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Biomedical subjects

K Gelsthorpe

Publications and source records attributed to K Gelsthorpe.

At least 19 recordsLinked to original sources
PubMed1997

Isolation and molecular characterization of spontaneous mutants of lymphoblastoid cells with extended loss of heterozygosity.

The human major histocompatibility complex comprising the HLA class I and II genes provides a versatile source of natural heterozygous loci. This polymorphic genetic system allows analysis of the mechanistic aspects of loss of heterozygosity (LOH), a major phenomenon observed at tumor suppressor genes in human cancer cells. Four lymphoblastoid cell lines, ORI, TK6, WI-L2-NS and VH, were used to adjust current HLA immunoselection protocols to quantify loss of HLA-A2 in human lymphoblastoid cell lines. The modified selection protocol was used to isolate independent spontaneous HLA-A2 mutants from the lymphoblastoid cell line ORI. The frequency of spontaneous loss of HLA-A2 in ORI was 1.7 x 10(-5). By HLA typing 35 spontaneous HLA-A2 mutants, we showed that 74% of the HLA-A2 mutants also lost expression of the HLA-B allele, which is located on the same haplotype as HLA-A2. Microsatellites on both arms of chromosome 6 were used for molecular characterization of the spontaneous HLA-A2 mutants. Loss of heterozygosity at various loci on the p-arm or loss of an entire chromosome 6 was found in 80% of the mutants. Surprisingly, it appeared that a presumed mitotic recombination event in the cell line ORI itself had resulted in homozygosity of all markers distal from the HLA locus up to the telomere. This greatly limited the detection of mitotic recombination, resulting in LOH up to the telomere, on the short arm of chromosome 6 in this cell line. However, gene dosage analysis detected two copies of the remaining D6S265 allele in mutants which showed LOH at various loci along the p-arm. This suggested that recombination resulted in LOH in these mutants. The lymphoblastoid cell line TK6 did contain informative microsatellites along the complete chromosome 6. Mutants of TK6 either retained heterozygosity of all p-arm markers, showed LOH of all p-arm markers or showed loss from a breakpoint up to the telomere. These data indicate that recombination and chromosome loss both are important mechanisms involved in loss of the HLA-A2 allele in vitro. Such mechanisms may be involved in LOH in vivo and contribute to loss of tumor suppressor alleles.

Cell Line↗
PubMed1997

An immunocytochemical study of MHC class I expression on human Langerhans cells and melanocytes.

Classical MHC class I glycoproteins (HLA-A, B, and C) present endogenous cytosolic peptide antigen fragments to CD8-positive T-cells. CD8-positive T-cell recognition and destruction of virus-infected cells are dependent on adequate cellular MHC class I expression. Constitutive MHC class I expression is ubiquitous, but known to be deficient on specific differentiated cell types which include hepatocytes, neurones, chondrocytes and myocytes. Although enabling assessment of MHC class I expression on individual cells, limitations of immunocytochemistry were encountered with this assessment on Langerhans cells and melanocytes. These dispersed intraepidermal cells were obscured by adjacent keratinocytes in sections immunostained for MHC class I glycoproteins. Initiatives designed to resolve the issue have included immunoelectron microscopy, cell culture techniques, and animal bone marrow chimera models. Despite the elegance of these techniques, the issue of MHC class I expression on Langerhans cells and melanocytes remains unresolved. In this immunocytochemical study, an alternative strategy was based upon the recognized deficiency of epithelial MHC class I expression within pilosebaceous adnexal units. Langerhans cells and melanocytes were therefore studied within this microenvironment of deficient MHC class I expression, using monomorphic and polymorphic MHC markers. Langerhans cells and melanocytes were demonstrated within pilosebaceous units of scalp skin by immunocytochemistry. Differentiation markers OKT6 (CD1a) and TMH1 defined Langerhans cells and melanocytes, respectively. Monomorphic MHC markers W6/32 and TAL IB5 defined invariant epitopes of HLA class I and II, respectively. Polymorphic MHC class I markers defined the HLA-Bw4 and HLA-Bw6 supertypic determinants. Constitutive MHC class I expression was shown to be deficient on Langerhans cells and melanocytes.

Hair Follicle↗
PubMed1995

A simple method for extracting DNA from old skeletal material.

Extraction of DNA from old skeletal material is of great importance in the identification of human remains, but is particularly difficult because the methods currently employed, especially those using phenol/chloroform, are not always satisfactory. A simple technique based on the removal of non-nucleic acid material by salting out (precipitation) with saturated sodium acetate is described; the presence of DNA in the extract being confirmed by amplification of selected sequences of the HLA-DRB1 gene using the polymerase chain reaction (PCR). The method was applied to fresh bone (five femoral heads and six vertebral bodies) and to bone from two forensic cases, 3 and 9 months post-mortem, respectively. Parallel extractions using the phenol/chloroform technique were performed on all samples in order to compare the efficiency of the two methods. Using sodium acetate precipitation, amplifiable DNA was consistently extracted from fresh bone, as well as from the two forensic cases. With the phenol/chloroform method, amplification was successful in only 7 out of 11 instances with the fresh bone samples and failed in both forensic cases. The studies also showed that an effective way of removing PCR inhibitors is to subject the extract to agarose gel electrophoresis, isolate the high molecular weight area and re-extract the DNA from the gel by boiling. It was concluded that the sodium acetate method is a valid alternative to established techniques for extracting DNA from bone and that it offers the advantages of being simple, quick, inexpensive and avoids using hazardous reagents.

Acetates↗
PubMed1995

Expression of an unusual Bw4 epitope by a subtype of HLA-B8 [B*0802].

The primary structure of a variant HLA-B8 antigen has been determined by cDNA cloning and sequencing. The variant, B*0802 differs, from the common B*0801 subtype at positions 77-83 of the alpha 1 helix that determine the Bw4 and Bw6 public epitopes. Whereas B*0801 has the common Bw6 motif, B*0802 has the Bw4 motif found in B*13 and B*44 allotypes. Serological analysis of B cell lines expressing B*0802 and of a B*0802 transfectant made with the HLA-A,B negative cell line 721.221 shows that B*0802 reacts with Bw4-specific antibodies, but at a level much lower than expected for Bw4 positive HLA-B allotypes.

Alleles↗
PubMed1992

Reliable identification of human albumin in ancient bone using ELISA and monoclonal antibodies.

In order to help reconstruct ancient dietary, domestic, and ritual behaviour, a method was developed to identify the blood protein albumin in ancient skeletal material. This was an inhibition enzyme-linked immunosorbent assay (ELISA) using a monoclonal antibody of IgG class against human albumin. With fresh material, the technique gave consistent and specific results and could detect as little as 10 ng of albumin. Extracts of bone from the English Civil War (A.D. 1644), Mediaeval (A.D. 1100-1400), Early Saxon (A.D. 450-600), Roman (A.D. 100-200), Iron Age (ca 400 B.C.) and Bronze Age (2200-1700 B.C. cal.) periods were then tested, samples of fresh human and animal bone being included as positive and negative controls, respectively. Albumin was demonstrated in human bone from all periods; there was no evidence of cross-reactivity with animal material. Detection seemed to depend largely on amount of sample and chronological age; other factors, such as physical integrity of the specimen and soil characteristics, appeared to be less important. Preliminary studies of other ancient skeletal remains showed that animal species could be readily identified and that albumin was probably still detectable in cremated material. It is concluded that our method provides a tool specific and sensitive enough for the reliable identification of the species-origin of small fragments of bone (and possibly of blood stains) and will thus allow insight into past behaviour patterns. ELISA may also be suitable for identifying other molecules (such as HLA and ABO) which would help determine racial affiliation and disease predisposition among ancient populations.

Albumins↗
PubMed1992

Detection of human proteins in buried blood using ELISA and monoclonal antibodies: towards the reliable species indentification of blood stains on buried material.

The survival of human proteins in blood stains on fragments of cloth buried in exposed soil was examined in a 15-month investigation carried out from September 1990 to December 1991. During this period there was a wide variety of weather conditions. Samples were exhumed at 4-weekly intervals for 16 weeks and finally at 65 weeks; extracts of the stains were tested for albumin and IgG using a highly specific and sensitive enzyme-linked immunosorbent assay (ELISA) performed with monoclonal antibodies. Human albumin survived well throughout the 15 months of study, but IgG could be detected only in the 4- and 8-week samples. The reactions for IgG were weaker than those for albumin, although the method's sensitivity (10 ng) was the same for each protein. Appropriate buried and non-buried control experiments were carried out using cloth, either unstained or stained with human blood or animal sera; there was no cross-reactivity between human and the other species investigated and soil did not affect the assay; under laboratory conditions, IgG and albumin survived equally well. The system's versatility was illustrated by using monoclonal anti-bovine-albumin to detect specific albumin in the extracts of buried cloth which has been stained with bovine serum. It was concluded that ELISA performed with monoclonal antibodies could be of great value in identifying blood stains for forensic purposes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Loss of HLA-A,B,C allele products and lymphocyte function-associated antigen 3 in colorectal neoplasia.

The expression of HLA-A,B,C antigens and lymphocyte function-associated antigen 3 in human colorectal adenomas and adenocarcinomas was studied by immunohistochemistry. None of 10 adenomas and only 1 of 30 carcinomas had lost expression of all HLA-A,B,C molecules. On the other hand, focal loss of an HLA-B product was seen in 2 of the adenomas, and complete losses of tumor cell HLA-A2 (in 7 of 13 cases), HLA-Bw4 (in 4 of 13 cases), and HLA-A3 (in 1 of 6 cases) were seen in the carcinomas. No complete losses of HLA-A1 (in 6 cases) or HLA-Bw6 (in 22 cases) occurred in the carcinomas. In addition, 1 of 20 adenocarcinomas totally lacked lymphocyte function-associated antigen 3. Because a loss of tumor cell HLA-A,B,C antigen or lymphocyte function-associated antigen 3 could be selected for through an advantage in escape from cytotoxic T-lymphocyte attack, our results suggest that immunoselection may be a more important mechanism in tumor progression than has previously been assumed.

Adenocarcinoma↗
PubMed1988

Loss of polymorphic A and B locus HLA antigens in colon carcinoma.

In the present study we have confirmed that approximately one third of human colorectal carcinomas fail to express the HLA - A,B,C monomorphic determinant reactive with the W6/32 MAb, and 44% express class II HLA antigens as shown by reactivity with NFK-1 MAb. Reduced staining with the W6/32 MAb was not always associated with the loss of beta 2m. In addition, the expression of HLA-A2 and Bw4 class I specific haplotypes on normal colon epithelium and tumour biopsy tissue was assessed. All normal colonic epithelia stained positively with MAb against A2 and Bw4 antigens, but a loss of these determinants was shown on tumour biopsies from patients tissue typed for the respective specificities. Loss of the A2 haplotype was shown in 4 of 15 tumour tissue samples, and loss of Bw4 specificities in 5 out of 7 tissue samples. The failure to detect specific loci determinants was not necessarily associated with loss of reactivity with W6/32 MAb.

Adenocarcinoma↗
PubMed1987

Insulin dependent diabetes in Asians.

Type 1 diabetes is said to be extremely rare in children in India, where diabetes treated with insulin may be due to chronic pancreatic disease or malnutrition. To see whether typical type 1 diabetes occurred in Asian children in the United Kingdom, all known Asian children with diabetes in industrial West Yorkshire were ascertained. A total of 17 such children were studied; of these, seven were from three multiplex families and two fathers from these families had diabetes. All children were ketosis prone and developed diabetes while resident in the UK. There were significant increases in HLA-B8 and HLA-DR3 and increases in HLA-DR4 and HLA-DR3/DR4, while HLA-B15 was absent. Islet cell antibodies, either IgG or complement fixing, were present in four of 18 subjects tested, all of whom had disease of short duration. The prevalence of type 1 diabetes in Asian children aged 15 years or less in West Yorkshire was 36/100,000, assuming complete ascertainment. It is concluded that typical type 1 diabetes may occur in Asian children and this condition may be more common in families who have migrated to the UK.

Adolescent↗
PubMed1987

Cell-mediated immunological status and association of genetic markers in hereditary cerebellar ataxia.

Sixteen unrelated patients with hereditary cerebellar ataxia (HCA) were studied for genetic association with HLA and sixteen other genetic markers. Cell mediated immunological status of these patients was also studied by in vitro lymphocyte transformation tests. HLA typing was done in five three-generation families of patients with autosomal dominant cerebellar ataxia (ADCA). Linkage between HLA and ADCA loci was analysed using LIPED. Negative lod scores were observed in all five families. This lack of evidence for linkage between the HLA and ADCA loci is attributed to genetic heterogeneity of the disease in the families studied. No significant deviation was found in lymphocyte function to mitogen/antigen stimulation. A possible association of B12 (B44) antigen with ADCA is suggested.

Cerebellar Ataxia↗
PubMed1986

Hereditary cerebellar ataxia and genetic linkage with HLA.

Five families with at least three generations of members affected with autosomal dominant spinocerebellar ataxia (SCA) were studied. HLA typing was carried out and the coded HLA haplotypes were used to calculate the likelihood of linkage using the LIPED computer program. The combined lod scores from these five families does not, by itself, support linkage. Negative lod scores were observed in all five families, however, when pooled with the previously published data significant lod scores were obtained [Z = 3.343 (theta = 0.20) and +4.286 (theta = 0.30)]. In four families, affected members had clinical features consistent with autosomal dominant cerebellar ataxia (ADCA) type I while in the fifth, ADCA type II was suggested. Clinical heterogeneity within ADCA raises doubts about the significance of summed lod scores. In view of the previous reports probably two genetically heterogeneous types of ADCA exist -- HLA linked and nonlinked.

Adolescent↗
PubMed1985

Monoclonal antibodies to detect A, B and H blood group isoantigens in superficial transitional cell carcinoma of the bladder: a means of predicting invasive recurrences.

Monoclonal antibodies to A, B and H blood group isoantigens (BGI) have been used in an indirect immunoperoxidase technique to study the expression of BGI in both normal and malignant transitional epithelium in patients of all blood groups. BGI was detectable in all 15 biopsies of normal transitional epithelium and in 28 of 50 (56%) stage pTa and pTl transitional cell carcinomas of the bladder (TCCB). In the tumours failing to express BGI there was a 55% invasive recurrence rate at 5 years compared with 14% in those tumours expressing BGI. The difference was significant at the P less than 0.01 level. The recent availability of a monoclonal antibody to H isoantigen expressed in group O patients, in addition to monoclonal anti A and anti B, has made possible the inclusion of patients of all blood groups into a study using a standard immunoperoxidase technique without the need for specially processed biopsy material. The advantages of this technique compared with the earlier methods are discussed with reference to its possible role in identifying those patients at highest risk of developing invasive recurrence of their tumour.

ABO Blood-Group System↗