Isoform-specific effects of ApoE on HSV immediate early gene expression and establishment of latency

Alzheimer's disease (AD) is a common and devastating neurodegenerative disease in which most cases are of unknown, sporadic origin. In addition to age, the most prevalent known risk factor for developing AD is carriage of the epsilon4 allele of Apolipoprotein E (ApoE). Carriage of the epsilon2 or epsilon3 allele of ApoE confers protection or no change in risk for AD, respectively. Latent herpes simplex virus type 1 (HSV-1) infection in the brain concurrent with ApoE4 carriage exacerbates risk for AD, suggesting that these two factors interact to promote neuronal dysfunction and degeneration in selective brain areas. Indeed, HSV-1 DNA has been found in regions primarily affected by AD, such as the temporal lobes, hippocampus, and neocortex. We hypothesize that HSV-1 infection in the background of ApoE4, but not ApoE2 or ApoE3, promotes an environment more conducive to neuronal degeneration. To investigate this idea, we have utilized transgenic mice that express human ApoE2, 3, or 4 alleles from astrocytes in a murine ApoE -/- background. We find that carriage of the different ApoE alleles dramatically affects HSV-1 immediate early gene expression as well as the establishment of latency. Both of these factors are poised to impact neuronal viability, inflammation, and viral spread. Our data support the concept that HSV-1 and ApoE4 interact to provide an environment conducive to the development and/or spread of AD.     

IFN gamma and CXCR-1 gene polymorphisms in idiopathic bronchiectasis

Idiopathic bronchiectasis is a disease of chronic, bacterial lung infection, unresolving inflammation and progressive lung damage. Bronchiectasis can be associated with autoimmune diseases including ulcerative colitis. Defects of both innate and adaptive immunity have been proposed. The airway inflammation is characterized by interleukin-8 (IL-8) expression and infiltration by neutrophils and T cells. Here we investigated two candidate gene polymorphisms that may contribute to disease susceptibility: a CXCR-1 (+2607 G/C) gene polymorphism that is implicated in IL-8 binding and neutrophil trafficking as well as the interferon-gamma (IFNgamma) (+874 T/A) polymorphism which is linked to levels of IFNgamma production. These polymorphisms were distributed similarly in the idiopathic bronchiectasis group and controls, suggesting that these two candidate gene polymorphisms are not associated with disease susceptibility.     

Evidence of DNA: protein interactions that mediate HSV-1 immediate early gene activation by VP16

The viral genes first expressed upon lytic infection by herpes simplex virus type 1 (HSV-1) encode the five immediate early (IE) proteins. IE gene expression is potently and specifically induced by a virion protein termed VP16. Previous studies have shown that the activating properties of VP16 are IE gene specific and mediated by upstream regulatory elements common to each IE gene. Paradoxically, however, VP16 does not appear to be a sequence-specific DNA-binding protein. To understand the specificity of VP16 activation, we identified the cis-regulatory sequences of an IE gene that mediate VP16 response. Two distinct DNA sequence motifs enable the ICP4 gene to respond to VP16. Biochemical fractionation of nuclear proteins from uninfected cells revealed the existence of cellular proteins that bind directly to each of these VP16 cis-response elements. These observations, in concert with the identification of functional domains of the VP16 protein, lead to the hypothesis that VP16 achieves activation specificity via protein: protein, rather than protein: DNA, interactions.     

Equine herpesvirus type 1 infected cell polypeptides: evidence for immediate early/early/late regulation of viral gene expression

EHV-1 polypeptide synthesis was examined in productively infected rabbit kidney and hamster embryo cells. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of extracts from [35S]methionine- and 3H-amino acid-labeled-infected and mock-infected cultures revealed the presence of 30 infected cell-specific polypeptides (ICPs) which ranged in apparent molecular weights from 16.5K to 213K. Twenty-two of these ICPs comigrated with virion structural proteins. Four ICPs (203K, 176K, 151K, 129K) were detected in extracts of infected cultures labeled in the presence or absence of actinomycin D (Act D) immediately after release from a 4-hr treatment with cycloheximide (CH). These polypeptides, which were designated as EHV-1 immediate early (alpha) ICPs, were not detected in unblocked (non-CH-treated) infected cells. The most abundant ICP was a 31.5K nonstructural protein which, in addition to a 74K protein, was detected in unblocked infected cells at 2-3 hr postinfection. These proteins appeared to be regulated as early (beta) ICPs, since neither protein was observed in Act D-treated cultures released from CH block. Twelve ICPs were classified as late (gamma) polypeptides on the basis of their reduced synthesis in cultures in which viral DNA replication was inhibited by phosphonoacetic acid. All but one (40K) of these late ICPs corresponded to virion structural proteins.     

Inducing gene expression in barrel cortex--focus on immediate early genes

Using only their vibrissae, rats and mice are able to recognize and differentiate surfaces not distinguishable by primates using their fingertips. It has been shown that sensory stimulation elicits the expression of immediate-early genes (IEG), e.g., c-fos and zif268, in the sensory cortex of rats and mice. Though most of these findings come from visual system, mice and rats rely more on their vibrissal system which also offers many advantages for designing precise and precisely controlled experiments. In this review, new models for the selective and simple stimulation of vibrissae are presented and discussed. The data demonstrating IEG expression in the vibrissal system is also reviewed.     

Sex hormone modulation of interferon (IFN) alpha/beta and gamma production by mouse spleen cell subsets following picornavirus infection

Replication of the diabetogenic variant of encephalomyocarditis virus (EMCV-D) in spleen cells and its association with subpopulations of spleen cells (L3T4+, Lyt-2+, Mac 1+, 33D1+ and AGM1+ cells) from both sexes of ICR Swiss mice were examined. Virus replication was limited to less than 0.5 log in suspensions of whole spleen cells, nonadherent cells or a B cell subfraction from both sexes of ICR Swiss mice following infection with EMCV-D at an MOI of 10; no virus replication was seen in adherent spleen cells from either sex. After 1 hour adsorption of EMCV-D onto spleen cells at a multiplicity of infection (MOI) of either 10 or 0.1, virus-associated cells were isolated using a monoclonal murine anti-EMCV-D and anti-mouse IgG conjugated to magnetic beads. Using an MOI of 0.1, less than 1% of spleen cells bound virus particles after 1 hour adsorption at 4 degrees C. Among the virus-positive cells, relatively higher percentages of adherent cell populations (Mac 1+ and 33D1+ cells) of both sexes bound virus particles within the first hour post-infection (PI) than did the other spleen cell subpopulations. Interferon (IFN) alpha/beta production was detected as early as 4 hours PI in female spleen cell cultures infected with EMCV-D at an MOI of 0.1 while no IFN alpha/beta activity was found in comparably infected male spleen cell cultures. Inhibiting IFN alpha/beta activity in the virus-infected spleen cell cultures during the first 20 hours of infection using polyclonal rabbit anti-mouse IFN alpha/beta serum eliminated production of IFN gamma as well as IFN alpha/beta. Spleen cell cultures depleted of adherent cells were unable to produce IFN alpha/beta or IFN gamma in the first 24 hours PI. The capacity to produce IFN gamma at 12 hours after virus infection of spleen cells from both sexes of mice was restored to adherent cell-depleted cultures by addition of mouse IFN alpha/beta at the time of infection. These results suggest that IFN alpha/beta and adherent cells play critical roles in the early production of IFN gamma (less than 16 hours PI) characteristic of the infected spleen cell cultures of females. Production of IFN alpha/beta and IFN gamma by spleen cells from both sexes of ICR Swiss mice was enhanced by administrating estrone to donor mice during the week before harvesting spleen cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differential effects of neonatal norepinephrine lesions on immediate early gene expression in developing and adult rat brain

Activity regulated cytoskeletal protein (Arc), c-fos and zif268 are immediate early genes (IEGs) important for adult brain plasticity. We examined developmental expression of these IEGs and the effect of neonatal noradrenergic lesion on their expression in developing and mature brain. N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride (DSP-4), a specific noradrenergic neurotoxin, was administered to rats on postnatal day (PND) 3 and in situ hybridization was used to assay Arc, c-fos and zif268 mRNA on PND 13, 25 and 60. In contrast to decreases in Arc, c-fos and zif268 expression produced by noradrenergic lesions of mature brain, lesions on PND 3 yield a strikingly different effect. Neonatal lesions produce increases in c-fos and zif268 expression in specific frontal cortical layers on PND 13, while Arc shows no change. These lesions lead to increases in zif268 expression in frontal cortical layers on PND 25, with no changes in c-fos or Arc expression, and on PND 60 they produce a significant increase in c-fos expression in hippocampus with no significant changes in Arc or zif268 expression. 2-[2-(2-Methoxy-1,4-benzodioxanyl)]imidazoline hydrochloride (RX821002), an alpha-2 adrenergic receptor (A2AR) antagonist, administered to control PND 60 animals produces elevations of Arc, zif268 and c-fos mRNAs. This response was eliminated in animals lesioned with DSP-4 on PND 3. These data indicate that norepinephrine regulation of IEG expression differs in developing and mature brain and that loss of developmental norepinephrine leads to abnormally high postnatal IEG expression. Previous studies have shown an important role for norepinephrine in brain development. Our data support the idea that norepinephrine plays an important role during CNS development and that changes in noradrenergic signaling during development may have long lasting effects, potentially on learning and memory.     

Preliminary studies on the interaction of TNF alpha and IFN gamma with alpha 2-macroglobulin.

The binding of 125I-labelled recombinant human TNF alpha and IFN gamma to isolated human blood alpha 2-macroglobulin has been investigated using molecular sieving procedures and non-denaturing PA gel electrophoresis in combination with autoradiography. These studies revealed that both cytokines readily bind to the electrophoretically fast form of alpha 2M generated by methylamine or protease treatment of this protein. PAGE/SDS gel investigations indicated that TNF alpha bound non-covalently while the IFN gamma interaction was covalent in nature. Preliminary competition studies also indicate that cold TNF alpha and IL-2 are more effective than cold IFN gamma at inhibiting the binding of labelled IFN gamma to alpha 2M. Bioassays revealed that "native" alpha 2M or its derivatives at 2 mg/ml concentration did not impair the antiproliferative effects of TNF alpha and IFN gamma on susceptible bladder tumour cell lines. Furthermore they did not interfere in the induction of Class II antigen expression by IFN gamma on inducible cell lines or in a 2-site ELISA assay for TNF.     

A simple procedure for expression and purification of selected non-structural (alpha and beta) herpes simplex virus 1 (HSV-1) proteins

The expression and isolation of herpes simplex virus 1 (HSV-1) immediate early (alpha) IE63 (ICP27) and of the early (beta) thymidine kinase (Tk) polypeptides in Escherichia coli JM 109 cells transformed with the PinPoint Xa-1 (Promega) plasmid construct carrying either the HSV-1 UL54 or UL23 genes are described. The resulting biotinylated fusion protein(s) could be easily induced and were purified in appropriate amounts by means of a monomeric avidin-conjugated resin (SoftLink Soft Release Avidin Resin, Promega) provided that: (1) the exponential growth of the selected transformed cells was monitored carefully; (2) the post-induction harvest interval was properly chosen; and (3) the period for adsorption to the avidin resin suitably adjusted. The isolated protein(s), although partially digested in the case of the IE63 polypeptide, were suitable antigen(s) for immunization of various animal species. Co-purification of trace amounts of endogenous biotinylated protein(s) produced in E. coli was eliminated by shortening the duration of adsorption to the avidin resin.     

Host cell and virus strain differences in synthesis of immediate early polypeptides in HSV-1 infected cells

The synthesis of the immediate early (IE) polypeptides was analysed in primary rabbit kidney (RK) cells and a stable line of rabbit lung (ZP) cells infected with the syncytial (syn) strain HSZP and the non-syn strain KOS of herpes simplex virus type 1 (HSV-1). Results showed the following: After cycloheximide reversal the infection of RK and ZP cells with HSZP strain led to synthesis of five IE polypeptides (175K, 136K, 87K, 68K, and 63K), while infection of both cell cultures with the KOS strain led to synthesis of significantly reduced amounts of the IE polypeptides. The ability to switch on the expression of non-alpha viral genes was impaired in RK cells infected with the HSZP strain. The IE polypeptides were still detectable without any sign of the non-IE polypeptide synthesis 4 hr after cycloheximide reversal. The observed failure of the IE HSZP polypeptides to undergo posttranslational modification in ZP cells may be the consequence of this phenomenon. In contrast to the KOS IE mRNAs, the HSZP IE mRNAs exhibited a pronounced functional stability in both cell cultures. The IE polypeptides were still synthesized in HSZP-infected cells which had been incubated for 19 hr after cycloheximide reversal in the presence of actinomycin D (Act D). The HSZP strain failed to suppress the host polypeptide synthesis in RK but not in ZP cells. However, the HSZP strain, in contrast to the KOS strain, proved to be defective with respect to the early shutoff of host polypeptide synthesis in both cell cultures.(ABSTRACT TRUNCATED AT 250 WORDS)     

HSV-1gC糖蛋白真核表达载体的构建及表达

目的:构建单纯疱疹病毒1型(HSV-1)囊膜糖蛋白C(gC)哺乳动物表达载体pCI-mCMV-gC-1-IRES-DHFR-L22R,实现其在中国仓鼠卵巢细胞(CHO-K1)中的稳定表达.方法:利用基因重组技术构建同时克隆有gC-1基因和中国仓鼠二氢叶酸还原(DHFR-L22R)基因的真核表达载体pCI-mCMV-gC-1-IRES-DHFR-L22R,按Lipofectamine(TM)2000说明书转染野生型CHO-K1细胞,在G418和氨甲喋呤(MTX)双筛选压力下筛选稳定表达细胞株,Slot blot方法测定重组蛋白的表达,并用His-Ni琼脂糖填料进行目的蛋白纯化,纯化后West-ern blot检测.结果:成功构建了真核表达载体pCI-mCMV-gC-1-IRES-DHFR-L22R.经过两轮筛选获得稳定表达HSV-1型邪基因的细胞系,Western blot成功检测到目的蛋白.结论:HSV-1 gC在CHO-K1细胞中成功表达,并具有良好的特异性结合活性,为进一步的实验研究和临床应用提供了基础.     

Developmental T cell receptor gene rearrangements: relatedness of the alpha/beta and gamma/delta T cell precursor.

To examine the relationships between T cell populations at various stages of development, T cell receptor (TcR) gene rearrangements were compared between the four murine populations of (a) early thymocytes, (b) early splenocytes, (c) adult thymocytes and (d) adult splenocytes. TcR alpha gene rearrangements were shown to progress from 5' to 3' regions of the J alpha locus and from 3' to 5' regions of the V alpha locus during the development of T cells in both the thymus and spleen. Thus, the gene rearrangement potentials of proximal genes varied with age, yielding a biased repertoire in the young vs. adult animal. As evidence that gamma/delta and alpha/beta gene rearrangements appeared concomitantly in individual precursors, it was found that: (a) multiple adult thymocytes bore alpha gene rearrangements on one chromosome and delta gene rearrangements on the homologous chromosome, and (b) V gamma 3-J gamma 1 rearrangements, prominent joins in the early gamma/delta T cell population, were also prominent in the early alpha/beta T cell subset. These data illustrate the non-random nature of the developmental TcR gene rearrangement and suggest that alpha/beta and gamma/delta T cell populations derive from related, if not identical, T cell precursor populations.     

Endotoxin-induced cytokine gene expression in vivo. II. Regulation of tumor necrosis factor and interleukin-1 alpha/beta expression and suppression.

Tumor necrosis factor alpha (TNF alpha) mRNA is present in a preformed intracellular pool in the spleen, liver, and small bowel of naive rats. Endotoxin (Salmonella typhus lipopolysaccharide) injected intravenously induces little or no increase in whole-organ TNF mRNA levels at 15', 30', 1 degree, 2 degrees, or 4 degrees, whereas serum TNF levels are markedly elevated at 1 and 2 hours. Dexamethasone pretreatment of rats suppresses LPS-induced serum TNF concentrations, but does not suppress TNF mRNA levels in the spleen or bowel. Tachyphylaxis experiments demonstrate that a second injection of endotoxin 2 hours after an initial injection fails to induce a second peak of serum TNF, although TNF mRNA levels in the spleen and bowel remain at the levels found in naive rats. Corynebacterium parvum upregulates endotoxin-induced serum TNF release and intravenous injection of IL-1 induces the release of serum TNF but neither alters whole-organ TNF mRNA levels. Interleukin-1 alpha (IL-1 alpha) mRNA was not constitutively detected in whole-organ RNA preparations of the spleen, liver, and small bowel of naive rats. Endotoxin induces IL-1 alpha mRNA most easily appreciated in the spleen beginning at 1 hour, peaking at 2 to 4 hours, and disappearing by 6 hours. Interleukin-1 beta (IL-1 beta) mRNA was not constitutively detected in the organs examined or was present in small amounts. Endotoxin induces IL-1 beta mRNA beginning at 0.5 hours, peaking at 1 hour, and disappearing by 6 hours. Dexamethasone pretreatment prevents the LPS-induced appearance of IL-1 alpha mRNA and suppresses but does not completely inhibit the appearance of IL-1 beta mRNA. C. parvum upregulates endotoxin-induced IL-1 mRNA expression. Intravenous injection of TNF or IL-1 both induce IL-1 mRNA expression. In conclusion, TNF mRNA is constitutively expressed and TNF mRNA levels as analyzed in whole-organ RNA preparations do not change in concert with serum TNF protein levels during conditions of endotoxemia, dexamethasone treatment, tachyphylaxis, priming with C. parvum, or after injection of IL-1. In contrast, IL-1 mRNA expression during endotoxemia, dexamethasone treatment, priming with C. parvum, or after injection of TNF or IL-1 shows clear increases and decreases in whole-organ RNA preparations.     

Chlamydiae modulate gamma interferon, interleukin-1 beta, and tumor necrosis factor alpha receptor expression in HeLa cells

Chlamydia psittaci was found to modulate receptor expression for the cytokine receptors that are involved in the synergistic induction of indoleamine dioxygenase in epithelial cells. Increases in receptor expression were seen even with inactivated Chlamydia, suggesting that chlamydial antigens and not products of infection are important for up-regulating cytokine receptor expression.     

Stoichiometric analysis of the TM2 6' phenylalanine mutation on desensitization in alpha1beta2 and alpha1beta2gamma2 GABA A receptors

The presence of phenylalanine (F) at the 6' position of transmembrane domain 2 (TM2) in the alpha4 subunit of alpha4beta2 nicotinic receptors enhances desensitization. As the GABA A receptor affords the ability to study the influence of as few as one and as many as five Fs at this position, we have used it to investigate potential subunit- and stoichiometry-dependent effects of the TM2 6'F mutation on desensitization. Whereas the presence of one F at this position decreased extent of desensitization, desensitization was increased in all configurations that included two or more Fs at the TM2 6' position; desensitization was particularly rapid with 3 or 4 F residues present. Our results demonstrate the ability of F residues at the TM2 6' position to modulate desensitization is likely conserved in the cys-loop family of ligand-gated ion channels. Moreover, our findings demonstrate both stoichiometric- and subunit-dependent effects of the ability of this mutation to regulate desensitization in GABA A receptors.     

An analysis of alpha/beta TCR V gene expression in the human thymus.

We have previously analysed alpha/beta T cell receptor (TCR) V gene usage in CD4+ and CD8+ subpopulations from human peripheral blood lymphocytes (PBL) and umbilical cord blood, and described a biased usage of some of the TCR V beta genes towards the CD4+ subpopulation. In this report, the TCR V gene usage in single positive (SP) CD4+ or CD8+ human thymocytes was analysed. Three previously described mAb with a biased usage in PBL and umbilical cord blood also had a skewed reactivity towards the CD4+ subpopulation in SP human thymocytes. Thus, in all 12 cases V beta 5.1 and V beta 6.7, and in 11/12 cases V beta 12 were preferentially used in the CD4+CD8-, compared to the CD4-CD8+ thymic subpopulation. Altogether, these results suggest a selection process in the thymus, supposedly through the positive influence of MHC class II determinants, to be responsible for this non-random, skewed TCR V gene usage.     

Functional role of alpha 2/beta 1 and alpha 4/beta 1 integrins in leukocyte intercellular adhesion induced through the common beta 1 subunit.

Whereas all of the integrins in the VLA protein subfamily are involved in cell-extracellular matrix interactions, only VLA-4 (through the alpha 4 subunit) has been implicated in the triggering of intercellular adhesion. Here we describe that the VLA protein beta 1 subunit (CD29) is also involved in the induction of homotypic cell aggregation. We have obtained three novel anti-beta 1 monoclonal antibodies (mAb) with the ability to induce cell aggregation on different leukocyte cell types. These mAb recognize an antigenic site on the common beta 1 chain of VLA proteins which is topographically and/or functionally distinct from other epitopes previously defined by several prototype anti-beta 1 mAb. Induction of cell aggregation by anti-beta 1 mAb is epitope specific, isotype and Fc independent, and displays kinetics similar to alpha 4-mediated aggregation. This cell aggregation requires an intact cellular metabolism, the presence of divalent cations in the extracellular medium, and the integrity of the cytoskeleton. We also have found that the Na+/H+ antiporter may be essential for this process. For Ramos cells, which bear only the VLA alpha 4/beta 1 heterodimer, intercellular adhesion induced through the VLA-beta 1 chain could be selectively inhibited by other anti-beta 1 mAb as well as by anti-alpha 4 mAb. Interestingly, anti-beta 1 mAb which induced strong aggregation of VLA-alpha 2- or VLA-alpha 4-transfected K562 cells, had minimal effect on the alpha 2- alpha 4- alpha 5+ K562 cell line. Furthermore, the beta 1-mediated induction of cell aggregation on alpha 2-K562- and alpha 4-K562-transfected cells was blocked by preincubation with either anti-alpha 2 or anti-alpha 4 mAb, respectively, as well as by other anti-beta 1 mAb. Interestingly, parental K562 cells were able to interact with both alpha 2- and alpha 4-transfected K562 cells, thus suggesting that counter-receptors for both integrins (VLA-2 and VLA-4) might exist on these cells. Together these results provide strong evidence supporting the involvement of alpha 2/beta 1 and alpha 4/beta 1 heterodimers in intercellular interactions and underline the pivotal role of the common beta 1 chain of VLA proteins in the integrin-mediated induction of cell aggregation.