p53-independent NOXA induction overcomes apoptotic resistance of malignant melanomas

Once melanoma metastasizes, no effective treatment modalities prolong survival in most patients. This notorious refractoriness to therapy challenges investigators to identify agents that overcome melanoma resistance to apoptosis. Whereas many survival pathways contribute to the death-defying phenotype in melanoma, a defect in apoptotic machinery previously highlighted inactivation of Apaf-1, an apoptosome component engaged after mitochondrial damage. During studies involving Notch signaling in melanoma, we observed a gamma-secretase tripeptide inhibitor (GSI; z-Leu-Leu-Nle-CHO), selected from a group of compounds originally used in Alzheimer's disease, induced apoptosis in nine of nine melanoma lines. GSI only induced G2-M growth arrest (but not killing) in five of five normal melanocyte cultures tested. Effective killing of melanoma cells by GSI involved new protein synthesis and a mitochondrial-based pathway mediated by up-regulation of BH3-only members (Bim and NOXA). p53 activation was not necessary for up-regulation of NOXA in melanoma cells. Blocking GSI-induced NOXA using an antisense (but not control) oligonucleotide significantly reduced the apoptotic response. GSI also killed melanoma cell lines with low Apaf-1 levels. We conclude that GSI is highly effective in killing melanoma cells while sparing normal melanocytes. Direct enhancement of BH3-only proteins executes an apoptotic program overcoming resistance of this lethal tumor. Identification of a p53-independent apoptotic pathway in melanoma cells, including cells with low Apaf-1, bypasses an impediment to current cytotoxic therapy and provides new targets for future therapeutic trials involving chemoresistant tumors.

Notch and NOXA-related pathways in melanoma cells

Notch receptor-mediated intracellular events represent an ancient cell signaling system, and alterations in Notch expression are associated with various malignancies in which Notch may function as an oncogene or less commonly as a tumor suppressor. Notch signaling regulates cell fate decisions in the epidermis, including influencing stem cell dynamics and growth/differentiation control of cells in skin. Because of increasing evidence that the Notch signaling network is deregulated in human malignancies, Notch receptors have become attractive targets for selective killing of malignant cells. Compared with proliferating normal human melanocytes, melanoma cell lines are characterized by markedly enhanced levels of activated Notch-1 receptor. By using a small molecule gamma-secretase inhibitor (GSI) consisting of a tripeptide aldehyde, N-benzyloxycarbonyl-Leu-Leu-Nle-CHO, which can block processing and activation of all four different Notch receptors, we identified a specific apoptotic vulnerability in melanoma cells. GSI triggers apoptosis in melanoma cells, but only G2/M growth arrest in melanocytes without subsequent cell death. Moreover, GSI treatment induced a pro-apoptotic BH3-only protein, NOXA, in melanoma cells but not in normal melanocytes. The use of GSI to induce NOXA induction overcomes the apoptotic resistance of melanoma cells, which commonly express numerous cell survival proteins such as Mcl-1, Bcl-2, and survivin. Taken together, these results highlight the concept of synthetic lethality in which exposure to GSI, in combination with melanoma cells overexpressing activated Notch receptors, has lethal consequences, producing selective killing of melanoma cells, while sparing normal melanocytes. By identifying signaling pathways that contribute to the transformation of melanoma cells (e.g. Notch signaling), and anti-cancer agents that achieve tumor selectivity (e.g., GSI-induced NOXA), this experimental approach provides a useful framework for future therapeutic strategies in cutaneous oncology.

Polycaprolactone-based scaffold plus recombinant human bone morphogenetic protein (rhBMP-2) in an ovine model of anterior spinal fusion

Synthetic scaffolds combined with growth factors have the potential to replace allograft or autograft as a graft material for spinal interbody fusion. Such tissue engineering approaches may be useful in Adolescent Idiopathic Scoliosis (AIS) surgery, however there are no studies to date examining the use of such biodegradable implants in combination with biologics in a thoracic spine model. This in vivo study examines the use of biodegradable polycaprolactone (PCL) based scaffolds with rhBMP-2 as a bone graft substitute in a sheep thoracic fusion model, where an anterior approach is used to simulate minimally invasive surgical deformity correction in the setting of AIS.

An introduction to biologics

An introduction to biologics 23.1 Introduction: Principles of biologics and their use as medicines 23.2 Protein biologics used as drugs 23.2.1 Proteins that function through enzymatic or regulatory activity. 23.2.1.1 Biologics as replacement of a deficient or abnormal protein. 23.2.1.2 Proteins that augment an existing biological process. 23.2.1.3 Proteins that provide a novel function or activity. 23.2.2. Proteins that function through specific targeting activity. 23.2.2.1. Monoclonal antibody nomenclature. 23.2.2.2. Naked monoclonal antibodies. 23.2.2.3. Conjugated monoclonal antibodies. 23.2.3. Recombinant protein vaccines.

Genetic analysis of maize streak virus isolates from Uganda reveals widespread distribution of a recombinant variant

Maize streak virus (MSV) contributes significantly to the problem of extremely low African maize yields. Whilst a diverse range of MSV and MSV-like viruses are endemic in sub-Saharan Africa and neighbouring islands, only a single group of maize-adapted variants - MSV subtypes A1 -A6 - causes severe enough disease in maize to influence yields substantially. In order to assist in designing effective strategies to control MSV in maize, a large survey covering 155 locations was conducted to assess the diversity, distribution and genetic characteristics of the Ugandan MSV-A population. PCR-restriction fragment-length polymorphism analyses of 391 virus isolates identified 49 genetic variants. Sixty-two full-genome sequences were determined, 52 of which were detectably recombinant. All but two recombinants contained predominantly MSV-A1-like sequences. Of the ten distinct recombination events observed, seven involved inter-MSV-A subtype recombination and three involved intra-MSV-A1 recombination. One of the intra-MSV-A1 recombinants, designated MSV-A1 UgIII, accounted for >60% of all MSV infections sampled throughout Uganda. Although recombination may be an important factor in the emergence of novel geminivirus variants, it is demonstrated that its characteristics in MSV are quite different from those observed in related African cassava-infecting geminivirus species. © 2007 SGM.

Polycaprolactone-based scaffold plus recombinant human bone morphogenic protein rhBMP-2) in a sheep thoracic spine fusion model

Adolescent idiopathic scoliosis is a complex three dimensional deformity affecting 2-3% of the general population. The resulting spinal deformity consists of coronal curvature, hypokyphosis of the thoracic spine and vertebral rotation in the axial plane with posterior elements turned into the curve concavity. The potential for curve progression is heightened during the adolescent growth spurt. Success of scoliosis deformity correction depends on solid bony fusion between adjacent vertebrae after the intervertebral (IV) discs have been surgically cleared and the disc spaces filled with graft material. Recently a bioactive and resorbable scaffold fabricated from medical grade polycaprolactone has been developed for bone regeneration at load bearing sites. Combined with rhBMP-2, this has been shown to be successful in acting as a bone graft substitute in a porcine lumbar interbody fusion model when compared to autologous bone graft alone. The study aimed to establish a large animal thoracic spine interbody fusion model, develop spine biodegradable scaffolds (PCL) in combination with biologics (rhBMP-2) and to establish a platform for research into spine tissue engineering constructs. Preliminary results demonstrate higher grades of radiologically evident bony fusion across all levels when comparing fusion scores between the 3 and 6 month postop groups at the PCL CaP coated scaffold level, which is observed to be a similar grade to autograft, while no fusion is seen at the scaffold only level. Results to date suggest that the combination of rhBMP-2 and scaffold engineering actively promotes bone formation, laying the basis of a viable tissue engineered constructs.

Bovine colostrum modulates cytokine production in human peripheral blood mononuclear cells stimulated with lipopolysaccharide and phytohemagglutinin

Bovine colostrum has been shown to influence the cytokine production of bovine leukocytes. However, it remains unknown whether processed bovine colostrum, a supplement popular among athletes to enhance immune function, is able to modulate cytokine secretion of human lymphocytes and monocytes. The aim of this investigation was to determine the influence of a commercially available bovine colostrum protein concentrate (CPC) to stimulate cytokine production by human peripheral blood mononuclear cells (PBMCs). Blood was sampled from four healthy male endurance athletes who had abstained from exercise for 48 h. PBMCs were separated and cultured with bovine CPC concentrations of 0 (control), 1.25, 2.5, and 5% with and without lipopolysaccharide (LPS) (3 microg/mL) and phytohemagglutinin (PHA) (2.5 microg/mL). Cell supernatants were collected at 6 and 24 h of culture for the determination of tumor necrosis factor (TNF), interferon (IFN)-gamma, interleukin (IL)-10, IL-6, IL-4, and IL-2 concentrations. Bovine CPC significantly stimulated the release of IFN-gamma, IL-10, and IL-2 (p < 0.03). The addition of LPS to PBMCs cocultured with bovine CPC significantly stimulated the release of IL-2 and inhibited the early release of TNF, IL-6, and IL-4 (p < 0.02). Phytohemagglutinin stimulation in combination with bovine CPC significantly increased the secretion of IL-10 and IL-2 at 6 h of culture and inhibited IFN-gamma and TNF (p < 0.05). This data show that a commercial bovine CPC is able to modulate in vitro cytokine production of human PBMCs. Alterations in cytokine secretion may be a potential mechanism for reported benefits associated with supplementation.

Biological performance of a polycaprolactone-based scaffold plus recombinant human morphogenetic protein-2 (rhBMP-2) in an ovine thoracic interbody fusion model

Introduction. We develop a sheep thoracic spine interbody fusion model to study the suitability of polycaprolactone-based scaffold and recombinant human bone morphogenetic protein-2 (rhBMP-2) as a bone graft substitute within the thoracic spine. The surgical approach is a mini- open thoracotomy with relevance to minimally invasive deformity correction surgery for adolescent idiopathic scoliosis. To date there are no studies examining the use of this biodegradable implant in combination with biologics in a sheep thoracic spine model. Methods. In the present study, six sheep underwent a 3-level (T6/7, T8/9 and T10/11) discectomy with randomly allocated implantation of a different graft substitute at each of the three levels; (i) calcium phosphate (CaP) coated polycaprolactone based scaffold plus 0.54µg rhBMP-2, (ii) CaP coated PCL- based scaffold alone or (iii) autograft (mulched rib head). Fusion was assessed at six months post-surgery. Results. Computed Tomographic scanning demonstrated higher fusion grades in the rhBMP-2 plus PCL- based scaffold group in comparison to either PCL-based scaffold alone or autograft. These results were supported by histological evaluations of the respective groups. Biomechanical testing revealed significantly higher stiffness for the rhBMP-2 plus PCL- based scaffold group in all loading directions in comparison to the other two groups. Conclusions. The results of this study demonstrate that rhBMP-2 plus PCL-based scaffold is a viable bone graft substitute, providing an optimal environment for thoracic interbody spinal fusion in a large animal model.