Analysis of motile oligodendrocyte precursor cells in vitro and in brain slices

Oligodendrocyte precursor cells are purported to migrate over long distances into the various brain regions where they differentiate into oligodendrocytes and fulfill their appropriate tasks, i.e., myelination of axons. Here we characterize motile oligodendrocyte precursor cells in detail. Video–time lapse analysis was performed on isolated precursor cells in single cell cultures, in co-culture with cerebellar microexplants, and in living brain slices. Motility analysis of individual cells was combined with electrophysiological, immunological, and morphological characterizations. Translocation of the cell bodies was not continuous but occurred in waves. All motile cells exhibited a simple morphology and most, but not all, of them expressed the A2B5 epitope in vitro. Patch clamp analysis of the motile cells confirmed that they belong to the O-2A lineage. The percentage of motile cells, as well as their velocities, were enhanced on substrate-coated laminin in comparison to poly-L-lysine. Motility was not influenced by the presence of cerebellar microexplants. O-2A progenitor cells did not migrate strictly along neurite fascicles which were projected from the microexplants. Glial progenitor cells in situ also did not strictly migrate along the main direction of the axonal fibers of the corpus callosum but rather traversed the fibers with an overall direction toward the cortex. After Lucifer Yellow filling of the motile progenitor cells in situ, we could demonstrate that they were dye-coupled to yet unidentified cells of the corpus callosum. GLIA 20:284–298, 1997. © 1997 Wiley-Liss, Inc.     

Isoproterenol exacerbates a long QT phenotype in Kcnq1-deficient neonatal mice: possible roles for human-like Kcnq1 isoform 1 and slow delayed rectifier K+ current

To determine whether the neonatal mouse can serve as a useful model for studying the molecular pharmacological basis of Long QT Syndrome Type 1 (LQT1), which has been linked to mutations in the human KCNQ1 gene, we measured QT intervals from electrocardiogram (ECG) recordings of wild-type (WT) and Kcnq1 knockout (KO) neonates before and after injection with the beta-adrenergic receptor agonist, isoproterenol (0.17 mg/kg, i.p.). Modest but significant increases in JT, QT, and rate-corrected QT (QTc) intervals were found in KO neonates relative to WT siblings during baseline ECG assessments (QTc = 57 +/- 3 ms, n = 22 versus 49 +/- 2 ms, n = 28, respectively, p < 0.05). Moreover, JT, QT, and QTc intervals significantly increased following isoproterenol challenge in the KO (p < 0.01) but not the WT group (p = 0.57). Furthermore, whole-cell patch-clamp recordings show that the slow delayed rectifier K+ current (IKs) was absent in KO but present in WT myocytes, where it was strongly enhanced by isoproterenol. This finding was confirmed by showing that the selective IKs inhibitor, L-735,821, blocked IKs and prolonged action potential duration in WT but not KO hearts. These data demonstrate that disruption of the Kcnq1 gene leads to loss of IKs, resulting in a long QT phenotype that is exacerbated by beta-adrenergic stimulation. This phenotype closely reflects that observed in human LQT1 patients, suggesting that the neonatal mouse serves as a valid model for this condition. This idea is further supported by new RNA data showing that there is a high degree of homology (>88% amino acid identity) between the predominant human and mouse cardiac Kcnq1 isoforms.     

Interleukin-1 and interleukin-6 gene polymorphisms and the risk of breast cancer in caucasian women.

PURPOSE: Genetic polymorphisms of cytokine-encoding genes are known to predispose to malignant disease. Interleukin (IL)-1 and IL-6 are crucially involved in breast carcinogenesis. Whether polymorphisms of the genes encoding IL-1 (IL1) and IL-6 (IL6) also influence breast cancer risk is unknown. EXPERIMENTAL DESIGN: In the present case-control study, we ascertained three polymorphisms of the IL1 gene cluster [-889 C/T polymorphism of the IL1alpha gene (IL1A), -511 C/T polymorphism of the IL1beta promoter (IL1B promoter), a polymorphism of IL1beta exon 5 (IL1B exon 5)], an 86-bp repeat in intron 2 of the IL1 receptor antagonist gene (IL1RN), and the -174 G/C polymorphism of the IL6 gene (IL6) in 269 patients with breast cancer and 227 healthy controls using PCR and pyrosequencing. RESULTS: Polymorphisms within the IL1 gene cluster and the respective haplotypes were not associated with the presence and the phenotype of breast cancer. The IL6 polymorphism was significantly associated with breast cancer. Odds ratios for women with one or two high-risk alleles versus women homozygous for the low-risk allele were 1.5 (95% confidence interval, 1.04-2.3; P = 0.04) and 2.0 (95% confidence interval, 1.1-3.6; P = 0.02), respectively. No association was ascertained between presence of the IL6 polymorphism and various clinicopathologic variables. CONCLUSIONS: Although polymorphisms within the IL1 gene cluster do not seem to influence breast cancer risk or phenotype, presence of the -174C IL6 allele increases the risk of breast cancer in Caucasian women in a dose-dependent fashion.     

Diagnostik von Metamorphopsien bei Netzhauterkrankungen unterschiedlicher Genese

We investigated the ability of preferential hyperacuity perimeter (PHP) and Amsler grid testing to detect metamorphosia in patients with macular hole (MH), central serous retinopathy (CSR), epiretinal membranes (EM), intermediate AMD (iAMD), classic and occult choroidal neovascularization (CNV) due to AMD, and compared the results. A total of 147 patients (n =153 eyes) with classic (35 eyes) and occult (38 eyes) CNV, iAMD (13 eyes), MF (23 eyes), RCS (11 eyes), EM (13 eyes) and control group (20 eyes) were involved. All of these patients underwent corrected visual acuity and eye examinations inclusive of the Amsler grid. The PHP test was performed after pupil dilation. In all patients, fundus photography and optical coherence tomography (OCT) (Humphrey/Zeiss OCT III) were performed. In patients with CNV and CSR a fluorescein angiography was also performed. Metamorphopsia detection rates by Amsler grid and PHP were compared statistically. The sensitivity of PHP vs Amsler grid in detecting metamorphosia was 69% vs 85% in patients with MH, for CSR 64% vs 73%, EM 77% vs 100%, iAMD 85% vs 100%, classic CNV 83% vs 94% and occult CNV 81% vs 71%. The results for patients with occult CNV were significant (P =0.046), using the chi(2)-test. The PHP-test showed high sensitivity for diagnosing CNV. In occult CNV, PHP was superior to the Amsler grid in detecting metamorphopsia. In the other diseases involving the macular (MH, EM, CSR, iAMD), the detection rate and sensitivity of the Amsler grid was superior to PHP.     

Selective retina therapy in patients with central serous chorioretinopathy

Background Central serous chorioretinopathy (CSC) is a disease with a localized breakdown of the outer blood–retinal barrier located within the retinal pigment epithelium (RPE) causing subretinal fluid accumulation. Selective retina therapy (SRT) is a new, minimally invasive laser technology that has been designed to selectively target the RPE. SRT spares retinal tissue.Methods Twenty-seven eyes of 27 patients with active CSC were treated with SRT using a pulsed double-Q-switched Nd-YLF prototype laser (λ=527 nm, t=1.7 μs). At baseline, best-corrected visual acuity was determined and fluorescein angiography and optical coherence tomography were performed. The patients were followed for up to 3 months.Results After 4 weeks 85.2% of patients showed complete resolution of subretinal fluid and in 96.3% there was no leakage visible on fluorescein angiography. After 3 months 100% of patients demonstrated no subretinal fluid and 100% of patients had no leakage activity on fluorescein angiography. Visual acuity, 20/40 at baseline, improved to 20/28 after 4 weeks and to 20/20 after 3 months.Conclusion SRT is a safe and effective treatment for active CSC. Especially if the RPE leak is located close to the fovea, SRT is the favoured therapeutic option. We recommend earlier treatment of patients with acute CSC in order to prevent development of chronic changes due to CSC with irreversible anatomical and functional damage. SRT might be considered as a first-line treatment for active CSC.     

Vascular infarction by subcutaneous application of tissue factor targeted to tumor vessels with NGR-peptides: activity and toxicity profile

tTF-NGR consists of the extracellular domain of the (truncated) tissue factor (tTF), a central molecule for coagulation in vivo, and the peptide GNGRAHA (NGR), a ligand of the surface protein aminopeptidase N (CD13). After deamidation of the NGR-peptide moiety, the fusion protein is also a ligand for integrin αvβ3 (CD51/CD61). Both surface proteins are upregulated on endothelial cells of tumor vessels. tTF-NGR showed binding to specific binding sites on endothelial cells in vitro as shown by flow cytometry. Subcutaneous injection of tTF-NGR into athymic mice bearing human HT1080 fibrosarcoma tumors induced tumor growth retardation and delay. Contrast enhanced ultrasound detected a decrease in tumor blood flow in vivo after application of tTF-NGR. Histological analysis of the tumors revealed vascular disruption due to blood pooling and thrombotic occlusion of tumor vessels. Furthermore, a lack of resistance was shown by re-exposure of tumor-bearing mice to tTF-NGR after regrowth following a first cycle of treatment. However, after subcutaneous (s.c.) push injection with therapeutic doses (1-5 mg/kg bw) side effects have been observed, such as skin bleeding and reduced performance. Since lethality started within the therapeutic dose range (LD10 approximately 2 mg/kg bw) no safe therapeutic window could be found. Limiting toxicity was represented by thrombo-embolic events in major organ systems as demonstrated by histology. Thus, subcutaneous injection of tTF-NGR represents an active, but toxic application procedure and compares unfavourably to intravenous infusion.     

Comparative genetic profiling aids diagnosis and clinical decision making in challenging cases of CUP syndrome

Cancer of unknown primary (CUP) denotes cancer cases where metastatic spread is histologically confirmed, but no respective primary tumor can be identified. The challenging diagnosis of CUP is further complicated in cases with previously identified malignancies or with dubious clonal relationship between metastatic sites due to ambiguous histology. Our study aims at elucidating clonal relationships by comparing the respective mutational spectra. Targeted next-generation sequencing (NGS) employing formalin-fixed and paraffin-embedded (FFPE) tumor tissue was performed on 174 consecutive CUP patients. Among these, 43/174 (24.7%) patients had a documented prior malignancy. Data on pairwise targeted NGS testing to address clonal relationships between the previous malignancy and the presumed CUP (n = 11) or between different CUP metastatic sites (n = 7) was available in 18 patients. NGS could clarify clonal relationships in 16/18 cases. Among the 11 CUP patients with antecedent malignancies, four cases were clonally independent of the previous malignancy but harbored deleterious germline mutations in BRCA/BAP1/ATM genes. Seven CUP cases were clonally related to the antecedent malignancy, changing the CUP diagnosis to relapse of the prior malignancy. In the seven CUP cases, with doubtfully related metastatic sites, NGS confirmed clonal relationship in five cases and was inconclusive in two. In conclusion, NGS proved an efficient tool to elucidate clonal relationships in clinically challenging CUP cases. Our study cautions against a premature diagnosis of CUP. Relapses of antecedent malignancies should be carefully considered. CUPs clonally independent from the antecedent malignancy should raise a red flag of a potential cancer-predisposing germline mutation.     

Early Epileptiform Discharges and Clinical Signs Predict Nonconvulsive Status Epilepticus on Continuous EEG

Background

Unilateral anterior spinal artery (ASA) occlusion resulting in bilateral medullary pyramidal (BMP) infarction is a rare and devastating stroke subtype. We present two cases highlighting the diagnostic and clinical challenges of BMP infarction.

Methods

Case reports and literature review.

Results

A 57-year-old man rapidly had severe vomiting and diarrhea 2 h after a meal. Examination revealed bulbar weakness and areflexic tetraplegia. Respiratory failure developed, requiring intubation and mechanical ventilation. Brain magnetic resonance imaging (MRI) showed a heart-shaped region of diffusion abnormality, characteristic of BMP infarction. Cerebral angiography showed an occluded left vertebral artery with unilateral left-sided origin of ASA. The patient required tracheostomy and percutaneous gastrostomy tube and was discharged to rehabilitation, with little improvement of his tetraplegia at 3-month follow-up.A 43-year-old woman presented to the emergency department with acute onset of lower-extremity paresthesia and history of upper respiratory infection 2 weeks prior. Initial examination findings included bulbar weakness, dysphagia, hyporeflexia, and generalized weakness. After admission, she had severe respiratory distress and required intubation. Lumbar puncture was evaluated for Guillain–Barré syndrome, but cerebrospinal fluid protein concentration was normal. Changes on diffusion-weighted MRI of the brain showed the characteristic heart-shaped BMP infarction, indicating occlusion of a unilateral ASA. She required tracheostomy and percutaneous gastrostomy tube placement, with no paralysis resolution.

Conclusion

Acute BMP infarction may present with flaccid tetraplegia mimicking neuromuscular disorders. When the infarction is recognized early, intravenous thrombolysis can be considered to reduce morbidity of this rare stroke subtype.

     

Micronucleus formation in human cancer cells is biased by chromosome size

Chromosomal instability is one of the hallmarks of cancer and caused by chromosome missegregation during mitosis, a process frequently associated with micronucleus formation. Micronuclei are formed when chromosomes fail to join a daughter nucleus during cell division and are surrounded by their own nuclear membrane. Although it has been commonly assumed that the gain or loss of specific chromosomes is random during compromised cell division, recent data suggest that the size of chromosomes can impact on chromosome segregation fidelity. To test whether chromosome missegregation rates scale with chromosome size in primary human cancer cells, we assessed chromosome sequestration into micronuclei in patient‐derived primary NCH149 glioblastoma cells, which display high‐level numerical chromosome instability (CIN), pronounced spontaneous micronucleus formation but virtually no structural CIN. The cells were analyzed by interphase fluorescence in situ hybridization using chromosome‐specific painting probes for all chromosomes. Overall, 33% of early passage NCH149 cells harbored micronuclei. Entrapment within a micronucleus clearly correlated with chromosome size with larger chromosomes being significantly more frequently missegregated into micronuclei than smaller chromosomes in primary glioblastoma cells. These findings extend the concept that chromosome size determines segregation fidelity by implying that size‐specific micronucleus entrapment occurs in primary human cancer cells as well.