Barbara N. Walter

The cellular response in neuroinflammation: The role of leukocytes, microglia and astrocytes in neuronal death and survival

Neuroinflammation is a complex integration of the responses of all cells present within the CNS, including the neurons, macroglia, microglia and the infiltrating leukocytes. The initiating insult, environmental factors, genetic background and age/past experiences all combine to modulate the integrated response of this complex neuroinflammatory circuit. Here, we explore how these factors interact to lead to either neuroprotective versus neurotoxic inflammatory responses. We specifically focus on microglia and astrocytic regulation of autoreactive T cell responses.

Differential biologic effects of CPD and 6-4PP UV-induced DNA damage on the induction of apoptosis and cell-cycle arrest

BackgroundUV-induced damage can induce apoptosis or trigger DNA repair mechanisms. Minor DNA damage is thought to halt the cell cycle to allow effective repair, while more severe damage can induce an apoptotic program. Of the two major types of UV-induced DNA lesions, it has been reported that repair of CPD, but not 6-4PP, abrogates mutation. To address whether the two major forms of UV-induced DNA damage, can induce differential biological effects, NER-deficient cells containing either CPD photolyase or 6-4 PP photolyase were exposed to UV and examined for alterations in cell cycle and apoptosis. In addition, pTpT, a molecular mimic of CPD was tested in vitro and in vivo for the ability to induce cell death and cell cycle alterations.MethodsNER-deficient XPA cells were stably transfected with CPD-photolyase or 6-4PP photolyase to specifically repair only CPD or only 6-4PP. After 300 J/m2 UVB exposure photoreactivation light (PR, UVA 60 kJ/m2) was provided for photolyase activation and DNA repair. Apoptosis was monitored 24 hours later by flow cytometric analysis of DNA content, using sub-G1 staining to indicate apoptotic cells. To confirm the effects observed with CPD lesions, the molecular mimic of CPD, pTpT, was also tested in vitro and in vivo for its effect on cell cycle and apoptosis.ResultsThe specific repair of 6-4PP lesions after UVB exposure resulted in a dramatic reduction in apoptosis. These findings suggested that 6-4PP lesions may be the primary inducer of UVB-induced apoptosis. Repair of CPD lesions (despite their relative abundance in the UV-damaged cell) had little effect on the induction of apoptosis. Supporting these findings, the molecular mimic of CPD, (dinucleotide pTpT) could mimic the effects of UVB on cell cycle arrest, but were ineffective to induce apoptosis.ConclusionThe primary response of the cell to UV-induced 6-4PP lesions is to trigger an apoptotic program whereas the response of the cell to CPD lesions appears to principally involve cell cycle arrest. These findings suggest that CPD and 6-4 PP may induce differential biological effects in the UV-damaged cell.

Production of lymphotoxin (LTα) and a soluble dimeric form of its receptor using the baculovirus expression system

Human LT alpha and a fusion protein (p60:Fc) comprised of the extracellular domain of the 60 kDa TNF receptor (TNFR60) fused to the Fc portion of human IgG1 were produced in insect cells infected with recombinant baculoviruses. The p60:Fc fusion produced in insect cells accumulates in culture supernatants to levels > 2 mg/l. Purified p60:Fc binds human TNF and LT alpha with high affinity (200-600 pM) and neutralizes TNF cytolytic activity at equimolar stoichiometric concentration. The data show that p60:Fc is an effective ligand-precipitating reagent which recognizes recombinant LT alpha produced in mammalian or insect cells and naturally occurring LT alpha produced in T cells. The levels of human LT alpha produced in baculovirus-infected insect cells is estimated to be approximately 20 mg/l. Insect cell-derived human LT alpha is biologically active in an L929 cytotoxicity assay and is efficiently neutralized by p60:Fc. These data demonstrate that the baculovirus system is useful for overexpressing biologically active LT alpha and p60:Fc and therefore, may be applicable to other oligomeric cytokines and soluble dimeric cytokine receptors.

Trans-epithelial immune cell transfer during suckling modulates delayed-type hypersensitivity in recipients as a function of gender.

Introduction Breast feeding has long term effects on the developing immune system which outlive passive immunization of the neonate. We have investigated the transfer of milk immune cells and examined the result of transfer once the recipients were adult. Methods Non-transgenic mouse pups were foster-nursed by green fluorescent protein (GFP) transgenic dams for 3 weeks and the fate of GFP+ cells was followed by FACS analysis, immunohistochemistry and RT-PCR for GFP and appropriate immune cell markers. Pups suckled by non-transgenic dams served as controls. Results Despite a preponderance of B cells and macrophages in the stomach contents of the pups, most cells undergoing trans-epithelial migration derived from the 3–4% of milk cells positive for T lymphocyte markers. These cells homed to the spleen and thymus, with maximal accumulation at 3–4 weeks. By sensitizing dams with an antigen which elicits a T cell-mediated delayed-type-hypersensitivity (DTH) response, we determined that nursing by a sensitized dam (compared to a non-sensitized dam) amplified a subsequent DTH response in females and yet suppressed one in males. Discussion These results suggest that clinical evaluation weighing the pros and cons of nursing male versus female children by mothers with genetically-linked hypersensitivity diseases, such as celiac disease and eczema, or those in regions of the world with endemic DTH-eliciting diseases, such as tuberculosis, may be warranted.

DNA-sequence specificity of mutations at the human thymidine kinase locus

We have established a system for the study of DNA-sequence specificity at a functionally heterozygous thymidine kinase (tk) locus in a human lymphoblastoid cell line (TK6). Characterization of the parental locus demonstrated that the 2 tk alleles were fortuitously distinguished by differential gene expression. One round of PCR amplification yielded a specific tk cDNA product only for the functional parental allele. Analysis of cDNA from newly mutated alleles which retain substantial levels of expression is thus simplified. Amplification and sequencing of tk genomic sequences was used for analysis of low expression mutants, and in order to distinguish and characterize deletion and splicing mutations. DNA-sequence analysis of the parental locus identified a frameshift in tk exon 4 of the non-functional parental allele, and surprisingly, an exon 7 frameshift mutation in the functional tk allele. This exon 7 frameshift results in a predicted alteration of the final 21 amino acids of the TK protein, and a C-terminal extension of 131 additional amino acids. Since TK6 is phenotypically TK+, we can infer that this major C-terminal modification does not eliminate enzymatic activity. The system was utilized for the analysis of 36 spontaneous TK- mutants. Loss of heterozygosity accounted for 58% of the mutations, 11% were attributable to intragenic deletions, and the remainder involved point mutations, primarily G:C to A:T transitions.

Insertioanl inactivation of the tk locus in a human B lymphoblastoid cell line by a retroviral shuttle vector

Insertional mutagenesis represents an inherent risk in retrovirally mediated gene therapy, but it may be a useful experimental strategy for identification and isolation of novel cellular loci. In this investigation we have established a model system using a heterozygous thymidine kinase (tk) marker locus in a human B lymphoblastoid cell line, and a M-MuLV based shuttle vector. The frequency of TK- mutants in cells carrying 1-2 proviruses per genome is approximately 2 x 10(-5), a 5-fold increase as compared to an uninfected control population. Southern analysis of a set of 13 retrovirus infected TK- mutants revealed a predominance of rearrangements among those mutants which had not undergone loss of heterozygosity. No consistent relationship was found to exist between the occurrence of a rearrangement and tk gene expression as detected by northern analysis. The mechanisms of retroviral shuttle vector insertional mutagenesis were characterized in more detail by focusing on a single TK- mutant, T2. The single proviral insert in T2 was found to lie within tk intron 2, in parallel orientation to the direction of tk transcription. DNA sequence analysis of tk cDNA revealed the presence of an aberrantly spliced product from which exon 4 is excluded. Aberrant splicing could sufficiently account for the low level of functional tk transcript and thus the TK- phenotype in T2, although potential contributions from other mechanisms cannot be excluded.