Shoshana Greenberger

Melanoma miRNA trafficking controls tumour primary niche formation

Melanoma originates in the epidermis and becomes metastatic after invasion into the dermis. Prior interactions between melanoma cells and dermis are poorly studied. Here, we show that melanoma cells directly affect the formation of the dermal tumour niche by microRNA trafficking before invasion. Melanocytes, cells of melanoma origin, are specialized in releasing pigment vesicles, termed melanosomes. In melanoma in situ, we found melanosome markers in distal fibroblasts before melanoma invasion. The melanosomes carry microRNAs into primary fibroblasts triggering changes, including increased proliferation, migration and pro-inflammatory gene expression, all known features of cancer-associated fibroblasts (CAFs). Specifically, melanosomal microRNA-211 directly targets IGF2R and leads to MAPK signalling activation, which reciprocally encourages melanoma growth. Melanosome release inhibitor prevented CAF formation. Since the first interaction of melanoma cells with blood vessels occurs in the dermis, our data suggest an opportunity to block melanoma invasion by preventing the formation of the dermal tumour niche.

The effectiveness of combined Chinese herbal medicine and acupuncture in the treatment of atopic dermatitis.

BACKGROUND Patients with atopic dermatitis increasingly use complementary medicine. OBJECTIVE The objective of this study was to assess the effectiveness of the combination of Chinese herbal medicine and acupuncture for the treatment of atopic dermatitis. METHODS Twenty (20) patients between the ages of 13 and 48 who had mild-to-severe atopic dermatitis were given a combined treatment of acupuncture and Chinese herbal medicine and were followed prospectively. The patients received acupuncture treatment twice a week and the Chinese herbal formula 3 times daily for a total of 12 weeks. Assessments were performed before treatment, and at weeks 3, 6, 9, and 12 of treatment. The primary outcomes were defined as the changes in the Eczema Area and Severity Index (EASI), Dermatology Life Quality Index (DLQI), and patient assessment of itch measured on a visual analogue scale (VAS). RESULTS After 12 weeks of treatment, an improvement in EASI was noted in 100% of patients, when compared with the baseline. The mean EASI fell from 4.99 to 1.81; the median percentage of decrease was 63.5%. Moreover, 78.8% of patients experienced a reduction in DLQI and VAS, as compared with the baseline. The mean DLQI decreased from 12.5 to 7.6 at the end of treatment, with 39.1% improvement. Mean VAS decreased from 6.8 to 3.7, with 44.7% improvement. No adverse effects were observed. CONCLUSIONS The results of this study suggest that the combination of acupuncture and Chinese herbal medicine have a beneficial effect on patients with atopic dermatitis and may offer better results than Chinese herbal medicine alone.

Endothelial-targeted gene transfer of hypoxia-inducible factor-1α augments ischemic neovascularization following systemic administration

Hypoxia-inducible factor-1α (HIF-1α) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1α targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1α molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1α administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.Hypoxia-inducible factor-1alpha (HIF-1alpha) is a key regulator of the response to low oxygen levels and has been used for therapeutic angiogenesis. Various routes of administration have been used for delivering genes to the ischemic region including the intramuscular (IM) and intraarterial routes. When compared with these delivery methods, the intravenous (IV) route confers many advantages, including less invasiveness and lower cost. However, its use is hampered by the fact that it does not result in specific and robust tissue expression of the genes. Our aim was to determine the feasibility, safety, and therapeutic efficacy of systemic administration of adenoviral-mediated HIF-1alpha targeted to the endothelium. Using confocal microscopy and biodistribution studies we demonstrated that a modified murine preproendothelin-1 promoter (PPE1-3x) can target gene expression specifically to endothelial cells within ischemic muscle following systemic IV administration in C57BL/6 mice. Accordingly, an adenovirus expressing a PPE1-3x-regulated stabilized HIF-1alpha molecule, further activated by constitutive activation of its C-transactivation domain (C-TAD), was created. Systemic tail-vein administration of this adenovirus in a mouse hindlimb ischemia model resulted in enhanced blood perfusion, improved clinical outcome, and increased capillary density without systemic toxicity, in contrast to the profound systemic side effects and lack of therapeutic efficacy following cytomegalovirus (CMV)-regulated HIF-1alpha administration. Collectively, these data suggest that transcriptionally controlled systemic proangiogenic gene therapy is feasible, safe, and efficacious.

Leishmania tropica in children: A retrospective study

BACKGROUND Limited data are available regarding topical and systemic therapies for Leishmania tropica in children. OBJECTIVE We sought to characterize the clinical presentation and evaluate the efficacy and safety of topical and systemic treatments in pediatric patients infected with L tropica. METHODS A retrospective study was performed on 47 children with L tropica cutaneous leishmaniasis. Treatments included topical or systemic therapy with liposomal amphotericin B or pentavalent antimony. RESULTS Seventy patients with L tropica cutaneous leishmaniasis were treated at our center between 2008 and 2012, of which 47 (67%) were children. The average age of the pediatric population was 8.8 years, and the face was the most common site of involvement (76%). The average number of lesions was 2.6. 24 children (51%) required systemic therapy. The patients were treated with 3 to 5 mg/kg/d of intravenous liposomal amphotericin B, and a response was observed in 83% of the patients within 3 months. LIMITATIONS This was a retrospective study. CONCLUSION The disease burden of L tropica in children is high, and because of facial involvement and a low response to topical therapies, systemic therapy is often required. In our experience, liposomal amphotericin B treatment in children is safe and effective and is required for a considerably shorter duration than treatment with pentavalent antimony.

Vascular Wall Maturation and Prolonged Angiogenic Effect by Endothelial-Specific Platelet-Derived Growth Factor Expression

Background: The implementation of angiogenic gene therapy at clinics is hindered by the transience of the therapeutic effect. Recruiting vascular wall smooth muscle cells, a process termed ‘maturation’, can stabilize newly formed vessels. Objective: To induce angiogenesis followed by vessel maturation in a murine ischemic limb model by endothelial cell-specific promoter regulated expression of vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB). Methods: We constructed adenoviral vectors containing angiogenic factors VEGF and PDGF-B regulated by a modified preproendothelin-1 (PPE-1-3x) promoter and investigated their angiogenic effect in a murine ischemic limb model. Results: VEGF gene therapy increased perfusion and the vessel density in the limb shortly after expression with PPE-1-3x promoter or cytomegalovirus (CMV) promoter vectors, but only PPE-1-3xVEGF treatment exhibited a sustained effect. Expression of PDGF-B by PPE-1-3x promoter resulted in morphological maturation of the vasculature and further increased the perfusion, while nonspecific expression of PDGF-B with CMV promoter had no therapeutic effect. Regulation of dual therapy with VEGF and PDGF-B by PPE-1-3x promoter resulted in an early-onset, sustained angiogenic effect accompanied by vessel maturation. Conclusions: Systemic gene therapy with the angiogenic factors VEGF and PDGF-B under angiogenic- endothelial cell-specific regulation was effective in inducing functionally and morphologically mature vasculature.

Methotrexate is an effective and safe adjuvant therapy for pemphigus vulgaris

BACKGROUND Pemphigus vulgaris (PV) is a chronic, autoimmune blistering disease. Most patients require long term therapy with systemic steroids as a first line of treatment. Immunosuppressive agents such as methotrexate (MTX) are administrated as second line therapy. Only a few reports have assessed MTX efficacy, with contradictory results. OBJECTIVE The aim of this study was to evaluate MTX as an adjuvant therapy in patients with PV. METHODS A retrospective study of 30 PV patients treated with MTX as an adjuvant therapy. Disease severity score and prednisone dosage served as assessing measures. RESULTS All patients were treated with 15 mg MTX per week. Of the 25 patients defined as severe or moderate disease at the beginning of treatment, 21 (84%) improved and downgraded their severity status at 6 months of treatment. In 21 patients (76.6%) we were able to reduce the prednisone dose. There was a significant improvement in the severity score (p=0.00001) and in prednisone dose (p=0.0001). Four patients (13%) suffered from mild side effects. CONCLUSION MTX treatment is safe and beneficial as a steroid-sparing agent in PV.

Specific induction of tumor neovasculature death by modified murine PPE-1 promoter armed with HSV-TK.

Background: One strategy to increase tissue specificity of gene therapy is to use promoters or enhancers. Objectives: (1) To enhance the selectivity of a murine preproendothelin-1 (PPE-1) promoter in tumor angiogenesis by using a positive endothelial transcription-binding element. (2) To test the specificity and efficiency of the modified PPE-1 promoter [PPE-1(3X)] in vitro and in vivo by using reporter genes, and the therapeutic gene herpes simplex virus-thymidine kinase (HSV-TK) in a mouse model of Lewis lung carcinoma (LLC). Results: The modified PPE-1 promoter specifically induced expression in the tumor angiogenic vascular bed with a 35-fold higher expression compared to the normal vasculare bed of the lung. Thus, when the HSV-TK gene controlled by the modified PPE-1 promoter was used systemically, it induced tumor-specific necrosis, apoptosis and mononuclear infiltrates, leading to massive destruction of the neovasculature of the pulmonary metastasis, which suppressed metastasis development. Conclusions: These results show that an adenoviral vector armed with HSV-TK controlled by the endothelial-selective murine PPE-1(3X) promoter is efficient and safe to target tumor neovasculature.

Antiangiogenic systemic gene therapy combined with doxorubicin administration induced caspase 8 and 9-mediated apoptosis in endothelial cells and an anti-metastasis effect.

Ad-PPE-Fas-c is an adenovector that expresses Fas-c under the control of the modified pre-proendothelin-1 (PPE-1) promoter. Fas-c is a chimeric death receptor containing the extracellular portion of tumour necrosis factor 1 receptor (TNFR1) and the transmembrane and intracellular portion of Fas. We recently demonstrated that Ad-PPE-Fas-c induced Fas-receptor-mediated endothelial cell apoptosis. Previously, doxorubicin was shown to enhance Fas-receptor clustering and the induction of its cascade. Therefore, the goal of this work was to test whether doxorubicin augments the capacity of Ad-PPE-Fas-c to induce endothelial cell apoptosis and to elucidate whether either the death-receptor-mediated apoptotic cascade or the mitochondria-associated apoptotic cascade is involved in the combined treatment effect. We found that a combined treatment of Ad-PPE-Fas-c and doxorubicin synergistically induced a reduction in endothelial cell viability and apoptosis. z-IETD-FMK, a caspase-8 inhibitor, and z-LEHD-FMK, a caspase-9 inhibitor, significantly decreased apoptosis induced by the combined treatment. Systemically administered combined therapy significantly reduced the lung metastases burden (70%) in mice as compared to each treatment alone. Thus, a combined treatment of Ad-PPE-Fas-c gene therapy and chemotherapy may be effective in the treatment of metastatic diseases and both the Fas cascade and the mitochondria-associated cascade are essential for this effect.

9-cis–Rich β-Carotene Powder of the Alga Dunaliella Reduces the Severity of Chronic Plaque Psoriasis: A Randomized, Double-Blind, Placebo-Controlled Clinical Trial

Background: Synthetic retinoids are one of the mainstay treatments of psoriasis. However, their use is occasionally limited by adverse effects, especially mucocutaneous, hepatic, and lipid profile toxicity. Thus, a search for retinoid metabolites that are both safe and active is essential. The alga Dunaliella bardawil is a natural source of the retinoid precursor 9-cis β-carotene that has a good adverse effect profile. Objective: To test the effect of the alga Dunaliella bardawil on psoriasis. Methods: Thirty-four adult patients with mild, chronic, plaque-type psoriasis were included in this monocentric, prospective, randomized, double-blinded pilot study. Patients received either capsules of the alga D. bardawil or starch powder capsules, as the placebo, for 12 weeks. The response to treatment was evaluated by changes in Psoriasis Area and Severity Index (PASI) and Dermatology Life Quality Index (DLQI) scores. Safety of the treatment was evaluated. Results: At the end of 6 weeks, the reduction in the mean PASI score was significantly higher in the Dunaliella group than in the placebo group (61.3% vs 34%, respectively, p = 0.002). The DLQI change did not reach significance (8.5% and 5.9% in the Dunaliella and in the control group, respectively, p = 0.9). We observed no significant change in the liver function tests or in the lipid profile. Conclusions: 9-cis β-carotene, in the form of D. bardawil, is an effective and safe treatment for patients with mild, chronic, plaque-type psoriasis. A larger study is warranted.

Systemic administration of radiation-potentiated anti-angiogenic gene therapy against primary and metastatic cancer based on transcriptionally controlled HSV-TK.

Transcription-targeted gene delivery directed against angiogenic endothelial cells is a new approach against advanced cancer. Moreover, the herpes simplex virus-thymidine kinase (HSV-TK) gene coupled with low dose radiotherapy is an efficient and externally controlled cytotoxic system. We have previously demonstrated enhanced endothelial-specific cell expression and killing using the modified murine pre-proendothelin-1 promoter (PPE1-3x) to direct adenoviral expression of a pro-apoptotic gene. The purpose of this study was to create an externally potentiated systemic antiangiogenic gene delivery system based on an adenoviral vector expressing HSV-TK under the regulation of PPE1-3X promoter combined with radiotherapy for eradicating metastatic cancer. Ad-PPE1-3x-TK induced endothelial-specific cell killing in-vitro upon introduction of the prodrug ganciclovir (GCV). BALB/c mice bearing a primary CT-26 colon carcinoma tumor showed tumor growth suppression and diminished tumor angiogenesis when the vector was administered intravenously, activated with GCV and potentiated with a single sub-therapeutic and non-toxic radiation dose. Moreover, intravenous administration of the vector, activated with GCV and potentiated with chest aimed radiation, to C57BL/6 mice bearing Lewis lung carcinoma metastases resulted in prolongation of mice survival. PPE1-3x-regulated HSV-TK expression was detected only in lung metastases in contrast to CMV-regulated expression. This novel system may benefit patients with metastatic disease.