William D. Lyman

Induction of human β-defensin-2 expression in human astrocytes by lipopolysaccharide and cytokines

Defensins are cationic peptides with broad‐spectrum antimicrobial activity. They are members of a supergene family consisting of α and β subtypes and each subtype is comprised of a number of different isoforms. For example, human α‐defensin (HAD) has six isoforms, which are expressed by polymorphonuclear leukocytes and Paneth cells. In contrast, human β‐defensin (HBD) has two isoforms that are expressed by epithelial cells of the skin, gut, respiratory and urogenital tracts. Recently, HBD‐1 was detected in human brain biopsy tissue. However, little is known about the expression of HBD‐1 or HBD‐2 in the CNS and whether neural cells can secrete these peptides. For the present study, human astrocyte, microglial, meningeal fibroblast and neuronal cultures were probed for the expression of HBD‐1 and HBD‐2 mRNA and protein. Each cell type was either maintained in tissue culture medium alone or in medium containing lipopolysaccharide (LPS) at concentrations ranging from 0.1 to 1 μg/mL, interleukin‐1 beta (IL‐1β) at 1–50 ng/mL, or tumor necrosis factor alpha (TNF‐α) at the same concentrations. The expression of HBD‐1 and HBD‐2 mRNAs was monitored by RT‐PCR. The cDNA products were sequenced to characterize the gene product. HBD‐2 protein was detected by immunoblot, immunoprecipitation and immunocytochemistry. Results of these studies showed that HBD‐1 mRNA was detected in all cell cultures except in those enriched for neurons. In contrast, HBD‐2 mRNA was detected only in astrocyte cultures that were treated with LPS, IL‐1β or TNF‐α. The detection of the respective proteins correlated positively with the mRNA results. As such, these data represent the first demonstration of HBD‐2 expression by astrocytes and suggest that this peptide may play a role in host defense against bacterial CNS pathogenesis.

HIV infection of fetal human astrocytes: the potential role of a receptor-mediated endocytic pathway

HIV infects microglia and astrocytes both in vivo and in vitro. Although there is a significant amount of information about microglial infection, data regarding astrocytes are more limited. For example, little is known about the initial membrane events occurring between HIV and astrocytes. Also, the mechanism by which HIV enters these cells remains to be determined. To address these questions, we exposed human astrocyte cultures to either HIV or to the HIV glycoprotein gp120. The cultures were analyzed for viral infection and gp120 binding to cultured cells by light and electron microscopy (EM) with and without immunocytochemistry, respectively; ligand-receptor biochemistry; and, Western, Northern and Southern blot analyses. The results of these studies showed that HIV binds to astrocytes via gp120 and a cell surface molecule weighing approximately 65 kDa that is neither CD4 nor galactocerebroside. Furthermore, binding of gp120 to astrocytes was concentration dependent and displayed a curve consistent with ligand-receptor binding. Additionally, radiolabeled gp120 binding was displaced by unlabeled gp120 but not by deglycosylated gp120, suggesting that the binding was specific. By EM, HIV virions were seen in clathrin-coated pits and in cytoplasmic vacuoles. This suggests linkage, in astrocytes, between a plasma membrane-associated protein that can act as a receptor for HIV and an endosomal pathway.

An efficient method for the cryopreservation of fetal human liver hematopoeitic progenitor cells.

The use of human hematopoietic progenitor cells (HPC) for transplantation requires efficient recovery methods and cryopreservation procedures. The purpose of this study was to determine cryopreservation techniques for fetal human liver (FHL) CD34+ cells. We assessed FHL HPC recovery efficiency after freezing and thawing by viability testing, fluorescence‐activated cell sorting analysis, and colony‐forming ability under different conditions. We also determined optimal cell freezing concentrations and the effect of rate‐controlled freezing on cell recovery. Lastly, cell recovery after varying freezing time periods was examined. Our results indicated that optimal cell recovery occurs when: A) cryopreservation medium consists of either 5% dimethylsulphoxide (DMSO) or 10% DMSO in combination with either 20% fetal bovine serum (FBS) or 70% FBS and when Iscoves modified Dulbeccos medium consists of not more than 10% DMSO; B) a rate‐controlled freezing device container is used; C) CD34+ cells are frozen at a concentration of 1 × 106/ml, and D) a thawing temperature of 37°C is used. These observations indicate that cryopreservation of FHL HPC is possible for up to 18 months in optimal conditions without losing hematopoietic activity.

Cytokine regulation of CD40 expression in fetal human astrocyte cultures

CD40 can participate in inflammatory processes after binding its cognate ligand (CD40L). We found that fetal human astrocytes constitutively express CD40 mRNA and protein. Upon incubating cultures with proinflammatory cytokines (TNF-alpha, IL-1beta and IFN-gamma) or with lipopolysaccharide (LPS), CD40 expression was increased. No change in CD40 expression was noted in astrocyte cultures incubated with IL-6, HIV or gp41. Astrocytes also showed increased release of proinflammatory cytokines TNF-alpha, IL-1beta and IL-6 after incubation with CD40L peptide. These observations suggest a role for CD40 in central nervous system (CNS) inflammation and that CD40/CD40L autocrine or paracrine pathways may mediate this role.

Increased Prevalence of G1P[4] Genotype among Children with Rotavirus-Associated Gastroenteritis in Metropolitan Detroit

ABSTRACT The G and P genotypes of rotavirus stool isolates from 100 children were determined by reverse transcription-PCR and nucleotide sequencing. G1P[4] was the most prevalent genotype(41%), followed by G1P[8] (16%) and G4P[4] (14%). The G genoypes detected were G1 (73%), G4 (17.4%), G9 (6.3%), and G2 (2.8%). The P genotypes were P[4] (71%) and P[8] (29%). Coinfection with more than one G genotype occurred in 12 patients, and coinfection with more than one P genotype occurred in 11 patients.

Ethanol Alters Cell Fate of Fetal Human Brain-Derived Stem and Progenitor Cells

BACKGROUND Prenatal ethanol (ETOH) exposure can lead to fetal alcohol spectrum disorder (FASD). We previously showed that ETOH alters cell adhesion molecule gene expression and increases neurosphere size in fetal brain-derived neural stem cells (NSC). Here, our aim was to determine the effect of ETOH on the cell fate of NSC, premature glial-committed precursor cells (GCP), and premature neuron-committed progenitor cells (NCP). METHODS NSC, GCP, and NCP were isolated from normal second-trimester fetal human brains (n = 3) by positive selection using magnetic microbeads labeled with antibodies to CD133 (NSC), A2B5 (GCP), or PSA-NCAM (NCP). As a result of the small percentage in each brain, NSC were cultured in mitogenic media for 72 hours to produce neurospheres. The neurospheres from NSC and primary isolates of GCP and NCP were used for all experiments. Equal numbers of the 3 cell types were treated either with mitogenic media or with differentiating media, each containing 0 or 100 mM ETOH, for 120 hours. Expression of Map2a, GFAP, and O4 was determined by immunoflourescence microscopy and western blot analysis. Fluorescence intensities were quantified using Metamorph software by Molecular Devices, and the bands of western blots were quantified using densitometry. RESULTS ETOH in mitogenic media promoted formation of neurospheres by NSC, GCP, and NCP. Under control conditions, GCP attached and differentiated, NSC and NCP formed neurospheres that were significantly smaller in size than those in ETOH. Under differentiating conditions, Map2a expression increased significantly in NSC and GCP and reduced significantly in NCP, and GFAP expression reduced significantly in GCP and NCP, and Gal-C expression reduced significantly in all 3 cell types in the presence of ETOH compared to controls. CONCLUSIONS This study shows that ETOH alters the cell fate of neuronal stem and progenitor cells. These alterations could contribute to the mechanism for the abnormal brain development in FASD.

Taurine enhances the growth of neural precursors derived from fetal human brain and promotes neuronal specification.

Taurine is present at high concentrations in the fetal brain and is required for optimal brain development. Recent studies have reported that taurine causes increased proliferation of neural stem/progenitor neural cells (neural precursor cells, NPCs) obtained from embryonic and adult rodent brain. The present study is the first to show that taurine markedly increases cell numbers in cultures and neuronal generation from human NPCs (hNPCs). hNPCs obtained from 3 fetal brains (14-15 weeks of gestation) were cultured and expanded as neurospheres, which contained 76.3% nestin-positive cells. Taurine (5-20 mM) increased the number of hNPCs in culture, with maximal effect found at 10 mM and 4 days of culture. The taurine-induced increase ranged from 57 to 188% in the 3 brains examined. Taurine significantly enhanced the percentage of neurons formed from hNPCs under differentiating conditions, with increases ranging from 172 to 480% over controls without taurine. Taurine also increased the cell number and neuronal generation in cultures of the immortalized human cell line ReNcell VM. These results suggest that taurine has a positive influence on hNPC growth and neuronal formation.

Resistance of stem-like cells from neuroblastoma cell lines to commonly used chemotherapeutic agents†

Cancer stem cell theory suggests that the presence of tumor initiating stem‐like cells in cancers may be responsible for cancer progression and relapse. CD133 cell surface maker expression has been used to identify stem‐like cells in cancer cell lines. Our goal was to identify such cells in neuroblastoma cell lines and to study the cytotoxicity of common anticancer drugs for those cells.

Comparison of neural precursor cell fate in second trimester human brain and spinal cord.

Abstract Neural transplantation holds promise for the treatment of traumatic brain and spinal cord injury by replacing lost cellular elements as well as repairing neural damage. Fetal human stem cells derived from central nervous system (CNS) tissue are potential transplantable sources for all cell types found in the mature human nervous system including neurons, astrocytes and oligodendroglia. Although nearly all areas of the fetal human neuraxis contain undifferentiated neural precursor cells, the phenotypic fate of the daughter cells might vary from one region to another during a specific developmental period. The purpose of this study was to compare the various cell types derived from neural precursors cultured from second trimester fetal human brain and spinal cord. To this end, brains (n = 8) and spinal cords (n = 8) of 15-24 week fetuses were dissociated and grown in culture medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (FGF) and leukemia inhibitory factor (LIF). The proliferating precursor cells from both brain and spinal cord grew as spherical masses that were plated on laminin-coated dishes after seven days in culture. During the next 5-7 days, the cells that emerged from these spheres were fixed and processed for immunocytochemistry. Brain derived spheres gave rise to cells expressing antigens specific for neurons (MAP-2ab and neuron specific-intermediate filaments), astrocytes (GFAP) and oligodendrocytes (A007). In contrast, cells that emerged from spinal cord derived spheres were only immunoreactive for GFAP. These data suggest that neuroepithelial precursor cells from different CNS regions, although similar in their responsiveness to proliferative growth factors, might differ in their ability to generate different cell types in the adult CNS. [Neurol Res 2001; 23: 260-266]

An efficient method for the culturing and generation of neurons and astrocytes from· second trimester human central nervous system tissue

Abstract The isolation culturing and expansion of human neural progenitors cells has important potential clinical applications in cellular transplantation strategies as well as in developmental studies involving the central nervous system (CNS). This study describes an efficient method to culture neurons and astrocytes as primary cultures, as well as from proliferative progenitor cells derived from second trimester fetal CNS tissue. Second trimester fetal human tissue was mechanically dissociated and subjected to trypsindissociation and trituration. The resulting suspension was passed over a Percoll density gradient. The middle (second) fraction of cells was centrifuged to yield a homogenous population of cells with 80%-90% viability. These cells were either cultured directly on laminin coated dishes with defined medium supplemented with fetal bovine serum or in defined medium supplemented with growth factors including epidermal growth factor, basic fibroblast growth factor and leukemia inhibitory factor. The primary cell cultures yielded neurons and astrocytes after 3-5 days in vitro verified by immunostaining with MAP2ab and GFAP. Cells exposed to growth factor supplemented medium formed free-floating spheres within one week. Upon growth factor removal and plating on laminin-coated dishes, brain derived spheres gave rise to neurons, astrocytes and oJigodendrocytes; spinal cord derived spheres generated only astrocytes. This protocol describes an efficient method to generate and culture neurons and astrocytes from second trimester human CNS tissue that may be useful in transplantation. and developmental studies.