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Biomedical subjects

P MacKay

Publications and source records attributed to P MacKay.

At least 19 recordsLinked to original sources

Stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct processes.

The action of repaglinide, a novel insulin secretagogue, was compared with the sulfonylurea glibenclamide with regard to the hypoglycemic action in vivo, binding to betaTC-3 cells, insulin secretion from perifused mouse islets, and capacity to stimulate exocytosis by direct interaction with the secretory machinery in single voltage-clamped mouse beta-cells. Two binding sites were identified: a high-affinity repaglinide (KD = 3.6 nmol/l) site having lower affinity for glibenclamide (14.4 nmol/l) and one high-affinity glibenclamide (25 nmol/l) site having lower affinity for repaglinide (550 nmol/l). In contrast to glibenclamide, repaglinide (in concentrations as high as 5 micromol/l) lacked the ability to enhance exocytosis in voltage-clamped beta-cells. Repaglinide was more potent than glibenclamide in stimulating insulin release from perifused mouse islets (EC50 29 vs. 80 nmol/l). The greater potency of repaglinide in vitro was paralleled by similar actions in vivo. The ED50 values for the hypoglycemic action were determined to be 10.4 and 15.6 microg/kg after intravenous and oral administration, respectively. The corresponding values for glibenclamide were 70.3 microg/kg (intravenous) and 203.2 microg/kg (oral). Further, repaglinide (1 mg/kg p.o.) was effective (P < 0.001) as an insulin-releasing agent in a rat model (low-dose streptozotocin) of type 2 diabetes. These observations suggest that the insulinotropic actions of repaglinide and glibenclamide in vitro and in vivo are secondary to their binding to the high-affinity repaglinide site and that the insulinotropic action of repaglinide involves both distinct and common cellular mechanisms.

Animals↗

Adoptive transfer of diabetes to and from old normoglycaemic BB rats.

Approximately 4% of diabetes-prone BB/Mol rats escape overt diabetes which occurs in other rats between 56 and 130 days of age. The ability of preactivated spleen cells from older non-diabetic and from acutely diabetic rats to adoptively transfer diabetes into young diabetes-prone rats was compared, and it was found that they transferred disease with similar incidence and with overlapping onset times in the recipients. Old non-diabetic rats were themselves susceptible to diabetes adoptively transferred from acutely diabetic or from old nondiabetic donors. Lymphocytic insulitis and pancreatic insulin content in unmanipulated old non-diabetic rats were both intermediate between those seen in acutely diabetic and in diabetes-resistant rats. In vivo treatment with polyinosinic-polycytidylic acid induced diabetes with faster onset in old non-diabetic rats than in young diabetes-prone rats. Adoptive transfer of fresh, whole spleen cells from old nondiabetic rats did not protect young BB rats against spontaneous diabetes, while cells from diabetes-resistant rats did. Spleens from old non-diabetic rats contained significantly lower percentages of T cells than spleens from acutely diabetic rats but not lower than spleens from age-matched diabetic rats, suggesting that this reduction was age-related. Finally, spleens from both old non-diabetic and from acutely diabetic rats were negative for the regulatory RT6+ T-cell subset. It is concluded that quiescent beta-cell autoimmunity seen in a fraction of BB/Mol rats can be reactivated upon non-antigen-specific immune stimulation.

Animals↗

Pancreatic exocrine function and necrotising enterocolitis.

Pancreatic protease deficiency may be an aetiological factor in enteritis necroticans, a disease sharing some features of necrotising enterocolitis (NEC). Using faecal chymotrypsin measurement we have prospectively studied pancreatic exocrine function in infants at risk of NEC. No significant difference was found comparing those infants who subsequently developed NEC and those who did not.

Chymotrypsin↗

Enterobacteriaceae and neonatal necrotising enterocolitis.

A comparative study of bowel colonisation and incidence of necrotising enterocolitis in neonates admitted to an intensive care unit is reported. Neonates of less than 33 weeks gestational age requiring mechanical ventilation for respiratory distress syndrome were randomised during the first week of life to receive either vancomycin and aztreonam or vancomycin and gentamicin for episodes of suspected sepsis after the first week of life. A higher proportion of neonates who received vancomycin and gentamicin had faecal colonisation with enterobacteriaceae at the end of the second, third, and fourth weeks of life. Treatment with vancomycin and aztreonam was associated with a rapid quantitative reduction in faecal colonisation with enterobacteriaceae, whereas there was no quantitative reduction in colonisation with enterobacteriaceae associated with treatment with vancomycin and gentamicin. There were no differences between the two groups in faecal colonisation with anaerobes, Enterococcus sp, Staphylococcus sp, or yeasts. Six (14.6%) of 41 who received vancomycin and gentamicin compared with 0 of 40 who received vancomycin and aztreonam subsequently developed necrotising enterocolitis.

Aztreonam↗

Quantitative changes in faecal microflora preceding necrotising enterocolitis in premature neonates.

Quantitative studies of faecal bacterial flora were carried out during the week preceding the clinical onset of 12 episodes of neonatal necrotising enterocolitis. There were considerable quantitative changes in the faecal flora preceding the clinical onset of both definite and possible episodes of necrotising enterocolitis. There was a decline in the numbers of some species from up to 72 hours before the clinical onset of the disease. Enterobacteriaceae were isolated from samples collected during the 48 hours preceding the clinical onset of all four definite episodes of necrotising enterocolitis. These were 'new' isolates in two episodes, and considerably increased numbers in another. The changes that we found are probably the result of changes in intraluminal conditions that precede the clinical onset of necrotising enterocolitis.

Bacteria↗

Direct isolation of coagulase-negative staphylococci from neonatal blood samples.

Coagulase-negative staphylococci (CNS) were isolated by direct inoculation of anticoagulated whole blood onto agar from 14 (41.2%) of 34 episodes (30 neonates) of suspected bacterial infection, associated with isolation of CNS from the same blood sample by broth-dilution blood cultures. The equivalent of more than 1,000 cfu ml-1 were isolated from four samples (four neonates); the range of counts was 1-103 cfu 25 microliters-1. There was a statistically significant association between isolation of CNS by direct agar inoculation and a raised C-reactive protein level and/or a positive nitroblue tetrazolium test and with the use of an intravascular catheter when compared to those episodes where CNS were isolated from broth blood cultures only. This simple procedure provides an estimate of the number of CNS in the blood of a neonate with suspected bacteraemia and may help to corroborate a diagnosis of CNS bacteraemia.

Coagulase↗

Spontaneous diabetes mellitus in the Bio-Breeding/Worcester rat. Evidence in vitro for natural killer cell lysis of islet cells.

We sought direct evidence for anti-islet cellular cytotoxicity in diabetic Bio-Breeding/Worcester (BB/W) rats by comparing the effects of splenic lymphoid cells from BB/W diabetic (D), diabetes-prone (DP), and diabetes-resistant (DR) rats on the release of 51Cr from damaged islet cells in vitro. D and DP splenic lymphoid cells were cytotoxic to major histocompatibility complex (MHC)-compatible Wistar-Furth (WF) rat islet cells and also to MHC-incompatible Lewis rat islet cells and a rat islet cell line (RIN 5F), whereas WF and Lewis rat spleen cells and a rat pituitary cell line (GH3) were not lysed by lymphoid cells from D or DP rats. The cytotoxic cells were identified as natural killer (NK) cells since NK-sensitive cells (G1-TC and YAC-1 cell lines) were lysed by D and DP spleen cells, YAC-1 cells competed for the lysis of RIN islet cells by D spleen cells, lysis of RIN cells was increased by using D spleen cells from the low density fraction (large lymphocytes/monocytes) of a Percoll density gradient, and incubation of D spleen cells with an antiserum to NK cells (anti-asialo GM1 serum) and complement decreased monoclonal antibody-defined subsets containing NK cells (W3/13+ OX19- and OX8+), and this was accompanied by similar decreases in cytotoxicity to YAC-1, RIN, and WF islet cells. These studies demonstrate that NK cell activity is increased in BB/W diabetic and DP rats, and that islet cells can serve as targets for these NK cells. The findings suggest that NK cells may participate in the islet-directed cellular cytotoxic response leading to beta cell destruction and diabetes.

Animals↗

Lymphoid cells of BB/W diabetic rats are cytotoxic to islet beta cells in vitro.

Assays were developed to detect cell-mediated immune destruction of pancreatic islet beta cells by lymphoid cells isolated from diabetic BioBreeding/Worcester (BB/W) rats. Splenic lymphoid cells from diabetic (D), diabetes-prone (DP), and diabetes-resistant (DR) BB/W rats were incubated for 2 days with monolayer cultures of major histocompatibility complex (MHC)-compatible Wistar-Furth (WF) rat islet cells or a rat islet cell line (RIN), and islet beta cell survival was determined by measuring insulin content in the cultures. D splenic lymphoid cells significantly decreased insulin content in WF rat islet (-32%) and RIN cultures (-77%). DP cells also significantly reduced the insulin content in WF rat islet (-20%) and RIN cultures (-63%), whereas DR cells had no significant effect. Islet-directed cytotoxicity was detected also by the release of 51Cr from RIN cells incubated for 8 h with BB/W spleen cells. Cytotoxicity was linearly related to the number of effector spleen cells. At a target: effector of 1:20, lysis (mean +/- SEM) of RIN target cells by spleen cells from D rats (21.6 +/- 2.0%) and DP rats (16.5 +/- 4.1%) was significantly greater than the effect of DR spleen cells (5.4 +/- 1.0%). D and DP splenic lymphoid cells activated in vitro for 2 days with concanavalin A exhibited a doubling of cytotoxicity to RIN islet cells. These results provide direct evidence for lymphoid cell-mediated immune damage to islet beta cells in diabetic BB rats.

Animals↗

Islet cell antibodies in insulin-dependent (type 1) diabetic children treated with plasmapheresis.

Plasma levels of islet cell cytoplasmic and cytotoxic antibodies were determined in 10 children with insulin-dependent diabetes mellitus (IDDM) treated with plasmapheresis shortly after diagnosis, and in 9 children with IDDM treated by conventional means alone. Islet cell cytoplasmic antibody (ICA) titers were determined by indirect immunofluorescence using unfixed sections of human pancreas, and islet cell cytotoxic antibody levels were determined in a complement-dependent antibody-mediated cytotoxicity (C'AMC) assay using a human fetal cloned insulin-producing cell line (JHPI-1) as target. Before plasmapheresis, ICA was present in 7 out of 10 children and C'AMC was positive in 4. Four successive treatments with plasmapheresis did not consistently decrease plasma levels of ICA or C'AMC. ICA was present in 15 out of the total 19 children at diagnosis, and titers of ICA decreased in 12 out of 15 subjects by at least 1 degree of dilution (1:3) at 18-30 months follow-up, whether or not they had been treated with plasmapheresis; C'AMC was positive in 6 out of the 18 children at diagnosis and decreased in 2 out of 6. Plasma levels of C-peptide did not differ at diagnosis but remained higher in the plasmapheresis treated diabetic children at 3 and 18-30 months follow-up. Neither ICA titers nor C'AMC levels correlated with plasma C-peptide responses at 18-30 months. It is concluded that plasmapheresis decreases ICA and C'AMC but is followed rapidly by a rebound effect, and does not affect the rates at which these islet cell antibodies decrease with increasing duration of IDDM.

Adolescent↗

A prospective analysis of islet-cell cytotoxic antibodies in insulin-dependent diabetic children. Transient effects of plasmapheresis.

We determined the effects of plasmapheresis on cytotoxic antibodies to islet cells in 10 children (aged 11-16 yr) with newly diagnosed insulin-dependent diabetes mellitus (IDDM), as well as the plasma levels of antibodies over the next 30 mo and their relation to serum C-peptide concentrations. Complement-dependent, antibody-mediated cytotoxicity (C'AMC) in plasma was measured in a 51Cr release assay using monolayers of the rat islet cell line RINm5F. Cytotoxic antibodies decreased in most IDDM subjects treated by plasmapheresis four times within 2 wk of diagnosis; however, the decreases were small and lasted less than 2-3 days. Thus, both before and after plasmapheresis, 7 of the 10 IDDM children were C'AMC-positive (51Cr release greater than 2 SD above mean for 13 healthy children). After 18-30 mo, only 2 of the original 7 IDDM children with C'AMC-positive plasmas were still positive, and 1 of the original 3 IDDM children without significant cytotoxicity had become positive. Meal-stimulated serum C-peptide responses, measured from diagnosis to 18-30 mo later, did not correlate with C'AMC values. We conclude that (1) plasmapheresis has only transient effects on islet-cytotoxic antibody levels in children with IDDM; (2) these antibodies decrease in most, but not all, subjects over the first 18 mo after diagnosis; and (3) the level of cytotoxic antibodies in IDDM plasma at diagnosis has no predictive effect on residual B-cell function.

Adolescent↗

Hepatitis B virus core antigen: synthesis in Escherichia coli and application in diagnosis.

Fragments of hepatitis B virus DNA cloned in plasmid pBR322 carrying the gene for the viral core antigen have been placed under the control of the lac promoter of Escherichia coli. Several of the new recombinants direct higher levels of synthesis of the antigen, but the degree of enhancement varies with the different structures of the plasmids and hence the mRNAs produced. The antigen in crude bacterial lysates is a satisfactory diagnostic reagent for antibodies to the core antigen in serum samples.

Antibodies, Viral↗

The conversion of hepatitis B core antigen synthesized in E coli into e antigen.

The antigen (HBeAg) of hepatitis B virus (HBV) is a polypeptide of 17-20,000 daltons closely associated with the core antigen (HBcAg) of Dane particles, from which it is released by a variety of disruptive procedures. HBeAg could be a unique component of HBV core particles or a derivative of HBcAg. To resolve this question immunodiffusion experiments were carried out with preparations of HBcAg synthesized in E coli carrying a recombinant plasmid from which the HBcAg, but no other HBV gene, was expressed. HBcAg was converted into HBeAg by proteolytic degradation under dissociating conditions, thus confirming at the molecular level that HBeAg is a component of HBcAg. This offers a new route to the detection of HBeAg and antibodies to the antigen.

Cloning, Molecular↗

Production of immunologically active surface antigens of hepatitis B virus by Escherichia coli.

Several plasmids have been constructed which direct the synthesis of hepatitis B virus surface antigens in Escherichia coli either as the native polypeptide or fused to other plasmid encoded polypeptides. When injected into rabbits, extracts from bacteria carrying some of these plasmids induced the synthesis of antibodies to the antigens even though the extracts did not give satisfactory positive results in radioimmunoassay for them. Either the NH2-terminal segment or the COOH-terminal segment of the surface antigens alone was sufficient to elicit the immune response, but antibodies against the two segments showed different specificities. The results emphasize the value of an in vivo assay for the presence of antigens in crude cell extracts and illustrate the feasibility of this type of screening with laboratory animals.

Antibody Specificity↗

Hepatitis B virus genes and their expression in E. coli.

A composite DNA sequence of regions of hepatitis B virus, determined from a series of recombinant plasmids, reveals the genes for the surface antigen and the core antigen of the virus. The sequence of the core antigen shows it to be a DNA binding protein. The core antigen gene is expressed in Escherichia coli and when injected into rabbits the bacterial product induces antibodies which react with core antigen isolated from human sources.

Amino Acids↗