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Biomedical subjects

C Bird

Publications and source records attributed to C Bird.

At least 37 records · Page 2Linked to original sources
PubMed1997

Immunochemical assessment of aflatoxin M1 in milk powder consumed by infants in São Paulo, Brazil.

Aflatoxin M1 was surveyed in 300 samples of whole milk powder consumed by infants at municipal schools and nurseries in São Paulo, Brazil. The analyses were performed by using commercially available test systems of a direct competitive enzyme-linked immunosorbent assay (ELISA). Samples were reconstituted in water (1:8), centrifuged at 1630 x gav for 15 min, and submitted directly to the assay without clean-up procedures. Results showed 33 (11%) positive samples for aflatoxin M1 at levels of 0.10-1.00 ng/ml (mean: 0.27 +/- 0.20 ng/ml). By using data on milk consumption patterns for 4-month-old children (highest intake), a mean daily intake of 3.7 ng/kg body weight/day was estimated. The implications of these data on human health are discussed.

Aflatoxin M1↗
PubMed1997

Production and characterization of monoclonal antibodies to human interleukin-12.

Interleukin-12 is a cytokine that plays a central role in mediating cell mediated immunity via enhancement of a TH1 cell response. IL-12, unusually for a cytokine, has a heterodimeric structure made up of 35 kDa and 40 kDa subunits. The aim of this study was to produce and characterize monoclonal antibodies to recombinant human IL-12. Twenty-two monoclonal antibodies to IL-12 were successfully produced and subunit specificity determined using recombinant human IL-12 and chimeric murine/human IL-12. All antibodies were shown to react with the p40 subunit by ELISA and immunoblotting with three of the MAbs being found to cross-react with murine IL-12. Using two individual bioassays for IL-12, seven of the MAbs were shown to neutralize biological activity of IL-12. Ten of the antibodies were found to be of use in immunocytochemistry, reacting with LPS-stimulated peripheral blood monocytes. The approaches and difficulties encountered in characterizing antibodies to a heterodimeric cytokine are discussed together with possible reasons for the failure to generate antibodies to the p35 subunit.

Animals↗
PubMed1996

Proteinase inhibitor 6 cannot be secreted, which suggests it is a new type of cellular serpin.

We have recently described a new serine proteinase inhibitor, proteinase inhibitor 6 (PI-6). This serpin has features that suggest it may function intracellularly, but its close resemblance to ovalbumin serpins like plasminogen activator inhibitor 2 (PAI-2) raises the possibility that it is secreted to regulate an extracellular proteinase. To determine whether PI-6 is secreted, we have examined its cellular distribution by immunohistochemistry and have attempted to induce its release from platelets and from cultured cells. We find that PI-6 is present in endothelial and epithelial cells, but it is apparently cytoplasmic and it is not released from cells in response to phorbol ester, dibutyryl cAMP or tumor necrosis factor alpha treatment. It is also not released from activated platelets. The addition of a conventional signal peptide to the amino terminus of PI-6 directed its translocation into the endoplasmic reticulum (ER), resulting in glycosylation but not secretion of the molecule. By contrast, the addition of the same signal peptide to PAI-2 markedly enhanced its translocation and secretion. Glycosylated PI-6 was sequestered in the ER and was incapable of interacting with thrombin. The failure of PI-6 to move along the secretory pathway, and the loss of inhibitory function of ER-localized PI-6, demonstrates that unlike PAI-2, PI-6 is not naturally secreted. Taken together, these results suggest that PI-6 has evolved to fulfil an intracellular role and that it represents a new type of cellular serpin.

Amino Acid Sequence↗
PubMed1996

Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays.

Some adverse reactions to the transfusion of platelet concentrates (PCs) cannot be attributed to antibodies against blood cells or to subclinical microbial agents. It has been suggested that leucocyte-derived inflammatory cytokines such as interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF) may contribute to a larger number of unexplained non-antibody-mediated adverse reactions. Three types of PCs, containing different levels of leucocytes, are currently produced. Filtration is used on demand to further reduce leucocyte contamination of these components. we have monitored the plasma of PCs prepared by the platelet-rich plasma method (PRP), the buffy-coat method or by apheresis for IL-6, IL-1, transforming growth factor-beta (TGF-beta), TNF and interferon gamma (IFN gamma). Biologically active IL-6 increased in stored PRP-PCs from a mean of 140 pg/ml on day 1 to 2395 pg/ml on day 5/6. Elevated levels of IL-8, as detected by immunoassay, were evident in PRP-PCs during routine storage under blood bank conditions. Small amounts of immunoreactive IL-1 with only minimal biological activity were present in some PRP-PCs by day 5/6. No significant increase in the levels of IL-8, IL-6 or IL-1 were seen in buffy-coat PCs during storage for 5/6 d. For apheresis PCs, an increase in IL-8 content, but not in IL-6 over 6 d was observed. In all three types of PCs, elevated amounts of both bioactive and immunoreactive TGF beta were present, but there was no evidence of any biologically active or immunoreactive TNF alpha. Pre-storage filtration of PRP-PCs for depletion of leucocytes prevented the increase in IL-8 and IL-6 levels of these PCs. Our results show that leucocyte reduction by buffy-coat method reduces cytokine levels to a comparable level to filtered or apheresis PCs, containing low levels of leucocytes, but use of these PCs in minimizing the severity and incidence of reactions in recipients will require clinical evaluation. This is the first comprehensive and comparative study which, on the basis of biological activity of cytokines, directly indicates that the mode of platelet production grossly influences the levels of cytokines.

Biological Assay↗
PubMed1996

Production of neutralizing granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies in carcinoma patients following GM-CSF combination therapy.

In this study, the development of neutralizing and non-neutralizing GM-CSF antibodies and the clinical consequences related to the induction of these antibodies were analysed in 20 patients with metastatic colorectal carcinoma receiving a combination therapy of Escherichia coli-derived GM-CSF and a colon carcinoma-reactive MoAb in the absence of any concomitant chemotherapy. The recombinant human GM-CSF was administered subcutaneously for 10 days every month for 4 months. Following the first cycle of treatment, no GM-CSF antibodies were detected, but during subsequent therapy, 19 of the 20 patients studied developed GM-CSF binding antibodies. However, only a proportion (40%) of the 19 antibody-positive patients developed antibodies that neutralized the biological activity of GM-CSF in an in vitro bioassay. The presence of GM-CSF neutralizing antibodies was associated with a significant reduction in GM-CSF-induced expansion of leucocytes, neutrophils and eosinophils. Such clinical effects were not apparent in patients with non-neutralizing antibodies. Further characterization of sera from patients with neutralizing antibodies showed that, in most cases, the antibodies neutralized the biological activity of GM-CSF preparations derived using different expression systems (Chinese hamster ovary cells and yeast), suggesting that these antibodies may have the potential to cross-react with endogenously produced GM-CSF. These effects should be considered before therapeutic use of cytokines, particularly in patients who are not immunosuppressed, and therefore capable of mounting an effective immune response. Our results indicate that assessment of production of neutralizing antibodies induced during cytokine therapy can be used to predict diminished clinical response to further therapy.

Adolescent↗
PubMed1996

Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits.

The influenza virus A/Japan/305/57 hemagglutinin (HA) can be converted from a protein that is essentially excluded from coated pits into one that is internalized at approximately the rate of uptake of bulk membrane by replacing the HA transmembrane and cytoplasmic sequences with those of either of two other glycoproteins (Roth et al., 1986. J. Cell Biol. 102:1271-1283). To identify more precisely the foreign amino acid sequences responsible for this change in HA traffic, DNA sequences encoding the transmembrane (TM) or cytoplasmic (CD) domains of either the G glycoprotein of vesicular stomatitis virus (VSV) or the gC glycoprotein of herpes simplex virus were exchanged for those encoding the analogous regions of wild type HA (HA wt). HA-HA-G and HA-HA-gC, chimeras that contain only a foreign CD, resembled HA wt in having a long residence on the cell surface and were internalized very slowly. HA-HA-gC was indistinguishable from HA in our assays, whereas twice as much HA-HA-G was internalized as was HA wt. However, HA-G-HA, containing only a foreign TM, was internalized as efficiently as was HA-G-G, a chimeric protein with transmembrane and cytoplasmic sequences of VSV G protein. Conditions that blocked internalization through coated pits also inhibited endocytosis of the chimeric proteins. Although the external domains of the chimeras were less well folded than that of the wild type HA, denaturation of the wild type HA external domain by treatment with low pH did not increase the interaction of HA with coated pits. However, mutation of four amino acids in the TM of HA allowed the protein to be internalized, indicating that the property that allows HA to escape endocytosis resides in its TM. These results indicate that possession of a cytoplasmic recognition feature is not required for the internalization of all cell surface proteins and suggest that multiple mechanisms for internalization exist that operate at distinctly different rates.

Amino Acid Sequence↗
PubMed1996

Microfocal techniques in quantitative radiography: measurement of cancellous bone organization.

Microfocal radiography records, with unusually good resolution, the detailed structural organization of cancellous bone. A textural imaging method, fractal signature analysis (FSA), was used to quantify the horizontal and vertical trabecular organization recorded within macroradiographic images of the spine of post-menopausal women and the tibia in osteoarthritic knees, and the analysis of variance method was applied to the wrist and hand of rheumatoid patients. Changes in trabecular structure were found to correlate with (i) body weight, age and bone mineral density in the lumbar spine of post-menopausal women; (ii) the degree of cartilage loss and age in the tibia of patients with knee OA; and (iii) analysis of variance quantified the extent of 'normal', osteopaenic and eroded bone in rheumatoid joints. Quantitation of cancellous bone organization can add significantly to our understanding of disease processes and effect of therapy in diseased joints.

Aged↗
PubMed1996

Physician responses to a hospital policy allowing them to not offer cardiopulmonary resuscitation.

OBJECTIVE: To determine whether physicians followed a hospital policy permitting them to not offer cardiopulmonary resuscitation (CPR) to patients considered unlikely to benefit from this therapy. DESIGN: Prospective series. SETTING: San Francisco General Hospital, a university-affiliated public hospital. PATIENTS: Attending physicians were interviewed about patients admitted over a period of 5 consecutive months for whom do-not resuscitate (DNR) orders were written. Only the first patient for whom each physician wrote a DNR order was included. MAIN OUTCOME MEASURES: Responses of physicians to questions regarding their initiation of DNR orders for these patients. RESULTS: Sixty-nine physicians responded to questions about DNR orders written for 69 patients. Fifty-seven of these physicians said that they were aware of the hospital policy, and 49 of the 57 physicians said that they generally agreed with it. Thirty-three physicians felt that CPR should be offered only to patients likely to benefit from it. In contrast, 36 physicians said that CPR should be offered to all patients, regardless of benefit. CPR was offered to 41 patients and 15 surrogates; 27 of these patients were considered to be unlikely to benefit from CPR. CONCLUSION: Despite a policy that allows them to do otherwise, physicians usually offered CPR to patients, regardless of benefit. Most physicians believed that CPR should be offered to all patients. These findings suggest that policies such as that of San Francisco General Hospital may not be compelling because physicians hold attitudes that are inconsistent with the policies.

Attitude of Health Personnel↗
PubMed1995

Sequence comparison and classification of beet luteovirus isolates.

Three distinct sequence groups were found among partial nucleotide sequences of 38 isolates of beet western yellows virus (BWYV) and beet mild yellowing virus (BMYV) from Europe, Iran and the USA. The first group contains both sugar beet and oilseed rape specific isolates, and the differentiating characteristic linked to this host range specificity are 2 single base pair changes in a 1,200 nucleotide region of the genome. It is proposed that the European BWYV strains that can be transferred at low frequency between rape and sugar beet belong to this group. Also belonging to this group are the published BWYV sequences of Veidt et al. and of the California BWYV-ST9 isolate. The second group contains mostly rape-derived isolates which have an intergenic region highly distinct from that of group-1 isolates but similar polymerase and coat protein regions. It is proposed that the rape-specific BWYV isolates which cannot be transmitted to sugar beet belong to this group. The third group contains mostly beet-specific isolates from Southern Europe and Iran, and may be adapted to the Mediterranean climate and flora. It is distinct from groups 1 and 2 in all three genome regions investigated and its polymerase and intergenic regions are as much related to those of potato leafroll virus (PLRV) and curcurbit aphid borne yellows virus (CABYV) as they are to those of group-1 and group-2. On the basis of sequence similarities and established nomenclature it is proposed to use BWYV for groups 1 and 2 (BWYV-1 and BWYV-2 respectively) and to use BMYV for group-3 isolates, which are distinct enough from the other two groups to merit a separate nomenclature.

Amino Acid Sequence↗
PubMed1994

Expression of a tomato cDNA coding for phytoene synthase in Escherichia coli, phytoene formation in vivo and in vitro, and functional analysis of the various truncated gene products.

Full length and truncated cDNA expression constructs of the phytoene synthase (psy) gene from tomato have been ligated into a pUC8 cloning vector. One of the truncated constructs was introduced into Escherichia coli carrying the Erwinia uredovora GGPP synthase gene. This transformant produced 15,15'-cis-phytoene, which was identified on the basis of its UV and IR spectral data, from geranylgeranyl diphosphate. The function of this gene product was further confirmed by in vitro assay using cell-free extract of E. coli harboring the construct. On transformation with the above constructs together with a plasmid containing the carotenoid gene cluster from E. uredovora devoid of the phytoene synthase (crtB) gene, yellow, carotenoid-containing, E. coli colonies were produced. The amounts of carotenoids synthesized by the transformed cells, related to the steady-state levels of psy mRNA, varied depending upon the psy constructs. The full-length psy clone produced 16-fold less carotenoids per unit amount of RNA than cells containing phytoene synthase without the first 114 N-terminal amino acids. Removal of further amino acids from the N-terminus caused a large decrease in carotenogenesis. A Western blot of ripe fruit stroma with a monoclonal antibody raised against phytoene synthase revealed a single protein band of apparent molecular mass 38 kDa. Based upon this immunological evidence, we conclude that the size of the transit peptide of phytoene synthase from ripe tomato fruit is approximately 9 kDa, corresponding to about 80 amino acid residues.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkyl and Aryl Transferases↗
PubMed1994

A collaborative trial of a semi-automatic system for slide preparation and screening in cervical cytopathology.

We report a test of an experimental system for machine-aided screening in cervical cytology, comprising the 'CYTOPRESS' semi-automatic slide preparation system (Nijmegen) and the 'CERVIFIP' interactive scanner (Edinburgh). Material from women attending clinics in Edinburgh and Nijmegen was stratified according to the severity of the conventional laboratory diagnosis and selected randomly within strata for inclusion in the test. Monolayered slides were prepared by CYTOPRESS from cervical scrape material remaining after preparation of conventional smears and scanned by CERVIFIP to determine the positions of the most 'suspicious' objects. The test was based on a set of 701 monolayers, divided equally between 'negatives' and 'abnormals' of various grades, of which 585 (83.4%) were passed automatically as adequate for machine-aided analysis. Approximately 15% of adequate slides were passed as 'negative' without operator interaction. In the remaining 85%, the suspicious objects were inspected by a human operator and a decision was then made either to refer each monolayer for conventional microscopic analysis, or to pass it as 'negative'. Where discrepancies occurred between the conventional laboratory and the system results, a consensus diagnosis was reached by taking into account all relevant information including clinical data. Of those with a consensus diagnosis of CIN 3 or worse an estimated 9.3 +/- 4.1% were passed by the system as 'negative'. Closer investigation of these false-negatives revealed that most, and perhaps all, were preventable by system improvements either planned or in progress. Corresponding false-negative rates for those graded 'CIN 1 or 2' and 'negative-early recall' were estimated, respectively as 18.9 +/- 5.3% and 22.9 +/- 3.1%. Of those with a 'negative-routine recall' consensus, 19.4 +/- 2.5% were referred for conventional microscopic analysis, a level well within acceptable limits for cost-effectiveness. Women whose initial laboratory smears were negative, but whose consensus diagnosis was 'negative-early recall' or CIN, are being investigated further to determine whether cervical abnormalities were in fact present. Over two-thirds of this group were referred by the machine-aided system for conventional microscopic analysis.

Diagnosis, Computer-Assisted↗
PubMed1993

Increased interleukin 6, but reduced interleukin 1, in delayed pressure urticaria.

Interleukin 1 (IL-1) and interleukin 6 (IL-6) were measured by bioassays in suction-blister exudates from lesional skin, from skin immediately following a pressure challenge, and from control skin (not subjected to pressure) of patients with delayed pressure urticaria. IL-6 activity in lesional exudates was significantly higher than in exudates from the other two sites. IL-1 activity in lesional exudates was not significantly higher than in the control exudates, but significantly less IL-1 activity was found immediately after pressure challenge than from the control site.

Adult↗
PubMed1992

The cytoplasmic domain of the polymeric immunoglobulin receptor contains two internalization signals that are distinct from its basolateral sorting signal.

The C-terminal cytoplasmic domain of the polymeric immunoglobulin receptor (pIgR) contains two tyrosine residues, Tyr668 and Tyr734. Previous work identifying Tyr734 as a critical residue in the endocytosis of the pIgR in Madin-Darby canine kidney (MDCK) cells also suggested that a second functional internalization signal was present (Breitfeld, P. P., Casanova, J. E., McKinnon, W. C., and Mostov, K. E. (1990) J. Biol. Chem. 265, 13750-13757). To test this hypothesis, Tyr668 and Tyr734 were mutated singly or together by oligonucleotide-directed mutagenesis of pIgR cDNA, and the mutants were expressed in MDCK cells. The amount of ligand internalized within 5 min from the basolateral membrane by the pIgR in which cytoplasmic tyrosines were mutated separately to Cys668 or Ser734 or together to Cys668, Ser734 was 58, 39, and 20%, respectively, of the internalized by the wild-type pIgR. The cytoplasmic and transmembrane domains of the pIgR, when joined to the external domain of the influenza virus hemagglutinin, retained the capacity to mediate rapid internalization. As with the full-length pIgR, mutation of either tyrosine in the chimera resulted in impairment of endocytosis, with mutation of Tyr734 having a significantly greater effect than mutation on Tyr668 on the initial rate of endocytosis (3 and 44% of control values, respectively). However, unlike the full-length pIgR, mutation of both tyrosines together in the chimera did not reduce internalization further. The two tyrosines in the cytoplasmic sequence of the pIgR, although widely separated in the linear amino acid sequence, both contribute to internalization of the protein, suggesting that both can function as internalization signals. In addition, the correlation between endocytosis and basolateral targeting of the pIgR in MDCK cells was investigated. Neither tyrosine of the cytoplasmic domain was necessary for basolateral targeting of the pIgR.

Amino Acid Sequence↗
PubMed1992

Stable expression of the human immunodeficiency virus type 1 envelope glycoprotein in transfected L cells.

An SV40-based expression vector was used to generate CD4-negative murine L cell lines which stably expressed the human immunodeficiency virus envelope glycoprotein (env). Despite the presence of abundant intracellular envelope glycoprotein, the expression of env gp120/41 was not detected on the cell surface. Pulse-chase studies showed that the majority of the gp120 detected at the end of a 20-h chase was in the culture medium. Therefore gp120 was shed and/or secreted from these cells. Transfected L cells (H-2k) served as targets for specific lysis by CTL raised against vaccinia virus-encoded env gp160. The discrepancy in relative levels of intracellular versus surface expression of env was probably due to the highly inefficient processing of newly synthesized gp160, as well as the apparent instability of the gp120/41 complex in the transfected cell lines. Digestion of immunoprecipitated gp120 and gp160 with endoglycosidase H and peptide N-glycosidase F revealed that the envelope glycoprotein in transfected L cells possessed both high mannose and complex N-glycans, analogous to the posttranslational modification of the mature envelope glycoprotein in infected T cells. These studies indicate that the relatively inefficient processing of env gp160 occurs in the absence of CD4, and that the stable surface expression of envelope gp120/41 complex may require additional factors not present in transfected cells.

Animals↗