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The serum kinetics of vancomycin was studied in two patients aged 3 and 15 years during antibiotic therapy for catheter related sepsis associated with Staphylococcus epidermidis. Vancomycin was administered, simultaneously, by parenteral conventional doses (30 mg/kg/day div q 8 h) and using the antibiotic-lock technique in the infected catheter at a high concentration (150 mg/ml) during one hour, 3 hours after each infusion. Pharmacokinetics data did not show any significant change in the serum kinetics of the antibiotic. The results suggest that delivering a high concentration of vancomycin in the infected catheter using the lock technique may be useful to sterilize infected catheter without toxic effect.

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The authors (pediatricians and psychiatrists) report on a three-year-experience of joint interviews with parents of children with malignant diseases. These meetings, held at diagnosis and a few months afterwards, throw a light on the familial environment and help parents in expressing their feelings towards their traumatic situation.

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Micronuclei have been induced by colchicine in rat kangaroo (Potorous tridactylis) PtK1 cells. The synthesis of RNA was investigated both in isolated micronuclei by quantifying RNA polymerase activities at different ionic strengths with or without inhibitors, and in micronucleated cells by radioautography after [3H]uridine pulse labeling. In vitro transcription shows that isolated micronuclei are able to take up [3H]UTP. The rate curves of incorporation are close to those of isolated diploid nuclei, though the level of incorporation was relatively lower (65-70%) than control nuclei. This indicates that micronuclei react to the ionic environment and to inhibitors in the same manner as described for many species of isolated diploid nuclei. The labelling distributions plotted from radioautographs show that micronuclei were able to efficiently incorporate the hot precursor. Furthermore, for short pulses there is no homogeneity in the labelling density among the different micronuclei and there is no correlation between the labelling intensity and the size of micronuclei. After 60-min pulse time, there is an enhanced uptake of [3H]uridine and all the micronuclei exhibit considerable labelling, although less than control cells. Thus, the micronuclei exhibit some characteristic RNA transcriptional activity in situ as well as after isolation. This material should be a particular interesting model with which to study the physiological activity and the role of each individual interphasic chromosome.

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A 5 year 9 month-old boy has received a bone marrow allograft for beta-thalassaemia major. Conditioning included busulfan: 16 mg/kg, cyclophosphamide 200 mg/kg and a (6 Gy) thoracoabdominal irradiation. After a 16 months follow-up, the child is currently in complete remission without treatment with all the markers of his donor. His 9 year-old sister has been allografted for beta-thalassaemia major, with the same conditioning regimen. After engraftment, rejection occurred at day 85 with severe aplastic anaemia. A second graft was performed with the same donor without engraftment and the patient died at day 18 of pneumonitis. A review of the literature is proposed and the ethical choices are discussed.

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A 13 years old girl is admitted for severe chronic anemia. Few blood sac looking like lesion are discovered. A Blue Rubber Bleb Nevus Syndrome is confirmed discovery of multiple intestinal angioma. No deep lesion is discovered otherwise. Clinical characteristics, nosology and evolutive trend of this rare syndrome are recalled.

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Anti HbS antibody levels were retrospectively studied in 31 children who received at least 4 immunisations with HEVAC vaccine. Results were as follow: 305 mUI/l (6 immunocompetent patients), 263 mUI/l (4 patients with solid tumors of therapy), 26 mUI/l (16 acute leukemias receiving intermittent reinduction therapy), 0.7 mUI/l (3 acute leukemias on maintenance therapy), 0 mUI/l (2 patients allografted for severe aplastic anemia, on immunosuppressive therapy). Antibody levels of patients who received immunisation during reinduction therapy were not correlated with intervall between immunisation and previous or next reinduction, not correlated with intervall from start of therapy, but seemed inversely correlated to age at time of diagnosis and at time of immunisation.

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An appendix removed 15 days before onset of symptoms of subacute sclerosing panencephalitis was examined retrospectively for measles virus ribonucleic acid (RNA). Tissue sections hybridised in situ to a cloned measles virus probe of deoxyribonucleic acid specific for nucleocapsid protein showed that many cells of the lymphoid tissue contained measles virus RNA. In contrast, only a few infected lymphoid cells were detected in three out of six seropositive controls and none in three seronegative infants. A widespread chronic viral infection of the immune system, established after measles, may promote or even initiate nerve cell infection in subacute sclerosing panencephalitis.

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Morphological and immunocytological studies have demonstrated the presence of paramyxovirus antigens in Paget's bone disease tissue and in particular antigens related to measles virus and respiratory syncytial virus. To examine the relationship between measles virus and Paget's bone disease we used in situ hybridization and a cloned measles virus DNA probe specific for the nucleocapsid protein to detect and locate measles virus RNA sequences in Paget's bone tissue. In five patients with the disease, measles virus RNA sequences were detected not only in 80 to 90% of the multinucleated osteoclasts where there is morphological and immunocytological evidence of measles virus activity but also in 30 to 40% of mononucleated bone cells, mainly osteoblasts, osteocytes, fibroblasts and lympho-monocytes. In contrast, no hybridization was observed in bone tissue from three control patients without signs of Paget's bone disease. These results indicate that the host cell range for measles virus in Paget's disease is more widespread than has been supposed. They also demonstrate the usefulness of the in situ hybridization method to detect viral genetic information in cells where viral antigenic activity is not detectable. These observations further support the hypothesis that measles virus is involved in the pathogenesis of Paget's bone disease.

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We report here a procedure allowing to select micronuclei corresponding to defined individualized chromosomes in conditions which preserve their synthetic activity. The mammalian PtK1 cells, which possess six chromosome pairs, were micronucleated by colchicine. DNA of the micronucleated cells was labeled by the Hoechst 33342 fluorochrome under vital conditions. The micronuclei were isolated by a gentle procedure and their fluorescence was analysed by flow cytometry. The flow-cytometry parameters were determined for the analysis of non-fixed subdiploid fractions. We obtained five distinct peaks of fluorescence which have been sorted. The sorted micronuclei are different in each peak exhibiting different fluorescence intensity. Peak 3 contains the micronuclei with nucleoli and chromocenters that correspond to the X chromosome in this cell line.

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The expression of the DNA of hepatitis B virus (HBV) was analysed in three cell lines (PLC/PRF/5, Hep 3B, L6EC3) which contain the HBV DNA integrated in their genome and release the viral surface antigen (HBsAg) in relation to cell growth. Using the in situ hybridisation technique and a cloned DNA probe specific for hepatitis B virus (PTKH9), the intracellular viral RNA localisation showed that for the three cell lines, HBV RNA are present in the different cell compartments according to the age of the culture. The nucleolar and nuclear localisation are visible in the early stages of the cell growth, whereas in the later stages viral RNA are found in the cytoplasm corresponding to the maximal production of the HBsAg. These observations suggest that the nucleolus is implicated in the expression of the integrated form of HBV genetic information, the regulation of which is linked to cell growth.

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Colchicine induces the formation of small nuclei called micronuclei which contain limited parts of the genome. Some of them exhibit a DNA content equivalent to that of a single chromosome. Our purpose was to study the preservation of chromosome integrity during this micronucleation in PtK1 cells. Observation of karyotypes obtained after 3 days of cell cycle restoration revealed that micronucleation did not affect chromosome integrity or the presence of each chromosome pair in the surviving cells. In 'early restoration' cells, all the chromosomes included a centromere and were represented in the karyotype, but at variable rates. Furthermore, flow cytometry analysis of micronucleated cells, intermediate in DNA rate between control PtK1 cells in G1 and those in G2/M phases, led us to consider the possibility of selective replication of some chromosomes during micronucleation. Using antibodies against the kinetochore proteins, we derived the presence of one centromeric region (1-2 spots) in the smallest micronuclei. Therefore, these data (karyotypes, number of chromosomes, DNA content and kinetochore proteins) seem to indicate that micronucleation does not induce chromosome damages or translocations. Micronuclei are a convenient tool for investigation of the role of the different chromosomes in the organization of the interphase nuclei.

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Co-isolated residual nuclear shells and residual nucleoli from membrane-depleted rat liver nuclei were spread according to Kleinschmidt's method. Comparison of the spread residual structures isolated from nuclear shells and spread pore complex-lamina isolated from nuclear envelopes showed that these residual structures are morphologically identical. Furthermore, our nuclear shell isolation procedure allowed visualization of DNA strands bound to a granular component of the lamina. The fragmentation of nuclear shells allowed us to obtain well-spread nucleolar remnants, in which we observed DNA strands anchored on a residual nucleolar network attached to the lamina. The different molecular features revealed by the spreading of residual nucleolar structures suggest that both non-transcribing nucleolar DNA and active ribosomal genes are linked to the nucleolar network. Although the exact nature of this network remains to be defined, the results of the present study strongly suggest that the DNA molecules of the chromosomes bearing ribosomal genes have many sites of attachment to a non-chromatin nucleolar network which can be referred to as a nucleolar skeletal complex.

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A quantitative electron microscopy autoradiography method was used to locate the sites of initial rRNA transcription and to determine whether there was a variability of fibrillar centres size related to nucleolar transcription in nucleoli of sympathetic neurons of a rat killed during the dark span of its light-dark cycle. Indeed, in this model two types of fibrillar centres (nucleolar organizing regions) were observed: a single large type characteristic of this phase of the photoperiod and a small type composed by several small fibrillar centres of normal size. The present results clearly demonstrated that: there was no incorporation into the fibrillar centres themselves; the rRNA transcription sites were restricted to the ribonucleoprotein (RNP) fibrillar component and the products thereafter migrated into the RNP granular component. There was no variability of fibrillar centres size related to nucleolar transcription. The level of transcription was found to be identical around the two types of fibrillar centres. They might be considered as equivalent. The results provide evidence that at least in this model, the size of fibrillar centres and the transcriptional activity are not correlated. Other factors must therefore be responsible for the difference in size.

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To clarify the relation between lymphocytes and measles virus in subacute sclerosing panencephalitis, we used in situ hybridization and a cloned measles virus DNA probe, specific for nucleocapsid protein, to detect measles virus RNA sequences in circulating lymphocytes and brain perivascular cuffs of patients with subacute sclerosing panencephalitis. Seventy to 90 per cent of peripheral mononuclear cells from three such patients were found to contain measles virus RNA sequences. In contrast, only a few infected cells were observed in four seropositive adults (0.1 to 5 per cent) and three age-matched children (10 to 15 per cent) used as controls. In one sample of brain tissue from a patient with subacute sclerosing panencephalitis, viral RNA sequences were also detected in nerve cells and in numerous cells from the perivascular infiltrates. In contrast, no hybridization was observed in brain tissue from a patient with herpetic encephalitis and from a patient with postlymphoma encephalitis. We conclude that measles virus has a strong tropism for lymphocytes and nerve cells in subacute sclerosing panencephalitis and that lymphocytes may be involved in the pathogenesis of the disease.

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Micronuclei are small interphase nuclei containing part of the genome; the DNA content of the smallest micronuclei is equivalent to one chromosome. For analysis by biochemical method and by cytofluorometry of interphase micronuclei containing a single chromosome, several isolation and purification procedures were tested and checked by fluorescent microscopy using the DNA dye Hoechst 33 342 and electron microscopy. Micronucleation of rat kangaroo epithelial cells was induced by colchicine treatment for three days. Micronuclei were isolated in a low ionic strength buffer containing collagenase, with concomitant mechanical shocks. Eighty % of the micronuclei were released after 3 to 7 min, with minimum nuclear breakage. Subsequent filtration through several polycarbonate filters 12, 8 and 5 micron in diameter enabled purification of the smallest micronuclei without aggregates or debris. Micronuclear morphology was well preserved, as shown by electron microscope observations. Therefore, we established the optimal conditions allowing gentle mass isolation of individual micronuclei of cultured PtK1 cells, compatible with flow cytometry analysis.

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The ultrastructure and the polypeptide composition of residual nuclear substructures including nuclear matrices, nuclear ghosts, and residual envelopes were investigated by means of electron microscopy and two-dimensional gel electrophoresis. Nuclear matrices were prepared by digesting isolated nuclei with DNase I alone, followed by high-salt extraction in 2 M NaCl. Nuclear ghosts were obtained by high-salt extraction of nuclei previously digested with DNase and RNase in MgCl2-containing buffers. To prepare residual envelopes, nuclei were first digested with RNase in the presence of EDTA, then digested with Mg2+ -activated DNase I, and extracted in 2 M NaCl. The results of this comparative study support the conclusion that the intranuclear matrix is made of two distinct RNA-containing elements. One of these elements appears on ultrathin sections as a thin fibrillar network. It disappears from RNase-digested nuclei, together with numerous basic proteins, whatever the conditions of digestion. Although this element is present in extranucleolar territories, it is a major component of residual nucleoli. The second element appears as coarse-beaded fibers absent from the nucleolar areas. Its preservation in residual nuclear substructures depends on the presence of Mg2+ ions during RNase digestion of nuclei. It is enriched in two minor basic proteins of relative mass 49 000 and 70 000. The involvement of this fibrogranular element in heterogeneous nuclear RNA attachment to the nuclear matrix is discussed.

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Nucleolar organizer region (NOR) silver staining was applied to sections of fixed material. A positive reaction on cryo-ultrathin sections was found as well as on semithin and ultrathin Lowicryl sections. Repeatable staining that was easy to control was obtained by a one-step procedure after aldehyde-Carnoy fixation. Fixation of the material by formaldehyde and glutaraldehyde alone in cacodylate buffer also maintained reaction selectivity when ammonium chloride was used after fixation. Enzymatic digestion by pronase, RNase A, DNase I, or micrococcal nuclease was applied to ultrathin Lowicryl sections. Pronase digestion removed the silver-stained proteins, whereas digestion by the nucleases did not. A routine procedure is proposed for easy NOR silver staining of sections that preserves a good tissue ultrastructure and is also compatible with cytochemical and immunological investigations.

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