In situ evaluation of growth fraction determined by monoclonal antibody Ki-67 and ploidy in surgically resected non-small cell lung cancers.

Ploidy and growth fraction were analyzed by means of a computer-assisted image processor in surgically resected non-small cell lung cancer (NSCLC). This study was done in order (a) to evaluate the distribution of anti-Ki-67 immunostaining and (b) to correlate this distribution to ploidy status and pTNM stage of NSCLC. Thirty-two patients underwent a surgical resection for primary NSCLC following complete staging. Indirect immunoperoxidase reactions of monoclonal antibody Ki-67 were done on frozen tissue sections. Integrated optical density and index of stained nuclear surface were calculated by means of a computer-assisted image processor in 120 fields of each preparation in order to quantify the Ki-67 immunostaining. DNA content was determined by means of cytometry of Feulgen-stained cytological prints. The ploidy status was defined for each tumor by DNA index, percentage of hypodiploid cells, and type of DNA content histogram (near diploid, hyperdiploid, hypodiploid, and multiploid). Reproducibility of immunostaining quantitative analysis was demonstrated by iterative measurements of the same slide. Intratumoral heterogeneity of Ki-67 immunostaining induced integrated optical density variation assessed on six nonconsecutive tissue sections from at least two regions of the same tumor. This intratumoral variability was 15 times lower than integrated optical density variability between tumors. The Ki-67 immunostaining varied significantly according to the DNA content histogram type (P less than 0.05, Kruskal-Wallis test); most of the specimens with high Ki-67 immunostaining were multiploid or hypodiploid. Moreover, Ki-67 immunostaining correlated to the percentage of hypodiploid cells. Ki-67 immunostaining and ploidy status did not vary significantly according to the tumor-nodes-metastasis stage. We conclude that (a) quantitative analysis of Ki-67 immunostaining is a reliable evaluation of growth fraction in NSCLC if a large number of fields are analyzed to take into account intratumoral variability, (b) hypodiploidy and multiploidy are frequent abnormalities of DNA content, (c) Ki-67 immunostaining is significantly higher in hypodiploid and multiploid tumors. Thus, determination of growth fraction and ploidy in surgically resected NSCLC specimens may be considered as complementary prognostic parameters independent of the stage of the disease.

Tumor cell membrane as a potential target for methyl-beta-cyclodextrin.

The purpose of this work was to determine the role of methyl-beta-cyclodextrin (MEBCD) in combination with doxorubicin (DOX) on DOX intracellular accumulation and efflux, in comparison to verapamil in a sensitive parental and multidrug-resistant human cancer cell line (HL-60 S and HL-60 R). Moreover, cell membrane and nuclear modifications induced by MEBCD were investigated. At concentration of 10 mumol for 10(6) cells, MEBCD combined with doxorubicin (DOX), was able to significantly enhance the intracellular concentration of DOX in HL-60 S and HL-60 R cell lines during the period of exposure. In the resistant subline, MEBCD activity was higher than that of verapamil. Moreover, treatment of cells with MEBCD resulted in a modification in cell membrane integrity and cell morphology, but had no own activity in the distribution of the cells within cell cycle.

[Should folic acid be given to women treated with valproic acid and/or carbamazepine? Folic acid and pregnancy in epilepsy]. / Faut-il prescrire de l'acide folique aux femmes traitées par acide valproïque et/ou carbamazépine? Acide folique et grossesse chez l'épileptique.

Fetal exposure to valproic acid or carbamazepine increases the risk of neural tube defect (NTD). The risk of a mother having a baby with spina bifida has been estimated at 1-2 p. 100, close to the rate of risk of recurrent cases. No study has evaluated the effect of folic acid in neonates of women treated with valproic acid or carbamazepine although the protective effect against NTD has been proven in other populations. Periconceptional folic acid supplementation, 0.4 to 1 mg/day, for at least one month prior to conception and until the date of the second missed menstrual period or later decreases the incidence of a first occurrence of neural tube defect. Periconceptional folic acid supplementation, 4 mg/day, decreases the recurrence of NTD in women who had previously had a child with NTD. It seems pertinent to recommend periconceptional folic acid supplementation in women treated with carbamazepine or valporic acid. There are very few data in women on which to base a decision to advise taking 4 mg/day (as used in recurrence prevention) or low doses of 0.4 mg/day (used in primary prevention).

[Adverse effects of glycolic irrigation solutions]. / Effets indésirables des solutions d'irrigation glycocollées.

The aim of this study was to evaluate the frequency and the gravity of Trans Urethral Resection of the Prostate Syndrome or 'TURP Syndrome,' which occurs when irrigating fluids containing 1.5 per cent glycocolle are used. All the adverse effects that occurred with 5 products containing 1.5 per cent glycocolle which were notified to the pharmacovigilance structures in France were reviewed. The adverse effects notified comprised 24 cases of TURP Syndrome and 5 of renal failure. TURP Syndrome consisted of neurological signs (92 per cent); cardiovascular signs (54 per cent); visual disturbance (42 per cent) and digestive signs (25 per cent). Hyponatraemia occurred in all patients (mean 113 +/- 6 mmol.l-1) and 25 per cent of patients died. In 27 per cent of cases TURP Syndrome occurred when glycocolle was in contravention of AMM guidelines. The Drugs Agency has requested that modification be included in the Vidal pharmacopoeia (warnings and adverse reactions) and on glycocolle bags and that surgeons and anaesthetists be informed.

[Adverse effects of the vaccines Tétracoq, IPAD/DTCP and DTCP. A French study of regional drug monitoring centers]. / Effets indésirables des vaccins Tétracoq, IPAD/DTCP et DTCP. Enquête française des centres régionaux de pharmacovigilance.

UNLABELLED: On request of the French Drug Agency, the Regional Pharmacovigilance Center (RPVC) of Tours has been in charge of the analysis of adverse events (AEs) associated with tetravalent vaccines IPAD/DTCP, DTCP and Tétracoq, and reported to the RPVC or to the pharmaceutical companies that produce them. METHODS: All AEs spontaneously reported during use of one of these vaccines to one of the French Pharmacovigilance Centers or to the responsible firms between January 1, 1986 and December 31, 1990 were take into account. An AE was noted as "serious" in accordance with the European criteria. The incidence of adverse effects was estimated by evaluating the ratio of adverse effects and the number of sales of the vaccine for the same period. RESULTS: From 1986 to 1990, 631 AEs (with 19 duplicate cases) associated with tetravalent vaccines in 606 children (75% < 1 year) were reported. The most frequent AEs were: local AEs at the site of injection (43%), neurologic disorders (12%), hyperthermia (10%) and allergic reactions (10%). Serious AEs represented 25% of all AEs and were similar to those usually described with these vaccines, particularly persistent crying (23), febrile seizures (12), apyretic seizures (14), uneasiness (28) and, rarely, shock (3). CONCLUSION: Incidences of AEs reported with pentavalent vaccines are very low, probably underestimated because of the under-notification by prescribers of AEs of vaccines licensed some time ago. It will be interesting to compare these data with AEs of penta- and hexavalent vaccines since they have replaced tetravalent vaccines.

-Skin eruption after the first dose of antitubercular quadri-therapy: consideration of pyrazinamide-. / Eruption après une première prise d'une quadrithérapie antituberculeuse: penser au pyrazinamide.

UNLABELLED: We report a rash after the first dose of antituberculosis polytherapy which raises questions concerning procedures to be followed. CASE REPORT: An 8-year-old child presented with a pruritic rash 1.5 hours after the first dose of isoniazide, rifampicine, pyrazinamide and ethambutol was simultaneously administered, which did not recur after successive re-administration of isoniazide and rifampicine. Pyrazinamide, which was re-administered last, induced a recurrence. Slower pyrazinamide re-administration allowed continuation of treatment. CONCLUSION: A protocol for re-administration of the four drugs is suggested.

[Eruption after the 1st dose of standard antitubercular chemotherapy. Thoughts on pyrazinamide]. / Eruption après la première prise d'une chimiothérapie standard antituberculeuse. Penser au pyrazinamide.

UNLABELLED: We report 3 cases of rash after the first dose of antituberculosis polytherapy, thus raising questions concerning the procedures to be followed. CASE REPORT: Three patients developed a pruritic rash 1 hour after the first dose of isoniazide, rifampicine, pyrazinamide and ethambutol given simultaneously. The eruption did not recur after readministration of isoniazide and rifampicine successively. Pyrazinamide, which was readministered last (at the full dose in one case and at progressive doses in the two others), induced a recurrence in two of them. Pyrazinamide was definitively withdrawn in one patient with recurrence and slower pyrazinamide readministration allowed continuation of treatment in the other two patients. CONCLUSION: Since pyrazinamide appeared to be responsible for rash following the first administration of antituberculosis polytherapy, a protocol for readministration of the 4 drugs is suggested. If the responsibility of pyrazinamide is confirmed it should be readministered very slowly.

[Isotretinoin (Roaccutane) in women of childbearing age: failure of following prescription guidelines]. / Isotrétinoïne (Roaccutane) chez la femme en age de procréer: insuffisance de suivi des recommandations de prescription.

BACKGROUND: Despite prominent warnings, pregnancies continue to be reported in women exposed to isotretinoin. PATIENTS AND METHODS: We report results of the analysis of 318 questions asked to pharacovigilance structures in France from 1987 to 1995 because of an exposition to isotretinoin during the risk period and of a prospective inquiry concerning isotretinoin prescription in women conducted among pharmacists. RESULTS: These 318 pregnancies began during the month after Roaccutane withdrawal (n = 104, 33 p. 100), during Roaccutane treatment (n = 163, 51 p. 100) or before Roaccutane treatment (n = 51, 16 p. 100). Of the 267 women with pregnancies conceived during treatment with isotretinoin (n = 104) or during the month after its discontinuation (n = 163), contraception was not prescribed in 28 (15 p. 100) or prescribed but with poor compliance in 109 (60 p. 100). Pregnancy was terminated voluntarily in 199 women (81 p. 100). In the 173 women who were interviewed in pharmacies, 49 (28 p. 100) did not use contraception and among them contraception was prescribed in only 59 p. 100. Only 14 p. 100 had received full information about isotretinoin and pregnancy. The teratogenic effects of isotretinoin were known by 98 p. 100 of the women and the need of contraception during treatment and for one month after discontinuation by 70 p. 100. DISCUSSION: Insufficient compliance with warnings is the main reason for pregnancies in women receiving isotretinoin therapy. A pregnancy prevention program is needed before prescription to ensure comprehension and to obtain informed consent of patients.

[Determination, by in situ hybridization on interphasic nucleus, a cytogenetic DNA index; application to breast cancer; comparison of this DNA index to DNA indexes determined by imaging and flow cytometry]. / Détermination, par hybridation in situ sur noyau interphasique, d'un index d'ADN cytogénétique; application au cancer du sein; comparaison de cet index d'ADN aux index d'ADN determinés par cytométrie en image et en flux.

In oncology, flow cytometry (FCM) and image cytometry (ICM) are commonly used to detect DNA aneuploid cell populations in solid tumors. Agreement between these two approaches is good. The use of both techniques in association minimizes the rate of FCM and ICM false negatives and gives better DNA pattern characterization, particularly for detection of any tumoral component in the FCM DNA diploid peak. Nevertheless, discrepancies exist between the FCM and the ICM DNA index values: the ICM DNA index is often greater than the FCM DNA index. The aim of the present study was to establish a cytogenetic DNA index by determining the chromosomal ploidy using a molecular cytogenetic approach and to compare it to the FCM and ICM DNA indexes. We present here the fluorescence in situ hybridization (FISH) technique we have adapted to the study of breast cancer in order to count the number of copies of the 22 + X human chromosomes in interphasic nuclei. This was achieved using a panel of 21 indirect FITC labeled probes which recognize specific chromosomic DNA sequences. Preliminary results obtained from DNA diploid and DNA aneuploid tumors are discussed.

Differential infectivity and immunopathology in murine experimental infections by two natural clones belonging to the Trypanosoma cruzi I lineage.

Immunopathology of Chagas' disease in Balb/c mice infected with 2 Trypanosoma cruzi clones, belonging to the T. cruzi I lineage and presenting different in vitro virulence (P/209 cl1 > SO34 c14) was compared. In the acute phase, evading mechanisms such as parasite-induced lymphocyte polyclonal activation and T cell immunosuppression were higher in mice infected with the clone giving a higher parasitaemia (P/209 cl1). A similar increase of non-specific isotypes was observed in both infections with IgG2a prevalence. Interestingly, CD8+ cell hypercellularity and lymphocyte immunosuppression were observed during the chronic phase (245 days post-infection) in mice infected by the most virulent clone. In the same way, the parasite-specific antibody response was more intense in P/209 cl1-infected mice over the acute phase. During the chronic phase this response remarkably dropped down in SO34 cl4-infected mice exclusively. Finally, P/209 cl1-infected mice presented a more severe inflammation and tissue damage in heart and quadriceps than SO34 cl4-infected mice. This comparative study showed differences between the two clones: a higher virulence in vivo being clearly associated with a greater ability to induce evasion mechanisms and severe tissue damage.

Distribution of T-cell receptor-bearing lymphocytes in the synovial membrane from patients with rheumatoid arthritis.

Using immunohistology and monoclonal antibodies directed to the T-cell receptor (TCR) chains, we have analysed the distribution of TCR-bearing lymphocytes within the membrane of rheumatoid arthritis (RA) patients. Alkaline phosphatase staining for TCR alpha beta-bearing lymphocytes showed a distribution paralleling that of the total T cells. Staining for the TCR gamma delta chains revealed a moderate and rather homogeneous distribution of T gamma delta lymphocytes within the RA synovium. As evidenced by simultaneous staining for alpha beta and gamma delta receptors, the relative count of T gamma delta to alpha beta-expressing cells is close to the peripheral count (e.g.5%), and lower than that previously observed in the synovial fluid. Interestingly, the peripheral type V gamma 9-J gamma P rearrangement using the T gamma delta cell subset was relatively decreased in the synovial membrane, as compared to synovial fluid and peripheral blood, suggesting that the T gamma delta distribution in the rheumatoid synovium resembles a thymic-like situation.

Characterization of proliferative cells in malignant melanomas and their inflammatory infiltrates.

Distinct changes in the antigenic phenotypes of mononuclear cells infiltrating primary and metastatic malignant melanomas (MM) have been shown to characterize distinct steps of melanoma progression. The purpose of our study is to establish whether the growth fraction of malignant melanoma cells is related to the mononuclear cell subtypes. Using monoclonal antibody Ki67, the presence of a nuclear antigen in proliferating cells of both tumor and inflammatory infiltrate cells was established in 20 primary recurrent and metastatic cutaneous melanomas. Monoclonal antibodies against lymphocyte and macrophage subsets were also applied in situ. Numerous CD8 and CD4 positive cells and natural killer (NK) cells were detected in all the infiltrates. A low CD4+/CD8+ ratio was observed in most tumors with a high proliferative activity. The presence of positive CD1+ cells seemed also to be correlated with a high activity. Our data suggest a correlation between inflammatory cell subsets and proliferative activity of MM.

Use of concanavalin A to analyse the mechanism of memory cell differentiation into killer cells.

The renewing by ConA of the cytolytic activity evaluated in a short-term chromium release assay in a population of memory cells obtained in a long-term mixed lymphocyte culture is shown to be largely dependent upon the dose of ConA used; a three staged phenomenon in terms of dose response and kinetics is analysed and suggests that at least for low concentration of ConA (0.5 micrograms/ml) the lectin acts on the same subpopulation and through the same mechanism as the specific antigen, as shown by DNA-synthesis inhibition experiments. Preincubation with ConA at doses giving the best secondary-like response strongly inhibits further response to the primary alloantigen. Experiments using mixtures of ConA and alloantigens as stimulators show that both agents can compete in differentiating memory cells into killer cells. All these data suggest an important overlap of the structures on memory cells which are triggered by ConA or specific antigen.