Phospholipid matrix as a target for sulfur mustard (HD): NMR study in model membrane systems.

Although the interactions of sulfur mustard (HD) with nucleic acids and proteins have been well studied, the toxic interactions with the membrane matrix and specially the phospholipid bilayer have so far been poorly investigated. We have used several NMR techniques to study these interactions: 1H NMR to observe the localization of HD in membranes of small unilamellar vesicles (SUV) of lecithin; 31P NMR to verify the hypothesis of pore formation in membranes of large unilamellar vesicles (LUV); and pseudo solid state 31P and 2H NMR to analyze the dynamic consequences of the presence of HD in multilayer dispersions of dimyristoylphosphatidylcholine (DMPC). Immediate and late modifications of the DMPC-HD complexes have been observed at the macroscopic and microscopic levels. After intoxication, HD is spontaneously incorporated into the membrane and locates at the level of the chain methylene groups. This incorporation occurs without formation of pores in the membrane. The presence of HD in the phospholipid dispersion differentially increases the membrane fluidity depending upon the level involved. Weak at the superficial level (phosphate group), this increase is dose-dependent on progression into the membrane. This increase is related to a lowering of transition temperature when measured at the chain level. Macroscopically, HD induces dose- and time-dependent modifications of the DMPC-HD complexes, leading to the formation of an optically transparent gel. This gel formation is confirmed at a microscopic level, where all structures disappear after intoxication.

Long-term regeneration of human epidermis on third degree burns transplanted with autologous cultured epithelium grown on a fibrin matrix.

BACKGROUND: Extensive third degree burn wounds can be permanently covered by the transplantation of autologous cultured keratinocytes. Many modifications to Green and colleagues' original technique have been suggested, including the use of a fibrin matrix. However, the properties of the cultured cells must be assessed using suitable criteria before a modified method of culture for therapeutic purposes is transferred to clinical use, because changes in culture conditions may reduce keratinocyte lifespan and result in the loss of the transplanted epithelium. METHODS: To evaluate the performances of human keratinocytes grown on a fibrin matrix, we assay for their colony-forming ability, their growth potential and their ability to generate an epidermis when grafted onto athymic mice. The results of these experiments allowed us to compare side by side the performance for third degree burn treatment of autologous cultured epithelium grafts grown according to Rheinwald and Green on fibrin matrices with that of grafts grown directly on plastic surfaces. RESULTS: We found that human keratinocytes cultured on a fibrin matrix had the same growth capacity and transplantability as those cultured on plastic surfaces and that the presence of a fibrin matrix greatly facilitated the preparation, handling, and surgical transplantation of the grafts, which did not need to be detached enzymatically. The rate of take of grafts grown on fibrin matrices was high, and was similar to that of conventionally cultured grafts. The grafted autologous cells are capable of generating a normal epidermis for many years and favor the regeneration of a superficial dermis. CONCLUSION: We have demonstrated that: 1) fibrin matrices have considerable advantages over plastic for the culture of skin cells for grafting and that it is now possible to generate and transplant enough cultured epithelium from a small skin biopsy to restore completely the epidermis of an adult human in 16 days; and 2) the generated epidermis self-renews itself for years. The use of fibrin matrices thus significantly improves the transplantation of cultured epithelium grafts for extensive burns as recently demonstrated in a follow-up work.

Modulation of a fibrotic process induced by transforming growth factor beta-1 in dermal equivalents.

Why initially normal wound healing sometimes shifts toward an impaired cicatrization is poorly understood. Collagen gels with incorporated fibroblasts constitute valuable in vitro models to study mechanisms of connective tissue reorganization. Such 1-week-old, partially contracted normal dermal equivalents were treated with concentrations of TGF-beta1 ranging from 1 to 10 ng/ml. The cytokine was applied in a single dose or four times at regular intervals, over a 2-week period. Dose-dependent activation of fibroblasts was observed after treatment. The cytokine induced a myofibroblastic transformation of dermal cells, significantly enhanced the process of dermal contraction, and stimulated synthesis of such proteins as cellular fibronectin, tenascin and smooth-muscle actin. Our approach is more informative than models using pathological or pretreated dermal cells, since it demonstrates newly induced modulation of fibrotic transformation in an initially normal dermal equivalent. This in vitro assay will enable us to study mechanisms involved in the shift between normal and impaired fibrotic transformation during wound healing.

In vitro dermal intoxication by bis(chloroethyl)sulfide. Effect on secondary epidermization.

Skin intoxication by bis(beta-chloroethyl)sulfide (BCES; sulfur mustard) induces cutaneous lesions similar to thermal burns, characterized by slowness of skin healing. We have developed an in vitro model of skin equivalent to investigate mechanisms involved in this delay. Direct intoxication of dermal equivalent produced dose- and time-dependent cytotoxicity. A decrease of macroscopic retraction of collagen gels was observed, parallel to the toxic concentration with, at histological level, absence of collagen fiber reorganization. Fibroblast synthesis of fibronectin was also inhibited by intoxication, as demonstrated at an immunobiochemical and immunohistochemical level. These dermal alterations were correlated with secondary modifications of epithelial maturation of nonintoxicated normal human keratinocytes. Cellular adhesion was perturbed, as visualized by a delay in expression and reorganization of basement membrane components, laminen, collagen IV, and fibronectin. Epidermal terminal differentiation was also affected, as shown by the absence of profilaggrin/filaggrin biosynthesis. We demonstrated in vitro, that direct dermal alterations secondarily induce disturbance of epithelial maturation. Taken together, these data show the fundamental role of dermal-epidermal interactions in a normal skin reconstruction. Clinical slowness of wound healing observed after cutaneous intoxication by BCES may thus be explained by direct alkylation of some structures and further disturbances of their biosynthesis.

[Nephropathy and filariasis from Loa loa. Apropos of 1 case of adverse reaction to a dose of ivermectin]. / Néphropathie et filariose à Loa loa. A propos d'un cas de réaction adverse à la prise d'ivermectine.

A patient with a filarial infection due to Loa loa, received single oral dose of ivermectin A few hours after, he has presented a severe renal impairment. Native of the Cameroons, he was also treated for malaria. A renal biopsy has been done, because of chronic proteinuria. It has showed microfilariae in glomeruli, thickening of the basement membrane and a segmental and focal mesangial hyalinosis. By immunofluorescence microscopy there were granular deposits of IgM and C3 along this membrane. Electron microscopy has showed subepithelial electron- dense deposits. This suggests that the renal disease is immunologically induced. During the therapy, adverse reactions seemed to be related with massive liberation of filarial antigens. This is well known with diethylcarbamazine but also now, sometimes with ivermectin.

Inhibitory effects of bFGF, VEGF and minoxidil on collagen synthesis by cultured hair dermal papilla cells.

Dermal papilla cells of rat vibrissa follicles cultivated in monolayers and in three-dimensional collagen gels show a different morphology in these culture systems. Dermal papilla cells cultured in lattices tend to express morphological features resembling those seen in vivo. Quantification of total collagen by incorporation of 3H-proline in monolayer cultures and in collagen lattices show that the amount of collagen found in dermal papilla cells is higher than that secreted. Moreover, collagen synthesis measured in lattices is reduced to about 50% of that found in monolayer cultures. The influence of growth factors on collagen synthesis by hair dermal papilla cells was investigated. We studied the effects of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF) and minoxidil on collagen synthesis in monolayers and in lattices. VEGF, bFGF and minoxidil significantly decreased the total amount of collagen. In monolayer cultures, there was approximately a 30% inhibition of collagen production with 5 ng/ml bFGF, 0.1 ng/ml VEGF and 100 ng/ml minoxidil. However, in the lattices this inhibition was reduced to about half. These results suggest that both culture substrate and growth factors influence collagen production by rat hair dermal papilla cells.

Adenoid cystic (cylindromatous) carcinoma associated with squamous cell carcinoma of the cervix uteri: cytologic presentation of a case with histologic and ultrastructural correlations.

BACKGROUND: Adenoid cystic (cylindromatous) carcinoma is a rare tumor. It accounts for approximately < 1-3% of primary adenocarcinomas of the cervix uteri. Its origin is debatable. It has a higher incidence in postmenopausal women but can develop in patients under 40. An association of adenoid cystic carcinoma with squamous cell carcinoma has been reported. CASE: A case of invasive adenoid cystic (cylindromatous) carcinoma associated with an in situ squamous cell carcinoma of the cervix detected on Papanicolaou smears is reported. Ultrastructural studies provided findings that confirmed that the hyaline material corresponded to the remnants of the lamina densa of the basement membrane of malignant cells. CONCLUSION: The concurrence of these two tumors supports the hypothesis that adenoid cystic carcinoma of the cervix may develop from multipotent reserve cells.

[Tumor of the peripheral nerve sheath with rhabdomyoblastic differentiation arising in a ganglioneuroma: neurilemmoma? A fortuitously detected case]. / Tumeur des gaines des nerfs périphériques à différenciation musculaire survenant sur un ganglioneurome: tumeur de Triton?

An otherwise healthy 21-year-old man with no evidence or family history of Von Recklinghausen's neurofibromatosis presented a posterior mediastinal mass detected on routine chest radiographs. The findings of standard light microscopy, ultrastructural examination and immunohistochemical studies suggested the working hypothesis of an unusual malignant "Triton" tumour: -arising in a pre-existing de novo ganglioneuroma; -fortuitously detected; -predominantly made of multinucleated spindle cells with only ultrastructural and immunohistochemical rhabdomyoblastic differentiation.

Clinical interest of cutaneous models reproduced in vitro for severe burn treatment: histopathological and ultrastructural study.

The healing of minimal skin lesions is usually obtained by epidermal migration and proliferation from peripheral wound margins. However, cutaneous grafts or reconstituted skin are necessary for severe injuries. Various models have recently been reproduced for this purpose. The aim of this work is to report the histopathologic evolution of burn lesions treated two years ago by autologous epidermis (Genzyme Tissue Repair, Boston, USA). Fifteen patients with severe burns (more than 80% of surface) have been treated. These observations have been based exclusively on biopsies of grafted wounds. Cultured epidermis is rapidly fully differentiated after grafting with temporary hyperplasia and normal strata. At 18 months, rete ridges formation is present only in young patients. Melanocytes and Langerhans' cells repopulated grafts rapidly. The use of cultured epidermis nowadays represents an important improvement in burn treatment.

[Malignant triton tumor of the posterior mediastinum treated by thoracoscopic approach. Apropos of a case]. / Tumeur maligne de Triton du médiastin postérieur abordée par thoracoscopie. A propos d'un cas.

A tumour of the posterior mediastinum was removed via thoracoscopy. The histology report was a triton tumour. This is a rare (less than 80 cases reported in the literature) malignant tumour of the nervous system: malignant schwannoma with rhabdomyoblastic differentiation. The characteristics of this rare tumour and the consequences of videoassisted surgery in this specific case are discussed: a second access is required to widen the thoracotomy and complementary radiotherapy is needed.

[Adenoid cystic carcinoma (cylindroma) of the uterine cervix. Histology, immunohistochemistry, ultrastructure in a case, with review of the medical literature]. / Carcinome adénoïde kystique (cylindrome) du col utérin. Histologie, immunohistochimie, ultrastructure d'un cas avec revue de la littérature médicale.

The authors report a case of adenoid cystic (cylindromatous) carcinoma of the endocervical canal associated with an intraepithelial squamous cell carcinoma of the cervical os in a 60-year-old woman suffering from metrorrhagia. Immunohistochemical study demonstrated labeling of cylindromatous cells with anti-actin, anti-vimentin and anti-keratin antibodies. Ultrastructural study provided significant findings to confirm that the hyaline material surrounded by tumor cells was derived from the remnants of the lamina densa of the basement membrane of tumor lobules. These morphological data are similar to those observed in adenoid cystic carcinomas (cylindromas) of salivary glands. They support the hypothesis that adenoid cystic carcinoma of the cervix may develop from the multipotent reserve cells.

[Progressive multifocal leukoencephalopathy localized in the thalamic grey matter in an AIDS-patient]. / Leuco-encéphalopathie multifocale progressive localisée en substance grise thalamique chez un patient atteint de SIDA.

Involvement of the deep grey matter is unusual in Progressive Multifocal Leukoencephalopathy (PML), which is disease of the white matter. Basal ganglia involvement is usually secondary to extension from lesions of adjacent white matter and easy to recognize. We report a biopsy-proven case of PML in which lesions were mainly thalamic.

In vitro infection of human macrophages with human T-cell leukemia virus type 1.

The tropism of the human T-cell leukemia virus type 1 (HTLV-1) for the cells of monocyte-macrophage lineage was evaluated by the coculture of blood monocyte-derived macrophages, with irradiated cells of HTLV-1 producing cell lines MT2 or C91/PL. The susceptibility to HTLV-1 was assessed by the detection of viral DNA using the polymerase chain reaction method. HTLV-1 gene expression in the cells was detected using in situ hybridization and by immunofluorescent staining of viral antigen. The presence of type C virus-like particles detected by electron microscopy and the ability to infect normal cord blood lymphocytes demonstrated that the infected macrophages produced infectious virus. These results indicate that human macrophages are susceptible in vitro to productive HTLV-1 infection, and thus might be involved in the pathogenesis of HTLV-1-related diseases.

Action of bis(betachloroethyl)sulphide (BCES) on human epidermis reconstituted in culture: Morphological alterations and biochemical depletion of glutathione.

Human keratinocyte cultures were treated with bis(betachloroethyl)sulphide (BCES), an alkylating and vesicant agent. At concentrations of 5 x 10(-4) to 5 x 10(-3)m, spontaneous detachment of the epithelium from the culture plate was observed, reproducing in vitro the cutaneous vesication observed in vivo. Progressive cellular alterations were shown with increasing concentrations of BCES (5 x 10(-5) to 5 x 10(-3)m). At low concentrations (5 x 10(-5)m), lesions of the nucleus, a significant target for BCES, were observed, along with lesions in the cytoplasmic organelles. An acute, dose-dependent depletion of cellular glutathione was observed, which occurred within 1 hr of treatment. Mechlorethamine, an analogue of BCES, induced at equivalent doses the same glutathione depletion and similar spontaneous detachment in vitro. We suggest that BCES, in addition to its genetic effects, acts by direct metabolic toxicity and induces glutathione depletion by direct conjugation. The lesions obtained in vitro reproduced those observed in vivo. Human keratinocyte cultures can be proposed as a good model for the study of the mechanisms of action of BCES.

Adult-type rhabdomyoma of the palate. Cytologic presentation of two cases with histologic and immunologic study.

The cytologic presentations of two adult-type rhabdomyomas of the soft palate are reported. Fine needle aspiration smears of the lesions contained globular tumor cells that had striations and intracytoplasmic rod-shaped inclusions. These inclusions, corresponding to hypertrophied Z bands, were pathognomonic of rhabdomyoma tissue. The cytologic diagnosis was confirmed by histologic and immunologic studies in both cases. The cytologic preparations fixed in alcohol or air dried did not show the "spiderweb cells" observed in the histologic sections of tissue fragments fixed in formaldehyde; this clearly illustrates that the morphology of the cells of this tumor depends primarily on the specimen preparation technique used. These cases suggest that aspiration cytology can establish the diagnosis of this benign tumor of striated muscle, for which total surgical removal is usually sufficient to effect a cure. The differential diagnosis is discussed in detail.

In vitro cytophotometry evaluation of gamma-radiation effects on human sperm cell by acridine orange staining.

Effects of gamma-radiations on human sperm nuclei in vitro were studied by cytophotometrical quantification on fixed smears stained by acridine orange some of which had undergone heat-treatment. Following irradiations at different doses (0.0, 2.5, 5.0, 7.5 gy) using a cobalt source, changes in distribution of acridine orange fluorescences emitted by nuclei (total fluorescence greater than 515 nm versus green fluorescence = 525 nm) appeared to be dose-dependent. Heat-treatment of irradiated heads was confirmed to be a complementary procedure necessary for a good evaluation of the initial degree in chromatin compactness. Chromatin alterations related to changes in emitted fluorescence could be due to the initial fragility of the DNA-nucleoprotein complex. These preliminary results suggest that the normality of sperm chromatin could be used as a biologic dosimeter.

[Adult type rhabdomyoma of the soft palate: pathology of 2 cases with review of the medical literature]. / Rhabdomyome de type adulte du palais mou: anatomie pathologique. A propos de deux cas avec revue de la littérature médicale.

In this paper, the authors report two cases of adult type rhabdomyomas of the soft palate. This tumor is composed of striated muscle cells containing striations and intracytoplasmic rod-shaped inclusions. In specimens already fixed in aqueous fixators (formalin...), some tumor cells presented a fixation artefact and displayed the morphology of so-called "spiderweb cells". Adult type rhabdomyoma is a real tumor. Total surgical removal is sufficient to cure the patient of this tumor in most cases.

[Reconstitution in vitro of human gingiva]. / Reconstitution in vitro de la gencive humaine.

A model of human gingiva to be used in pharmacological, basic and clinical research was performed in vitro. This model was obtained through a method of low density seeding epithelialization, from a seeding made up of dissociated human gingiva epithelial cells, of a connective tissue equivalent composed of human fibroblasts included in a collagen gel. The histological and ultrastructural data show a multilayered epithelium and the biochemical analysis (two dimensional gel electrophoresis known as NEPHGE) of the cytokeratins used as molecular markers for epithelial differentiation shows the precise differentiation state of the epithelium thus reconstituted. Even though this model has less of a differentiation than that of an in vivo gingival epithelium, it does actually reproduce exactly the structures of the human gingiva namely a multilayered epithelium lying on a connective tissue. It also offers the advantage of cellular elements which are compatible with gingival graftings.

[The value of measuring membrane potentials of urothelial cells using cytofluorometry in the study of tumors of the bladder. Preliminary analysis]. / Intérêt de la mesure du potentiel de membrane des cellules urothéliales en cytofluorométrie dans l'étude des tumeurs de vessie. Analyses préliminaires.

In the context of investigation of a prognostic marker applicable to bladder tumours, the authors propose the cytofluorometric study of the membrane potential of malignant urothelial cells using a molecular probe, 3,3' dihexyloxacarbocyanine. This preliminary study demonstrated a significant increase in the membrane potential of malignant urothelial cells in comparison with normal cells, which leads the authors to propose this cellular parameter as a new tool in the prognostic evaluation of bladder tumours.