Suppression of reaginic antibodies with modified allergens. IV. Induction of suppressor T cells by conjugates of polyethylene glycol (PEG) and monomethoxy PEG with ovalbumin

Administration of multiple injections of conjugates of ovalbumin (OA) and polyethylene glycol (PEG) or its monomethoxy derivative (mPEG) into mice which had been sensitized with 2,4-dinitrophenylated OA (DNP3-OA) abrogated both the anti-OA and anti-DNP IgE responses, in spite of additional injections of the sensitizing dose of DNP3-OA in the presence of A1(OH)3. Treatment of mice with OA-PEG in A1(OH)3 stimulated preferentially helper T cells, whereas injection of mice with OA-PEG in the absence of adjuvant elicited predominantly suppressor T cells. The unresponsive state of mice which had been treated 21 days earlier with OA-PEG could not be broken by the transfer of normal spleen cells and an additional sensitizing dose of DNP3-OA. Transfer of spleen cells from tolerized animals to normal mice dampened the capacity of the latter to mount both anti-DNP and anti-OA IgE responses; however, the suppressive effect of these cells was eliminated by treatment of the normal recipients with cyclophosphamide, which is a procedure known to inactive suppressor T cells, and hence it may be concluded that this effect was not due to the carryover of the tolerogen with the transferred cells. All these results provide strong support for the conclusion that the suppressor cells induced by the treatment of mice with OA-PEG and OA-mPEG conjugates belonged to a T cell subpopulation, and that the B cells of these mice were devoid of suppressive activity.     

Genetic heterogeneity in familial hyperinsulinism [published erratum appears in Hum Mol Genet 1998 Sep;7(9):1527]

Familial hyperinsulinism (HI) is a disorder characterized by dysregulation of insulin secretion and profound hypoglycemia. Mutations in both the Kir6.2 and sulfonylurea receptor (SUR1) genes have been associated with the autosomal recessive form of this disorder. In this study, the spectrum and frequency of SUR1 mutations in HI and their significance to clinical manifestations of the disease were investigated by screening 45 HI probands of various ethnic origins for mutations in the SUR1 gene. Single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses of genomic DNA revealed a total of 17 novel and three previously described mutations in SUR1 . The novel mutations comprised one nonsense and 10 missense mutations, two deletions, three mutations in consensus splice-site sequences and an in- frame insertion of six nucleotides. One mutation occurred in the first nucleotide binding domain (NBF-1) of the SUR1 molecule and another eight mutations were located in the second nucleotide binding domain (NBF-2), including two at highly conserved amino acid residues within the Walker A sequence motif. The majority of the remaining mutations was distributed throughout the three putative transmembrane domains of the SUR1 protein. With the exception of the 3993-9G-->A mutation, which was detected on 4.5% (4/88) disease chromosomes, allelic frequencies for the identified mutations varied between 1.1 and 2.3% for HI chromosomes, indicating that each mutation was rare within the patient cohort. The clinical manifestations of HI in those patients homozygous for mutations in the SUR1 gene are described. In contrast with the allelic homogeneity of HI previously described in Ashkenazi Jewish patients, these findings suggest that a large degree of allelic heterogeneity at the SUR1 locus exists in non-Ashkenazi HI patients. These data have important implications for genetic counseling and prenatal diagnosis of HI, and also provide a basis to further elucidate the molecular mechanisms underlying the pathophysiology of this disease.      

Mapping of antibody binding epitopes of a recombinant Poa p IX allergen.

Antibody binding epitopes of a recombinant Poa p IX allergen were delineated using recombinant DNA and solid-phase peptide synthesis procedures. The full-length cDNA clone KBG60 and its four overlapping recombinant fragments, KBG60.1, KBG60.2, KBG8.3 and KBG10 which spanned the entire molecule were synthesized in E. coli with aid of the plasmid expression vector, pWR590.1. The antigenic and allergenic sites of these recombinant proteins were analyzed by ELISA using human IgE and murine IgG antibodies. It was thus demonstrated that although the epitopes were found on all the fragments tested, the majority of these were located on a C-terminal fragment, rKBG8.3. Furthermore, synthetic peptides were also employed to identify the epitopes of rKBG60 protein. The use of antisera raised against native KBG pollen extract and the recombinant KBG8.3 protein to scan a total of 56 overlapping deca-penta peptides, covering the entire rKBG60 protein, revealed that 10 positive peptides involved in the antibody-binding site(s). Taken together, the results of these studies indicate that rKBG60 protein possesses at least 10 antibody binding epitopes.     

Measured haplotype analysis of the angiotensin-I converting enzyme gene

Linkage and segregation analysis have shown that circulating angiotensin-I converting enzyme (ACE) levels are influenced by a major quantitative trait locus that maps within or close to the ACE gene. The D variant of a 287 bp insertion/deletion (I/D) polymorphism in intron 16 of the gene is associated with high ACE levels and may also be related to increased risk of cardiovascular disease. Multiple variants that are in linkage disequilibrium with the I/D polymorphism have been described, but it is unknown if any of these are directly implicated, alone or in combination with as yet undiscovered variants, in the determination of ACE levels. An analysis of 10 polymorphisms spanning 26 kb of the ACE gene revealed a limited number of haplotypes in Caucasian British families due to strong linkage disequilibrium operating over this small chromosomal region. A haplotype tree (cladogram) was constructed with three main branches (clades A-C) which account for 90% of the observed haplotypes. Clade C is most likely derived from clades A and B following an ancestral recombination event. This evolutionary information was then used to direct a series of nested, measured haplotype analyses that excluded upstream sequences, including the ACE promoter, from harbouring the major ACE-linked variant that explains 36% of the total trait variability. Residual familial correlations were highly significant, suggesting the influence of additional unlinked genes. Our results demonstrate that a combined cladistic/measured haplotype analysis of polymorphisms within a gene provides a powerful means to localize variants that directly influence a quantitative trait.      

Maternal L‐carnitine supplementation improves glucose and lipid profiles in female offspring of dams exposed to cigarette smoke

Sex differences in disease susceptibility due to maternal programming have been reported. We previously observed that maternal smoking induced renal disease and neurological changes are restricted to males, while both male and female offspring develop metabolic disorders. We have also found that maternal L‐carnitine supplementation during gestation and lactation can significantly improve glucose intolerance and hyperlipidaemia in male offspring. This study aimed to determine whether such beneficial effects can also occur in female offspring. Balb/c female mice were exposed to cigarette smoke (SE) 6 weeks prior to gestation, during gestation and lactation. A subgroup of the SE dams was given L‐carnitine (1.5 mmol/L in drinking water) during gestation and lactation. Female offspring were studied at 20 days (weaning) and 13 weeks (adulthood). Maternal smoking increased liver weight (%) and blood glucose levels at 20 days, as well as glucose intolerance and plasma triglycerides levels at adulthood (P < .05). The hepatic lipid metabolic marker adipose triglyceride lipase was downregulated in the SE offspring at 20 days (P < .05). At 13 weeks, the hepatic pro‐inflammatory markers IL‐1β and TNF‐α mRNA expression were upregulated, while the anti‐inflammatory marker IL‐10 mRNA expression was downregulated in the SE offspring (P < .05). Liver fibrosis was apparent at 20 days and 13 weeks. Maternal L‐carnitine supplementation either normalised or suppressed the detrimental effects induced by maternal smoke exposure (P < .05). We conclude that maternal L‐carnitine supplementation improves metabolic parameters in the female offspring of SE dams.     

Developing and utilising a new funding model for home‐care services in New Zealand

Worldwide increases in the numbers of older people alongside an accompanying international policy incentive to support ageing‐in‐place have focussed the importance of home‐care services as an alternative to institutionalisation. Despite this, funding models that facilitate a responsive, flexible approach are lacking. Casemix provides one solution, but the transition from the well‐established hospital system to community has been problematic. This research seeks to develop a Casemix funding solution for home‐care services through meaningful client profile groups and supporting pathways. Unique assessments from 3,135 older people were collected from two health board regions in 2012. Of these, 1,009 arose from older people with non‐complex needs using the interRAI‐Contact Assessment (CA) and 2,126 from the interRAI‐Home‐Care (HC) from older people with complex needs. Home‐care service hours were collected for 3 months following each assessment and the mean weekly hours were calculated. Data were analysed using a decision tree analysis, whereby mean hours of weekly home‐care was the dependent variable with responses from the assessment tools, the independent variables. A total of three main groups were developed from the interRAI‐CA, each one further classified into “stable” or “flexible.” The classification explained 16% of formal home‐care service hour variability. Analysis of the interRAI‐HC generated 33 clusters, organised through eight disability “sub” groups and five “lead” groups. The groupings explained 24% of formal home‐care services hour variance. Adopting a Casemix system within home‐care services can facilitate a more appropriate response to the changing needs of older people.