Nocardia farcinica Meningitis Masquerading as Central Nervous System Metastasis in a Child With Cerebellar Pilocytic Astrocytoma.

Juvenile pilocytic astrocytoma, the most common pediatric central nervous system (CNS) neoplasm, characteristically displays an indolent growth pattern and rarely demonstrates metastatic dissemination. Reports of infections mimicking CNS metastatic disease are also rare and can impact treatment. We report the youngest known case of a child with a CNS Nocardia farcinica infection who had a known cerebellar pilocytic astrocytoma, review other infections that may masquerade as CNS neoplasms, and discuss N. farcinica CNS infections.

Usefulness of McRAPD for typing and importance of biofilm production in a case of nosocomial ventriculoperitoneal shunt infection caused by Candida lusitaniae.

A case report of ventriculoperitoneal shunt infection caused by Candida lusitaniae in a 6-year-old patient with cerebral astrocytoma and obstructive hydrocephalus is presented briefly with emphasis on the course of antifungal treatment. Seven isolates recovered subsequently from the cerebrospinal fluid were studied retrospectively. To confirm identity, isolates were typed using pulsed-field gel electrophoresis and melting curve of random amplified polymorphic DNA (McRAPD). Further, the ability to form biofilm and its susceptibility to systemic antifungals were evaluated. Using McRAPD, identity of C. lusitaniae isolates showing slight microevolutionary changes in karyotypes was undoubtedly confirmed; successful application of numerical interpretation of McRAPD for typing is demonstrated here for the first time. The strain was also recognized as a strong biofilm producer. Moreover, minimum biofilm inhibitory concentrations were very high, in contrast to low antifungal minimum inhibitory concentrations of isolates. It can be concluded that McRAPD seems to be a simple and reliable method not only for identification but also for typing of yeasts. A ventriculoperitoneal shunt colonized by C. lusitaniae was revealed as the source of this nosocomial infection, and the ability of the strain to form biofilm on its surface likely caused treatment failure.

Pattern recognition by TREM-2: binding of anionic ligands.

We recently described the cloning of murine triggering receptor expressed by myeloid cells (TREM) 2, a single Ig domain DNAX adaptor protein 12-associated receptor expressed by cells of the myeloid lineage. In this study, we describe the identification of ligands for TREM-2 on both bacteria and mammalian cells. First, by using a TREM-2A/IgG1-Fc fusion protein, we demonstrate specific binding to a number of Gram-negative and Gram-positive bacteria and to yeast. Furthermore, we show that fluorescently labeled Escherichia coli and Staphylococcus aureus bind specifically to TREM-2-transfected cells. The binding of TREM-2A/Ig fusion protein to E. coli can be inhibited by the bacterial products LPS, lipoteichoic acid, and peptidoglycan. Additionally, binding can be inhibited by a number of other anionic carbohydrate molecules, including dextran sulfate, suggesting that ligand recognition is based partly on charge. Using a sensitive reporter assay, we demonstrate activation of a TREM-2A/CD3zeta chimeric receptor by both bacteria and dextran sulfate. Finally, we demonstrate binding of TREM-2A/Ig fusion to a series of human astrocytoma lines but not to a variety of other cell lines. The binding to astrocytomas, like binding to bacteria, is inhibited by anionic bacterial products, suggesting either a similar charge-based ligand recognition method or overlapping binding sites for recognition of self- and pathogen-expressed ligands.

An infectious aetiology for childhood brain tumours? Evidence from space-time clustering and seasonality analyses.

To investigate whether infections or other environmental exposures may be involved in the aetiology of childhood central nervous system tumours, we have analysed for space-time clustering and seasonality using population-based data from the North West of England for the period 1954 to 1998. Knox tests for space-time interactions between cases were applied with fixed thresholds of close in space, <5 km, and close in time, <1 year apart. Addresses at birth and diagnosis were used. Tests were repeated replacing geographical distance with distance to the Nth nearest neighbour. N was chosen such that the mean distance was 5 km. Data were also examined by a second order procedure based on K-functions. Tests for heterogeneity and Edwards' test for sinusoidal variation were applied to examine changes of incidence with month of birth or diagnosis. There was strong evidence of space-time clustering, particularly involving cases of astrocytoma and ependymoma. Analyses of seasonal variation showed excesses of cases born in the late Autumn or Winter. Results are consistent with a role for infections in a proportion of cases from these diagnostic groups. Further studies are needed to identify putative infectious agents.

Differences in the interactions of Nocardia asteroides with macrophage, endothelial, and astrocytoma cell lines.

An in vitro model for studying host cell interactions with Nocardia asteroides was developed. Thus, macrophage cell lines J774A.1 and P388D1, pulmonary artery endothelium cell line CPAE, rat glial tumor cell line C6, and human astrocytoma cell lines CCF-STTG1 and U-373 MG were infected with either log- or stationary-phase cells of N. asteroides GUH-2, and the host cell-nocardia interactions were determined by light microscopy and electron microscopy. Polyclonal antinocardial antibody did not enhance uptake of nocardiae by any of these cell lines; however, log-phase cells of GUH-2 infected a higher percentage of J774A.1 and P388D1 than did stationary-phase organisms. When cells infected with stationary-phase GUH-2 were incubated for 6 h, filaments developed, which indicated that nocardial growth had occurred. In J774A.1 and P388D1, only 31 to 57% of the total stationary-phase coccobacillary cells that were phagocytized formed filaments within 6 h. This indicated that there was some inhibition of growth of the phagocytized nocardiae within these macrophage cell lines; however, the nocardiae grew within the endothelial (> 87% filaments) and astrocytoma (100% filaments) cell lines. Microfilament inhibitor cytochalasin B inhibited uptake of GUH-2 by macrophages and other cell lines, except that there was no effect on uptake of nocardial cells by astrocytoma cell line U-373 MG. Scanning and transmission electron microscopy showed phagocytosis of GUH-2 by the different cell lines. In cytochalasin B-treated cells, nocardiae were shown to penetrate through the cell surface and become internalized in a manner distinct from typical phagocytosis, suggesting that filamentous forms of this organism have a phagocytosis-independent invasion factor. The extent of this cytochalasin-resistant cellular penetration by the nocardiae differed in the different cell lines.

The effects of cytomegalovirus on human immunodeficiency virus replication in brain-derived cells correlate with permissiveness of the cells for each virus.

Human cytomegalovirus (HCMV) is commonly found in the brains of patients with AIDS and in some cases can be detected in the same cells as can human immunodeficiency virus type 1 (HIV-1). In this study, we analyzed the patterns of replication of HIV-1 and HCMV in singly infected cells and the effects of dual infection in human brain-derived cell lines of three different origins: neuroblastoma cell lines SK-N-MC and SY5Y; astrocytoma/glioblastoma cell lines U373-MG and Hs 683; and undifferentiated glioblastoma cell lines A172 and T98G. To bypass the restriction at the adsorption/penetration step in these CD4-negative cells, we used HIV-1 (amphotropic retrovirus) pseudotypes. These HIV-1 pseudotypes infected the majority of the cells in the cultures and expressed high levels of HIV-1 gene products in all except the SY5Y cells. The cell lines differed in the ability to support HCMV infection, but coinfection with HIV-1 had no effect on HCMV replication. The A172 cells were completely nonpermissive for HCMV gene expression, while HCMV replication in the singly infected T98G and SK-N-MC cell lines was restricted at the level of some early gene products. This resulted in complete and partial inhibition, respectively, of viral DNA synthesis. Dual infection of the A172, T98G, and SK-N-MC cells had no effect on HIV-1 replication. The other three cell lines, U373-MG, Hs 683, and SY5Y, were fully permissive for HCMV replication. In the U373-MG and Hs 683 cells, HCMV markedly inhibited the synthesis of HIV-1 gene products. In contrast, a transient stimulation of HIV-1 production followed by a repression was observed in the dually infected SY5Y cells. We conclude from these results that under conditions in which both HIV-1 and HCMV can undergo fully permissive infection, HCMV can repress HIV-1 gene expression. In cells in which HCMV replication is limited but HIV-1 replicates well, there is no effect on HIV-1 gene expression. However, activation of HIV-1, at least transiently, may occur in cells in which HIV-1 gene expression is limited. These studies suggest that a threshold level of some HIV-1 gene product(s) may obscure activation or promote repression of HIV replication by HCMV.

Comparison of MRC-5 and U-373MG astrocytoma cells for detection of cytomegalovirus in shell vial centrifugation cultures.

U-373MG astrocytoma cells are susceptible to human cytomegalovirus (CMV) infection and offer the advantage of a continuous cell line for clinical laboratory use. U-373MG to MRC-5 cells for detection of CMV by centrifugation culture were therefore compared. At 20 h, 10 (6.1%) versus 12 (7.4%) of 163 clinical specimens were positive for CMV, and at 40 h, 12 (7.4%) versus 17 (10.4%) were positive in U-373MG and MRC-5 cells, respectively. Substantial toxicity was found in U-373MG cells (84%) when inoculated with blood specimens. For detection of CMV in centrifugation culture, MRC-5 cells are superior due both to higher sensitivity and lesser toxicity.

Target cells for HIV in the central nervous system: macrophages or glial cells?

Infection of foetal or embryonic brain cells and cell lines from human astrocytomas and gliomas with HIV1 derived from T-lymphoma cultures leads to the expression of HIV in about 1 to 2% of the cells in culture. Single-cell cloning of astrocytoma cells shortly after infection resulted in the establishment of persistently HIV1-infected cell lines. These cultures were characterized by low production of virus and moderate intra- and extracellular expression of structural proteins. However, high expression of the nef regulatory protein was found. The virus could be rescued by cocultivation with T cells and primary macrophages giving rise to typical syncytia formation. In contrast to infection with HIV-infected T-lymphoma lines, cocultivation with HIV1-infected primary macrophages or monocytic cell lines induced a reduction in the growth of astrocytes and failed to induce productive infection. These in vitro observations support the hypothesis that astrocytes and glial cells may be a reservoir for HIV in the central nervous system and that macrophages may not carry the virus to the brain, but rather may be infected in the brain after having penetrated the blood-brain barrier.

Owl monkey astrocytoma cells in culture spontaneously produce infectious JC virus which demonstrates altered biological properties.

A tumor cell suspension of an explanted JC virus (JCV)-induced owl monkey glioblastoma was inoculated intracranially into four recipient juvenile owl monkeys. Twenty-eight months following inoculation one owl monkey developed a glioblastoma, which was explanted into tissue culture. DNA from both the tumor tissue and tumor cells in culture hybridized to a JCV DNA probe by Southern analysis, indicating that free, as well as integrated, viral DNA may be present. At the time of the second culture passage, viral JCV DNA was extracted from these cells and cloned into a plasmid vector. Nucleotide sequencing of the regulatory region of the cloned DNA demonstrated homology with the prototype Mad-1 strain of JCV and revealed a 19-base-pair deletion in the second 98-base-pair tandem repeat that eliminated a second TATA box. This deletion is characteristic of the Mad-4 strain of JCV, which is highly neurooncogenic. By the third culture passage, 100% of the cells were T-antigen positive. Approximately one-third of the cells in culture hybridized to a biotinylated JCV DNA probe when in situ hybridization was used, a technique that only detects high-copy-number of replicating viral sequences. By the culture passage 5 and continuing through culture passage 14, viable JC virions could be recovered. The T protein synthesized by this virus, now termed JCV-586, differed from both the Mad-1 and Mad-4 strains in that it formed a stable complex with the cellular p53 protein in the tumor cells. Also, the JCV-586 T protein reacted to several monoclonal antibodies made to the simian virus 40 T protein that were not recognized by either the Mad-1 or Mad-4 strains.

Immunohistochemical detection of the gene product of Rous sarcoma virus in human brain tumors.

We have studied 108 cases of brain tumors by immunohistochemical staining utilizing an antiserum against the src gene product and GFA protein in order to elucidate the relationship between the brain tumors and the transforming genes. While the normal human brain tissues were not positive with the antiserum against the src gene product, several astrocytomas were clearly positive and other brain tumors were negative with the same. Src-gene product related peptide was detected in some of the human astrocytomas.

Cloning of SV40 genomes from human brain tumors.

From two human brain tumors SV40 genomes were isolated by recombinant DNA techniques. The SV40 genome cloned from a human meningioma DNA was shown to be indistinguishable from wild-type SV40. In contrast, the SV40 genome cloned from a human astrocytoma proved to be a nonviable deletion mutant with a truncated early region removing most of the large T-coding region. In addition, this mutant also carries a tandem duplication of an intact origin of replication.

Amplification of the murine leukemia virus Mr 70,000 glycoprotein gene product by human xenografts in athymic mice.

Passage of human tumors in athymic mice is accompanied by an increase in serum levels of the Mr 70,000 murine leukemia virus envelope protein, gp70. Elevated levels of gp70 can be detected in tissues of the hematopoietic systems of mice bearing human xenografts, but there is no evidence of synthesis of gp70 in these tissues. By far, the highest concentration of gp70 is in the human xenografts themselves. When assayed for gp70, 8 human xenografts and 12 cell lines established from human xenografts were all positive. In the plasma membrane of the human astrocytoma xenograft, T24, the gp70 was found to be approximately 10% of the total membrane protein. In contrast, the concentration of the Mr 30,000 viral core protein, p30, was 17-fold less. Only trace amounts of complete infectious virus could be detected. A human prostate carcinoma line that had not been grown in the athymic mice was found to have no gp70, but was shown to be able to synthesize gp70 after a single passage in the athymic mice.

New world primates as a model of viral-induced astrocytomas.

Owl and squirrel monkeys are susceptible to the oncogenic effects of JCV. These species of New World monkeys can be safely inoculated intracerebrally. Care must be taken with owl monkeys since they have an inherited clotting abnormality. Incubation times for the development of tumors range from 14 to 30 months. Anorexia was the first clinical sign of tumor development. The clinical course is rapid with death within two to three days. This model provides a means for studying diagnostic, virological, immunological and therapeutic techniques which are applicable to human patients with astrocytomas.

Viral-induced astrocytomas in squirrel monkeys.

Saimiri sciureus, the squirrel monkey, is susceptible to the oncogenic effects of JCV following intracerebral inoculation. As in owl monkeys, tumor development follows an incubation time of 14 to 30 months. Four of six virus-inoculated monkeys developed cerebral tumors, three of which were astrocytomas grade 4 and the remaining tumor was a poorly differentiated astrocytoma. All tumors showed high cellularity, mitotic figures, and cellular pleomorphism. In the astrocytoma grade 4, neovascularization was a prominent feature. The blood vessels in the poorly differentiated astrocytoma appeared normal. Multinucleated giant cells were present in all four astrocytomas. Antemortem hemorrhage was seen in one astrocytoma grade 4. Other tumor types were not seen. The occurrence of astrocytomas in a second species of New World monkeys confirms the oncogenicity of JCV for nonhuman primates.

Glial and divergent cells in primate central nervous system tumors induced by JC virus isolated from human progressive multifocal leukoencephalopathy (PML).

Immunofluorescent stains for fibronectin (FN) and glial fibrillary acidic protein (GFAP) were used in conjunction with routine histologic stains to study tumors induced in squirrel and owl monkeys by JC virus from progressive multifocal leukoencephalopathy (PML). Three varieties of glioma were identified. The first and most common variety was a neoplasm similar to grade 4 astrocytoma in humans. The second had thin, normal-appearing FN-positive vessel walls and a vastly expanded neuroectodermal parenchyma which could not be characterized by routine histologic stains. Anti-GFAP revealed the glial nature of the parenchyma. Isolating glial parenchymal cells from divergent FN-positive cells has become important to neurooncology. This type of tumor may be of particular interest for such isolations due to its high ratio of glial cells to divergent cells. The third variety was not a homogeneous neoplasm. It occurred as focal regions within tumors of the first type, and consisted of giant cells with huge nuclei. These cells resemble the cells of a human giant cell glioblastoma and bear a slight similarity to the bizarre glial cells seen in PML. The rare human giant cell glioblastoma might have an association with JC virus or with PML.

Neuroradiological studies of JCV-induced astrocytomas in nonhuman primates.

Astrocytomas in nonhuman primates following JC virus inoculation provides a model which can be used to evaluate diagnostic and therapeutic techniques used in humans. The CT scan appearance of astrocytomas in nonhuman primates closely resembles that seen in humans. Our studies have shown that tumors may be detected in asymptomatic monkeys. Serial scans have shown astrocytomas to grow rapidly with breakdown of the blood-brain barrier. CT scanning has demonstrated the presence of tumor which was undetectable by gross examination at necropsy but confirmed by light microscopy. Studies are in progress to further define the radiological appearance of gliomas, to evaluate contrast-tagged anti-tumor antibodies as a diagnostic tool in evaluating gliomas by computerized tomography, and to evaluate metabolic parameters of actrocytomas by positron emission tomography.

Differences in early ultrastructural aspects of the replication of measles and subacute sclerosing panencephalitis viruses in a cell culture from a human astrocytoma.

Monolayer cultures from a human astrocytoma were infected with small amounts of Mantooth Subacute Sclerosing Panencephalitis (SSPE) and Edmonston measles viruses. The infected cells were studied with an electron microscope 48 hours and 96 hours post-inoculation (PI). By 48 hours PI, both viruses produced syncytia and cytoplasmic inclusions of granular nucleocapsids 20 to 25 nm in diameter which did not differ in appearance. With the Edmonston measles virus granular nucleocapsids assembled into budding particles were found just under the cell membrane while nucleocapsids of Mantooth SSPE virus spared the area under the cell membrane and were not incorporated into budding particles. Inclusions of smooth nucleocapsids, 15 nm in diameter, could be seen within the nuclei of Mantooth SSPE virus infected cells 96 hours PI; such nuclear inclusions were not found in the Edmonston measles virus infected cells. These results are compared with those obtained in other cell systems and are discussed with respect to recent findings in the field of SSPE.

The fine structure of intracranial neoplasms induced by the inoculation of avian sarcoma virus in neonatal and adult rats.

Groups of F-344 rats were inoculated with the Bratislava-77 strain of avian sarcoma virus (B-77 ASV) within 24 hours of birth, at 9 days of age, or between 97 and 119 days of age. Intracranial tumors developed in each age group. Multiple tumors with mixed histologic patterns developed in rats inoculated at 1 or 9 days of age. Solitary tumors with a uniform histologic pattern developed in rats inoculated as adults. On the basis of light and electron microscopic study, the majority of tumors in each age group were classified as astrocytomas and divided into either poorly differentiated, gemistocytic, pilocytic, or polymorphic varieties. The polymorphic astrocytomas were most common among neonatally inoculated rats, while the pilocytic astrocytomas were most common among rats inoculated as adults. Ultrastructural characteristics of astrocytes, including gap junctions and 7- to 9-nm filaments, were present in the majority of tumors in each age groups. Astrocytomas induced in adult rats were remarkable for the presence of extensive basement membrane alone the astrocytic cell surfaces. Intracytoplasmic virus-like particles (R particles) were common in the tumor cells. These virus-like particles are morphologically distinct from C-type B-77 ASV, and no morphologic evidence of C-type virus replication was observed in any of the tumors.