Oesophageal involvement in 26 consecutive patients with mucous membrane pemphigoid.

BACKGROUND: Oesophageal involvement of mucous membrane pemphigoid (MMP) has not yet been thoroughly described. OBJECTIVES: To characterize systematically the endoscopic lesions of a series of patients with oesophageal symptoms seen at a referral centre for autoimmune bullous diseases. METHODS: Clinical, endoscopic and immunological findings of consecutively referred patients with MMP with oesophageal involvement, systemic and endoscopic treatments, and follow-up are described. RESULTS: Of 477 consecutive patients with MMP consulting between 2002 and 2012, 26 (5·4%) had symptomatic oesophageal involvement. Dysphagia, observed in 23 (88%) patients, was the most frequent symptom. Oesophageal symptoms could be the first sign of MMP. Patients with oesophageal involvement had a mean of three other involved sites. At initial oesophageal endoscopy, 17 of 26 patients had active lesions (intact bullae, erosions and/or erythema), 15 had stricture(s) and 12 had other cicatricial lesions. Systemic therapy alone achieved oesophageal symptom relief for five patients. Dilatation was combined with systemic therapy for 12 patients and was successful in nine; one perforation occurred. CONCLUSIONS: Symptomatic oesophageal involvement affected 5·4% of patients with MMP. Dermatologists and gastroenterologists should be aware of these mucocutaneous diseases and their oesophageal involvement, as it could lead to earlier diagnosis and better care. Oesophageal dilatation could be a therapeutic option for symptomatic stricture not relieved by optimized systemic therapy alone.

Oral cyclophosphamide without corticosteroids to treat mucous membrane pemphigoid.

BACKGROUND: Mucous membrane pemphigoid (MMP) still represents a potentially life- and sight-threatening disease. Immunosuppressants, such as cyclophosphamide (CYC), are indicated for patients with severe and/or refractory MMP. OBJECTIVES: To evaluate the efficacy and safety of daily oral CYC without corticosteroids as therapy for severe MMP. METHODS: Thirteen patients with severe refractory MMP, who received oral CYC at an initial dose of 2 mg kg(-1) without corticosteroids, were retained. Previous treatments, for example dapsone, sulfasalazine or topical agents, were maintained during CYC treatment. Initial clinical severity and response to treatment were assessed by scoring. CYC was stopped after complete remission (CR), or when MMP progressed or lymphopenia (< 0·7 × 10(9) cells L(-1) ) occurred. RESULTS: After 52 weeks of CYC treatment, the overall response rate was 69% (9/13 patients) with a median time to disease control of 8 weeks (range 4-52 weeks). Seven patients (54%) entered CR with a median time to CR of 24 weeks (range 16-52 weeks), all remaining in CR at week 52. The mean duration of CYC administration was 12 weeks (range 2-52 weeks). The most common side effect was lymphopenia (10/13 patients), which led to CYC withdrawal for six patients. No sepsis was observed. CONCLUSIONS: CYC without corticosteroids had rapid efficacy in patients with severe refractory MMP and was safe.

Superparamagnetic bifunctional bisphosphonates nanoparticles: a potential MRI contrast agent for osteoporosis therapy and diagnostic.

A bone targeting nanosystem is reported here which combined magnetic contrast agent for Magnetic Resonance Imaging (MRI) and a therapeutic agent (bisphosphonates) into one drug delivery system. This new targeting nanoplatform consists of superparamagnetic γFe(2)O(3) nanoparticles conjugated to 1,5-dihydroxy-1,5,5-tris-phosphono-pentyl-phosphonic acid (di-HMBPs) molecules with a bisphosphonate function at the outer of the nanoparticle surface for bone targeting. The as-synthesized nanoparticles were evaluated as a specific MRI contrast agent by adsorption study onto hydroxyapatite and MRI measurment. The strong adsorption of the bisphosphonates nanoparticles to hydroxyapatite and their use as MRI T2(∗) contrast agent were demonstrated. Cellular tests performed on human osteosarcoma cells (MG63) show that γFe(2)O(3)@di-HMBP hybrid nanomaterial has no citoxity effect in cell viability and may act as a diagnostic and therapeutic system.

[Acquired cutis laxa and myeloma: large vacuolated cells in the dermis]. / Cutis laxa acquise généralisée associée à un myélome: découverte de grandes cellules vacuolisées dermiques.

INTRODUCTION: Cutis laxa is a rare disorder characterized by loss of elastic tissue. Several organs are often involved such as the skin, lungs, heart, digestive system or genitourinary tract. It may be inherited or acquired, generalized or localized. Its pathogenesis is unclear. Association of acquired cutis laxa with myeloma or plasma cell dyscrasia is very rare. We report a case of acquired cutis laxa associated with a myeloma. CASE REPORT: A 59 year-old woman was admitted for skin hyperlaxity present for a number of years. Light microscopic examination of a skin sample revealed fragmented elastic fibers. Electron microscopic examination of the elastic network demonstrated numerous large vacuolated cells with the appearance of macrophages around abnormal elastic and collagen fibers of the reticular dermis. In addition, a stage-1 IgG lambda myeloma was detected. The patient was treated by thalidomide for one year. After this treatment, electron microscopy examination did not reveal any large vacuolated cells in the dermis, and elastic and collagen fibers were not modified and skin laxity seemed to be stabilized. DISCUSSION: Acquired cutis laxa may be associated with many systemic diseases or can appear after inflammatory skin diseases. Seven cases of generalized cutis laxa associated with myeloma and four cases associated with plasma cell dyscrasia have been reported in the literature. In our case, as in 2 previously described cases, large vacuolated cells resembling macrophages were seen in the dermis. They were thought to play a role in cutis laxa.

A hydroxyethylated cholesterol-based cationic lipid for DNA delivery: effect of conditioning.

We have synthesised a novel cholesterol-based cationic lipid to promote DNA transfer in cells. This lipid, dimethyl hydroxyethyl aminopropane carbamoyl cholesterol iodide (DMHAPC-Chol) contains a biodegradable carbamoyl linker and a hydroxyethyl group in the polar amino head moiety and is characterised by NMR. Liposomes prepared from this lipid and dioleoyl phosphatidyl ethanolamine (DOPE) in equimolar proportion showed a weak cytotoxicity as revealed by MTT assays and are efficient to deliver plasmids DNA evaluated by the expression of reporter genes in vitro and in vivo. In this paper, we present an original method to determine the lipid concentration based on the colorimetric detection of the colipid DOPE and the measure of the molar ratio DOPE/cationic lipid in the liposome by FTIR spectroscopy. The liposomes and lipid/DNA complexes structures were characterized by transmission electron microscopy (TEM) and by quasi-elastic light scattering (QLS). TEM indicated that the complexes correspond to aggregates containing globular substructures with liposomes size. The method of immuno-gold labelling was used to detect plasmid in the complex and reveals the presence of DNA inside the aggregates. Transfection results showed efficient DNA transfer depending on the charge ratio and liposomes conditioning. Gel retardation results indicated that at a molar charge ratio between X = 1.5 and X = 2.5 (depending on the liposome conditioning), all DNA was taken by liposomes. We showed that conditioning by freeze-drying (lyophilization) facilitates storage and improves transfection efficiency. When the liposomes were lyophilized prior to DNA addition or when the complexes were subjected to freeze-thawing cycles, the obtained complexes showed a transfection with levels enhanced up to four and five-fold respectively for the lyophilized liposomes and freeze-thawed complexes. NMR was used to characterize the modifications under freezing which showed an effect on 31P spectra.

Evaluation and optimization of DNA delivery into gliosarcoma 9L cells by a cholesterol-based cationic liposome.

This paper reports results concerning the transfection of gliosarcoma cells 9L using an original cholesterol-based cationic liposome as carrier. This cationic liposome was prepared from triethyl aminopropane carbamoyl cholesterol (TEAPC-Chol) and a helper lipid, dioleoyl phosphatidyl ethanolamine (DOPE). The used concentration of liposome was not cytotoxic as revealed by the MTT test. TEAPC-Chol/DOPE liposomes allowed the plasmids encoding reporter genes to enter the nucleus as observed both by electron microscopy and functionality tests using fluorescence detection of green fluorescent protein (GFP) and luminometric measurements of luciferase activity. By changing the cationic lipid/DNA molar charge ratio, optimal conditions were determined. Further, improvement of the transfection level has been obtained by either precondensing plasmid DNA with poly-L-lysine or by adding polyethylene glycol (PEG) in the transfection medium. The optimal conditions determined are different depending on whether the transfection is made with cells in culture or with tumors induced by subcutaneous (s.c.) injection of cells in Nude mice. For in vivo assays, a simple method to overcome the interference of haemoglobin with the chemiluminescence intensity of luciferase has been used. These results would be useful for gaining knowledge about the potential for the cationic liposome TEAPC-Chol/DOPE to transfect brain tumors efficiently.

In vitro and in vivo transfection of melanoma cells B16-F10 mediated by cholesterol-based cationic liposomes.

In vitro and in vivo transgene expression in B16-F10 melanoma cells has been investigated using an original cationic liposome prepared with triethyl aminopropane carbamoyl cholesterol iodide (TEAPC-Chol) as carrier. TEAPC-Chol/DOPE (dioleoyl phosphatidyl ethanolamine) liposomes are unilamellar, very stable and not toxic in the used concentration range. The yield in complexation with plasmid DNA can reach 100% even in the presence of fetal calf serum. The transfection level has been evaluated by luminometric measurements of luciferase expression. With TEAPC-Chol/DOPE (1:1) liposomes, a relatively high transfection level in B16-F10 cells has been observed comparing to commercial reagents. For in vivo assays, the transfection level in tumors induced in Nude mice has been optimized by studying the effects of charge ratio, of the helper lipid and of the injection volume. Results showed that TEAPC-Chol/DOPE (1:1) liposomes have improved 10-fold transfection level versus direct gene transfer of free DNA.

Familial sperm polyploidy induced by genetic spermatogenesis failure: case report.

We report a case of oligoasthenoteratozoospermia in a 40 year-old patient with a familial history that revealed multiple cases of infertility and perinatal deaths. The patient's semen sample contained 2x10(6) spermatozoa/ml, with an overall progressively motile population of <5%. Cytological analysis revealed a teratozoospermia with 100% of abnormal macrocephalic sperm heads and an irregular acrosomal cap in 38% of cells. Moreover, 72% of spermatozoa carried multiple flagella (2-5). The midpiece was elongated and/or enlarged with cytoplasmic droplets in 15% of cells. The multiple anomalies index (MAI) was 3.3 (normal value = 1.6), reflecting the high incidence of spermatozoal morphological abnormalities in this patient. Ultrastructural analysis revealed the presence of 2 or 3 vacuolated nuclei per sperm head. The acrosome was abnormal and the chromatin, partially packaged, appeared rough. In some cases, a large amount of cytoplasm containing vacuoles was observed around the nucleus and the acrosome. The mitochondrial helix was disorganized. Chromosome analysis performed on blood cells revealed a normal karyotype. Three-colour fluorescence in-situ hybridization (FISH) analysis of 1148 spermatozoa showed 21.6% to be diploid, 62.4% triploid, 13.3% quadriploid and 2.7% hyperploid (<4n). In conclusion, we suggest that this case could result from a genetically induced spermiation failure, the origin of which is discussed.

Mouse offspring after microinjection of heated spermatozoa.

The thermostability of the mammalian sperm genome was previously reported, but no live offspring after conception with heated spermatozoa had yet been obtained. In the present study, mouse spermatozoa were heated at 56 degrees C for 30 min and microinjected into mouse oocytes. Fertilization did not occur unless activation was induced by incubation in a calcium-free medium containing strontium. Under these conditions fertilization and cleavage rates were comparable to those obtained after microinjection of control spermatozoa, but the developmental rate to the blastocyst stage was lower. When transferred to foster mothers, embryos derived from heated sperm developed into phenotypically normal offspring, which grew and reproduced normally. In the mouse, heated spermatozoa can therefore support full embryonic development after microinjection into oocytes.

Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol.

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.

Interactions of lymphokine-activated killer cells and A549 lung carcinoma nodules maintained in three-dimensional culture.

Lymphokine-activated killer (LAK) cells were cocultured in the presence of interleukin-2 (IL-2), with pulmonary surfactant secreting A549 lung carcinoma nodules and maintained in continuous three-dimensional culture for 2-6 days in an attempt to test the response of tumor cells which produce LAK cell inhibitory substances. The A549 nodules secrete mucus which envelops them. This mucus is also secreted inside pseudoalveolar structures characteristic of these nodules. The mucus contains the pulmonary surfactant and sialomucins, both LAK cell inhibitory substances. The spontaneous infiltration into the neoplastic tissue and the membrane contacts established between the two cell types were studied by means of histological, immunohistochemical and electron-microscopic methods. Free-floating LAK cells were allowed to sediment and adhere freely to the nodule surface. The cytostatic and cytolytic effects of LAK cells were tested using thymidine incorporation into DNA and flow cytometry. Despite the presence of a mucus envelope, LAK cells adhered to the A549 nodule surface and penetrated spontaneously into them in the presence of IL-2; they settled mainly in the pseudoalveolar structures where they became apoptotic. According to electron-microscopic observations performed on the second day of coculture, the LAK cells, which remained between the cancer cells, established mostly pinpoint contacts with the carcinoma cells, forming cytoplasmic fusions. These fusions indicate the induction of pores in both the cancer cell and the LAK cell membranes. Electron-microscopic observation also displayed LAK-cell-associated apoptotic and necrotic carcinoma cells. However, at this stage of the coculture (day 2), the DNA synthesis rate of the A549 nodules still remained unchanged; it diminished by approximately 3 times on day 4 and almost stopped on day 6: nodule disintegration was then complete. In the free-floating LAK cell component of the cocultures, DNA synthesis was already strongly inhibited (26x) by the second day. Nevertheless, their cytolytic effect remained unaltered, as was tested on A549 monolayer cells. The presence of tumor necrosis factor (TNF) in the coculture supernatant has been demonstrated, and when coculturing took place in the presence of monoclonal TNF antibody, nodule proliferation was significantly enhanced (up to 145%). Our results indicate that, despite the presence of pulmonary surfactant and sialomucins containing mucus, LAK cells were capable of killing lung carcinoma cells in three-dimensional culture at an early stage of coculture (day 2) by direct cell-to-cell contact. Total nodule disintegration, however, was complete much later (on day 6), and taking into account the low amount of LAK cells in the cancer tissue, this seemed to be the result of an indirect effect implying, in particular, the presence of soluble TNF.

Effects of lymphokine-activated killer-cells on melanoma nodules maintained in 3-dimensional culture.

Lymphokine activated killer (LAK) cells were cocultivated from 2 to 6 days with WM266 metastatic melanoma cells maintained as nodules in organotypic culture. The LAK cells in suspension were allowed to deposit freely on the nodule surface from where they could infiltrate spontaneously into the nodules. Immunohistochemical studies were done to localize the LAK cells as well as electron microscopical observations for effector/target membrane contacts. Proliferation of the nodules was tested and also that of the LAK cells after coculturing using tritiated thymidine incorporation into DNA. Cell death was determined by arrest of thymidine incorporation and total nodule disintegration. Infiltration rate of LAK cells into the nodules was low: after coculturing 5% of the nodule cells were LAK cells. Although close membrane contacts and cytoplasmic fusions between effector and target cells leading to tumor cell apoptosis were observed, this direct cytolytic process seemed to be too infrequent for the induction of total nodule disintegration at day 6. Therefore, the indirect pathway to cytolysis might be predominant implying, among other cytokines, soluble TNF. On the other hand, LAK cell proliferation diminished strongly after coculturing (down to 11%) but the cytotoxicity was significantly enhanced (18% higher) suggesting an enhancement of differentiation. This might account for the peculiar efficacy of LAK cells on melanomas in vivo and it would be of interest to study this phenomenon further.

[Effects of LAK cells activated by IL-2 on MCF-7 human breast cancer cell line maintained in organotypic culture]. / Effets des cellules LAK en présence d'IL-2 sur la lignée carcinomateuse mammaire humaine MCF-7 maintenue en culture organotypique.

Lymphokine Activated Killer (LAK) cells, stimulated by interleukin 2 (IL-2) have a pronounced antitumor effect in the therapy of melanoma and renal cancers. LAK cells were cultivated in presence of the nodules of the human breast adenocarcinoma cell line MCF-7 maintained in organotypic culture to study the interactions between lymphocytes and breast tumor cells. After two days of co-culture, the proliferation of MCF-7 nodules and that of LAK cells was diminished about five folds. The cytotoxic effect of the latter, appreciated by Chrome 51 release was unchanged after the coculture. In histological sections, the penetration of the LAK cells into the MCF-7 nodules was accompanied by an increase of tumor necrosis but also by a glandular differentiation of cancerous tissue. Polarized epithelial cell formations bording neoplasic lumens with intracytoplasmic vacuoles filled with mucus, appeared in the nodules. The immunohistochemistry underlines the presence of T lymphocytes marked by UCHL1 and CD3 antibodies and of Natural Killer (NK) cells marked by IOT10, located between the MCF-7 cancer cells. In electron microscopy, the membrane contacts were tight and were accompanied by the appearance of secondary lysosomes and nuclear alterations. The relatively low infiltration level of the nodules may lead to the supposition that an indirect mechanism will intervene in this dual action of a LAK cells: increase of necrosis, although partially, and development of glandular and functional differentiation.