[Haemophilia and dental procedures; a complex combination]. / Hemofilie en tandheelkundige behandelingen; een complexe combinatie.

A 28-year-old patient with severe haemophilia A presented to the emergency department with significant and painful swelling of the left cheek, an extensive haematoma extending from the left ear to the anterior thoracic region, an intraoral haematoma over the soft palate with deviation of the uvula to the right, and complaints of shortness of breath when lying down. Three days prior, his dentist had performed a restoration of the 36 molar under local anaesthesia. Due to pain, the general practitioner had administered an intramuscular injection of a non-steroidal anti-inflammatory drug (NSAID) two days post-procedure. The patient was admitted for treatment with coagulation factors and pain management. Dental procedures and local anaesthesia in patients with a severe coagulation disorder require specific preparatory measures, such as administration of coagulation factors. Collaboration and consultation with a patient's haematologist or haemophilia treatment centre are essential requirements for safe dental care.

Are NIRS-derived cerebral autoregulation and ABPopt values different between hemispheres in hypoxic-ischemic brain injury patients following cardiac arrest?

PURPOSE: Near-infrared spectroscopy (NIRS) has been suggested as a non-invasive monitoring technique to set cerebral autoregulation (CA) guided ABP targets (ABPopt) in comatose patients with hypoxic-ischemic brain injury (HIBI) following cardiac arrest. We aimed to determine whether NIRS-derived CA and ABPopt values differ between left and right-sided recordings in these patients. METHODS: Bifrontal regional oxygen saturation (rSO2) was measured using INVOS or Fore-Sight devices. The Cerebral Oximetry index (COx) was determined as a CA measure. ABPopt was calculated using a published algorithm with multi-window weighted approach. A paired Wilcoxon signed rank test and intraclass correlation coefficients (ICC) were used to compare (1) systematic differences and (2) degree of agreement between left and right-sided measurements. RESULTS: Eleven patients were monitored. In one patient there was malfunctioning of the right-sided optode and in one patient not any ABPopt value was calculated. Comparison of rSO2 and COx was possible in ten patients and ABPopt in nine patients. The average recording time was 26 (IQR, 22-42) hours. The ABPopt values were not significantly different between the bifrontal recordings (80 (95%-CI 76-84) and 82 (95%-CI 75-84) mmHg) for the left and right recordings, p = 1.0). The ICC for ABPopt was high (0.95, 0.78-0.98, p < 0.001). Similar results were obtained for rSO2 and COx. CONCLUSION: We found no differences between left and right-sided NIRS recordings or CA estimation in comatose and ventilated HIBI patients. This suggests that in these patients without signs of localized pathology unilateral recordings might be sufficient to estimate CA status or provide ABPopt targets.

Association between Bone Mass and Dental Hypomineralization.

The aim of this study was to examine the association between the bone mass (bone mineral content [BMC]) and hypomineralized second primary molars (HSPMs)/molar incisor hypomineralization (MIH) in 6-y-old children. This cross-sectional study was embedded in the Generation R Study, a population-based prospective cohort study, starting from fetal life until adulthood in Rotterdam, Netherlands. The European Academy of Pediatric Dentistry criteria were used to score the intraoral photographs on the presence or absence of HSPMs and MIH. Bone mass was measured with a dual-energy x-ray absorptiometry (DXA) scan. Intraoral photographs and DXA scans were available in 6,510 6-y-old children. Binary logistic regression models were used to study the association between the bone mass and HSPMs/MIH. In total, 5,586 children had their second primary molars assessed and a DXA scan made; 507 children were diagnosed with HSPM. Of 2,370 children with data on their permanent first molars, 203 were diagnosed with MIH. In the fully adjusted model, children with lower BMC (corrected for bone area) were more likely to have HSPMs (odds ratio, 1.13; 95% confidence interval, 1.02 to 1.26 per 1-standard deviation decrease). A lower BMC (corrected for bone area) was not associated with MIH (odds ratio, 1.02; 95% confidence interval, 0.87 to 1.20 per 1-standard deviation decrease). We observed a negative association between BMC (corrected for bone area) and HSPMs. No association was found between BMC (corrected for bone area) and MIH. Future research should focus on investigating the mechanism underlying the negative association between the bone mass and HSPMs. Our study, in a large population of 6-y-old children, adds the finding that BMC (corrected for bone size) is associated with HSPMs but not with MIH in childhood.

Environmental risk assessment of musk ketone and musk xylene in The Netherlands in accordance with the EU-TGD.

An environmental risk assessment has been carried out for musk ketone and musk xylene according to the EU Technical Guidance Document for Environmental Risk Assessment for New and Existing Substances [1]. Musk ketone and musk xylene are used in fragrances for cosmetics and household products. For the fragrance industry these are important fragrance ingredients because of their excellent substantivity as well as for their unique smell, which determines largely the odor of a product. The initial environmental risk assessment is based on information provided by the fragrance industry as represented in the Netherlands by its association NEA, by the Research Institute for Fragrance Materials (RIFM) and data reported in the international open literature. The risk assessment includes and evaluation of the risks for aquatic organisms in surface water and sediment and for soil organisms in soil after application of sewage sludge. Secondary poisoning of fish-eating birds and mammals is considered as well. For each compartment the Predicted Environmental Concentration (PEC) is compared to the Predicted No Effect Concentration (PNEC) to obtain PEC/PNEC ratios. Since monitoring data are available in water, sediment and fish, similar ratios are obtained with measured concentrations instead of the predicted ones. For both substances, PEC/PNEC ratios are at or below 0.1 for organisms in the aquatic environment, including sediment organisms. PEC/PNEC ratios for fish-eating predators are 0.01. Ratios based on monitoring data are below 0.01 for all of these organisms. For soil organisms the PEC/PNEC ratio is 0.5 for musk ketone and 1.3 for musk xylene. Although in the Netherlands (as well as in some other European countries), sewage sludge presently finds no application as fertilizer on agricultural soil, the aim of environmental policy is to upgrade the sludge quality to enable future applications on agricultural and grassland. The reliability of the predicted soil concentrations can be greatly improved by obtaining experimental data on fate and behaviour of musk ketone and musk xylene in digested sludge and soil. The risk assessment provides reassurance for the aquatic compartment while pointing the way for obtaining additional data for the soil compartment.

Lethal body burden of triphenyltin chloride in fish: preliminary results.

1. Guppies exposed to several triphenyltin chloride (TPTC) concentrations in water died as soon as a body burden of 20 +/- 10 nmol/g fish was reached. 2. Accumulation of TPTC during exposure in acute toxicity experiments can be predicted by using the kinetic parameters of TPTC. 3. The lethal body burden is two orders of magnitude lower than for narcotic organic compounds such as chlorobenzenes.

Effect of a Trichinella spiralis infection on the distribution of mast cell precursors in tissues of thymus-bearing and non-thymus-bearing (nude) mice determined by an in vitro assay.

The frequency of precursor cells capable of giving rise to cells with characteristics of mucosal mast cells in tissues from thymus-bearing and non-thymus-bearing (nude) mice orally infected with Trichinella spiralis was determined with an in vitro assay. Analysis of the frequency of mast cell precursors in bone marrow, blood, spleen and small intestinal tissue revealed similar frequencies of mast cell precursors in bone marrow from both thymus-bearing and athymic mice. These frequencies in bone marrow were not affected by infection. However, in blood and spleen from thymus-bearing mice at Day 7 post-infection (p.i.), and in the gut at Day 14 p.i., significant increases of mast cell precursor frequencies were detected. In contrast, no significant increase was observed in the tissues of infected nude mice. These data are in accordance with in vivo findings, indicating that a mucosal mast cell response in the gut is both thymus and antigen dependent. It was concluded that a mucosal mast cell response to infection with T. spiralis is probably due to local proliferation and maturation of residing mast cell precursors, that this response might be amplified by an influx of precursor cells from the blood into the gut, and that both phenomena are T-cell dependent.

DNA and nuclear protein measurement in isolated nuclei of human endometrium.

Feulgen-DNA and nuclear light green-protein measurements have been performed in isolated nuclei of normal (nonmalignant) and malignant human endometrial homogenates. The DNA content of the G0/G1 fraction of malignant endometrium showed much overlap with that of normal endometrium, or was slightly increased. Two of the 18 carcinomas were clearly aneuploid. No correlation was found between the histological grade and the DNA content. The tumors of clinical stage II and higher all had a higher DNA content than that of normal endometrium. The percentage of cells present in the proliferative fraction was higher in proliferative endometrium than in secretory and post-menopausal atrophic endometrium. For malignant endometrium, percentages were found comparable to that of normal endometrium or higher. No correlation was found with the histological grade. Tumors of stage II and higher had intermediate values compared to those of carcinomas below stage II. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. However, for post-menopausal women, most values of the carcinomas exceeded that of normal, atrophic, endometrium. Within the tumor population, no correlation was found with the histological grade. Higher values were found with tumors of clinical stage II and higher.

DNA and nuclear protein measurement in columnar epithelial cells of human endometrium.

Propidium iodide DNA flow cytometry, Feulgen-DNA, and nuclear light green protein scanning cytometry were performed in columnar epithelial cells of normal, nonmalignant human endometrium and endometrial adenocarcinomas. Columnar cells were identified by immunohistochemical staining for cytokeratin 18, an intermediate filament protein specifically present in columnar cell epithelium. DNA measurements derived from flow and scanning cytometry showed comparable results. The DNA content of the G0/G1 fraction of the adenocarcinomas had a considerable overlap with that of normal endometrium, with that of the carcinomas shifted toward higher values. For the carcinomas, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas. Most of the clinical stage I tumors showed a DNA content in the normal diploid region. Three of the four carcinomas of clinical stage II and higher had an increased DNA content. For the carcinomas, the percentage of cells in the proliferative fraction, as determined from scanning cytometric derived DNA histograms, was comparable to that of normal endometrium, or higher. No correlation was found with the histological grade. Tumors of clinical stage II and higher had intermediate values compared to carcinomas of lower stages. The nuclear protein/DNA ratio of malignant endometrium completely overlapped that of normal endometrium. Within the tumor population, no correlation was found with the histological grade, with the exception of the adenosquamous carcinomas, and clinical stage. Based on the aforementioned parameters, no discrimination could be obtained between normal and malignant endometrium. However, when the DNA content of the G0/G1 fraction was combined with the coefficient of variation of the nuclear protein/DNA ratio, a clear discrimination could be obtained with only two false-positive cases.

Detection of metabolic changes in hepatocytes by quantitative cytochemistry.

Studies by means of quantitative histochemistry and cytochemistry have greatly contributed to the knowledge of metabolic changes in liver parenchymal cells. In the present paper recent work along this line is reviewed with emphasis on three topics, polyploidy as a source of metabolic heterogeneity, proteolysis in the regulation of hepatocyte cell mass and ischemic injury of hepatocytes. In all three fields, accuracy and precision of information obtained by quantitative histochemical means has been greatly enhanced by a thorough knowledge of the mechanisms of histochemical reactions obtained by fundamental work on matrix chemistry, and well-considered application of optical measuring tools and conditions of measurement. These are the principles put forward by van Duijn since the pioneer period of histochemistry and to whom this review is dedicated.

A visible DNA-protein stain: Feulgen-Pararosanilin(SO2) Light Green.

Feulgen-Pararosanilin(SO2) Light Green, a DNA-protein stain, is described that is suited both for visual analysis and quantitative cytochemical measurement. The stain has been applied on cervical cells and quantitative aspects have been studied on chicken erythrocyte and rat liver nuclei. In comparison to single staining, the Feulgen-Pararosanilin(SO2) DNA content of the nuclei remains unaltered after application of the combined staining. The Light Green protein content is reduced considerably, however, dependent on the degree of chromatin condensation in the nucleus.

In vitro studies on mast cell proliferation in N. brasiliensis infection.

We have previously shown that mast cells with the morphological and biochemical properties of mucosal mast cells (MMC) proliferate and mature in rat bone marrow cultures stimulated with factors from antigen or mitogen-activated T lymphocytes. Here we have used this system to explore the MMC hyperplasia which occurs in infections with gastrointestinal nematode parasites. Lymphocytes producing MMC-growth factor were present from day 10 onwards in N. brasiliensis-infected rats and mesenteric lymph nodes (MLN) were the major source of activated lymphocytes. When different tissues of normal rats were cultured in the presence of conditioned medium by far the greatest proliferation of MMC occurred in bone marrow, indicating an origin of MMC from haemopoietic precursors. Cultures of infected rat bone marrow yielded considerably greater numbers of MMC than cultures of normal rat bone marrow and experiments using semisolid culture media indicated that N. brasiliensis infection causes an increase in the frequency of MMC progenitors in the bone marrow. A scheme is put forward for the sequence of events occurring in vivo based on the results of these and other published experiments. The reasons for the restricted in vivo localization of MMC to the mucous membranes and associated lymph nodes is discussed. Finally we give the results of microspectrophotometric analysis which has shown that the cultured mast cell contain a non-heparin proteoglycan, thus adding a further feature to the list of MMC-like properties of these cells.

Glucose-6-phosphate dehydrogenase activity in individual rat hepatocytes of different ploidy classes. I. Developments during postnatal growth.

The glucose-6-phosphate dehydrogenase (G6PDH) activity of isolated male rat hepatocytes has been investigated in relationship to the ploidy classes of the cells during the first 20 weeks of postnatal growth. The G6PDH activity in the individual cells was measured with an improved quantitative cytochemical method. The data obtained showed that throughout the whole period of postnatal growth there existed a proportional relationship between the genome copies per cell and the amount of G6PDH activity per cell for binuclear diploid (BD), mononuclear tetraploid (MT) and binuclear tetraploid (BT) cells but not for mononuclear diploid (MD) cells. In the MD cells, which are the stem cells of the liver parenchyma, the activity measured was 1.5 times higher than expected. Furthermore, during postnatal growth, the G6PDH activity per hepatocyte was low at the age of 2 weeks, increased somewhat after weaning (5 weeks) and then more dramatically after 8 weeks to reach a maximum between 12 and 16 weeks. This development occurred in MT and BT cells at an earlier age than in MD and BD cells, in which the increase in enzyme activity followed some 3 weeks later. Castration of the rats before puberty did not influence the development of the amount of G6PDH activity per cell of any of the ploidy classes.

The use of Light Green and Orange II as quantitative protein stains, and their combination with the Feulgen method for the simultaneous determination of protein and DNA.

The protein dyes Light Green and Orange II were studied separately and in combination with the Feulgen-Pararosanilin(SO2) and -Thionin(SO2) method for the simultaneous determination of DNA and protein. - With polyacrylamide modelfilms the pH dependency, specificity and stoichiometry of Light Green and Orange II have been investigated. The results of both staining methods with different biological objects have been compared. - In addition, the Feulgen-Thionin(SO2) method was studied with model films with respect to its specificity and stoichiometry. In biological objects it has been compared with the Feulgen-Pararosanilin(SO2) method. - When combining the Light Green staining with the Feulgen-Pararosanilin(SO2) procedure and the Orange II staining with Feulgen-Thionin(SO2), both Feulgen-DNA stainings, which were first applied, proved to be unaffected by the following protein staining procedure. When the Feulgen procedure was carried out without the dye, followed by Light Green staining, the latter became reduced when a sulfite water rinse was included but was unaffected when a running tap water rinse was used. In the case of the Orange II staining a serious reduction in dye binding capacity was found in both situations. - When the Feulgen-Pararosanilin(SO2) Light Green procedure was carried out on isolated nuclei with all dyes present, a decrease of protein dye binding was observed, similar to that found with the well-known Feulgen-Pararosanilin(SO2) Naphthol Yellow S combination. It is concluded that in spite of this reduction the latter two combinations can be used for the cytophotometric analysis of DNA and protein in the same object.

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. II. Practical applications for light microscopy.

The application of the new nucleic acid dye Cuprolinic Blue to cell smears and tissue sections has been described. Without added cations, Cuprolinic Blue stains both DNA and RNA, whereas in the presence of 1 M MgCl2, Cuprolinic Blue specifically stains single-stranded RNA only. The total RNA can be stained after removal of DNA by DNAase digestion. Fixation in a modified Carnoy solution gave optimal staining results in all cases tested. By cytophotometry, a reliable and reproducible relative estimate can be obtained of the total nucleic acid content, the total RNA content and the amount of single-stranded RNA alone per cell.

Cuprolinic Blue: a specific dye for single-stranded RNA in the presence of magnesium chloride. I. Fundamental aspects.

Qualitative and quantitative aspects of the cationic dye Cuprolinic Blue were investigated with model films of polyacrylamide gel in which RNA, DNA and other biological polyanionic compounds had been incorporated. In the presence of 1 M MgCl2, Curpolinic Blue was found to bind specifically to single-stranded RNA, leaving native DNA, proteins, (acid) polysaccharides and phospholipids completely unstained. Under these conditions, Cuprolinic Blue is complexed by non-electrostatic bonds with non-stacke purine bases, mainly adenine. Optimal conditions for dye binding and differentiation have been defined. Both the Cuprolinic Blue-MgCl2 staining of single-stranded RNA and the Cuprolinic Blue staining of RNA and DNA in the absence of MgCl2 were found to obey the Lambert-Beer law. The advantages and possible applications of Cuprolinic Blue are compared with well-known (indirect) histochemical RNA staining procedures.

Cytophotometry of glucose-6-phosphate dehydrogenase activity in individual cells.

With the aid of thin films of polyacrylamide gel containing purified glucose-6-phosphate dehydrogenase subjected to cytochemical procedures for the enzyme using tetranitro blue tetrazolium, arbitrary units of integrated absorbance obtained with a Barr & Stroud GN5 cytophotometer were converted into units of enzyme activity. This conversion enabled cytochemical data to be compared directly with biochemical values. The conversion was applied to the cytochemical estimation of glucose-6-phosphate dehydrogenase activity in isolated rat hepatocytes, mouse oocytes, rabbit thymocytes, human granulocytes and human fibroblasts. Several control procedures were performed to confirm the admissibility of this conversion, such as: the estimation of the absorption characteristics of the formazans of tetranitro blue tetrazolium both in solution and precipitated in biological specimens; the linearity of the relationship between the increase of absorbance and incubation time; and the effect of different incubation conditions on the amount of specific formazan production.

On the DNA content and ploidy of trypanosomes.

We have determined the nuclear and kinetoplast DNA content of two trypanosomatids by quantitative absorption and fluorescence cytophotometry of individual Feulgen-pararosaniline stained cells. For the insect trypanosomatid Crithidia fasciculata we find nuclear and kinetoplast DNA contents of 0.095 and 0.032 pg per non-replicating cell. For the African trypanosome Trypanosoma brucei these values are 0.097 and 0.004 pg. A sub-population of T. brucei cells with two kinetoplasts and one nucleus was found to contain 0.181 pg/nucleus. The DNA values of bloodstream form T. brucei and the procyclic culture from were not significantly different. In DNA-DNA renaturation experiments the haploid amount of DNA in T. brucei was previously found to be 0.041 pg/nucleus (Borst, P., Fase-Fowler, F., Frasch, A.C.C., Hoeijmakers, J.H.J. and Weijers, P.J. (1980) Mol. Biochem. Parasitol. 1,221-246). Our data, therefore, indicate that T. brucei is diploid. No sub-population of haploid cells was observed in T. brucei grown in rats or in culture.