Determination of the ED95 of intrathecal hyperbaric prilocaine with sufentanil for scheduled cesarean delivery: a dose-finding study based on the continual reassessment method.

BACKGROUND: Scheduled cesarean section is routinely performed under spinal anesthesia using hyperbaric bupivacaine. The current study was undertaken to determine the clinically relevant 95% effective dose of intrathecal 2% hyperbaric prilocaine co-administered with sufentanil for scheduled cesarean section, using continual reassessment method. METHODS: We conducted a dose-response, prospective, double-blinded study to determine the ED95 values of intrathecal hyperbaric prilocaine used with 2,5 mcg of sufentanil and 100 mcg of morphine for cesarean delivery. Each parturient enrolled in the study received an intrathecal dose of hyperbaric prilocaine determined by the CRM and the success or failure of the block was assessed as being the primary endpoint. RESULTS: The doses given for each cohort varied from 35 to 50 mg of HP, according to the CRM, with a final ED95 lying between 45 and 50 mg of Prilocaine after completion of the 10 cohorts. Few side effects were reported and patients were globally satisfied. CONCLUSIONS: The ED95 of intrathecal hyperbaric prilocaine with sufentanil 2.5 µg and morphine 100 µg for elective cesarean delivery was found to be between 45 and 50 mg. It may be an interesting alternative to other long-lasting local anesthetics in this context. TRIAL REGISTRATION: The study was registered on January 30, 2017 - retrospectively registered - and results posted at the public database clinicaltrials.gov ( NCT03036384 ).

Structure and biochemical composition of desmosomes and tonofilaments isolated from calf muzzle epidermis.

Complexes of plasma membrane segments with desmosomes and attached tonofilaments were separated from the stratum spinosum cells of calf muzzle by means of moderately alkaline buffers of low ionic strength and mechanical homogenization. These structures were further fractionated by the use of various treatments including sonication, sucrose gradient centrifugation, and extraction with buffers containing high concentrations of salt, urea, citric acid, or detergents. Subfractions enriched in desmosome-tonofilament-complexes and tonofilament fragments were studied in detail. The desmosome structures such as the midline, the trilaminar membrane profile, and the desmosomal plaque appeared well preserved and were notably resistant to the various treatments employed. Fractions containing desmosome-tonofilament complexes were invariably dominated by the nonmembranous proteins of the tonofilaments which appeared as five major polypeptide bands (apparent molecular weights: 48,000; 51,000; 58,000; 60,000; 68,000) present in molar ratios of approx. 2:1:1:2:2. Four of these polypeptide bands showed electrophoretic mobilities similar to those of prekeratin polypeptides from bovine hoof. However, the largest polypeptide (68,000 mol wt) migrated significantly less in polyacrylamide gels than the largest component of the hoof prekeratin (approximately 63,000 mol wt). In addition, a series of minor bands, including carbohydrate-containing proteins, were identified and concluded to represent constituents of the desmosomal membrane. The analysis of protein-bound carbohydrates (total 270 microgram/mg phospholipid in desmosome-enriched subfractions) showed the presence of relatively high amounts of glucosamine, mannose, galactose, and sialic acids. These data as well as the lipid composition (e.g., high ratio of cholesterol to phospholipids, relatively high contents of sphingomyelin and gangliosides, and fatty acid pattern) indicate that the desmosomal membrane is complex in protein and lipid composition and has a typical plasma membrane character. The similarity of the desmosome-associated tonofilaments to prekeratin filaments and other forms of intermediate-sized filaments is discussed.

Blastic variant of mantle cell lymphoma: a rare but highly aggressive subtype.

The blastic variant (BV) form of mantle cell lymphoma (MCL) is considered to be a very aggressive subtype of non-Hodgkin's lymphoma (NHL). In order to determine its clinico-biological features and response to therapy we studied 33 patients (17%) out of 187 suffering from MCL who were diagnosed with a BV of MCL. Blastic variant was diagnosed according to histopathological patterns, immunophenotyping, and bcl1 gene rearrangement and/or cyclin D1 overexpression. Three patients initially diagnosed with large cell NHL were classified as BV. Patients received front-line therapy including CHOP-like regimen or CVP (n = 29), or chlorambucil (n = 4) and CHOP or ESAP as second-line therapy. High-dose intensification with stem cell transplantation (SCT) was performed in 11 cases (autoSCT, n = 8; alloSCT, n = 3). All but two patients were in complete remission (CR) at the time of transplant (CR1, n = 5; CR2, n = 4). Clinical and biological characteristics did not differ from those of the common form of MCL. The median age was 62 years (29-80), with a sex ratio (M/F) of 2.6:1. Of the 33 patients, 66% had extranodal site involvement, 85% had an Ann Arbor stage IV, and 82% had peripheral lymphadenopathy. Circulating lymphomatous cells were seen in 48% of cases. Twelve patients (36%) entered a CR1 with a median duration of 11 months. Fifteen patients (46%) failed to respond and rapidly died of progressive disease. Second-line therapy led to a 26% (6/23) CR2 rate. Nine patients relapsed after high-dose therapy. Twenty-two of the 33 patients (66%) died of refractory or progressive disease. Median overall survival (OS) time was 14.5 months for the 33 BV patients as compared to 53 months for the 154 patients with a common form of MCL, P <0.0001. In the univariate analysis, OS was influenced by age, extranodal site involvement, circulating lymphomatous cells, and international prognosis index (IPI). In the multivariate analysis, only IPI affected OS: patients with IPI > or =2 had 8 months median OS as compared to 36 months median OS for patients with IPI <2, P = 0.003. Blastic variant is one of the worst forms of NHL. An improved recognition of BV of MCL is required, particularly in high-grade CD5+ NHL using immunophenotyping and bcl1 molecular study. Standard therapy using anthracycline or even high-dose intensification produce poor results and an alternative treatment should be proposed to such patients.

Specific internalization of basal membrane domains containing the integrin alpha 6 beta 4 in dispase-detached cultured human keratinocytes.

The integrin alpha 6 beta 4 is polarized towards the basal side of basal keratinocytes and helps anchor these cells to the basement membrane components. We have found that cultured human epidermal keratinocytes, when detached from their culture substratum, as for grafting, using the enzyme dispase, rapidly internalize the basal membrane domains containing the integrin alpha 6 beta 4, while integrins of the very late antigen subtype remain on the cell surface. Detachment and incubation at 4 degrees C prevent this internalization, as well as the contraction of the detached sheet area. Subsequent incubation at 37 degrees C initializes this contraction and allows the basal integrin alpha 6 beta 4 to be internalized. We took advantage of this blockage to label upon detachment using immunogold techniques, the alpha 6 subunit present on the basal cell surface; then we studied its internalization with the electron microscope. This internalization pathway differs from classical receptor-mediated endocytosis, and intermediate filaments might possibly play a role in this process. Interestingly, 1 h after their internalization from the basal membrane, a third of the gold particles labeling the alpha 6 subunit was found between lateral membranes of basal cells, strongly suggesting that the integrin alpha 6 beta 4 can be partly recycled to the cell surface in these conditions.

Basal detachment of the epidermis using dispase: tissue spatial organization and fate of integrin alpha 6 beta 4 and hemidesmosomes.

Dispase has been utilized to produce basal detachment of the epidermis of human skin biopsies and to study the consequences induced afterwards during incubations of the detached tissue. Spatial reorganization of the epidermis is observed under these conditions and is characterized by disappearance of the typical basal keratinocyte layer. Immunofluorescent labelings reveal upward migration of several cells exhibiting the basal phenotype between suprabasal differentiating keratinocytes and demonstrate progressive intracellular expression of hemidesmosomal components: the integrin alpha 6 beta 4 and two plaque components, the 230-kDa bullous pemphigoid antigen and HD1, a 500-kDa protein. Using electron microscopy and immunogold techniques, we demonstrate that the hemidesmosome-containing basal membrane domains enter the cell cytoplasm after detachment of the epidermal tissue. Partial recycling of internalized hemidesmosomal components is also suggested. Our findings illustrate the processing of released hemidesmosomes in detached basal keratinocytes and suggest some heterogeneity between basal cells migrating towards a suprabasal position and those remaining in the basal layer. These results suggest that the dispase-detached epidermis is a self-remodeling tissue in which basal keratinocytes' and tissue's polarities observed in the anchored epidermis are progressively changing.

The use of colloidal gold for screening monoclonal antibodies to cell surface antigens.

An immunogold method in Terasaki plates is described which allows accurate and sensitive visualization of the binding of monoclonal antibodies to cell surface antigens and is suitable for large scale screening. Monolayers of fixed cells are prepared in the wells. The binding of monoclonal antibodies is detected by a protein A gold complex. The cell-bound gold can be visualized by either optical or transmission electron microscopy. The results obtained with various monoclonal antibodies are presented.

Utilization of human cultured epidermal keratinocytes: irreversibility of the inhibition of proliferation induced in stored detached cultures.

In order to look for the best conditions required for the utilization of cultured epithelium in the treatment of burn wounds, some experiments have been performed studying the storage of dispase-detached cultures. The viability of keratinocytes and, after trypsin dissociation of the cultures, the adhesion and proliferative potential of living keratinocytes were measured. Our laboratory investigations suggest that the storage period of detached cultures has to be kept as short as possible to preserve the keratinocytes' growth potential.

[Carcinoma of the cervical stump. Retrospective analysis of 43 cases]. / Carcinomes sur col restant. Analyse retrospective de 43 cas.

Seven to eight percent of cervix carcinomas are carcinomas of the cervical stump. The prognosis for these tumors has sometimes been considered more unfavourable than that for carcinomas on intact uterus. From 1976 to 1986 we treated 43 patients with carcinoma of the cervical stump. The mean age was 63.6 years. Staging system used was FIGO classification modified according to the criteria of Institut Gustave Roussy in Villejuif. There were 12 stage IB, 12 early stage II, 5 late stage II and 14 stage III. Twenty-four centropelvic tumors (IB and early stage II) were treated with radiotherapy and surgery, 2 with surgery alone and one with radiotherapy alone. Late stage II and stage III tumors were treated with radiotherapy alone (+ hysterectomy for two patients). Local control rate was 83% for centropelvic tumors and 53% for late stage II and stage III. Seven patients developed distant metastases. Uncorrected 5-year actuarial survival rate was 78% for centro-pelvic tumors and 46% for late stage II and stage III. Three patients developed severe complications (grade 3). Prognostic factors were: stage, nodal status and pathological status of the cervix after radiotherapy. For the same stage the results were similar to those observed for carcinomas on intact uterus treated at our institution during the same time period.

Inhibition of basal cell proliferation during storage of detached cultured epidermal keratinocyte sheets.

Grafting of human cultured keratinocytes is a promising possibility for the treatment of large burned skin areas. However, during the preparation of cultures for grafting, a necessary step of detachment from culture substratum is performed by incubation with Dispase. In the present paper, we report studies performed to determine whether prolonged storage after detachment would have a detrimental effect on keratinocyte proliferation. Our results show that the number of bromodeoxyuridine-incorporating cells located in the basal layer of the culture gradually diminishes during storage of detached cultured epidermal keratinocyte sheets. Furthermore, the rate of [3H]thymidine incorporation in such detached cultures is also progressively reduced. These observations indicate that the detachment and storage of cultured epidermal keratinocyte sheets for grafting impedes their cell proliferation capacity, as revealed by DNA-synthesis measurements.

Basal cell adhesion to a culture substratum controls the polarized spatial organization of human epidermal keratinocytes into proliferating basal and terminally differentiating suprabasal populations.

The contribution of adhesion to an extracellular matrix in the polarized spatial organization of keratinocytes was studied in dispase-detached cultures stored as floating sheets. Proliferating and terminally differentiating cell populations were, therefore, localized on tissue sections by their DNA-synthesizing ability and involucrin immunostaining, respectively. A progressive reorganization was induced from superposed proliferating and differentiating layers into clusters exhibiting differentiating cells on the outside. Measurements of proliferation and terminal differentiation in detached cultures revealed the progressive disappearance of proliferating cells, followed by an increase in involucrin-positive cells. Attempts to block the spatial reorganization by the addition of components of the extracellular matrix remained unsuccessful. These results suggest that basal anchorage is responsible for the superposition of proliferating and differentiating cells in keratinocyte cultures. They afford new arguments for the induction of terminal differentiation in non-adhesive keratinocytes which exhibit a concomitant modification of cell shape.

Fast neutron therapy for inoperable or recurrent sacrococcygeal chordomas.

Between 1981 and 1994, 13 patients were referred to the Orleans neutrontherapy department with inoperable or recurrent pelvic chordomas. One patient who had already been irradiated refused the treatment, fearing complications. Among the 12 patients suitable for evaluation, ten had undergone one to five previous surgical operations. The time lapse between the last surgical operation and neutrontherapy was 13 months. Neutrons were used alone or as a boost depending on the tumor volume or treatment purpose. At four years, crude survival and local control probability (Kaplan-Meier) were 61 and 54% respectively. This small series suggests that fast neutrontherapy can provide a good alternative for the treatment of inoperable sacral chordomas.

SEM morphological studies of phagocytosis by rat macrophages and rabbit polymorphonuclear leukocytes.

Peritoneal rat macrophages and rabbit polymorphonuclear leukocytes were obtained after thioglycollate or glycogen stimulation. Optimal conditions for phagocytosis were determined by a recently developed quantitative fluorimetric assay. We studied by serial SEM micrographs macrophages and polymorphonuclear leukocytes incubated either in medium or in the presence of different types of phagocytic particles. We compared the morphological aspects of adsorption and phagocytic processes for opsonized microorganisms (Micrococcus lysodeikticus and Candida albicans) with these for inert beads of iron and metallic mercury. Without phagocytosis, in the presence of fresh homologous serum, we observed a progressive development of microvilli or lamellipodia in ruffles and by the end, hypertrophied ruffles appeared at one pole of the cell. We noted extremely well developed ruffles during phagocytosis of opsonized microorganisms. These were practically absent on the macrophages incubated with inert particles. The mean number of adsorbed particles is more elevated in the case of iron and metallic mercury beads than for microorganisms. The rate of ingestion of inert particles was considerably higher than for microorganisms even when they were opsonized. In conclusion, at all stages of the phagocytic process, we observe different morphological features of the macrophages depending on the nature of the phagocytosed particles.

A new quantitative fluorimetric assay for phagocytosis of bacteria.

We describe a new quantitative fluorimetric assay for phagocytosis of bacteria. A suspension of fluorescein-labelled bacteria (Micrococcus lysodeikticus) is mixed and incubated with phagocytes. After fixation with paraformaldehyde, the excess non-phagocytosed and adsorbed bacteria are lysed with a solution of lysozyme in phosphate-buffered saline. After washing of the phagocytes, the fluorescence of those that have ingested the labelled bacteria is measured with a spectrofluorimeter. We report results obtained with different types of phagocytes which show that this method allows sensitivity, saturation and kinetic studies and the calculation of a phagocytic index.

Early secondary acute myelogenous leukemia in breast cancer patients after treatment with mitoxantrone, cyclophosphamide, fluorouracil and radiation therapy.

BACKGROUND: The topoisomerase II-targeted drugs, epipodophyllotoxins and anthracyclines, have been shown to induce therapy-related AML (t-AML) characterized by a short latency period after chemotherapy, the absence of prior myelodysplastic syndrome and stereotyped chromosome aberrations. Few reports have been published on patients treated with the anthracenedione mitoxantrone which also targets topoisomerase II. We observed 10 cases of such t-AML over a 7-year-period in breast cancer patients treated with mitoxantrone combined with fluorouracil, cyclophosphamide and regional radiotherapy, and in three cases with vindesine. PATIENTS AND METHODS: We retrospectively analyzed patients referred to our hospital for AML with a past history of polychemotherapy for breast cancer, including mitoxantrone, either as adjuvant (8 patients)/neoadjuvant (1 patient) therapy or for metastatic disease (1 patient). We studied the probability of developing t-AML in a prospective series of 350 patients treated with an adjuvant FNC regimen (mitoxantrone, fluorouracil, cyclophosphamide) and radiation therapy. RESULTS: The median age was 45 years (range 35-67). t-AML developed 13-36 months (median 16) after beginning chemotherapy for breast cancer, and 4-28 months (median 10.5) after ending treatment. As described in t-AML following treatment with epipodophyllotoxins or anthracyclines, we found a majority of FAB M4, M5 and M3 phenotypes (7 of 10), and characteristic karyotype abnormalities that also can be found in de novo AML: breakpoint on chromosome 11q23 (3 patients), inv(16)(p13q22) (2 patients), t(15;17)(q22;q11) (1 patient), t(8;21)(q22;q22) (1 patient) and del(20q)(q11) (1 patient). The prognosis was poor. All patients died of AML shortly after diagnosis. Since two patients had been enrolled in a prospective trial for the treatment of breast cancer which included 350 patients, the probability of developing t-AML was calculated to be 0.7% from 25-40 months, using the Kaplan-Meier method (95%, confidence interval (95% CI): 0.1-4.5). CONCLUSIONS: The combination of mitoxantrone with cyclophosphamide, fluorouracil, and radiation therapy can induce t-AML, as with other topoisomerase II-targeted drugs. Despite a low incidence, the prognosis appears to be poor.