Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT.

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.

Isostatic compression, a new process for incorporating vancomycin into biphasic calcium phosphate: comparison with a classical method.

Isostatic compression has rarely been used to load calcium-phosphate biomaterials with therapeutic agents. This report, concerning four processes associating vancomycin, compares isostatic compression with wet granulation, a classical method. In the wet granulation study, vancomycin was associated with biphasic calcium-phosphate (BCP) granules either by adsorption or incorporation with a new granulation. In the isostatic compression study, BCP powder was compressed at 100, 140 and 200 MPa. The blocks obtained were crushed and 200-500 microm, sieved; thus, the vancomycin solution was absorbed on these granules. Compaction of BCP and vancomycin powders gave, after crushing and sieving, granules loaded with vancomycin. In each study, 5% vancomycin was associated with BCP. Vancomycin release profiles were assessed by an in vitro culture chamber dissolution test. Physicochemical studies of BCP and vancomycin showed their structural integrity after isostatic compression. Isostatic compression prolonged vancomycin release time from 3 to 7 days and the release time became greater as isostatic pressure increased, probably because of the porosity decrease of the granules during compression.

Predictive factors for node involvement in papillary thyroid carcinoma. Univariate and multivariate analyses.

For patients with papillary thyroid carcinoma, lymph node involvement is a common complication, resulting in node dissection and its resulting morbidity. To determine means of limiting lymph node dissections, we attempted to define intra-operative criteria predictive of node metastasis and so identify the patients likely to benefit from this procedure. This retrospective study concerned 158 patients (118 female) treated between 1974 and 1996 for papillary thyroid carcinoma by total thyroidectomy associated with bilateral (central and lateral) (n = 119) or unilateral (n = 39) dissection. The following criteria were used to study the predictive value of node involvement: age, sex, tumour size, tumour site, uni- or multifocality, existence or not of a tumour capsule, existence or not of perithyroid involvement and presence or not of vascular invasion. 99 patients (63%) had node involvement. Four factors showed predictive value for node involvement in univariate analysis: vascular invasion (P = 0.02), male sex (P = 0.008), absence of a tumour capsule (P < 0.0001) and perithyroid involvement (P < 0.0001). Two factors were predictive in multivariate analysis: absence of a tumour capsule and perithyroid involvement. Our results enabled us to calculate the risk of node involvement for each patient as a function of the existence of a peritumoral capsule and/or perithyroid involvement and to determine the indication for dissection. When neither of these factors was present, the risk of node involvement was 38.3% and dissection was not considered essential. If both risk factors were found, the risk was 87.1% and dissection was considered necessary.

Long-term follow-up of Vena Tech-LGM filter: predictors and frequency of caval occlusion.

PURPOSE: To report the frequency of caval occlusion after Vena Tech-LGM filter placement and identify related factors and their potential clinical significance. MATERIALS AND METHODS: The filter was inserted into 243 patients, 142 of whom met inclusion criteria for this prospective study. Follow-up examinations performed every 2 years included clinical evaluation, plain frontal radiography of the abdomen, duplex scanning of the inferior vena cava (IVC), and/or phlebocavography. RESULTS: A progressive decrease in IVC patency was observed, reaching 66.8% at 9 years of follow-up. Complete caval occlusion occurred in 28 patients and was significantly (P < 10(-6)) associated with retraction in 24 cases. Caval occlusion was not related to age, sex, pulmonary embolism (PE), deep venous thrombosis level, underlying conditions predisposing to a thromboembolic disease before filter insertion, the level of filter placement, use of anticoagulant therapy, and death during follow-up. PE with anticoagulation failure was a predictive factor (P = .016) of subsequent filter occlusion during follow-up as compared to all other clinical indications for filter placement. Filter patency at 9 years of follow-up was 35.2% in the PE group with anticoagulation failure and 80% for other patients (odds ratio, 2.5; 95% confidence interval 1.16-5.4). CONCLUSION: PE with anticoagulation failure was the only factor predictive of subsequent caval occlusion observed in patients after Vena Tech-LGM filter placement. Caval occlusion was also related to Vena Tech-LGM filter retraction, which usually occurred at the time of occlusion.

[Using capture-recapture models to estimate transition rates between states in interval-censored data]. / Utilisation des modèles de capture-recapture pour l'estimation de taux de transitions entre etats en présence de censure par intervalles.

We introduce capture-recapture models, survival models developed in animal population biology to estimate survival / transition rates between sites within a population. We then show how to use these models in epidemiology or clinical research, to estimate survival / transition rates between stages of a disease (e.g. cancer, AIDS...), when patients enter a longitudinal study with imperfect follow-up resulting in interval-censoring. This method yields unbiased estimates, since the compliance (defined here as the probability of visiting the doctor at the planned date) is modelled jointly. We apply the method to cancer data, and show that "time" (elapsed since entry of a patient in the study) affects compliance in these data.

[Optimization of parasitological diagnosis of human intestinal microsporidiosis]. / Optimisation du diagnostic parasitologique des microsporidioses intestinales humaines.

The diagnosis of microsporidiosis by staining stools is known to be fast and cheap. To obtain a specific and sensitive result, two colorimetric methods must be used: staining by the fluorochrome Uvitex 2 B (VAN GOOL) and trichrome. Among the four staining methods of trichrome currently studied, the WEBER coloration could be considered as the most efficient. The density of microsporidia spores could be semi-quantitatively evaluated, because their distribution is homogeneous.

Achalasia: outcome of patients treated with intrasphincteric injection of botulinum toxin.

BACKGROUND: To evaluate the safety and clinical efficacy of botulinum toxin (BT) in patients with achalasia followed up for six months. METHODS: Fifty five symptomatic patients with manometrically proven achalasia were included in a multicentre prospective trial. Before and two weeks and two months after intrasphincteric injection of BT, symptoms of dysphagia, regurgitation, and chest pain were scored on a 0-3 scale, and lower oesophageal sphincter pressure (LOSP) was assessed. The symptom score was determined again at six months, clinical improvement being defined by < or = 3, relapse by > 3, and failure as a relapse after two injections or loss to follow up. RESULTS: Except for transient chest or epigastric pain (22%), no side effects were observed. There was a significant decrease in LOSP after treatment. Symptom scores were significantly improved at two weeks (2.0 (SD 1.6)), two months (1.7 (1.8)), and six months (1.9 (2.0)) compared with pretreatment values (5.1 (1.8), p < 0.001). At six months, 33 patients had clinical improvement (27 after one injection), 17 were considered failures, and five had just relapsed. Although there was a trend for age (older patients being more responsive), age, sex, prior duration of symptoms, initial symptom score, weight loss, LOSP, magnitude of oesophageal contractions, vigorous or non-vigorous achalasia, previous dilatations, and radiological features were not predictive of results. CONCLUSIONS: This multicentre series confirms that intrasphincteric injection of BT is a safe procedure, resulting in clinical improvement in 60% of patients with achalasia at six months. The therapeutic role of BT in achalasia needs further evaluation with regard to other alternatives.

Comparative targeting of human colon-carcinoma multicell spheroids using one- and two-step (bispecific antibody) techniques.

In the perspective of radioimmunotherapy (RIT) of micrometastases, we compared, in multicell spheroids (MS), the uptake and retention kinetics of 125I-F(ab)'2 F6 anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), and the affinity enhancement system (AES) using an anti-CEA/anti-DTPA-indium bispecific antibody (BsMAb) and a 125I-labeled di-DTPA-In-tyrosine-lysine bivalent hapten. We used MS of colorectal tumor cell lines expressing CEA strongly (LS 174T), weakly (HT-29) or not at all (HRT-18). Uptake and retention kinetics of 125I-F(ab)'2 F6 and 125I-BsMAb used alone gave similar results. The highest uptake values, obtained with LS 174T MS, were slightly lower with AES than with 125I-F(ab)'2 F6. However, effective retention half-lives were longer for AES than for 125I-F(ab)'2 F6 or for 111In-labeled monovalent hapten after pre-incubation of spheroids with BsMAb. Autoradiography showed the same slow and heterogeneous distribution of 125I-F(ab)'2 F6 and 125I-BsMAb. These results indicate that the 2-step technique is more favorable for RIT: uptake values were approximately the same but uptake kinetics were more rapid, and retention half-life was longer than with the one-step technique.

Comparative study of risk factors in patients undergoing coronary or femoropopliteal artery bypass grafting.

OBJECTIVE: To compare risk factors in two populations of patients with advanced atheroma requiring coronary or femoropopliteal artery bypass grafting to try to account for the different localisations of vascular disease. DESIGN: Cross sectional epidemiological study. SETTING: Cardiovascular surgery department of a university hospital. SUBJECTS: 464 men (mean age 59.25 (SD 8.57) years) undergoing coronary artery bypass grafting; 74 men (mean age 56.28 (13.3) years) undergoing femoropopliteal artery bypass grafting; and 204 control men (mean age 45.07 (6.59) years) who had been recruited in a preventive medicine department. INTERVENTIONS: Blood samples were drawn 24 hours before surgery. METHODS: Lipid and lipoprotein concentrations were measured for each patient and with adjustment for age were compared by analysis of covariance. The main risk factors (smoking, arterial hypertension, obesity, and diabetes) were determined by a standardised history, and the chi 2 test was used to compare the results in the two patient groups. Pairwise comparisons between the three populations were performed by logistic discriminant analysis. RESULTS: Both patient groups showed a significant rise in triglyceride concentration and in the ratio of total cholesterol to high density lipoprotein cholesterol (R1) and a drop in apolipoprotein AI and high density lipoprotein cholesterol concentrations. Disturbances were greater in patients undergoing coronary artery bypass grafting than in those undergoing femoropopliteal artery bypass grafting for the R1 ratio, apolipoprotein B concentration, and the ratio of apolipoprotein AI to apolipoprotein B (R2). A higher proportion of smokers was found in the femoropopliteal bypass group than in the coronary bypass group, whereas were often obese. Logistic discriminant analysis with adjustment for age and with the coronary bypass as the reference group selected three factors: smoking, the R2 ratio, and obesity. CONCLUSION: Disturbances in lipid and apoprotein concentrations varied with respect to bypass site. Other risk factors played a part in accelerating the atherogenic process, especially smoking in patients undergoing femoropopliteal artery bypass grafting and, to a lesser degree, obesity in patients undergoing coronary artery bypass grafting.

Modulation of associated ovarian carcinoma antigens by 5 cytokines used as single agents or in combination.

Optimization of intraperitoneal radioimmunotherapy of ovarian cancer depends on increasing the antigenic expression of tumor cells. For this purpose, we studied the effect of 5 cytokines (IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha and TGF-beta), used as single agents or in combination, on 4 ovarian cancer cell lines which present different antigenic profiles with the monoclonal antibodies (MAbs) tested (OC125, OVTL-3, MOv 18 and MOv 19). Analyses were performed by flow cytometry and the Scatchard technique in order to study antigenic modulation. The effect on proliferation was determined by cell counting. Expression of O3 antigen, recognized by the OVTL3 MAb, was increased up to 2.5 times after IFNs and TNF-alpha (used as single agent) on the 2 lines presenting low basal expression (SHIN-3 and IGROVI). The expression of CA125 antigen and the antigens recognized by MOv 18 and MOv 19 MAbs was not increased by any of the cytokines tested. The combination IFN-gamma+TNF-alpha was synergistic on cytotoxicity and enhanced O3 expression, providing 10 times as many sites per cell on the SHIN-3 line. For 3 other associations (IFN-alpha+IFN-gamma, IFN-beta+IFN-gamma and IFN-alpha+TNF-alpha), there was an additive effect on O3 expression and on cell cytotoxicity.

Haemophilus type B conjugate vaccine: postimmunization kinetics of IgM and IgG antibody responses in ten vaccinated children.

A new Haemophilus type b conjugate vaccine coupling capsular polysaccharide of Haemophilus influenzae b to tetanus toxoid is available in France and other countries. We have studied the kinetics of the immune response in ten children aged 17 to 50 months during the 4 weeks after immunization with one dose of Haemophilus type b conjugate vaccine. Eight serum samples were collected from each child at day 0 (D0), D2, D4, D7, D10, D14, D21 and D28. An ELISA method has been used to discriminate between IgM and IgG classes of anti-polyribosylribitol phosphate antibodies. A high level of IgM appeared at D7 and persisted until D28. The increase in IgG was regular and progressive from D7.

Effects of low doses of lorazepam on psychometric tests in healthy volunteers.

The effects of low oral doses of lorazepam on several cognitive and performance tasks were investigated in 50 healthy students. A double-blind, parallel group design was used to compare five treatments: placebo and lorazepam 0.5, 0.75, 1 mg and progressive doses up to 1.5 mg. After randomization, all subjects received placebo for 3 days in a single-blind procedure followed by five consecutive days of treatment. Subjects completed a battery of tests each day of the 5 days active treatment and the day after stopping the treatment. There were no significant differences between placebo and lorazepam on the free recall test and the critical flicker fusion frequency test, but lorazepam produced significant improvement on the digit symbol substitution test and the choice reaction time test. We suggest that low repeated doses of lorazepam in healthy subjects improve the psychomotor performance without sedation and memory impairment.

Cerebrospinal fluid neopterin levels in children with central nervous system leukemia.

Cerebrospinal fluid (CSF) neopterin levels were determined by high-pressure liquid chromatography in 48 normal children and in 15 children with meningeal relapse of hematologic malignancies (13 acute lymphoblastic leukemia and 2 high-grade lymphomas). When meningeal relapse was diagnosed, all patients had CSF neopterin levels higher than mean normal value +2 standard deviations. No significant correlation between the blast count in the CSF and neopterin levels was observed. CSF data before relapse were available in 10 children: the neopterin values at relapse were significantly higher than values observed at diagnosis. In 3 patients, elevated neopterin levels preceded the occurrence of neurologic signs and the detection of blast cells in CSF by 15 to 30 days. In the absence of infection, the rise of CSF neopterin levels in patients with hematologic malignancies indicates an active phase of the disease. This could reflect a cell-mediated immunologic process induced by malignant cells. The measurement of CSF neopterin should be helpful in the monitoring of patients to detect early meningeal relapse.

Magnesium, total calcium, phosphorus, copper, and zinc in plasma and erythrocytes of venous cord blood from infants of diabetic mothers: comparison with a reference group by logistic discriminant analysis.

Concentrations of magnesium (Mg), total calcium (Ca), phosphorus (P), copper (Cu), and zinc (Zn) were investigated in plasma (Pl) and erythrocytes (Erc) of venous cord blood of 44 infants of diabetic mothers (IDMs). These same concentrations plus total glycohemoglobin and fructosamine were determined at delivery in a subset of 15 mothers of these infants. Mineral results for IDMs were compared with those for 66 apparently healthy newborns. The duration of gestation in the two groups was significantly different (P < 0.001). After adjustment for gestational age, the mean (+/- SD) differences between groups were significant for birth weight, head circumference, Erc-Mg (1.71 +/- 0.17 for IDMs vs 1.76 +/- 0.15 mmol/L for control subjects), Pl-Ca (1.96 +/- 0.32 vs 2.48 +/- 0.22 mmol/L), Pl-P (1.99 +/- 0.40 vs 1.57 +/- 0.25 mmol/L), and Erc-Cu (10.9 +/- 2.41 vs 12.9 +/- 3.00 mumol/L), but not for Erc-Zn (33.0 +/- 18.3 vs 40.4 +/- 13.6 mumol/L). The variable that best discriminated between the two infant groups after adjustment for gestational age was Pl-Ca. In the 15 mothers, Pl-Mg (0.67 +/- 0.07 mmol/L) and Pl-Ca (1.66 +/- 0.21 mmol/L) concentrations were low, Pl-Zn (9.81 +/- 3.40 mumol/L) was normal, and Pl-Cu (33.5 +/- 10.7 mumol/L) was above normal. Correlations between total glycohemoglobin and mineral values of the mothers or paired IDM mineral values were not significant. The concentration of Pl-Ca was positively correlated with Erc-Cu (P < 0.001) and Pl-Cu (P < 0.05) in the comparison group newborns but not in the IDMs.

Soluble interleukin 2 receptor (Tac chain) is not a reliable marker in kidney transplant recipient monitoring.

T lymphocyte expansion is triggered through interaction of interleukin 2 (IL-2) with its high-affinity receptor (IL-2R). This molecule is a heterodimer comprising an antigen-inducible component, the Tac chain (P55). Activation of T lymphocytes also generates a soluble form of this P55 called S-IL-2R. S-IL-2R is elevated in many T-cell-related pathologies (leukemia, autoimmunity, etc.). In graft recipients, rejection is a result of T-cell activation by graft antigens and therefore might induce a release of S-IL-2R in the circulation; this parameter is now said to be a good indicator of rejection. We have performed a study in renal graft recipients in order to assess the usefulness of circulating S-IL-2R particularly to discriminate the origin of renal failure in cases of rejection or of cyclosporin-A (CsA)-induced nephrotoxicity. We demonstrated that there are no differences between isolated values in the clinical groups at the time of diagnosis. Variations in S-IL-2R are increased compared to steady-state periods during rejection and cytomegalovirus infections, although not in CsA toxicity episodes. However, at the individual level there are too many false-positive and false-negative results, making this parameter no more meaningful than serum creatinine levels alone or even in association (as tested in logistic discriminant analysis). In addition, it seems that the variations in S-IL-2R are partly related to renal function itself, as suggested by the correlation between S-IL-2R levels and serum creatinine levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Association between magnesium, calcium, phosphorus, copper, and zinc in umbilical cord plasma and erythrocytes, and the gestational age and growth variables of full-term newborns.

We determined reference values in umbilical cord plasma and erythrocytes for magnesium, total calcium, phosphorus, copper, and zinc, and then calculated correlations and stepwise-regression equations in 66 white full-term newborn infants (35 boys, 31 girls). Only infants meeting certain optimal criteria and benefiting from excellent maternal conditions and uncomplicated pregnancies were included. There were no significant sex-related differences at birth among the variables studied. Gestational age was positively correlated with erythrocyte zinc (P less than 0.001), and plasma calcium was positively correlated with erythrocyte copper (P less than 0.001). Plasma copper proved to be the most significant variable in the stepwise-regression equation for birth height as the dependent variable. The most significant regressors accounting for birth weight were erythrocyte zinc followed by plasma zinc.

Study of lipoprotein particles LpAI and LpAI:AII in patients before coronary bypass surgery.

Lipids, apolipoproteins, and LpAI and LpAI:AII particles were studied in 43 men (mean age 51, SD 7, years) 24 h before their coronary bypass surgery and in 54 control men (mean age 46 SD 9, years). LpAI and LpAI:AII were analyzed by electroimmunodiffusion and by a noncompetitive enzyme-linked immunoassay, respectively. Concentrations of LpAI and LpAI:AII in the bypass patients were significantly lower (P less than 0.001) than those in the controls. Apolipoprotein AI was significantly correlated with LpAI (P less than 0.001) and LpAI:AII (P less than 0.01) in controls, but only with LpAI:AII (P less than 0.001) in bypass patients. Discriminant analysis between controls and patients showed apolipoprotein AI to be the most powerful discriminant factor; the addition of LpAI and LpAI:AII did not improve discriminant power. We conclude that the determination of LpAI and LpAI:AII particles reflects metabolic disorders in patients but does not significantly influence the discrimination of such patients into risk groups.

Logistic discriminant analysis of lipids and apolipoproteins in a population of coronary bypass patients and the significance of apolipoproteins C-III and E.

The lipid and apolipoprotein states of 74 men (mean age 49.96 +/- 5.9 years) were studied 24 h before coronary bypass surgery and their results were compared with those of a control group of 78 men (mean age 48.88 +/- 5.41 years). Apolipoproteins C-III (apo C-III) and E (apo E) were determined in particles with (LpB) and without (nonLpB) apo B separated using a concanavalin A reagent. Apo C-III was significantly increased in LpB particles (P less than 0.001), and apo E in LpB (P less than 0.001) and nonLpB (P less than 0.001) particles. The significant variables selected in logistic discriminant stepwise analysis were total cholesterol/HDL-cholesterol, apo E-nonLpB and apo C-III-LpB. This last parameter, which is more discriminant than triglycerides, provides a more specific indication of dyslipoproteinemia in coronary bypass patients; in association with the other two variables, it significantly improved the percentage of correctly classified individuals.