High efficacy of photodynamic therapy on rat endometrium after systemic administration of benzoporphyrin derivative monoacid ring A.

BACKGROUND: The aim of this study was to evaluate the effect of the benzoporphyrin derivative monoacid ring A (verteporfin)-mediated photodynamic therapy (PDT) on rat endometrium and to determine the optimal drug concentration for endometrial ablation. METHODS: Five minutes after i.v. injection of different concentrations of verteporfin into 24 female Sprague-Dawley rats, 630 nm light treatment was delivered for 500 s (120 J/cm2) to the left horn of the uterus. The 24 rats were divided into six groups according to the drug dose injected, four rats per group: group I (2 mg/kg), group II (1 mg/kg), and groups III, IV, V and VI with 0.5, 0.25, 0.125 and 0.0625 mg/kg respectively. Four days later, the rat uteri were analysed by light microscopy. RESULTS: Endometrial destruction was seen in all six groups, with the most significant result in group I (P<0.008). Conservation of the myometrium was most significant in groups III, IV, V and VI. Acute inflammatory cells in the stromal endometrium were recorded mainly in groups I and II. However, the drug dosage that was most significant in destroying the glands with conservation of the myometrium and not causing severe inflammation was between 0.5 and 0.125 mg/kg. CONCLUSIONS: Verteporfin was effective in endometrial ablation in all our animal groups, and the dose range of 0.5-0.125 mg/kg appeared to be adequate. This observation will have to be scaled for clinical application.

[Preparation of a syrup with Bakis roots (Tinospora bakis) and evaluation of its choleretic activity in rats]. / Preparation d'un sirop a base de racines de Bakis (Tinospora bakis) et evaluation de son activite cholérétique chez le rat.

The aim of this work was to prepare a pharmaceutical using the aqueous extract of bakis roots and to check if the choleretic activity of this latter was maintained in cholestatic rats. So, a sirup was prepared and tested. The obtained results had shown that the aqueous extract maintained its choleretic activity. Indeed, when it was used at a dose of about 7.5 mg/100 g of weight, the sirup induced a significant increase of bilary secretion in healthy rats and cholestatic rats, confirming the results observed with the aqueous extract. Therefore, futher investigations in order to improve the quality of the sirup can be considered before performing clinical trials.

Gene therapy for cystic fibrosis with aerosolized adenovirus-CFTR: characterization of the aerosol and scintigraphic determination of lung deposition in baboons.

For cystic fibrosis (CF) gene therapy using an aerosolized adenovirus expressing the CFTR gene, optimization of the inhalation conditions is a prerequisite to obtain sufficient amount of CFTR protein expression in the target areas of the respiratory tract. For such a purpose, in vivo radioisotopic imaging of the radiolabeled virus is a unique strategy for a quantitative assessment of the actual deposition. In the present study, an adenovirus CFTR (AdCFTR) was labeled with 99m Technetium gamma emitting isotope in such conditions that its bioactivity was preserved. The 99mTc-AdCFTR aerosol was characterized using both laser diffraction and cascade impaction for sizing with further determination of nebulized and inhalable fractions. After administration to baboons, scintigraphic quantitation of the regional lung distribution was performed and the actual dose deposited in the target area was estimated and expressed as an equivalent viral titer. Since a virus scintigraphy is not realistic in a hospital setting, we have developed an approach using 99mTc-DTPA (diethylene triamino pentaacetic acid) that could be used to predict the virus deposition. Indeed, similarities observed between 99mTc-DTPA and 99mTc-adenovirus aerosol imaging patterns validates the use of the 99mTc-DTPA scintigraphy that we propose as a pretherapeutic test for each patient prior to gene transfer.

Radioisotopic imaging allows optimization of adenovirus lung deposition for cystic fibrosis gene therapy.

Cystic fibrosis is a common, heriditary disease resulting from mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Airway transfer of the CFTR gene is a potential strategy to treat or prevent the lung pathology that is the main cause of morbidity and mortality. Among the vectors used for gene therapy, adenoviruses have shown their ability to transfer the CFTR gene to respiratory epithelial cells, using either instillation or nebulization. Our objective was to characterize the lung deposition of aerosolized adenovirus by quantitative radioisotopic imaging, the only noninvasive technique allowing in vivo quantitation of inhaled drugs. We first labeled an adenovirus expressing human CFTR with the gamma-emitting radioisotope, technetium 99m (99mTc), and determined the best labeling conditions to allow preservation of virus bioactivity. We then administered the radioaerosol to baboons, determined lung regional deposition of 99mTc-labeled adenovirus, and compared the expression of CFTR transcripts 3 and 21 days after inhalation. The expression of vector-encoded mRNA ranged from 4 to 22% with respect to the endogenous CFTR mRNA depending on the lung segments. Moreover, we have developed a model using 99mTc-DTPA (diethylenetriamine pentaacetic acid), which can be used, as an alternative to adenovirus, to determine the profile of lung deposition of the vector. This study demonstrates that scintigraphy is a useful technique to achieve optimization of gene administration to the airways.

Variability of immunohistochemical reactivity on stored paraffin slides.

AIMS: To investigate the effects of slide storage on immunohistochemical staining, since recent reports have indicated that storage of unstained paraffin slides for up to 12 weeks may lead to false negative immunostaining of tumour markers. METHODS: 11 antibodies (anti-cytokeratin, epithelial membrane antigen (EMA), vimentin, smooth muscle actin, PS100, chromogranin, CD45, CD20, CD3, CD30, and oestrogen receptor (OR) were tested on unstained paraffin slides of breast carcinomas, lymphomas, and neuroendocrine tumours that had been stored for three to 10 years. All the paraffin blocks were recut less than one week before immunostaining. Immunostainings of years old slides were compared with those of recent slides in at least five cases for each antibody. For three antibodies (antichromogranin, anti-CD3, and anti-OR) we also tested one year old and three months old slides. RESULTS: Intensity of staining on years old slides was strikingly reduced for chromogranin and CD3 in several cases and was slightly stronger for vimentin. In some cases a significant decrease of OR positivity was observed after three months storage, and a complete loss of OR immunostaining after 12 months. No significant difference was noted with the other antibodies. CONCLUSIONS: Immunohistochemical detection of some antigens located either in the nucleus, in the cytoplasm, or on the cytoplasmic membrane could be impaired by storage of paraffin slides as short a time as three months. One should be cautious of doing retrospective immunohistochemical studies on stored unstained slides.

Aerosol administration of a recombinant adenovirus expressing CFTR to cystic fibrosis patients: a phase I clinical trial.

Ad CFTR, a replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator (CFTR), was administered by aerosolization in a single escalating dose to three pairs (cohorts) of cystic fibrosis (CF) patients. Buffer only was administered to the nose and lungs 9-14 days before nasal instillation of virus followed the day after by aerosolization of Ad CFTR to the lung. Nasal doses (defined in terms of viral plaque forming units, pfu) were 10(5), 10(7), and 4 x 10(8), whereas aerosolized doses were 10(7), 10(8), 5.4 x 10(8) for each cohort, respectively. No acute toxic effects were observed in the first 4 weeks after virus treatment. Shedding of infectious Ad CFTR was never detected, whereas detection of vector DNA sequences and CFTR expression demonstrated DNA transfer to the nose and airways of patients. No significant deviations in immunological and inflammatory parameters were observed in serum and in bronchoalveolar lavage (BAL). Importantly, for all patients, the serum anti-adenovirus antibody levels did not change significantly from baseline and no antibodies against adenovirus were found in BAL.

[Aerosol administration of a replication defective recombinant adenovirus expressing normal human cDNA-CFTR in the respiratory tractus in patients with cystic fibrosis]. / Administration par aérosol d'un adénovirus recombinant déficient pour la réplication contenant cADN-CFTR humain normal dans le tractus respiratoire de patients atteints de mucoviscidose.

At present it is conceivable to think that gene therapy represents a way to treat or even prevent the respiratory manifestations of cystic fibrosis. Consistent to such a concept, there is sufficient evidence that Ad-CFTR, a recombinant replication-deficient adenovirus expressing the human cystic fibrosis transmembrane conductance regulator cDNA, can vectorize the expression of a functional CFTR (cystic fibrosis transmembrane conductance regulator) to the nasal and airway epithelia. The clinical protocol was designed to assess the safety of single escalating doses of a replication defective adenovirus expressing the cystic fibrosis transmembrane conductance regulator gene (Ad-CFTR) when administered to the tracheobronchial portion of the airways and whether biological efficacy of CFTR delivery could be demonstrated. Six cystic fibrosis patients received nasal instillation and subsequent aerosol (Optineb, Air Liquide, Paris, France) administration of Ad-CFTR the following day. Doses (pfu) applied to the nose were 10(5) (patients SG and PB), 10(7) (patients FP and EP) and 4 x 10(8) (patients DS and FG), while aerosolised doses were 10(7) (patients SG and PB), 10(8) (patients FP and EP) and 5.4 x 10(8) (patients DS and FG), respectively. No acute toxic effects, no increase in the titer of anti-adenovirus antibodies and no spreading or shedding of Ad-CFTR were detected. In one patient Ad-CFTR DNA was found in the urine and blood two days after aerosolisation. Ad-CFTR DNA was detected in nasal and bronchial brush samples, in BAL, in saliva and tonsils 21, 8, 14 and 4 days post virus administration, respectively. Ad-CFTR mRNA (RT-PCR on bronchial cells) and CFTR protein (immunochemistry on nasal and bronchial cells) were detected up to 14 days following Ad-CFTR administration. These results show that the nebulisation of Ad-CFTR is a possible approach for treating the respiratory manifestation of cystic fibrosis.

Aerosol-mediated delivery of recombinant adenovirus to the airways of nonhuman primates.

At present, it is conceivable that gene therapy of the cystic fibrosis airway epithelium is possible using the direct transfer of a functional human cystic fibrosis transmembrane conductance regulator (CFTR) gene to a wide variety of patients' tracheo-bronchial cells. Here we describe a novel approach (aerosolization) to deliver a replication-deficient adenovirus carrying the CFTR gene (Ad.CFTR) to the airways. Results obtained in vitro and in Rhesus monkeys suggest that the delivery of recombinant adenovirus as an aerosol is feasible and is not associated with severe toxicity after single or double administration depending on the Ad.CFTR dose. This study supports the concept of aerosolization as a delivery method for adenovirus-mediated lung gene therapy.

[Defined media for hybridoma culture: do they really replace serum and why are they attractive?]. / Milieux définis pour la culture d'hybridomes: remplacent-ils réellement le sérum et en quoi sont-ils attractifs?

The cell culture efficiency of serum free media is still the main question which limits the use of such a product. However, the experience due to a long usage of serum free media allows to certify that for hybridomas the proliferation growth is often similar to the levels attained with serum. Antibodies secretion is also good or superior to the one obtained with classical media used in different culture devices like flasks and cytocultures. Finally the higher purity degree of monoclonal antibodies in the cell culture supernatant is also a major advantage of serum free media.

[Optimization of the purification of human plasma fibronectin by double affinity chromatography using gelatin and heparin Trisacryl LS]. / Optimisation de la purification de la fibronectine plasmatique humaine par double chromatographie d'affinité sur gélatine et héparine Trisacryl LS.

The authors described an optimized method of preparation to obtain pure human plasma fibronectin. The new performances related to the chromatographic and concentration steps, where the saving of time is about 40% and the lyophilization step where the saving of energy is also about 40%. The final product is without any less of either physical and biological activities. The size of the columns, the volumes of the chromatographic supports (gelatin and heparin-Trisacryl LS) and the quantity of the treated plasma are very much important and also the quantities of the final product are very increased.

[Purification of human plasma fibronectin by double affinity chromatography on gelatin and heparin-Trisacryl]. / Purification de la fibronectine plasmatique humaine par double chromatographie d'affinité sur gélatine et héparine-trisacryl.

In this work the human plasma fibronectin was purified by affinity chromatography using a tandem column system. The first affinity column was filled with gelatin-Trisacryl whereas the second one contained heparin-Trisacryl. This double affinity chromatography demonstrated its high efficiency in term of purity and yield. Several analytical methods (electrophoresis, immunoelectrophoresis, F.P.L.C. and adhesion assay on cultured eucaryotic cells) evidenced in fact the high purity of the preparation as well as its biological behaviour in term of cell adhesion and spreading. The performances of the sorbents used facilitate the scaling up when large quantities of FNP are needed.

Flow cytometric analysis of hepatoma tissue and HeLa cells grown on various types of microbeads using hydroxyurea, nocodazole and aphidicolin in succession.

Applying Hydroxyurea, Nocodazole and Aphidicolin in succession to obtain parasynchronous growth, the progression of HTC and HeLa cells through the cell cycle has been monitored by laser flow cytometry. The experimental results show that HTC cells behave identically whether grown in monolayer or attached to dextran-based microbeads but that the chemical nature of the micro-support itself plays an important role especially on the speed with which the cells pass from mitosis into G1, polyacrylamide-based microbeads being superior in this respect.

Culture of chondrocytes in medium supplemented with fetal calf serum or a serum substitute: Ultroser G.

Fetal calf serum and a serum substitute, Ultroser G, were compared for their effects on the growth curves, clonal growth and cell cycle progression of rabbit chondrocytes in primary culture and during at least three cell passages and included a screen for the maintenance of cartilage-like differentiation i.e. the presence of type II collagen. Proliferation was also compared with another serum substitute, Nu-Serum. Ultroser G is shown to be equivalent to fetal calf serum as far as chondrocyte proliferation is concerned, clonal growth is improved and biosynthesis of type II collagen is maintained in primary culture.

Liposome-mediated DNA transfer in eukaryotic cells. Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage.

The pBR322 plasmid containing the sequence encoding beta-lactamase, the enzyme conferring resistance to ampicillin, was encapsulated in liposomes of different phospholipid composition and incubated with synchronized cells. In mitotic cells as compared to cells synchronized in G1, twice as many exogeneous DNA molecules were found associated with the cell nuclear DNA, when fluid, neutral liposomes were used. These liposomes are taken up by the cells mainly via endocytosis. When fluid, negatively charged liposomes were used as carriers about the same number of exogenous DNA molecules were found associated with the nuclear DNA both in mitotic and in G1-synchronized cells. The efficiency for gene transfer of liposomes entering the cells by different mechanisms was further studied and expressed both by the fraction of the radioactive plasmid associated with the nuclear DNA and by the level of the beta-lactamase activity detected in the transfected cells. It appears that liposomes entering the cells mainly via an energy-dependent mechanisms are more efficient for this type of DNA transfer.

Cell surface receptors for wheat germ agglutinin and limulin in baby hamster kidney cells and ricin resistant variants.

The cell surface glycoconjugates of Baby Hamster Kidney cells and of four ricin resistant variants were investigated by the use of 125I-substituted ricin (Ricinus communis toxin) which binds galactose residues, and by the use of fluorescein labelled lectins which bind N-acetylneuraminic acid and/or N-acetylglucosamine: Limulin (Limulus polyphemus agglutinin), wheat germ agglutinin (Triticum vulgare agglutinin) and succinylated wheat germ agglutinin. Striking differences in the number of lectin and/or ricin receptors were found between the cell surface of wild type cells and that of ricin resistant variants. The results are discussed on the basis of the main glycopeptide structure, and of the specificity of the sugar binding proteins used. The ricin resistance of variant cells is concomitant to modifications of the concentration of certain glycoconjugate structures which are accessible to the sugar binding proteins. Depending on the variants, N-asparaginyl glycopeptide types and/or O-glycosidic glycopeptide types are affected.