Edo P. J. Arnoldus

Somatic pairing of chromosome 1 centromeres in interphase nuclei of human cerebellum

SummaryInterphase nuclei isolated from paraffin-embedded tissue of four normal brains were hybridized with biotinated repetitive DNA probes specific for the (peri) centromeric regions of chromosomes 1 and 7. Hybridization results were visualized with a peroxidase-DAB system after which the number of specific signals per nucleus was counted using bright field microscopy. Using the probe specific for chromosome 7 (p7t1), both the cerebral and the cerebellar samples showed 2 spots in 82% and 83%, respectively, of the nuclei. In situ hybridization with the chromosome 1 probe (pUC1. 77) showed two spots in 69% of the cerebral nuclei. In cerebellar samples, hybridization with pUC1.77 resulted in only one large spot per nucleus in 82% of the cells. The average spot size in nuclei with one signal was about 1.6 times as large as that in nuclei with two signals. These observations suggest that the single large spot in the cerebellar cells is not the result of monosomy of chromosome 1 but that it reflects somatic pairing of the two chromosome 1 centromeres. Based on the size and the fraction of nuclei with one large spot, the small granular neuron is the most likely candidate. The difference between cerebral and cerebellar samples indicates that this somatic pairing of chromosome 1 is a cell-type-dependent phenomenon.

Interphase cytogenetics reveals somatic pairing of chromosome 17 centromeres in normal human brain tissue, but no trisomy 7 or sex-chromosome loss

Nuclei isolated from normal human brain tissue, collected from six autopsies, were hybridized with a panel of nine satellite DNA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, and the X and Y chromosomes. The results did not confirm the recently reported trisomy 7 and loss of sex chromosomes observed in metaphases obtained from normal brain tissue after short-term cultures; however, cells of all six brains displayed somatic pairing of the chromosome 17 centromeres in approximately 50% of the nuclei.

Feasibility of in situ hybridisation with chromosome specific DNA probes on paraffin wax embedded tissue.

The feasibility was studied of in situ hybridisation using chromosome specific DNA probes on paraffin wax embedded normal and malignant tissues from different organs. Both isolated nuclei and 5 microns sections were used in in situ hybridisation experiments with biotinylated repetitive DNA probes specific for the centromeric regions of chromosomes 1 and 17. The hybridisation results were visualised with peroxidase-diaminobenzidine. The optimal pretreatments with sodium thiocyanate and pepsin were experimentally defined for the different tissues. Although interphase cytogenetics on paraffin wax embedded tissue is possible, the results indicate that it has its limitations, compared with investigations on fresh tumour tissue.

Polymerase chain reaction and viral culture techniques to detect HSV in small volumes of cerebrospinal fluid; an experimental mouse encephalitis study

A technique is described for the detection of HSV-DNA in very small volumes (5-10 microliters) of cerebrospinal fluid (CSF). The method was evaluated in CSF samples of 4-6-week-old mice inoculated with HSV-1 via the corneal route. The sensitivity of the PCR assay was compared with results of spin-amplified viral culture with immunofluorescent visualization (SAC/IF), routine viral culture (RVC) and radioactive dot-blot hybridization (DBA) in CSF samples obtained from other mice. The results show the PCR to be superior over the other techniques: infectious virus or viral DNA in CSF was demonstrated by PCR, SAC/IF, RVC and DBA in 68, 55, 20 and 2.5%, respectively. These results show the feasibility of the PCR for a rapid, non-invasive diagnosis of human HSV-encephalitis.

Diagnosis of progressive multifocal leucoencephalopathy by hybridisation techniques.

In situ hybridisation with acetyl-aminofluorene (AAF) and 35S-labelled DNA probes for polyomaviruses, was used to detect JC virus DNA in brain necropsy material in a patient with progressive multifocal leucoencephalopathy (PML). In a second patient PML was diagnosed from a brain necropsy specimen using the same technique. The main infected cell type were oligodendrocytes; dot hybridisation was used to estimate the number of viral copies in each infected cell. Southern blot hybridisation for further analysis of the viral genome was also carried out. In situ hybridisation with non-radioactive labelled polyomavirus DNA provides a simple and specific means for studying viral DNA in formaldehyde fixed tissue sections from patients with suspected PML. Even in small biopsy samples hybridisation results can be correlated with standard histopathological, immunocytochemical, and electron microscopic findings.

PCR in herpes simplex virus infections of the central nervous system

Abstract Herpes simplex virus (HSV) infections of the central nervous system (CNS) together comprise a special group of herpetic diseases with respect to residual damage and difficulties in diagnosis. The various syndromes and manifestations are presented, in particular the most common and severe form, herpes simplex virus encephalitis (HSE). The probable reasons for the failure of routine viral methods for establishing a rapid and early diagnosis in HSE are discussed. The technical problems of polymerase chain reaction (PCR) in the diagnosis of HSE and its use as a diagnostic method are reviewed.

Polymerase Chain Reaction Detection of Herpes Simplex Virus in Cerebrospinal Fluid

Herpes simplex encephalitis (HSE) is a serious disease with significant morbidity and mortality if left untreated. It can be treated with acyclovir, but for this to be effective it must be instituted early during the course of disease. Noninvasive, early diagnosis, however, is presumptive at best, and many patients are treated without a definitive diagnosis.

Interphase cytogenetic analysis of gliomas.

Interphase cytogenetics is the application of nonradioactive in situ hybridization with chromosome-specific DNA probes to interphase nuclei. The possibilities and limitations of this new technique for the study of chromosomal aberrations in gliomas are discussed.

Interphase cytogenetics on brain tumors

The development and application of a procedure for interphase cytogenetics on brain tumor material is described. Nuclei isolated from freshly removed brain tumor tissue were investigated for chromosomal aberrations by nonradioactive in situ hybridization with a panel of chromosome-specific probes. The panel consisted of nine satellite DNA probes specific for the centromeric regions of chromosomes 1, 6, 7, 10, 11, 17, 18, X, and Y. For each probe, the number of hybridization signals per cell was determined in 200 nuclei. It was inferred from the hybridization results that in 11 gliomas (seven astrocytomas grade II-IV, three oligodendrogliomas, and one ependymoma) the numerical aberrations were gains of chromosomes 1 (once), 7 (twice), 10 (once), 11 (twice), and X (twice); losses of chromosomes 1 (once), 10 (twice), 17 (twice), and Y (once); and complete tetraploidy (once). Among the 18 investigated meningiomas monosomy 18 and trisomy 17 were observed once and twice, respectively. An additional hybridization with a cosmid probe for the BCR gene on 22q11 indicated monosomy 22q in 11 meningiomas. These results show the value of interphase cytogenetics for the analysis of solid tumors for which it is relatively difficult to obtain sufficient metaphases of good quality for conventional cytogenetics.

Prolonged Coma and Severely Attenuated EEG After a Single Seizure

Summary: A 63‐year‐old woman presented with a comatose state after a fall. Results of cranial computed tomography (CT) and magnetic resonance imaging (MRI) scans were normal. An EEG recorded 5 h after admission was very severely attenuated and slowed. Consciousness and EEG were improved the next day. No cause was detected initially. After sleep deprivation, the patient had a generalized seizure followed by a similar coma and EEG. Even a single seizure may cause a prolonged coma with a very severely attenuated and slowed EEG.