Dysregulation of angiopoietin-1 plays a mechanistic role in the pathogenesis of cerebral malaria
Cerebral malaria is a leading cause of global morbidity and mortality. Interventions targeting the underlying pathophysiology of cerebral malaria may improve outcomes compared to treatment with antimalarials alone. Microvascular leak plays an important role in the pathogenesis of cerebral malaria. The angiopoietin (Ang)-Tie-2 system is a critical regulator of vascular function. We show that Ang-1 expression and soluble Tie-2 expression were associated with disease severity and outcome in a prospective study of Ugandan children with severe malaria and in a preclinical murine model of experimental cerebral malaria. Ang-1 was necessary for maintenance of vascular integrity and survival in a mouse model of cerebral malaria. Therapeutic administration of Ang-1 preserved blood-brain barrier integrity and, in combination with artesunate treatment, improved survival beyond that with artesunate alone. These data define a role for dysregulation of the Ang-Tie-2 axis in the pathogenesis of cerebral malaria and support the evaluation of Ang-Tie-2-based interventions as potential adjunctive therapies for treating severe malaria.
New Drug for Sepsis on the Cheap?
A novel cell-penetrating peptide to facilitate intercellular transport of fused proteins
Cell-based delivery of cell penetrating peptides (CPPs) could represent a new platform for intracellular peptide delivery to local tissues. Expressed CPPs, coupled to a secretory signal peptide (SP), can support intercellular transport. However, low secretion efficiency, which may correlate with the positive charge of most CPPs, has emerged as one of the main impediments for efficient intercellular transport. We have reported that a modified Tat-based CPP (Tatm) with reduced positive charge is secreted efficiently, but its transduction activity was greatly reduced. We now show that a triple repeat of Tatm (Tatm3x) with an elongated alpha-helical amphipathic structure enhances transduction activity and simultaneously retains its secretion efficacy, although passage through the secretory pathway reduces its cell-penetrating activity. SP-Tatm3x supports intercellular transport of fused fluorescent proteins, as well as cell entry and function of a pro-apoptotic peptide. In addition, SP-Tatm3x largely escapes RNA inhibition, which is identified as another potential impediment to CPP-mediated intercellular transport. Expression of SP-Tatm3x in heparan sulfate proteoglycan-negative cells further improves its transduction activity. These results demonstrate the feasibility of intercellular transport of proteins, but further work is needed to better understand the reduction of cell-penetrating activity associated with secretion of CPP-fusion proteins.
Interventions to stabilize endothelium improve survival in experimental cerebral Malaria [Meeting Abstract]
Cerebral malaria (CM) pathogenesis is associated with endothelial activation and perturbation of the blood brain barrier (BBB). Endothelial specific signalling pathways, including the Angiopoietin (Ang)-Tie-2 and Slit/ Roundabout (Robo)-4 systems, are key regulators of endothelial integrity and vascular leakage. Our lab and others have reported that adult and pediatric CM is associated with increased circulating levels of biomarkers of endothelial activation and dysfunction (e.g. Ang-2, sTie- 2, sICAM-1). We hypothesize that interventions to promote endothelial stability will prevent deleterious alterations to the BBB and improve outcome following Plasmodium infection. Using the murine model of Plasmodium berghei ANKA (PbA)-induced experimental CM (ECM), we show that alterations in protein and mRNA levels of angiopoietins are associated with disease severity in the ECM, similar to observations in human populations. Time course experiments established a temporal relationship where PbA-associated alterations in endothelial regulators directly precede the loss of BBB integrity and the onset of neurological symptoms of ECM, such as seizures and paralysis. Pro-Ang-1 treatment strategies (e.g. Adenoviral mediated expression of Ang-1) significantly improved survival in PbA-infected ECM-susceptible C57Bl/6 mice compared to empty adenoviral vector and vehicle controls (p=0.001). Pharmacological activation of the Slit-Robo pathway, using therapeutic administration of recombinant Slit2N, also significantly prolonged survival in PbA-infected C57Bl/6 mice compared to untreated controls (p=0.0007). This benefit was further increased when Slit2N was used as adjunctive therapy in combination with a sub-curative dose of artesunate. To establish direct experimental evidence for a causal role of angiopoietins in ECM, the effect of Ang-1 genetic deletion on disease outcome is currently under investigation using a conditional Cre/loxP system. In summary, we show that adjunctive treatment strategies based on promoti!
Expressed Cell-penetrating Peptides Can Induce a Bystander Effect, but Passage Through the Secretory Pathway Reduces Protein Transduction Activity
Despite advances in vector technology, inefficient gene transfer still limits clinical efficacy of cancer gene therapy. Cell-penetrating peptides (CPPs), such as the basic domain of the transactivator of transcription (Tat) protein of HIV-1, are internalized by intact cells and have been used to deliver purified recombinant proteins. A combination of gene therapy with protein transduction technology could induce a strong bystander effect and represent a platform to deliver proteins to target cells. However, whether expressed CPP can facilitate intercellular trafficking, i.e., a bystander effect, is controversial. Our data suggest that expressed fusion proteins that contain the basic domain of Tat do not induce a detectable bystander effect. However, Tat-fusion proteins that also contain a secretory signal peptide (SP) can induce a bystander effect in vitro, although the in vivo effect is small. Surprisingly, despite the presence of a SP, the bystander effect does not seem to be related to secretion of the fusion protein. In fact, Tat-fusion proteins are secreted very inefficiently, and protein transduction seems largely mediated by fusion proteins that are released by cell lysis. Modification of Tat can improve secretion efficacy and prevent cleavage by the endoprotease furin, but passage through the secretory pathway is associated with reduced transduction activity of Tat-fusion proteins
A novel protein transduction domain with improved secretion and transduction activity [Meeting Abstract]
Cell penetrating peptides (CPPs) are widely used to deliver proteins and other macromolecules into cells. Generally, CPPs are either synthesized and coupled to the therapeutic payload or expressed in bacteria as fusion proteins followed by various purification steps. However, despite great interest in this technology, difficulties with production, purification and unfavorable pharmacokinetics are still limiting its clinical success. As an alternative approach, expression of CPPs that support intercellular transfer of fused proteins could avoid production and purification needs and serve as a tool to deliver therapeutic proteins. We have previously reported that the basic domain of HIV Tat, fused to fluorescent proteins can support intercellular transfer, but only when coupled to a secretory signal peptide (SP). However, SP-Tat fusion proteins are very inefficiently secreted and transduction seems mostly mediated by fusion proteins released by means other than secretion through the classic pathway. We also demonstrated that a modified Tat-based CPP (Tatm) is secreted much more efficiently, but its transduction activity was greatly reduced compared to Tat. We now show that an elongated 3x repeat sequence of Tatm (Tatm3x) enhances transduction activity. Secretion activity is greatly improved compared to Tat, although somewhat reduced when compared to Tatm. Expressed SP-Tatm3x fusion proteins are localized in the ER and cytoplasm and are secreted mainly via the classical pathway. Fusion proteins that are released through cell lysis have greatly improved transduction activity compared to SP-Tatm. Based on co-culture and mixing experiments, expressed SP-Tatm3x supports intercellular transport of fused fluorescent proteins in vitro. As previously shown for SP-Tatm fusion proteins, travel through the secretory pathway also reduces transduction activity of SP-Tatm3x fusion proteins. In conclusion, expressed SP-Tatm3x displays improved secretion and transduction activity and represents an important step forward in the development of a CPP that supports intercellular transport of fused proteins
RELATIVE ANGIOPOIETIN LEVELS ALTER SUSCEPTIBILITY TO EXPERIMENTAL CEREBRAL MALARIA [Meeting Abstract]
Illness-related distress in women with clinically localized cutaneous melanoma [Meeting Abstract]
Women may experience significant distress following treatment of melanoma. We conducted a prospective cross-sectional survey to examine illness-related distress in 100 women treated for clinically localized melanoma. We assessed distress with seven validated measures, including general depressive symptoms, distress related to illness, quality of life, concerns about recurrence, appearance perceptions, body image, and reproductive concerns. Over 80% of patients approached agreed to participate. Mean participant age was 54 (range 21-89), 27% had a family history of melanoma, and 64% were married. Most had melanoma of the extremity (61%), with a mean depth of 1.1 mm (range 0-10.5 mm). Significant depressive symptomatology occurred in 10% of patients, and 12% reported a high level of intrusive thoughts related to melanoma. Quality of life scores indicated more disruption on psychological (M = 6.33 SE = 0.18), compared to social (M = 7.75 SE = 0.20) and physical (M = 8.10 SE = 0.16) functioning. Only 20% of women reported no body image distress, while 64% of patients rated their appearance as worse post-treatment. 23% were unsatisfied with the appearance of their surgical site and 38% changed what they wore post-treatment. Recurrence concerns indicated significant worry about health (M = 1.73 SE = 0.12) and death (M = 1.98 SE = 0.14), compared to womanhood (M = 0.57 SE = 0.09) or their roles in life (M = 1.17 SE = 0.11). Patients were worried that future children would have an increased risk of cancer (52%), but most (>85%) were not concerned about fertility. Women treated for clinically localized melanoma have significant levels of distress associated with their body image and fear of recurrence. Interventions to target these areas may reduce the distress associated with the diagnosis and treatment of melanoma
Essentials of medical genomics
Hoboken, N. J. : Wiley-Blackwell, 2009
Extent: xii, 439 p. ; 24 cm.
Angiopoietin-1 increases survival and reduces the development of lung edema induced by endotoxin administration in a murine model of acute lung injury
OBJECTIVE: To evaluate the effect of angiopoietin-1, an angiogenic growth factor, on lung capillary leakage and survival in a murine model of acute lung injury. DESIGN: Laboratory investigation. SETTING: Research laboratory at New York University School of Medicine and Department of Veterans Affairs, NY Harbor Healthcare System. SUBJECTS: C57BL/6 mice weighing 18-20 g, susceptible to endotoxin-induced acute lung injury. INTERVENTIONS: Acute lung injury was induced in C57BL/6 mice by the intraperitoneal administration of endotoxin. The effects of angiopoietin-1, expressed from a nonreplicating E1a-deleted adenovirus containing the angiopoietin-1 complementary DNA (AdAng1), on survival and lung injury were evaluated. An E1a-deleted adenovirus that does not contain a transgene (Ad312) and phosphate-buffered saline were used as controls. MEASUREMENTS AND MAIN RESULTS: Angiopoietin-1 protein was detected by immunoblotting in the serum of mice that received an intraperitoneal injection of AdAng1 but not in mice that received the control virus Ad312. When compared with control groups, mice that received AdAng1 5 days before endotoxin administration had improved survival and significantly less protein leakage from the circulation into the lungs, as detected by quantitative spectrophotometric measurements of Evans blue dye. Furthermore, when compared with controls, histopathology and immunostaining of lungs against CD31 and smooth muscle actin suggested preservation of vascular integrity and decreased tissue damage in mice pretreated with AdAng1. When endotoxin administration preceded infection with AdAng1 by 3 hrs, no benefit was observed. CONCLUSIONS: These data show that adenoviral mediated expression of angiopoietin-1 can protect against the development of lung capillary protein leak and decrease the mortality induced by endotoxin. However, the timing of AdAng1 administration in relation to the onset of lung injury may be critical