Biomedical subjects

R C Bleackley

Publications and source records attributed to R C Bleackley.

At least 73 records · Page 4Linked to original sources

PubMed1994

Split-virus influenza vaccines: do they provide adequate immunity in the elderly?

BACKGROUND: Senescence of T-cell function increases susceptibility to influenza with aging. In healthy elderly, we have found that inactivated whole-virus vaccine (WVV) effectively boosts helper T-cell (Th)-mediated immunity. Recently, however, the use of WVV has been superseded by split-virus vaccine (SVV) to questionably reduce adverse effects of vaccination. METHODS: Healthy young adults were compared to healthy elderly adults for their response to SVV. Peripheral blood mononuclear cells (PBMC) obtained pre-vaccination and 6 and 12 weeks post-vaccination, were cultured with live influenza virus, and supernatant IL2 activity was measured. RESULTS: Both groups showed an increase in in vitro IL2 activity by 6 weeks post-vaccination but IL2 decreased to pre-vaccination levels by 12 weeks. Young and elderly adults who had received WVV one year prior did not respond to SVV in this study. CONCLUSIONS: SVV does not provide Th-mediated immunity for the duration of the flu season and may actually suppress Th-mediated immunity in previous recipients of WVV.

Adult↗

PubMed1993

Activation of recombinant murine cytotoxic cell proteinase-1 requires deletion of an amino-terminal dipeptide.

Murine cytotoxic cell proteinase-1 (granzyme B) is a member of a family of novel serine proteinases that have been implicated to participate in destruction of target cells by cytotoxic T lymphocytes. Comparison of the sequence of the cDNA with the sequence of the protein isolated from cytoplasmic granules of cytotoxic T lymphocytes suggested that this protein may be synthesized as a preproenzyme containing an amino-terminal activation dipeptide. Here we show that this activation dipeptide regulates the activity of the enzyme in hydrolysis of its preferred substrate tert-butyloxycarbonyl-Ala-Ala-Asp-thiobenzyl ester. Lysates of COS cells transfected with a vector expressing the unmodified cytotoxic cell proteinase-1 cDNA were unable to hydrolyze this substrate, whereas lysates of cells transfected with a construct in which the activation dipeptide codons has been deleted were able to hydrolyze the substrate. In each case Western blotting of the lysates revealed a form of the proteinase with an apparent molecular weight of 27,000. We conclude that the activation dipeptide regulates activity of the enzyme. This is the first report of production of an enzymatically active recombinant cytotoxic T cell serine proteinase. The strategy for successful expression of an activated form of cytotoxic cell proteinase-1 may be applicable to other members of this proteinase family.

Amino Acid Sequence↗

PubMed1993

Cytotoxic cell proteinase gene expression and cytolytic activity by anti-CD3-activated cytotoxic T lymphocytes is sensitive to cyclosporin A but is not dependent on interleukin-2 synthesis.

We have examined the role of interleukin (IL) 2 in the expression of cytotoxic cell proteinases (CCP) 1 and 2, as well as in the induction of major histocompatibility complex (MHC)-unrestricted cytotoxic activity in murine T cell cultures following stimulation with anti-CD3 monoclonal antibody. A dramatic reduction in CCP-1 and CCP-2 gene expression and near absence of cytolytic activity was shown to occur in these cultures when the expression of IL-2 was inhibited by 10(-6) M cyclosporin A (CsA). The inhibitory effect of CsA could not be eliminated by the addition to culture of recombinant IL-2 at concentrations typically present in anti-CD3-stimulated T cell culture supernatants. Furthermore, when endogenous IL-2 (45-60 U/ml) present in anti-CD3-stimulated T cell cultures was neutralized with anti-mouse IL-2 antibody there was no effect on CCP-1 and CCP-2 mRNA expression and only a slight decrease in cytolytic activity. The expression of CCP-1 and CCP-2 gene products and the induction of MHC-unrestricted cytotoxic activity in anti-CD3-stimulated T cell cultures therefore occur independently of IL-2 synthesis but are regulated by a CsA-sensitive mechanism.

Animals↗

PubMed1993

Molecular cloning, sequencing, and analysis of the cDNA for rabbit muscle glycogen debranching enzyme.

Six peptides were isolated from glycogen debranching enzyme purified from rabbit muscle, and their sequences were determined. A cDNA library made from rabbit muscle using random hexamer primers was screened with oligonucleotide probes constructed in accordance with these peptide sequences. Seven cDNA clones comprising the open reading frame were found, whereas oligo(dT) cDNA libraries yielded no positive clones because of the long 3'-nontranslated region of 2.3 kb. The open reading frame of 4665 bases codes for a 1555-amino-acid protein of M(r) 177,542. Compared to the sequence from human muscle, there are an additional 40 amino acid residues upstream from the N-terminus, and the next 10 residues show no homology. For the remaining 1505 residues, the two sequences exhibit an identity of 93%. The four consensus sequences commonly found at the carboxy termini of beta-strands in the alpha/beta barrel domains of amylases and glucanotransferases are also found in the N-terminal half of the debranching enzyme, suggesting that this structural domain may be present. This and other evidence suggests that the N-terminal half may encompass the transferase activity, leaving the glucosidase activity for the C-terminal half. The latter shows no significant homology to known proteins. An unusual feature of the sequence is the presence of three pairs of adjacent cysteines, which may explain inhibition of the enzyme by organic arsenites.

Amino Acid Sequence↗

PubMed1993

DNA fragmentation induced by cytotoxic T lymphocytes can result in target cell death.

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.

Animals↗

PubMed1993

Induction of a proteoglycan core protein mRNA in mouse T lymphocytes.

The mouse T lymphocyte cell line EL4.E1 synthesizes a proteoglycan core protein (PGCP) mRNA which is identical to serglycin mRNA found in mouse bone marrow-derived mast cells and a mouse mastocytoma cell line. PGCP mRNA was strongly induced in EL4.E1 cells by phorbol myristate acetate, which also induces mRNAs for several cytokines in these cells. In contrast to the induction of cytokine mRNAs, however, the induction of PGCP mRNA was not inhibited by Cyclosporine. PGCP mRNA was also inducible by allogeneic stimulation of normal mouse spleen cells, and by Con A stimulation of an Interleukin 2-producing T hybridoma cell line. A number of other cell lines expressed an identical or similar, mRNA, including two cytotoxic T cell lines, and three tumor cell lines related to bone marrow-derived cells. The levels of several proteoglycans have previously been reported to increase in cells of bone marrow origin under activating conditions, but this appears to be the first report of an induction of the corresponding PGCP mRNA by immune stimulation of T lymphocytes.

Animals↗

PubMed1993

Antibody response to whole-virus and split-virus influenza vaccines in successful ageing.

The antibody response to influenza vaccination has been variably reported to be decreased in elderly individuals. To determine the effect of ageing alone on this antibody response, a group of carefully-screened healthy elderly subjects were compared with young adult controls. Antibody titres for several strains of influenza were measured before and after vaccination with whole-virus (WVV) and split-virus influenza vaccines (SVV) in two successive years. In general, the antibody response to WVV was greater than the response to SVV. Both groups showed a similar response to the H3N2 strain but the elderly group showed a lower response to the H1N1 and B strains of virus contained in the vaccine. Antibodies to older strains of influenza A but not B were stimulated by vaccination with SVV. In the elderly group, the response to older viral strains was relatively increased compared with newer strains. In contrast, the young control group had better antibody responses to the newer than to the older strains of influenza tested. Reductions in the antibody response to influenza vaccination may, therefore, be related to the phenomenon of original antigenic sin and the cohort effect of exposure to H1N1 during childhood in the elderly group studied. The increased immunogenicity of WVV must be considered in light of the current wide use of SVV in the elderly.

Adult↗

PubMed1992

Calreticulin in T-lymphocytes. Identification of calreticulin in T-lymphocytes and demonstration that activation of T cells correlates with increased levels of calreticulin mRNA and protein.

Ca2+ is an essential second messenger for T cell activation, but the exact mechanisms of its action are poorly understood. The cytosolic Ca2+ concentration is significantly increased upon the stimulation of T cells with either mitogen, cross-linking antibodies, or their cognate ligands. In this study, expression of calreticulin, a major Ca(2+)-binding (storage), KDEL protein of the endoplasmic reticulum was examined in resting and concanavalin A (ConA)-stimulated mouse and human T-lymphocytes. Both resting, mouse and human lymphocytes contain very low levels of calreticulin mRNA and protein. Mouse splenocytes stimulated with ConA exhibited an induction in calreticulin mRNA which peaked by Day 4. A 5-fold increase in the immunoreactive calreticulin protein band was also observed in the cells during this period of stimulation. Similarly when human lymphocytes were cultured with ConA a significant increase in the levels of the calreticulin mRNA and protein was observed. The peak of calreticulin mRNA was observed at Day 1 rather than Day 4 as seen for the mouse. These results clearly demonstrate the presence of calreticulin, a Ca(2+)-binding protein originally characterized in muscle tissue, in activated T-lymphocytes. Furthermore, we show that expression of calreticulin correlates with T-lymphocyte activation. Our results suggest that calreticulin may be involved in the signaling pathway for the induction of Ca(2+)-dependent processes and may represent one regulatory mechanism operating in activation of T-lymphocytes.

Amino Acid Sequence↗

PubMed1992

Two cytotoxic cell proteinase genes are differentially sensitive to sodium butyrate.

The 5'-flanking regions of two cytotoxic cell protease genes, CCP1 and 2, are sufficient to confer cytotoxic T lymphocyte-specific expression when fused to a reporter gene. The two regulatory regions are, however, differentially sensitive to treatment of the recipient cell, MTL 2.8.2, with sodium butyrate. With CCP1 a six-fold increase in cat expression was observed, whereas CCP2 was insensitive to the butyrate treatment. One major butyrate-sensitive regions was defined in the CCP1 5'-flanking sequence between -243 to -112 and another less effective one between-682 to -427. These fragments of DNA were also able to confer responsiveness to butyrate when ligated to a heterologous fos promoter. These sequences within the 5' flank of CCP1 share homology with other elements that have been defined as butyrate-responsive. We believe that our results argue against a pleiotropic affect of butyrate such as histone acetylation. More likely sodium butyrate is mediating a specific stimulation of transcription through modification of the activities of selected transcriptional regulatory proteins that in turn affect their interactions with proteins bound to the promoter.

Animals↗

PubMed1992

A comparison of the flanking regions of the mouse cytotoxic cell proteinase genes.

T lymphocyte activation correlates with the transcriptional induction of a variety of genes that encode proteins that are believed to play a role in specific effector functions of the mature cells. Transcripts corresponding to members of the cytotoxic cell proteinase (CCP) family of genes accumulate with different kinetics depending upon the nature of the T cell stimulus. The profile of expression for each family member is unique. Sequences corresponding to the 5' and 3' flanking regions of each of the CCP genes were isolated and sequenced. A comparison of these sequences reveal regions of conservation that are consistent with the differential expression observed and indicate potential regulatory elements.

Animals↗

PubMed1992

Quantitative polymerase chain reaction analysis of cytotoxic cell proteinase gene transcripts in T cells. Pattern of expression is dependent on the nature of the stimulus.

A quantitative polymerase chain reaction assay was developed that allowed us to monitor transcript levels corresponding to individual members of the cytotoxic cell proteinase (CCP) gene family during T cell activation. Selective expression was observed and shown to depend upon the mode of T cell antigen receptor stimulation. Mitogen or allogeneic stimulation of cells resulted in the appearance of transcripts corresponding to all the genes measured, whereas alpha CD3 antibody produced a response restricted to just two family members. This differential gene activation represents a heterogeneity in cytotoxic T lymphocytes that has not been recognized previously. It may indicate that the T cell branch of the immune system can distinguish between different forms of stimulation and respond by synthesizing a specific set of effector and ancillary molecules that is most appropriate for lysis of cells bearing that type of antigen. Only CCP1 transcripts correlated with cytotoxicity for all modes of stimulation. The patterns for the others are suggestive of distinct and ancillary, rather than direct effector, roles in the lytic mechanism.

Animals↗

PubMed1992

Peritoneal exudate lymphocyte and mixed lymphocyte culture hybridomas are cytolytic in the absence of cytotoxic cell proteinases and perforin.

We have utilized the sensitive polymerase chain reaction (PCR) to determine whether cytotoxic T lymphocyte (CTL) hybridomas generated from peritoneal exudate lymphocytes (PEL) and mixed lymphocyte cultures (MLC) express transcripts for perforin and the cytotoxic cell proteinases CCP1 to CCP5. We could readily detect less than one transcript per cell using this methodology. Cytolytic activity could be induced to varying levels in four of the five hybridoma clones tested. With the exception of low level CCP2 expression in the MLC hybridoma MD45 following antigen stimulation, all of the hybridomas could be stimulated to function as potent cytolytic cells in the complete absence of perforin or CCP transcripts. PCR analysis utilizing actin primers indicated that all samples contained material which could be reverse transcribed and PCR-amplified. These results support the argument that populations of lymphocytes do exist that are capable of target cell lysis by an alternative mechanism not involving perforin and CCP.

Animals↗

PubMed1992

The effect of influenza vaccination on IL2 production in healthy elderly: implications for current vaccination practices.

Age-related senescence of T-cell mediated responses is well recognized. This study was designed to determine how aging affects the T-cell mediated Interleukin 2 (IL2) response to influenza vaccination. A group of healthy elderly individuals were compared to a control group of healthy young adults for their response to the 1990 influenza vaccine. Cultures of peripheral blood mononuclear cells (PBMC) were prepared from venous blood samples taken prevaccination (pre) and 8 and 12 weeks post-vaccination (post). PBMC cultures stimulated with inactivated A/Shanghai/16/89 (contained in the 1990 vaccine) and A/Philippine/2/82 (not contained in the vaccine) were assayed for peak IL2 activity. We find that after influenza vaccination, there was an insignificant increase in IL2 activity when PBMC from the young control group were stimulated with A/Shanghai/16/89 (pre, 5.14 U/mL/10(6) PBMC; post, 6.64 U/mL/10(6) PBMC) but there was a significant increase in IL2 activity when stimulated with A/Phillippine/2/82 (pre, 1.5 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). In similar cultures of PBMC from the elderly group, there was a significant increase in IL2 response to both A/Shanghai/16/89 (pre, 1.6 U/mL/10(6) PBMC; post, 3.5 U/mL/10(6) PBMC) and A/Philippine/2/82 (pre, 0.86 U/mL/10(6) PBMC; post, 8.3 U/mL/10(6) PBMC). Measurements of CD4+/CD8+ populations were not affected by vaccination and were not significantly different in the two groups. Subgroup analysis of the elderly group revealed that previous influenza vaccination in 1989 did not significantly affect IL2 levels measured in the present study. This study shows that in healthy elderly, influenza vaccination effectively restores IL2 activity to normal. There appears to be an age-related decrease in the duration of T-cell memory.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1992

The strong correlation of cytotoxic T lymphocyte-specific serine protease gene transcripts with renal allograft rejection.

Several immune mechanisms are likely to be responsible for renal allograft rejection. The relative importance of delayed-type hypersensitivity versus cytotoxic T lymphocytes is controversial. We analyzed human renal allografts biopsies for intragraft expression of IL-1 beta, IL-6, and TNF alpha genes--putative mediators of DTH--as well as IL-2, IL-2 receptor (R) beta, and a CTL-specific serine protease gene. Total RNA was extracted from tissue samples and the mRNA fraction was converted to cDNA using oligo dT and reverse transcriptase. Then cDNA was amplified by the polymerase chain reaction (PCR) for 35 cycles using specific oligonucleotide primers. Each PCR analysis included beta-actin oligonucleotide primers to coamplify this constitutively expressed gene as an internal control. A total of 24 core allograft biopsies were studied and classified into a 3 histological categories: acute cellular rejection, equivocal components of rejection, and no evidence of rejection. There was no statistically significant difference in beta-actin expression among these histologic categories (P greater than 0.08). Interestingly, in this sample size, no significant difference was found between rejecting and nonrejecting samples for transcripts of any of the cytokines or IL-2R beta mRNAs. Apparently, DTH-like mechanisms are present in all allografts. However, detection of CTL-specific serine protease gene expression was almost exclusive to rejecting samples (P less than 0.003). These findings suggest that activation of CTLs play an active, but hardly exclusive, role as effectors of graft dysfunction in the rejection process. While this study does not define the relative importance of the genes examined, it does suggest that evidence of CTL-specific serine protease expression may provide a means of monitoring for rejection episodes or as a diagnostic aid when conventional diagnostic criteria are not conclusive.

Adolescent↗

PubMed1991

Transcription of two cytotoxic cell protease genes is under the control of different regulatory elements.

Precursor cytotoxic T lymphocytes are activated upon interaction with antigen and interleukin 2. The development of the mature killer cell phenotype is achieved by the transcription of a number of function related genes including those encoding a family of cytotoxic cell proteases (CCP). Two of these proteases, CCP1 and CCP2 encoded by C11 and B10, are shown in this report to contain cell-specific transcriptional regulatory elements within their 5'-flanking regions. Two positive regulatory sequences were mapped for C11 (-682 to -427 and -243 to -112) and one for B10 (-279 to -189). In addition each flanking region contains a region of DNA (B10 -1617 to -1049 and C11 -427 to -243) that has a negative influence on transcription. The positive regions do not appear to correspond to any previously characterized regulatory elements but do map to the same region as DNase I hypersensitive sites. When ligated to heterologous promoters these elements can still stimulate transcription but cell-specificity of expression is lost. In addition the conbination of positive regulatory region and promoter is important for the stimulatory effect. The ability of these regulatory sequences to function and to determine cell-specific transcription does not appear to be an intrinsic property but also depends upon the context.

Animals↗

PubMed1991

Structure and evolution of the cytotoxic cell proteinase genes CCP3, CCP4 and CCP5.

A family of serine proteinases is believed to be important in cell-mediated cytotoxicity. Presented here are the genomic sequences for three murine members of this cytotoxic cell proteinase (CCP) family: the CCP3, CCP4 and CCP5 genes. All three of these genes have introns inserted at the same codon sites and the same exon distribution of the active site residues. These characteristics are also shared with the CCP1 and CCP2 genes, the charter members of the CCP gene family. Phylogenetic analysis using intron and exon sequences suggests that all five genes arose by various duplication events. This analysis also indicates that the recently described HuCCPX and CCP2 genes originated from recombination events between genes of different lineages. A phylogenetic and Southern analysis of the recombinant HuCCPX gene suggests that the human genome contains an additional CCP gene that has yet to be described. Finally, evidence is presented suggesting that the cDNA clone originally describing the CCP5 gene was derived from an alternately spliced transcript.

Amino Acid Sequence↗

PubMed1991

Effects of hypothyroidism and high-fat feeding on mRNA concentrations for the low-density-lipoprotein receptor and on acyl-CoA:cholesterol acyltransferase activities in rat liver.

1. Induction of hypothyroidism in rats by feeding propylthiouracil (PTU) significantly increased serum cholesterol concentrations, and the effect was more pronounced for cholesterol in low-density lipoproteins (LDL) rather than high-density lipoproteins (HDL). The concentrations of serum triacylglycerol were decreased in hypothyroidism. These effects on serum lipids were also seen when the normal rats were pair-fed with the PTU-treated group. 2. Feeding a diet rich in saturated fat and cholesterol further increased cholesterol concentrations in LDL and also elevated that in very-low-density lipoprotein (VLDL) of hypothyroid rats. In euthyroid rats such a diet resulted in a relatively small increase in VLDL cholesterol, whereas LDL cholesterol was decreased. 3. Steady-state concentrations of mRNA for the hepatic LDL receptor were significantly decreased in the livers of hypothyroid rats, but were not significantly changed by high-fat feeding in euthyroid or hypothyroid rats. 4. The expression of the LDL receptor in hepatocytes cultured from hypothyroid rats was decreased relative to the euthyroid controls. 5. Whereas the esterification of cholesterol with oleate in hepatocytes cultured from hypothyroid rats was decreased, the activity of acyl-CoA:cholesterol acyltransferase (ACAT) in the livers of these animals was not changed. 6. High-fat feeding increased the hepatic ACAT activity in normal and hypothyroid rats. 7. Incubation of rat hepatocytes with 10 nM-tri-iodothyronine for 4 h increased the relative concentration of the mRNA for the LDL receptor by 25%. 8. It is therefore concluded that thyroid hormones stimulate the synthesis and expression of the hepatic LDL receptor. Elevated cholesterol concentrations in LDL in hypothyroidism probably result from a primary defect in the expression of the hepatic receptor, rather than indirectly via changes in ACAT activity.

Animals↗

PubMed1991

Factors influencing transient expression in cytotoxic T cells following DEAE dextran-mediated gene transfer.

A number of transfection protocols have been tested for the introduction of exogenous DNA into cytotoxic T cells. These included electroporation, lipofection, calcium phosphate coprecipitation, polybrene-assisted gene transfer, and DEAE dextran-mediated transfer. Only the latter gave significant and reproducible transfection efficiencies coupled with low toxicity. The DEAE dextran protocol was optimized for the transfection of a transcription reporter construct pRSVcat into a cloned cytotoxic cell line. Among the parameters investigated were cell density, amount of input DNA, concentration of DEAE dextran, DNA adsorption time, temperature, use of permeabilization and expression facilitators, and recovery time. The optimized protocol was then used to demonstrate the presence of cis-acting regulatory regions in the 5'-flanking sequences of two cytotoxic cell-specific serine protease genes and, in addition, was shown to be applicable to other cloned T-cell lines.

Adsorption↗