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Biomedical subjects

M W Turner

Publications and source records attributed to M W Turner.

At least 55 records · Page 3Linked to original sources
PubMed1996

In vitro expansion and characterization of dendritic cells derived from human bone marrow CD34+ cells.

Dendritic cells (DC), as professional antigen-presenting cells, play a major role in stimulating naive T cell responses in vivo and in vitro, and may exacerbate or modulate T lymphocyte-mediated reactions, such as interactions between a hematopoietic graft and the recipient, eg GVHD and graft-versus-leukemia. Here, we describe a two-stage cell culture system for expansion of functionally active human DC from CD34+ marrow precursors. Optimal outgrowth was achieved by initially culturing CD34+ cells for 5 days in medium containing GM-CSF, MGF and TNF-alpha. Substitution of CD40L and IL-4 for TNF-alpha during a subsequent 5-day subculture increased DC content, such that by 10 days the cultures contained approximately 40% DC as determined by immunophenotype and morphology. An increase in DC purity to 84% at 10 days was achieved by immunomagnetic separation for CD1a+ cells from 5-day cultures and subculturing these cells in medium with IL-4 and CD40L. Reversing the sequence of growth factors during culture and subculture decreased the yield and purity of DC. Expression of CD80 and CD86 was enhanced by adding CD40L and IL-4, and the DC showed stimulatory activity in MLC. In conclusion, we have described a simple two-stage culture system to generate functional DC from CD34+ marrow precursors.

Antigens, CD34↗
PubMed1995

Mannose binding protein gene mutations associated with unusual and severe infections in adults.

A defect in opsonisation can cause a common immunodeficiency. A mutation in mannose binding protein (MBP) caused by point mutations in the MBP gene will lead to such a defect. This type of syndrome can cause recurrent infections in infants between 6 and 18 months of age but is not generally believed to predispose to adult infections. We looked at 4 patients with severe and unusual infections in whom MBP gene mutations were the only identified cause of immunodeficiency and one patient with combined MBP and IgA deficiency. We analysed the MBP genotypes of all the patients in whom we suspected an immunodeficiency because of their clinical history. Infections seen were recurrent skin abscesses, chronic cryptosporidial diarrhoea, meningococcal meningitis with recurrent herpes simplex, and fatal klebsiella lobar pneumonia. Both sexes were affected and ages ranged from 15 to 56 years. Two patients were homozygous for codon 54 mutations, one patient had codon 52 and codon 54 mutations and was phenotypically homozygous, and two patients were heterozygous for codon 54 mutations. Individuals homozygous for MBP mutations are unusual in the general population (approximate frequency 0.3%). The occurrence of three homozygotes for MBP mutations among these five infected patients suggests that MBP deficiency may confer a life-long risk of infection.

Adolescent↗
PubMed1995

Experimental intestinal hypersensitivity. Effect of four anti-allergic drugs on protein uptake, permeability to sugars and mucosal mast-cell activation.

The ability of four drugs with anti-allergic action to modulate the uptake of bystander protein, lactulose/rhamnose permeability ratios and mast cell activation was studied in rats presensitized with egg albumin in alum and challenged intraduodenally with the same antigen. Beclomethasone dipropionate (BDP) and nedocromil both significantly reduced the uptake of the bystander protein, bovine serum albumin (P < 0.002 and P > 0.02 respectively). BDP also significantly reduced sugar permeability (P < 0.01). In animals with elevated lactulose/rhamnose permeability ratios we confirmed our earlier observation of a significant correlation between levels of the specific mucosal mast cell protease Rat Chymase II (RChyII-previously known as RMCPII) and the sugar ratios. None of the drugs had any influence on the levels of mast cell protease II released following challenge and there was no correlation between the histological light microscopic appearance of the mast cells and the experimental treatment administered. Our results suggest that in the gut the pharmacological effect of anti-allergic drugs may be complex. Some, such as nedocromil, appear to act only on the mechanisms underlying increased protein uptake whereas others, such as BDP, appear to abrogate both increased protein uptake and increased sugar permeability.

Animals↗
PubMed1995

Mannose-binding protein in preterm infants: developmental profile and clinical significance.

The aim of this study was to determine the developmental profile of mannose-binding protein (MBP) in preterm infants. MBP was measured in 885 longitudinally collected serum samples from 168 preterm infants, and 63 were genotyped with respect to the codon 54 mutation in the MBP gene. MBP level/codon 54 genotyping were also determined on the cord blood of 146/123 term infants and 138/123 adults, respectively. The best cut-off values of MBP for dividing preterm, term infants and adults into 'low' and 'high' MBP groups were 400 ng/ml (55 low, 113 high), 700 ng/ml (35 low, 111 high) and 750 ng/ml (33 low, 105 high), respectively, by achieving the least number of misclassifications according to the codon 54 mutation. The relative risk of the 'low' groups for presence of the codon 54 mutation compared with 'high' groups were 42.4, 67.9 and 22.9 for preterm, term infants and adults, respectively (P < 0.00001). The gestational age and birth weight of the 'low' (n = 55) and 'high' (n = 113) MBP groups of the 168 preterm infants were 29.5 +/- 2.8 weeks, 30.5 +/- 2.8 weeks (P = 0.03) and 1230 +/- 317 g, 1277 +/- 289 g (P = 0.35), respectively. The mean MBP levels of these two groups of preterm infants were different (P < 0.001) at all ages measured. As a whole group, the MBP level rose from a mean of 500 ng/ml at 25 weeks gestation to 1700 ng/ml at 20 weeks post full-term. The mortality rates of 'low' and 'high' MBP groups of preterm infants were 22% and 12%, respectively (P = 0.113). This difference in mortality was due to gestational age and birth weight standard deviation score (SDS) after adjusting for length of gestation and gender (P = 0.0001) rather than to low MBP levels (P = 0.65). MBP levels were not related to birthweight SDS score (P = 0.26). The mean +/- s.d. MBP levels for preterm, term infants and adults without the codon 54 mutation were 1225 +/- 701 ng/ml (n = 45), 2064 +/- 829 ng/ml (n = 88) and 2473 +/- 1395 ng/ml (n = 95), respectively; the corresponding values for those with the codon 54 mutation were 130 +/- 275 ng/ml (n = 18), 533 +/- 665 ng/ml (n = 35) and 330 +/- 225 ng/ml (n = 28), respectively. Intra-uterine growth retardation in preterm infants does not influence MBP levels. For those without the codon 54 mutation, there is a significant difference in MBP level between the three age groups. For those with the codon 54 mutation, there is a significant difference between preterm and term infants, but not between term infants and adults. We conclude that there is a maturation in MBP levels for preterm infants, and that a moderately low MBP phenotype does not affect survival. We cannot exclude an effect of profoundly reduced MBP levels (characteristic of individuals homozygous for the codon 54 mutation), since no such preterm infant was identified in this study.

Adult↗
PubMed1995

Distinct physicochemical characteristics of human mannose binding protein expressed by individuals of differing genotype.

Mannose binding protein (MBP) is a serum collectin (collagenous lectin) believed to be of importance in innate immunity. Three point mutations, in codons 52, 54 and 57 of exon 1 of the human MBP gene, have been predicted to affect the tertiary structure of the collagenous region of the protein, and are known to be associated with low serum concentrations of MBP. However, other groups working with recombinant mutant proteins have claimed that the proteins are expressed and assembled normally. The aim of the present investigation was to characterize the effects of these mutations on the physicochemical nature of MBP that is present in the circulation in vivo, and for this we used polyacrylamide gel electrophoresis, gel filtration and sucrose density gradient centrifugation, followed by immunoblotting and enhanced chemiluminescence. The circulating wild-type MBP appeared to comprise a mixture of polymers formed from two to eight subunits (each based on three identical 32,000 MW polypeptide chains) of apparent molecular weights 200,000-700,000, with dimers and trimers constituting the predominant forms. Individuals homozygous for the codon 54 or 57 mutation had dramatically reduced concentrations of serum MBP, mainly comprising material of an apparent molecular weight of 120,000-130,000. Heterozygous individuals showed characteristics of both phenotypes. In contrast to the results obtained with artificial expression systems, our data suggest that individuals homozygous for the MBP mutations have very little circulating protein and that this comprises mainly low molecular weight material.

Carrier Proteins↗
PubMed1995

Partial characterization of a circulating tolerogenic moiety which, after a feed of ovalbumin, suppresses delayed-type hypersensitivity in recipient mice.

Serum collected 60 min after feeding ovalbumin (OVA) to BALB/c mice transfers specific tolerance to naive recipients via an unknown mechanism. We have now identified a large fragment of the OVA molecule as the putative active moiety. Specific absorption of immunoreactive OVA from tolerogenic serum by immunoaffinity chromatography removed the tolerogenic activity; following elution of the bound OVA and subsequent injection into naive recipients it was possible to demonstrate tolerogenic activity equivalent to that seen with unmanipulated serum. Passage of OVA tolerogenic serum through a bovine serum albumin (BSA)-specific affinity column had no effect on in vivo OVA tolerogenic activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting using anti-OVA antibodies demonstrated the presence of two bands with apparent molecular weights (MW) of 21,000 and 24,000 in the OVA-related material absorbed from the tolerogenic serum. The 24,000 MW moiety was also visualized by silver staining. These moieties are putative candidate tolerogens as they were absent from normal BALB/c mouse serum analysed 5 min after feeding OVA and from severe combined immunodeficiency (SCID) mouse serum--without tolerising activity--analysed 60 min after a similar feed.

Animals↗
PubMed1994

Association of Gm allotypes with the antibody response to the outer membrane proteins of a common upper respiratory tract organism, Moraxella catarrhalis.

Previously, Gm allotypes have been shown to influence human serum Ig subclass levels as well as the Ab levels achieved after Ag stimulation. The majority of the latter studies have focused on Ab responses to polysaccharide Ags. In this study, we have investigated the relationship between Gm allotypes and naturally occurring serum Ab levels to a bacterial protein Ag, the outer membrane proteins of a common microorganism, Moraxella catarrhalis. In the sera of 160 patients having chronic/recurrent sinusitis, there was a highly significant correlation between the level of specific anti-M. catarrhalis IgG3 level and certain Gm phenotypes. After additional investigation, we found that the presence of G3m(21) homozygosity correlated significantly with lower levels of Ag-specific IgG3. Specific anti-M. catarrhalis IgG3 levels were found to be independent of total serum IgG3 concentrations, and there was no correlation between the serum level of IgG3 and any Gm phenotype. Total IgG and IgG2 that were specific for pneumococcal cell wall polysaccharide also were measured in this group of patients, and no correlation was found between the naturally occurring IgG2 subclass levels to pneumococcal cell wall polysaccharide and the interactive effect of G2m(23) (syn: G2m(n)) and Km(1). Gm allotypes may influence Ab responses other than the anti-carbohydrate responses and, therefore, should be taken into account when investigating IgG subclass responses to protein Ags.

Adolescent↗
PubMed1994

Mammalian lectins in activation and clearance mechanisms involving the complement system.

CRP and the mammalian lectins containing collagen-like structure (collectins) can clearly participate in a variety of antibody-independent recognition and clearance mechanisms which result in the neutralisation and elimination of pathogenic organisms (Fig. 2). Only CRP and MBP appear to have the capacity to activate complement, and while all the collectins (MBP, SP-A, SP-D and conglutinin) can utilise the C1q receptor, only conglutinin shows specificity for iC3b. The C-type lectin domains in the globular heads of the collectins have the capacity to recognise a range of specific carbohydrate structures which are found on the surfaces of pathogens commonly associated with infections in blood, lung and amniotic fluids. The proposed presentation of the carbohydrate-collectin complex to C1q receptors, via the collagen-like regions in the collectins is an attractive hypothesis for the triggering of protective mechanisms and there are already a number of publications which support this view. However, the precise manner by which these collagen 'stalks' interact with the C1q receptor and the complete characterisation of the receptor on a variety of different cell types remains to be elucidated. Similarly, whether or not there is a role for any of these lectins in autoimmunity/immunopathology has not, as yet, been addressed.

Animals↗
PubMed1994

Failure of SCID mice to generate an oral tolerogen after a feed of ovalbumin: a role for a functioning gut-associated lymphoid system.

The role of the mucosal immune system in the generation of circulating tolerogenic ovalbumin (OVA) moieties has been investigated after a single feed of the protein. Serum collected from SCID mice 1 hr after a 25-mg feed of OVA was unable to transfer tolerance of delayed-type hypersensitivity (DTH) into naive BALB/c recipients. This is in contrast to serum collected from BALB/c mice which was able to transfer DTH tolerance to naive BALB/c recipients. The levels of circulating OVA detected in the serum of SCID mice 60 min after feeding OVA were approximately half those detected in the serum of BALB/c mice at the same time-point. However even dose adjustment of SCID mouse serum to a level of immunoreactive OVA equivalent to that found in BALB/c serum was unable to induce DTH tolerance in BALB/c recipients. This failure of SCID serum to transfer tolerance was shown to be unrelated to the germ-free conditions under which SCID mice are kept. Serum from OVA-fed germ-free BALB/c mice transferred DTH tolerance at equivalent levels to serum from conventionally reared BALB/c mice. When the intestinal morphology and intraepithelial lymphocyte (IEL) numbers in the duodenum of SCID mice were compared to conventionally reared and germ-free BALB/c controls, SCID mice were characterized by a lower number of IEL with a different morphology from the majority of IEL found in BALB/c mice.

Administration, Oral↗
PubMed1994

IgG subclass levels in chronic rhinosinusitis.

Sera from seventy-four adult patients with chronic or recurrent rhinosinusitis (mean duration 10.3 years) were tested for levels of total immunoglobulin and IgG subclasses. Fourteen (19%) had low levels of one of the major immunoglobulin classes and 23 (31%) had one or more IgG-subclass deficiencies, i.e. values less than the mean minus 2 standard deviations of a control population sample. Nineteen patients had low IgG3 levels. The group as a whole showed significantly low mean levels of IgG3, 46.9 +/- 19.5 mg/dl compared to 76 +/- 21 mg/dl for the controls, p < 0.0005 (Student's t-test). Since there was no clinical difference between those with and without IgG3 deficiency, there exists the possibility of an underlying immune defect, possibly involving heavy chain switching, in all these patients.

Adult↗
PubMed1993

The role of pH in modified ELISA procedures used for the estimation of functional antibody affinity.

Solid phase assays for the measurement of functional antibody affinity are increasingly being used in both clinical and research settings. The majority of such assays employ a chemical reagent to disturb antibody binding but relatively little is known about the properties of such reagents and the basis of their effect on antigen-antibody binding. We have evaluated the diethylamine (DEA) ELISA procedure for the measurement of functional antibody affinity in two independent assays, one for functional human IgG subclass affinity to an organism, Moraxella catarrhalis, and the other for measuring functional affinity of mouse monoclonals specific for the cat allergen Fel d I. DEA was shown to increase the pH of the buffering solution and it was this rise in pH that affected antibody binding. Alkaline buffer and DEA were equally efficient in the inhibition of binding of both the human IgG subclasses and the two mouse monoclonal antibodies to the solid phase. In contrast, pH was shown to have no role in the chaotropic effect of the ion, thiocyanate.

Animals↗
PubMed1992

Identical point mutation leading to low levels of mannose binding protein and poor C3b mediated opsonisation in Chinese and Caucasian populations.

A common opsonic defect occurring in 7% of the Caucasian population is associated with low serum levels of the lectin mannose binding protein (MBP). This study sought to determine whether the deficiency was also present in a Chinese population using sera obtained from 100 healthy Chinese children (age range 6 weeks-16 years). The distribution profiles of MBP levels and C3b/C3bi fragments binding to mannan coated plates were both bimodal and similar to the corresponding Caucasian profiles. Serum MBP levels were low in 9% of the Chinese children and all of these sera generated low levels of C3b/C3bi fragments. Overall there was a high significant correlation between MBP levels and C3b opsonin generation (r = 0.77; P less than 0.001). By analogy with similar findings in a Caucasian population we believe this correlation to be a reflection of antibody independent complement activation by MBP. In a pilot study of DNA obtained from three adult Chinese with low MBP levels the point mutation causing MBP deficiency in Caucasians was identified in all three cases.

Adolescent↗
PubMed1992

High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene.

We have previously identified, in three British families having an index child with frequent infections, a point mutation (GGC-->GAC) in codon 54 of exon 1 of the gene for the human lectin mannose binding protein (MBP). This was associated with low serum levels of this complement activating protein and would be anticipated to impair opsonization of mannose rich microorganisms. We now report a second point mutation (GGA-->GAA) in Gambians from West Africa, involving codon 57 of exon 1. By substituting carboxylic acids for axial glycines in the translated proteins both mutations would be expected to disrupt the secondary structure of the collagenous triple helix of the 96 kDa MBP subunits. In the Gambians the codon 57 mutation was studied by PCR, sequence analysis and restriction analysis and found to be remarkably common (frequency of the mutant gene 0.29 in adults and 0.23 in newborns) whereas the codon 54 mutation was very rare (frequency 0.003). However, the codon 54 mutation was frequent in both a British Caucasian and a Hong Kong Chinese population (frequency of the mutant gene 0.17 and 0.11 respectively). It was predicted that both homozygous and heterozygous individuals would have profoundly reduced serum levels of the protein and this was confirmed by immunoassay as was the reduced capacity of such sera to activate complement through the MBP initiated classical pathway. Our data indicate that the two mutations have arisen independently since the divergence of African and non African populations and both have attained high frequencies.

Adult↗
PubMed1992

Role of cell wall polysaccharide in the assessment of IgG antibodies to the capsular polysaccharides of Streptococcus pneumoniae in childhood.

The interference of antibodies to pneumococcal cell wall polysaccharide (CWPS) in the measurement of antibodies to capsular polysaccharides in children was assessed after vaccination with pneumococcal polysaccharide vaccine. ELISAs were developed to measure IgG subclasses specific for pneumococcal types 3, 6, 19, and 23 and CWPS. Analysis of antibody levels to all four capsular polysaccharides was affected by the presence of antibodies to CWPS, and their removal altered both anti-capsular polysaccharide antibody levels and the interpretation of responses to the vaccine. Thus, it is likely that CWPS contaminating pure capsular polysaccharide reagents used in most standard immunoassays is responsible for falsely elevated measurements of antibodies to capsular polysaccharide and the incorrect assessment of anti-pneumococcal antibody status in childhood.

Adolescent↗
PubMed1991

Molecular basis of opsonic defect in immunodeficient children.

Low serum mannose-binding protein (MBP) concentrations are associated with a common opsonic defect. Sequence analysis of the MBP gene in three children with recurrent infections, the opsonic defect, and low serum MBP concentrations showed a point mutation at base 230 of exon 1 causing a change of codon 54 from GGC to GAC. The replacement of glycine with an aspartic acid residue disrupts the fifth Gly-Xaa-Yaa repeat in the collagen-like domain of each 32 kD MBP peptide chain and probably prevents the formation of the normal triple helix. Study of sixteen members of the three families showed autosomal dominant co-inheritance of the mutation and low serum MBP concentrations.

Alleles↗