Hybrid retroviral vector with MCK enhancers inserted in LTR for stable and specific expression of human factor IX in skeletal muscle

Background Retroviral vectors have been widely used to introduce foreign into various target cells in vitro, thus showing relatively high systemic delivery efficiency of various transgene products. The authors investigated the stability and efficiency of skeletal muscle-specific hybrid retroviral vectors in expression of human factor IX (FIX) in vitro and iv vivo.Methods FIX cDNA in LIXSN vector was replaced with a FIX minigene containing splicing donor and splicing acceptor sequence of first intron of human FIX gene. Two copies of muscle creatine kinase enhancer (MCK, Me2) were inserted in forward or reverse orientation at Nhel site of 3‘ long terminal repeat (LTR), resulting in two hybrid vectors, which were designated as LMe2lXm2SN (F) and LMe2lXm2SN(R), respectively. The vectors were tested in vitro and in vivo for stability and musclespecificity of factor IX expression with SCID mice.Results Muscle cells carrying vector with Me2 expressed significantly higher levels of FIX (up to 1800 ng/106.24h) than those without Me2, thus suggesting that Me2 could specifically increase expression level of FIX in muscle cells. Myoblasts transduced with LMe2lXm2SN(R) produced much less FIX in vivo in SCID mice than LMe2lXm2SN(F). One or two copies of Me2 sequence were deleted in myoblasts transduced with LMe2lXm2SN(R) without changing the orientation of Me2.Conclusions LTR inserted with MCK enhancers can specifically increase human FIX expression in skeletal muscle cells in vitro and in vivo, and MCK enhancer should be positioned in the same orientation as that of LTR promoter.     

The use of digitized endoscopic imaging of 5-ALA-induced PPIX fluorescence to detect and diagnose oral premalignant and malignant lesions in vivo

5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PPIX) fluorescence has shown an outstanding sensitivity for the assessment of oral lesions, but its application was hampered by low specificity due to the high false-positive rates. The purpose of our study was to explore the feasibility of quantifying PPIX fluorescence images to improve the diagnostic specificity for detecting early oral lesions in vivo. A digitized 5-ALA-mediated endoscopic imaging system was utilized to acquire PPIX fluorescence images from in vivo oral tissues. Forty-nine patients (118 biopsies) with known or suspected premalignant or malignant oral lesions were recruited for ALA-PPIX fluorescence endoscopic imaging. The red and blue channels of PPIX fluorescence images were digitized and stored for fluorescence quantification. The red-to-blue intensity ratios were calculated from the fluorescence images to correlate with histologic findings of the biopsies. The results showed that normal oral mucosa exhibited blue color of the back-scattered excitation light in the fluorescence images, whereas the suspicious lesions displayed bright reddish fluorescence. Applying the red-to-blue intensity ratio (I(R)/I(B)) as a diagnostic algorithm yielded a sensitivity of 92% and 98%, and specificity of 96% and 96%, for separating benign tissue from dysplasia, and cancer tissue, respectively, and a sensitivity and specificity of 98% and 92%, respectively, for differentiating cancer tissue from dysplasia in the oral cavity. Our study demonstrates that quantifying ALA-PPIX fluorescence endoscopic images associated with the red-to-blue intensity ratio as a diagnostic algorithm can provide good differentiation between the different stages of oral premalignancy and malignancy (p<0.0001, unpaired 2-sided Student's t-test), and thus has a potential to significantly improve the noninvasive diagnosis and evaluation of early oral neoplasia in vivo.     

Validation of a Tissue Microarray to Study Differential Protein Expression in Inflammatory and Non-Inflammatory Breast Cancer

AIMS: Inflammatory breast cancer (IBC) is an aggressive subtype of breast cancer with poor prognosis. The mechanisms responsible for the aggressive clinical evolution are incompletely understood. We constructed a tissue microarray (TMA) and validated its use in translational IBC research. Differential expression of proteins that might play a role in causing the IBC phenotype was studied. METHODS AND RESULTS: A TMA containing 34 IBC and 41 non-stage matched non-IBC tumours was constructed. Five core biopsies were taken for each IBC and three cores for each non-IBC tumour. The TMA was validated using three approaches: (1) the excellent concordance between immunohistochemical results of the initial pathological examination and the results obtained with the TMA for ER, PR and HER2/neu (kappa > 0.74); (2) the known differential expression between IBC and non-IBC for four bio-markers in IBC (ER, PR, p53 and HER2/neu) was confirmed ( p < 0.01); (3) the HER2/neu status using three different antibodies (CB11, TAB250 and HercepTest) was highly concordant (kappa > 0.75). Furthermore, the overexpression of E-Cadherin and RhoC GTPase in IBC ( p < 0.05) was confirmed. We did not find a differential expression pattern for carbonic anhydrase IX (CA IX) and EGFR. CONCLUSIONS: Using different approaches, we have validated the use of our TMA for studying differential protein expression in IBC and non-IBC. We confirm the overexpression of E-Cadherin and RhoC GTPase in IBC. The lack of differential expression for CA IX and EGFR might suggest the pathways are equally utilised in both types of breast cancer.     

Phototoxicity of exogenous protoporphyrin IX and delta-aminolevulinic acid in the photo hen's egg test

BACKGROUND: Oxygen, appropriate light sources, and special photosensitizers are necessary to induce photochemical damage in tumor cells via photodynamic therapy (PDT) delta-aminolevulinic acid (ALA) is increasingly used in PDT, because topical or systemic administration of ALA induces accumulation of endogenous porphyrins preferentially in neoplastic tissues. Subsequent radiation with light of approximately 630 nm leads to selective damage of tumor cells. PDT should optimally leave peritumoral tissues unaffected, but only few data are reported on the effects and the time course of ALA-induced porphyrins in tumor-free tissues. METHODS: Therefore, we studied the phototoxic effects of protoporphyrin IX (PP) and ALA-induced porphyrins in a recently established phototoxic model based on tumor-free tissue, the photo hen's egg test (PHET). RESULTS: Employing this test procedure, PP provoked strong phototoxic reactions when irradiated with Ultraviolet A immediately and up to 30 h after substance application. In contrast, ALA induced a significant phototoxic effect only if irradiated 24 h after application. CONCLUSION: Thus, we observed a delayed phototoxic effect of ALA in tumor-free tissue of the yolk sac (YS) blood vessel system. This delayed phototoxic response 24 h after ALA application is probably caused by endogenously synthesized porphyrins. In contrast, epithelial tumors show a maximum porphyrin accumulation 4-8 h after ALA application whereas in healthy human skin porphyrin synthesis is less intensive but prolonged with maximum levels 24-48 h after ALA application. Thus, ALA induced virtually the same delayed phototoxic effect in the tumor-free YS blood vessel tissue as in healthy human skin. These results show that the PHET is a useful model for the predictive preclinical risk assessment of exogenous or endogenous photosensitizers.     

Inhibitors in the Swedish population with severe haemophilia A and B: a 20-year survey

AIM: To survey the entire population (n = 116) afflicted with severe haemophilia A or B born in Sweden over a 20-y period (1980-1999), and to examine the epidemiological, genetic and clinical aspects of development of inhibitors to factors VIII and IX (FVIII/FIX). METHODS: One hundred of the subjects had haemophilia A and 16 had haemophilia B. All of these subjects had received prophylactic treatment and had a check-up of inhibitor status at least twice a year. Sixty-one were born between 1980 and 1989 and 55 between 1990 and 1999. RESULTS: Nineteen percent (19/100) of those with haemophilia A and 37% (6/16) with haemophilia B developed inhibitors at 12-18 mo of age, after exposure to FVIII/FIX concentrates for an average of 14 d in the case of haemophilia A and 16 d in haemophilia B. All patients with inhibitors carried mutations that impaired protein synthesis. The high incidence of FIX inhibitors may have been due to the large number of complete deletions (13%) in the Swedish haemophilia B population. Patients with haemophilia A showed no significant increase (p = 0.65) in incidence of inhibitors (n = 10/48, total incidence 21%) in the 1990s, when they were treated mainly with recombinant products, as compared to the 1980s (n = 9/52, 17%), when they received intermediate/high-purity plasma-derived concentrates. CONCLUSION: Our population-based study verifies that genotype has a general impact on the incidence of FVIII/FIX inhibitors, and that recombinant FIII/FIX concentrates are not a predisposing factor for inhibitor development.     

Acceleration of ALA-induced PpIX fluorescence development in the oral mucosa

BACKGROUND AND OBJECTIVES: The development of 5-aminolevulinic acid (ALA)-induced tissue fluorescence is optimal 2-4 hours after ALA application. Goal of this work was to develop a means of accelerating oral topical ALA-induced tissue fluorescence. STUDY DESIGN/MATERIALS AND METHODS: In 300 hamsters, DMBA (9,10 dimethyl-1,2-benzanthracene) cheek pouch carcinogenesis produced dysplasia in 3-5 weeks. Topical application of 20% ALA in Eucerin was followed by localized ultrasound treatment (1, 3.3 MHz) in 150 animals. In 75 animals, ALA was applied in an Oral Pluronic Lecithin Organogel (OPLO-an absorption enhancer) vehicle. Seventy-five animals received only topical ALA in Eucerin. Hamsters were sacrificed and cryosections underwent fluorescence measurements, histological evaluation, 20-180 minutes after ALA application. One-way ANOVA detected independent effects of pathology on laser-induced fluorescence (LIF). Two-way ANOVA tested for independent effect of pathology and of OPLO, ultrasound, and interaction effects. RESULTS: Ultrasound significantly (P < 0.05) accelerated tissue fluorescence development. CONCLUSIONS: Low-frequency ultrasound can accelerate ALA-induced fluorescence development.     

Effects of light fractionation and different fluence rates on photodynamic therapy with 5-aminolaevulinic acid in vivo

To improve efficacy of photodynamic therapy (PDT) with intravenously administered 5-aminolaevulinic acid (ALA) fractionating the light dose or reducing the light intensity may be a possibility. Therefore, Syrian Golden hamsters were fitted with dorsal skinfold chambers containing an amelanotic melanoma (n=26). PDT was performed (100 mW cm(-2), 100 J cm(-2), continuously or fractionated, and 25 mW cm(-2), 100 J cm(-2); continuously or fractionated) using an incoherent light source following i.v. application of ALA. Following fractionated irradiation, the light was paused after 20 J cm(-2) for 15 min. Prior to and up to 24 h after PDT tissue, pO(2) was measured using luminescence lifetime imaging. The efficacy was evaluated by measuring the tumour volume of amelanotic melanoma cells grown subcutaneously in the back of Syrian Golden hamsters (n=36). Only high-dose PDT resulted in a significant decrease of pO(2). Irrespective of the mode of irradiation only high-dose PDT induced complete remission of all tumours (13 out of 13). It could be shown that low-dose PDT failed to induce a significant decrease of pO(2). No significant effect of fractionated irradiation was shown regarding the therapeutic efficacy 28 days after PDT. Thus performing a fractionated PDT with ALA or reducing the light intensity seems not to be successful in clinical PDT according to the present data.     

Pharmacology of protoporphyrin IX in nude mice after application of ALA and ALA esters

Aminolevulinic acid (ALA), ALA methylester (ALA-Me) and ALA hexylester (ALA-Hex) were topically applied for 5 and 20 hr, respectively, on normal skin of mice. The distribution of protoporphyrin IX (PpIX) induced in 7 different tissues by these drugs was determined either by spectrofluorometric measurements with an optical fibre probe or by chemical extraction of PpIX from the tissues. The results from these 2 types of measurements were compared. Both methods showed that ALA and the esters induced similar amounts of PpIX at the skin spot where they were applied and that the esters produced much less PpIX at remote skin spots (i.e., spots outside the location where the drugs were applied) than ALA did, notably after 20 hr application. After 20 hr of drug application ALA produced much more PpIX in liver, intestine and lungs than the esters did. In contrast with the direct fluorescence measurements, the extraction method showed detectable amounts of PpIX in liver, intestine and lung after application of the esters, notably of ALA-Me. The discrepancy is probably related to the fact that the pigmented tissues absorb light and, therefore, the direct fluorescence readings are misleading. Notably in the liver, which contains high concentration of light-absorbing pigments, very weak direct fluorescence was seen. In no case there was any accumulation of PpIX in muscle tissue nor in brain. The esters seem to penetrate less into the circulation than ALA, and PpIX formed by them in the skin is faster cleared than PpIX formed from ALA. This is also true after oral and i.p. administration of the drugs.     

A protocol for the dental management of von Willebrand's disease, haemophilia A and haemophilia B

A guide for the dental management of the three inherited bleeding disorders, von Willebrand's disease, haemophilia A and haemophilia B, was established jointly by the Institute of Medical and Veterinary Science Transfusion and Haemostasis Unit in conjunction with the Medically Compromised Dental Unit at the Adelaide Dental Hospital. This protocol was subjected to a successful trial for 24 months.