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Supplementary Tables S1-S2.

Purpose: Unlike platelet-derived growth factor-B (PDGF-B), the role of PDGF-D in tumor progression or treatment is largely unknown. To this end, we determined the role of PDGF-D in breast cancer progression, metastasis, and response to chemotherapy.Experimental Design: We first examined PDGF-D expression in human breast carcinomas by immunohistochemical (IHC) staining. To mimic high PDGF-D expressing tumors, we stably transfected the breast cancer cell lines MDA-MB-231 and 4T1 with pdgf-d cDNA, and implanted these tumor cells orthtopically into nude mice. We monitored tumor growth by caliper measurement and bioluminescence imaging. We also used short hairpin RNA interference (shRNAi) and imatinib to block PDGF-D/PDGFRβ signaling. Finally, we studied the effect of PDGF-D on doxorubicin delivery and efficacy.Results: Human breast cancers express high levels of PDGF-D. Overexpression of PDGF-D promoted tumor growth and lymph node metastasis through increased proliferation, decreased apoptosis, and induction of CXCR4 expression. Blockade of CXCR4 signaling abolished PDGF-D–induced lymph node metastasis. Furthermore, overexpression of PDGF-D increased perivascular cell coverage and normalized tumor blood vessels. As a result, PDGF-D overexpression facilitated tissue penetration of doxorubicin and enhanced its treatment efficacy.Conclusions: PDGF-D is highly expressed in human breast cancer and facilitates tumor growth and lymph node metastasis, making it a potential target in breast cancer. At the same time, PDGF-D increases drug delivery and hence improves the efficacy of chemotherapy through vessel normalization. Therefore, judicious use of PDGF-D/PDGFRβ blockers would be necessary to minimize the adverse effects on concomitantly administered cytotoxic therapies. Clin Cancer Res; 17(11); 3638–48. ©2011 AACR.

Systemic sclerosis (SSc) is a fibrotic disease that initiates in the skin and progresses to internal organs, leading to a poor prognosis. Unraveling the etiology of a chronic, multifactorial disease such as SSc has been aided by various animal models that recapitulate certain aspects of the human pathology. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce many clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin (Spondin-2) in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing early inflammatory cytokine production and collagen secretion in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Fibrosis is a result of chronically activated fibroblasts leading to the overproduction of extracellular matrix (ECM), causing tissue hardening and loss of organ function. Systemic sclerosis (SSc) is a fibrotic skin disease marked by inflammation, autoimmunity and vasculopathy along with progressive fibrosis of the skin and internal organs. A major bottleneck in understanding the etiology of SSc has been the lack of a holistic animal model that can mimic the human SSc disease. We found that the transcription factor Snail is overexpressed in the epidermis of SSc patients and a transgenic mouse recapitulating this expression pattern is sufficient to induce hallmark clinical features of the human disease. Using this mouse model as a discovery platform, we have uncovered a critical role for the matricellular protein Mindin in fibrogenesis. Mindin is produced by Snail transgenic skin keratinocytes and aids fibrogenesis by inducing inflammatory cytokine and collagen production in resident dermal fibroblasts. Given the dispensability of Mindin in normal tissue physiology, targeting this protein holds promise as an effective therapy for fibrosis.

Fibrosis is a prevalent pathological condition arising from the chronic activation of fibroblasts. This activation results from the extensive intercellular crosstalk mediated by both soluble factors and direct cell-cell connections. Prominent among these are the interactions of fibroblasts with immune cells, in which the fibroblast-mast cell connection, although acknowledged, is relatively unexplored. We have used a Tg mouse model of skin fibrosis, based on expression of the transcription factor Snail in the epidermis, to probe the mechanisms regulating mast cell activity and the contribution of these cells to this pathology. We have discovered that Snail-expressing keratinocytes secrete plasminogen activator inhibitor type 1 (PAI1), which functions as a chemotactic factor to increase mast cell infiltration into the skin. Moreover, we have determined that PAI1 upregulates intercellular adhesion molecule type 1 (ICAM1) expression on dermal fibroblasts, rendering them competent to bind to mast cells. This heterotypic cell-cell adhesion, also observed in the skin fibrotic disorder scleroderma, culminates in the reciprocal activation of both mast cells and fibroblasts, leading to the cascade of events that promote fibrogenesis. Thus, we have identified roles for PAI1 in the multifactorial program of fibrogenesis that expand its functional repertoire beyond its canonical role in plasmin-dependent processes.

Adeno-associated virus (AAV)-mediated gene therapy holds great promise if challenges related to vector neutralization by pre-existing antibodies are circumvented. The use of autologous or allogeneic cells to shield the vector might offer the possibility of successful gene transfer in such a situation. In the present study, we evaluated the feasibility of AAV-transduced mesenchymal stromal cells (MSCs) as a vehicle for hepatic gene transfer in a murine liver injury model. In our initial studies to determine the most suitable vector, we observed that AAV1 (91%) and AAV6 (72%) serotypes are highly efficient in transducing MSCs. Subsequently, we generated a transient liver injury model to analyse the efficacy of MSCs homing to the liver, as well as their hepatic gene transfer efficiency; our data show that administration of acetaminophen (500 mg/kg) served as a cue for the homing of MSCs to the liver. Furthermore, sex-mismatched transplantation of AAV1-infected MSCs demonstrated a 3.5-fold (day 7) and 2.2-fold (day 28) higher hepatic gene transfer efficiency. To further corroborate this, we estimated the donor cell Y chromosome copies in the liver of recipient female mice. Our data revealed a 12.7-fold increase in average genome copies of male MSCs in the livers of recipient mice with injury compared to control, 60 days after transplantation. However, in vivo administration of AAV-transduced MSCs in the presence of neutralization antibodies (intravenous immunoglobulin, IVIG) was not beneficial. This is possibly due to the clearance of transplanted MSCs by circulating IVIG and underscores the need to develop suitable in vivo models to study such a mode of gene transfer. Copyright © 2015 John Wiley & Sons, Ltd.

Subcutaneous xenografts have been used for decades to study hepatocellular carcinoma (HCC). These models do not reproduce the specific pathophysiological features of HCCs, which occur in cirrhotic livers that show pronounced necroinflammation, abnormal angiogenesis and extensive fibrosis. As these features are crucial for studying the role of the pathologic host microenvironment in tumor initiation, progression and treatment response, alternative HCC models are desirable. Here we describe a syngeneic orthotopic HCC model in immunocompetent mice with liver cirrhosis induced by carbon tetrachloride (CCl4) that recapitulates key features of human HCC. Induction of substantial hepatic fibrosis requires 12 weeks of CCl4 administration. Intrahepatic implantation of mouse HCC cell lines requires 30 min per mouse. Tumor growth varies by tumor cell line and mouse strain used. Alternatively, tumors can be induced in a genetically engineered mouse model. In this setting, CCl4 is administered for 12 weeks after tail-vein injection of Cre-expressing adenovirus (adeno-Cre) in Stk4(-/-)Stk3(F/-) (also known as Mst1(-/-)Mst2(F/-); F indicates a floxed allele) mice, and it results in the development of HCC tumors (hepatocarcinogenesis) concomitantly with liver cirrhosis.

Key Clinical Message Ichthyosis prematurity syndrome (IPS) is reported mainly from Scandinavia where most of the cases are homozygous or compound heterozygous for the nonsense mutation c.504C>A (p.Cys168*) in exon3 indicating a common ancestor for this mutation. The occurrence of IPS in an Indian patient suggests that it is more widespread than previously reported.

Sorafenib—a broad kinase inhibitor—is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia—and its consequences on treatment resistance—remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1α) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen–positive (Gr-1+) myeloid cell infiltration. The SDF-1α/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1α expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1+ myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis—despite persistently elevated hypoxia—in part by reducing Gr-1+ myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. Conclusion: Blocking SDF-1α/CXCR4 or Gr-1+ myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment. (Hepatology 2014;59:1435-1447)

Unlike blood vessels in healthy tissue, the solid tumor microvasculature is dysfunctional and immature. Tumor vessels harbor structural and functional instability, which facilitates several distinct stages of tumor progression, including early sprouting angiogenesis, metastatic cell extravasation into distant organs, and the development of established tumor hypoxia, which fuels metastasis and impairs the efficacy of antitumor therapies (1–9). The endothelial cell (EC) receptor tyrosine kinase Tie-2/Tek is a critical regulator of vascular maturity. Tie-2 activation by its agonistic ligand angiopoietin 1 (Ang-1) increases perivascular cell (PVC) coverage, tightens EC junctions, reduces permeability, and increases vessel diameter (10–12). Angiopoeitin-2 (Ang-2) antagonizes the effects of Ang-1 in a context-dependent manner (6). Recent studies have shown a deleterious role for Ang-2 in mediating tumor vessel abnormalities and metastasis (13,14), suggesting that Tie-2 deactivation by Ang-2 in part mediates the unstable vessel phenotype seen within tumors. Current strategies targeting the Ang/Tie-2 axis in tumors have focused primarily on direct targeting of the angiopoietins, but such studies have not directly examined complex context-dependent interactions between Ang-1 and Ang-2 or the effects of angiopoietin blockade on Tie-2 activity (15–18). There are currently 10 agents targeting the Ang/Tie-2 axis under investigation in clinical trials (19). Vascular endothelial protein tyrosine phosphatase (VE-PTP) is an EC-specific receptor tyrosine phosphatase that dephosphorylates and consequently inactivates Tie-2 (20). The role of VE-PTP in the tumor vasculature is unknown. Recently, AKB-9778 has been developed as a novel, potent, and selective inhibitor of VE-PTP through structural modifications of 1,2,3,4-tetrahydroisoquinolinyl sulfamic acids (21). We hypothesized that pharmacological VE-PTP inhibition would activate Tie-2 signaling in ECs independent of Ang-1/Ang-2 ligands, stabilizing tumor vessels and mitigating the negative consequences of tumor vessel instability (20).

Efficient generation of competent vasculogenic cells is a critical challenge of human induced pluripotent stem (hiPS) cell-based regenerative medicine. Biologically relevant systems to assess functionality of the engineered vessels in vivo are equally important for such development. Here, we report a unique approach for the derivation of endothelial precursor cells from hiPS cells using a triple combination of selection markers—CD34, neuropilin 1, and human kinase insert domain-containing receptor—and an efficient 2D culture system for hiPS cell-derived endothelial precursor cell expansion. With these methods, we successfully generated endothelial cells (ECs) from hiPS cells obtained from healthy donors and formed stable functional blood vessels in vivo, lasting for 280 d in mice. In addition, we developed an approach to generate mesenchymal precursor cells (MPCs) from hiPS cells in parallel. Moreover, we successfully generated functional blood vessels in vivo using these ECs and MPCs derived from the same hiPS cell line. These data provide proof of the principle that autologous hiPS cell-derived vascular precursors can be used for in vivo applications, once safety and immunological issues of hiPS-based cellular therapy have been resolved. Additionally, the durability of hiPS-derived blood vessels in vivo demonstrates a potential translation of this approach in long-term vascularization for tissue engineering and treatment of vascular diseases. Of note, we have also successfully generated ECs and MPCs from type 1 diabetic patient-derived hiPS cell lines and use them to generate blood vessels in vivo, which is an important milestone toward clinical translation of this approach.

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T -> alanine A] or 9 K -> arginine R] single mutant or 2 double K -> R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T -> A and 7 K -> R vectors were significantly higher (similar to 63-90%) than the AAV2-WT vectors (similar to 30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated similar to 8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Purpose: Unlike platelet-derived growth factor-B (PDGF-B), the role of PDGF-D in tumor progression or treatment is largely unknown. To this end, we determined the role of PDGF-D in breast cancer progression, metastasis, and response to chemotherapy. Experimental Design: We first examined PDGF-D expression in human breast carcinomas by immunohistochemical (IHC) staining. To mimic high PDGF-D expressing tumors, we stably transfected the breast cancer cell lines MDA-MB-231 and 4T1 with pdgf-d cDNA, and implanted these tumor cells orthtopically into nude mice. We monitored tumor growth by caliper measurement and bioluminescence imaging. We also used short hairpin RNA interference (shRNAi) and imatinib to block PDGF-D/PDGFRβ signaling. Finally, we studied the effect of PDGF-D on doxorubicin delivery and efficacy. Results: Human breast cancers express high levels of PDGF-D. Overexpression of PDGF-D promoted tumor growth and lymph node metastasis through increased proliferation, decreased apoptosis, and induction of CXCR4 expression. Blockade of CXCR4 signaling abolished PDGF-D–induced lymph node metastasis. Furthermore, overexpression of PDGF-D increased perivascular cell coverage and normalized tumor blood vessels. As a result, PDGF-D overexpression facilitated tissue penetration of doxorubicin and enhanced its treatment efficacy. Conclusions: PDGF-D is highly expressed in human breast cancer and facilitates tumor growth and lymph node metastasis, making it a potential target in breast cancer. At the same time, PDGF-D increases drug delivery and hence improves the efficacy of chemotherapy through vessel normalization. Therefore, judicious use of PDGF-D/PDGFRβ blockers would be necessary to minimize the adverse effects on concomitantly administered cytotoxic therapies. Clin Cancer Res; 17(11); 3638–48. ©2011 AACR .

Clinical studies converge on the observation that circulating cytokines are elevated in most cancer patients by anti–vascular endothelial growth factor (VEGF) therapy. However, the source of these molecules and their relevance in tumor escape remain unknown. We examined the gene expression profiles of cancer cells and tumor-associated macrophages in tumor biopsies before and 12 days after monotherapy with the anti-VEGF antibody bevacizumab in patients with rectal carcinoma. Bevacizumab up-regulated stromal cell–derived factor 1α (SDF1α), its receptor CXCR4, and CXCL6, and down-regulated PlGF, Ang1, and Ang2 in cancer cells. In addition, bevacizumab decreased Ang1 and induced neuropilin 1 (NRP1) expression in tumor-associated macrophages. Higher SDF1α plasma levels during bevacizumab treatment significantly associated with distant metastasis at three years. These data show that VEGF blockade up-regulates inflammatory pathways and NRP1, which should be evaluated as potential targets for improving anti-VEGF therapy. [Cancer Res 2009;69(20):7905–10]

Fibrosis is a pervasive disease in which the excessive deposition of extracellular matrix (ECM) compromises tissue function. Although the underlying mechanisms are mostly unknown, matrix stiffness is increasingly appreciated as a contributor to fibrosis rather than merely a manifestation of the disease. Here we show that the loss of Fibulin-5, an elastic fibre component, not only decreases tissue stiffness, but also diminishes the inflammatory response and abrogates the fibrotic phenotype in a mouse model of cutaneous fibrosis. Increasing matrix stiffness raises the inflammatory response above a threshold level, independent of TGF-β, to stimulate further ECM secretion from fibroblasts and advance the progression of fibrosis. These results suggest that Fibulin-5 may be a therapeutic target to short-circuit this profibrotic feedback loop., Stiffness in the extracellular matrix is thought to contribute to pathological cutaneous fibrosis. Here, the authors identify the elastic fibre protein Fibulin-5 as a link and potential therapeutic target mediating the transition of cutaneous stiffening to fibrosis.

The discovery of human induced pluripotent stem cells (hiPSCs) might pave the way toward a long-sought solution for obtaining sufficient numbers of autologous cells for tissue engineering. Several methods exist for generating endothelial cells or perivascular cells from hiPSCs in vitro for use in the building of vascular tissue. We discuss current developments in the generation of vascular progenitor cells from hiPSCs and the assessment of their functional capacity in vivo, opportunities and challenges for the clinical translation of engineered vascular tissue, and modeling of vascular diseases using hiPSC-derived vascular progenitor cells.

Adeno-associated virus (AAV)-mediated gene therapy holds great promise if challenges related to vector neutralization by pre-existing antibodies are circumvented. The use of autologous or allogeneic cells to shield the vector might offer the possibility of successful gene transfer in such a situation. In the present study, we evaluated the feasibility of AAV-transduced mesenchymal stromal cells (MSCs) as a vehicle for hepatic gene transfer in a murine liver injury model. In our initial studies to determine the most suitable vector, we observed that AAV1 (91%) and AAV6 (72%) serotypes are highly efficient in transducing MSCs. Subsequently, we generated a transient liver injury model to analyse the efficacy of MSCs homing to the liver, as well as their hepatic gene transfer efficiency; our data show that administration of acetaminophen (500 mg/kg) served as a cue for the homing of MSCs to the liver. Furthermore, sex-mismatched transplantation of AAV1-infected MSCs demonstrated a 3.5-fold (day 7) and 2.2-fold (day 28) higher hepatic gene transfer efficiency. To further corroborate this, we estimated the donor cell Y chromosome copies in the liver of recipient female mice. Our data revealed a 12.7-fold increase in average genome copies of male MSCs in the livers of recipient mice with injury compared to control, 60 days after transplantation. However, in vivo administration of AAV-transduced MSCs in the presence of neutralization antibodies (intravenous immunoglobulin, IVIG) was not beneficial. This is possibly due to the clearance of transplanted MSCs by circulating IVIG and underscores the need to develop suitable in vivo models to study such a mode of gene transfer. Copyright © 2015 John WileySons, Ltd.

Understanding the association between the intrauterine hyperglycemic milieu and the development of adult diabetic vasculopathy is of particular relevance in India, where diabetes and vascular disease are prevalent. The gestational diabetes mellitus placenta is a valuable tool to examine blood vessels that have been exposed to hyperglycemic cues. We report an interesting observation in a cohort of gestational diabetes mellitus foetal placental vasculature from South India. Transmission electron microscopy demonstrated pericyte detachment and pericyte ghost cells reminiscent of adult type 2 diabetic retinopathy, in gestational diabetes mellitus foetal placental blood vessels that were not observed in non-gestational diabetes mellitus placentas ( p ≤0.001). Endothelial cell irregularity was observed in 76% gestational diabetes mellitus foetal blood vessels as compared with 10.4% non-gestational diabetes mellitus placental vasculature ( p ≤0.001). Other abnormalities noted in gestational diabetes mellitus placenta included mitochondrial abnormalities, increased micro vessel density and thickening of basement membranes. These results suggest that adult type 2 diabetic vasculopathy has developmental origins in utero.

Despite significant advancements with recombinant AAV2 or AAV8 vectors for liver directed gene therapy in humans, it is well-recognized that host and vector-related immune challenges need to be overcome for long-term gene transfer. To overcome these limitations, alternate AAV serotypes (1–10) are being rigorously evaluated. AAV5 is the most divergent (55% similarity vs. other serotypes) and like AAV1 vector is known to transduce liver efficiently. AAV1 and AAV5 vectors are also immunologically distinct by virtue of their low seroprevalence and minimal cross reactivity against pre-existing AAV2 neutralizing antibodies. Here, we demonstrate that targeted bio-engineering of these vectors, augment their gene expression in murine hepatocytes in vivo (up to 16-fold). These studies demonstrate the feasibility of the use of these novel AAV1 and AAV5 vectors for potential gene therapy of diseases like hemophilia.

Recombinant adeno-associated virus vectors based on serotype 8 (AAV8) have shown significant promise for liver-directed gene therapy. However, to overcome the vector dose dependent immunotoxicity seen with AAV8 vectors, it is important to develop better AAV8 vectors that provide enhanced gene expression at significantly low vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal machinery, we modified specific serine/threonine kinase or ubiquitination targets on the AAV8 capsid to augment its transduction efficiency. Point mutations at specific serine (S)/threonine (T)/lysine (K) residues were introduced in the AAV8 capsid at the positions equivalent to that of the effective AAV2 mutants, generated successfully earlier. Extensive structure analysis was carried out subsequently to evaluate the structural equivalence between the two serotypes. scAAV8 vectors with the wild-type (WT) and each one of the S/T -> Alanine (A) or K-Arginine (R) mutant capsids were evaluated for their liver transduction efficiency in C57BL/6 mice in vivo. Two of the AAV8-S -> A mutants (S279A and S671A), and a K137R mutant vector, demonstrated significantly higher enhanced green fluorescent protein (EGFP) transcript levels (similar to 9- to 46-fold) in the liver compared to animals that received WT-AAV8 vectors alone. The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response, and a concomitant two-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (106 vs. 7.7 vector copies/mouse diploid genome) when compared to WT-AAV8 vectors. To further study the utility of the K137R-AAV8 mutant in therapeutic gene transfer, we delivered human coagulation factor IX (h. FIX) under the control of liver-specific promoters (LP1 or hAAT) into C57BL/6 mice. The circulating levels of h. FIX: Ag were higher in all the K137R-AAV8 treated groups up to 8 weeks post-hepatic gene transfer. These studies demonstrate the feasibility of the use of this novel AAV8 vectors for potential gene therapy of hemophilia B.

Primary tracheal tumours are rare and less frequently observed than bronchial tumours. Primary neurogenic tumours of the trachea as schwannomas or neurilemmomas are extremely uncommon. We report a tracheal schwannoma in a female patient who presented with breathlessness and wheeze, and she was being treated for asthma. Flexible bronchoscopy revealed a large pedunculated tracheal mass and biopsy confirmed schwannoma. She was treated with laser ablation with partial reduction of the tumour. Subsequently, she was lost to follow-up, although resection of the tumour with tracheal reconstruction was planned.

Two percent of all malignant pancreatic tumors are metastases from other primaries, with small cell lung cancer, colorectal cancer, breast cancer and hematological neoplasms being the commonest. Renal cell carcinoma (RCC) metastasizing to the pancreas is rare and occurs in 2.8% of patients with metastatic RCC. However, RCC is the most common primary leading to solitary pancreatic metastasis. Metastases often present many years after nephrectomy for primary RCC (median time of 8 years) and should therefore be looked for on surveillance or when patients present with upper abdominal symptoms. Complete surgical resection when possible offers the best chance for cure.

This is a case report of a 29 year old woman who presented with painless and progressive obstructive jaundice. Imaging investigations of the abdomen revealed a tumour of the common bile duct. She was treated by complete excision of the bile duct and hepaticojejunostomy. The histopathology report of the tumour read as benign schwannoma.

Mast cell disease (MCD) is a clonal disorder of the mast cell and its precursor cells. Cardiac surgery in MCD is rarely described. We report an unusual case of a 14-year-old girl who was admitted for atrial septal defect closure and incidentally found to have hepatosplenomegaly with lymphadenopathy who underwent a successful open-heart surgery, after a diagnosis of MCD.

Retroperitoneal soft tissue sarcomas are difficult to treat because the retroperitoneal organs and great vessels are often involved by the time the patients come to a surgeon. We present the case of a 48 year old woman with a retroperitoneal leiomyosarcoma that had infiltrated the IVC and the renal veins.

Abstract 2045 Recombinant adeno-associated virus vectors based on serotype (AAV)-8 have shown significant promise for liver directed gene therapy of hemophilia B. However, in a recent clinical trial, two patients who received highest dose (2×1012 vg/kg) of the self-complementary (sc)AAV8 vector developed capsid specific T cells that required glucocorticoid therapy to attenuate this response [Nathwani et al, New Eng J Med, 2011]. Thus, the theme of AAV vector dose dependent immunotoxicity seen with AAV2 vectors earlier seem to re-emerge with AAV8 vectors as well. It is therefore important to develop novel AAV8 vectors that provide enhanced gene expression at significantly less vector doses. Since it is known that AAV vectors during intracellular trafficking are targeted for destruction in the cytoplasm by the host-cellular kinase/ubiquitination/proteasomal degradation machinery, we modified specific serine/threonine kinase or ubiquitination targets on AAV8 capsid to improve its transduction efficiency. To test this, point mutations at specific serine (S), threonine (T) or lysine (K) residues were generated on AAV8 capsid. scAAV8-EGFP vectors containing the wild-type (WT) and each one of the 5 S/T/K-mutant capsids were evaluated for their liver transduction efficiency at a dose of 5 × 1010 vgs/ animal in C57BL/6 mice in vivo. Two of the AAV8-S>A mutants (S279A and S501A) and a K137R mutant vector, demonstrated significantly higher EGFP expression (3.6 to 12.5 fold) in the liver compared to animals that received WT-AAV8 vectors alone (Figure 1). The best performing AAV8 mutant (K137R) vector also had significantly reduced ubiquitination of the viral capsid, reduced activation of markers of innate immune response [interleukin (IL)-6, IL-12, tumor necrosis factor α, Kupffer cells (KC) and innate immune responsive toll like receptors (TLR)-9] with a concomitant 2-fold reduction in the levels of neutralizing antibody formation in comparison to WT-AAV8 vectors. Vector bio-distribution studies also revealed that the K137R mutant had a significantly higher and preferential transduction of the liver (22 fold), lungs (9.7 fold) and muscle (8.4 fold) tissue when compared to WT-AAV8 vectors. Further on-going studies with the optimal mutant scAAV8 vector expressing human coagulation factor IX in murine models of hemophilia B, will demonstrate the feasibility of the use of these novel vectors for potential gene therapy of hemophilia B. Figure 1: Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Figure 1:. Efficacy of novel AAV8 S>A and K>R vectors (A) EGFP expression in hepatocytes 4 weeks post administration of AAV8 vectors in C57BL/6 mice, (B) Neutralization antibody levels against AAV8 vectors (C) Ubiquitination levels of K137R-AAV8 compared to the WT-AAV8 vector. Disclosures: No relevant conflicts of interest to declare.

Oral lesions of tuberculosis though uncommon, are seen in both the primary and secondary stages of the disease. In secondary tuberculosis, the oral manifestations may be accompanied by lesions in the lungs, lymph nodes, or in any other part of the body and can be detected by a systemic examination. Primary oral tuberculosis may present as a diagnostic challenge for the clinician. Here we report two patients with primary tuberculosis in the oral cavity who presented to the dental department, were diagnosed and referred for medical management.