Increased expression of osteopontin gene in atypical teratoid/rhabdoid tumor of the central nervous system.

The atypical teratoid/rhabdoid tumor, primary to the central nervous system, is a highly malignant and aggressive neoplasm of infancy and childhood. Although having distinct biological features and clinical outcomes, it is frequently misdiagnosed as primitive neuroectodermal tumor/medulloblastoma. To further distinguish the underlying pathogenesis and to identify biological markers for clinical use, an atypical teratoid/rhabdoid tumor-derived cell line was established and its gene expression pattern analyzed in comparison to the human astrocyte SVG12 cell line and the human DAOY medulloblastoma cell line using a complementary DNA microarray method. The osteopontin gene was found specifically upregulated in atypical teratoid/rhabdoid tumor cells. This specificity was confirmed by immunohistochemistry in pathological sections of tissues from atypical teratoid/rhabdoid tumor patients. Even though the role of osteopontin in the cytopathogenesis of atypical teratoid/rhabdoid tumor still needs to be determined, our data support that overexpressed osteopontin is a potential diagnostic marker for atypical teratoid/rhabdoid tumor.     

Overexpression of connexin43 alters the mutant phenotype of midgestational wnt-1 null mice resulting in recovery of the midbrain and cerebellum

The midbrain-hindbrain (MHB) junction plays a key role in the patterning of the embryonic neural tube and the formation of brain structures such as the cerebellum. The mitogen wnt-1 is critical for cerebellar development, as evidenced by the lack of MHB region and cerebellar formation in the wnt-1 null embryo. We have generated wnt-1 null embryos overexpressing the gap junction gene connexin43 by crossing wnt-1 null heterozygotes into the CMV43 mouse line. We have confirmed that these mice show an increase in gap junctional communication by dye coupling analysis. Two-thirds of wnt-1 null CMV43(+) mouse embryos at E18.5 have a cerebellum. In addition, changes in the wnt-1 null phenotype in mouse embryos overexpressing connexin43 are observed as early as E9.5. At this stage, one-quarter of wnt-1 null CMV43(+) embryos display extra or expanded tissue present at the MHB boundary (a wnt-1 null enlarged phenotype). In situ hybridization studies conducted on these embryos have indicated no changes in the expression of embryonic brain positional markers in this region. We conclude from these studies that overexpression of the connexin43 gap junction restores cerebellar formation by compensating for the loss of wnt-1.     

Demonstration of T Lymphocytes in Cerebrospinal Fluid

Cerebrospinal fluid (CSF) was allowed to drop straight into Hanks's balanced salt solution. After centrifugation the pellet was resuspended and mixed with sheep erythrocytes. The mixture was further handled as in the E-rosette test with peripheral blood lymphocytes. CSF from 20 individuals were investigated, and rosette-forming cells (RFC) were found in all. Six patients with normal fluid had between 46% and 83% RFC. Four patients with multiple sclerosis had increased numbers of RFC (94%-96%). Low numbers of RFC were found in one patient with cerebellar ataxia and in one of two patients with acute viral meningitis. With this technique RFC can be counted even in normal CSF with a 3-ml sample.     

Utilization review of the use of BACTEC PLUS high-volume blood culture bottles.

The BACTEC PLUS 26 (NR26) (Becton Dickinson, Towson, Md.) high-volume blood culture bottle replaced the less expensive smaller-volume NR6A bottle in our hospital. An audit carried out several months after their introduction revealed that only 17.5% of the NR26 bottles received the required blood volume. Several audits and educational programs were required in order to achieve a compliance rate of > 60%.     

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.     

Systemic autoimmune disease induced by dendritic cells that have captured necrotic but not apoptotic cells in susceptible mouse strains

Systemic lupus erythematosus (SLE) is an autoimmune disorder of a largely unknown etiology. Anti-double-stranded (ds) DNA antibodies are a classic hallmark of the disease, although the mechanism underlying their induction remains unclear. We demonstrate here that, in both lupus-prone and normal mouse strains, strong anti-dsDNA antibody responses can be induced by dendritic cells (DC) that have ingested syngeneic necrotic (DC/nec), but not apoptotic (DC/apo), cells. Clinical manifestations of lupus were evident, however, only in susceptible mouse strains, which correlate with the ability of DC/nec to release IFN-gamma and to induce the pathogenic IgG2a anti-dsDNA antibodies. Injection of DC/nec not only accelerated disease progression in the MRL/MpJ-lpr/lpr lupus-prone mice but also induced a lupus-like disease in the MRL/MpJ-+/+ wild-type control strain. Immune complex deposition was readily detectable in the kidneys, and the mice developed proteinuria. Strikingly, female MRL/MpJ-+/+ mice that had received DC/nec, but not DC/apo, developed a 'butterfly' facial lesion resembling a cardinal feature of human SLE. Our study therefore demonstrates that DC/nec inducing a Th1 type of responses, which are otherwise tightly regulated in a normal immune system, may play a pivotal role in SLE pathogenesis.