ischemic one, and the specificity of ApoSense targeting was demonstrated by its localization to regions of apoptotic/necrotic cell death, detected morphologically and by TUNEL staining. CONCLUSION: ApoSense technology should have significant clinical utility for real-time, noninvasive detection of renal parenchymal damage of various types and evaluation of its distribution and magnitude; it may facilitate the assessment of efficacy of therapeutic interventions in a broad spectrum of disease states.