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Biomedical subjects

M C Barnardo

Publications and source records attributed to M C Barnardo.

10 recordsLinked to original sources
PubMed2001

CD1 genotyping of patients with Mycobacterium malmoense pulmonary disease.

Mycobacterium malmoense is an opportunistic mycobacterium that occasionally causes disease in non-immunosuppressed individuals. As only a few individuals exposed to these organisms actually develop clinical disease, it is possible there is a genetic component to susceptibility. CD1 molecules are capable of presenting antigens from more virulent mycobacteria to T cells; therefore, we were interested in discovering whether recently described polymorphisms in CD1 molecules modulated susceptibility to M. malmoense pulmonary disease. The CD1 system comprises five genes (CD1A, -B, -C, -D, and -E) located on chromosome 1 (1q22-23). CD1 molecules are structurally and functionally related to major histocompatibility complex (MHC) class I molecules and are expressed on dedicated antigen-presenting cells. The primary function of CD1 molecules is to present lipid and glycolipid antigens to T cells. We have developed an allele-specific polymerase chain reaction-sequence-specific primer (PCR-SSP) method of CD1 genotyping. Using this method, we compared the allele and haplotype frequencies of CD1 in 49 HIV-negative patients with M. malmoense pulmonary disease with those in 342 normal controls. The CD1A and CD1E alleles were nominally identified as CD1A*01, CD1A*02, CD1E*01 and CD1E*02, and the control gene frequencies were found to be 5%, 95%, 67% and 33%, respectively. No significant difference was observed between the patient and control cohorts. Positive linkage disequilibrium values of 0.73 were observed between CD1A*02 and CD1E*01 (P<0.0001; chi2 test), and 0.94 between CD1A*01 and CD1E*02 (P<0.0001; chi2 test). Typing was also performed for two previously described CD1D alleles (CD1D*01 and CD1D*02), although only CD1D*01 was detected.

Antigens, CD1↗
PubMed2000

Detection of HLA-specific IGG antibodies using single recombinant HLA alleles: the MonoLISA assay.

BACKGROUND: Because of the presence of confounding antigens, the assignment of HLA antibody specificity is difficult in highly sensitized patients, and the definition of an acceptable HLA mismatch requires a significant workload per patient. We describe a new ELISA method, monoLISA, for detection of immunoglobulin (Ig)G HLA antibody using single recombinant HLA class I monomers bound to microtiter plates. METHODS: HLA-A2 and -B8 monomers were synthesized and used as screening targets for 85 sera from renal patients. The sera contained various IgG and IgM HLA-specific antibodies, including anti-A2 and anti-B8,defined in a conventional complement-dependent cytotoxicity test (CDC). Investigations were performed to determine possible effects on antibody binding of differential monomer peptide presentation as well as lack of glycosylation. RESULTS: A good correlation was found between CDC-defined specificities and the reactivity observed with HLA monomers. MonoLISA attained means of 100% sensitivity and 92.5% specificity compared with CDC. Neither the presence of different peptides, nor the absence of glycosylation of the monomer affected the ability of monoLISA to detect antibody. CONCLUSION: This study demonstrates that the mono-LISA method for HLA antibody detection is valid. Because this has the potential to reduce the work involved in screening sensitized patients awaiting transplantation for HLA antibodies, resources aimed at increasing the number of constructed monomers would be well targeted.

Alleles↗
PubMed1998

The PCR-SSP Manager computer program: a tool for maintaining sequence alignments and automatically updating the specificities of PCR-SSP primers and primer mixes.

An emerging problem of molecular typing methods such as PCR amplification using sequence-specific primers (PCR-SSP) is that they frequently require updating as new alleles are constantly being described which potentially affect the specificity of every PCR-SSP reaction. PCR-SSP uses pairs of primers to detect cis-linked polymorphisms and thus each new allele described must be compared to each individual primer pair. Furthermore, sequence homology between the various loci for class I and class II means that, for example, new HLA-A sequences have to be compared with HLA-B and HLA-C primer mixes to rule out cross-locus amplification. We have developed a computer program known as SSP Manager which is capable of aligning HLA class I and class II sequences obtained from Internet-accessible databases such as GenBank. The program then updates all individual primer specificities held in its database before updating the specificities of all primer mixes. Sets of primer mixes can then be combined from the primer mix directory to create PCR-SSP typing trays which are subsequently analysed by the program. A report is generated which stipulates whether all known sequences are amplified and the reason for apparent failure to test for individual alleles, e.g. a lack of relevant sequence information. SSP Manager has the flexibility to cope with unusual sequences (deletions and insertions), primers with internal mismatches and primers with a deliberate mismatch. The program also has many tools for developing new primer mixes, such as the facility to search for novel reactions using Boolean operators. The organisation and operational use of the SSP Manager program is described and its uses are illustrated with an updated allele list for our previously described Phototyping PCR-SSP class I and class II typing set. The SSP Manager is available on request from the authors.

Antisense Elements (Genetics)↗
PubMed1998

Allele-specific HLA-B*15 typing by PCR-SSP and its application to four distinct ethnic populations.

We present a set of primer mixes for the allele-specific typing of the HLA-B*15 group by PCR-SSP. The set comprises 46 primer mixes which are designed to unequivocally resolve all but two of the 666 possible combinations of the B*15 alleles, B*1501-37 (B*1536 sequence unavailable). A core subset of 34 of the 46 mixes can be used alone to give a high resolution B*15 typing set. This allows for the identification of each B*15 allele when present as the only B*15 allele and the majority of the possible B*15 homozygotic combinations. The method was validated using reference DNA samples and the B*15 allele frequency in 4 distinct ethnic populations was investigated. The results show that these populations contain predominantly mutually exclusive sets of B*15 alleles.

Alleles↗
PubMed1997

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology.

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed.

Alleles↗
PubMed1996

A correlation between HLA-C matching and donor antirecipient CTL precursor frequency in bone marrow transplantation.

A newly developed, reliable, DNA-based method for typing for alleles of the HLA-C locus has been applied in the context of unrelated, volunteer donors for bone marrow transplantation. Some donors matched for HLA-A, -B, -DR, and -DQ have been found to generate in vitro high frequencies of CTL reactive with the recipient's cells. Here we demonstrate that there is a highly significant correlation of the frequencies of CTL precursors and incompatibility at the HLA-C locus. These data indicate that HLA-C locus incompatibility should be avoided in unrelated donor bone marrow transplantation.

Bone Marrow Transplantation↗
PubMed1996

High resolution HLA-C typing by PCR-SSP: identification of allelic frequencies and linkage disequilibria in 604 unrelated random UK Caucasoids and a comparison with serology.

Recent evidence indicates that HLA-C molecules are biologically relevant by eliciting T-cell responses and exerting control over NK cell function. In addition, HLA-C is associated with susceptibility to various diseases, notably psoriasis vulgaris. Clarification of the full biological roles for HLA-C has however proved difficult because detection of HLA-C antigens by complement mediated cytotoxicity using alloantisera is inefficient. Up to 50% of individuals in every race have serologically undetectable HLA-C locus antigens due to a combination of relatively low expression, lack of serological reagents and a lack of information about the distribution of the HLA-C blank alleles. Recently, amplification of DNA using sequence-specific primers (PCR-SSP) has proved a reliable, accurate and rapid method for medium resolution HLA-C typing. We have now developed high resolution HLA-C typing by PCR-SSP utilizing allele and group-specific PCR-SSP reactions which can identify all HLA-C alleles (except non-coding change alleles) in most heterozygous combinations. Using this system we have typed 604 unrelated United Kingdom Caucasoids to generate accurate frequency and linkage disequilibrium data. To assess the validity of serology for HLA-C, PCR-SSP typings for 527 out of the 604 individuals were compared to serology. We find that the frequency of many HLA-C antigens has been underestimated by serology and some antigens such as Cw6 are consistently assigned incorrectly by serology. The overall discrepancy rate between serology and SSP was high at 37% (195/527). High-resolution HLA-C typing of 112 International Histocompatibility Workshop cell lines has also been performed.

Alleles↗
PubMed1995

Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP).

We have developed a single DNA typing method which uses 144 sequence-specific primer (SSP) reactions to simultaneously detect all known HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 and DQB1 specificities in an allele specific or group specific manner using the same method, reagents, PCR parameters and protocols for all loci. The results from this integrated class I & II method can be visualized on a single photographic or electronic image and hence is described as "Phototyping". Phototyping has an overall resolution greater than or equivalent to good serology and results can be obtained in under 3 hours making the method suitable for genotyping potential cadaver donor peripheral blood without serological backup. This in turn produces the potential for reducing cold ischaemia times in renal transplantation as well as the application of prospective matching to cardiac and liver transplantation. The method has capacity to detect new alleles, for example, novel amplification patterns suggestive of 4 new HLA-B alleles have been detected. The Phototyping set has been used as the sole method of HLA typing for over 1010 individuals. Phototyping is not problem-free; deviations from the standard protocol, poor quality DNA and unsuitable PCR machines can result in individual PCR failures or in incorrect assignment of antigens. Approximately 5% of genotypes were repeated (either partially or fully) because of incomplete or equivocal results.

Base Sequence↗