Direct observation of the gene organization of the complement C4 and 21-hydroxylase loci by pulsed field gel electrophoresis.

Pulsed field gel electrophoresis and enzymes that cut genomic DNA infrequently have been used to define large RFLPs at the human C4 loci. With the enzymes BssH II or Sac II, and C4 or 21-hydroxylase DNA probes, it has been possible to observe directly the number of C4 genes present on a haplotype, and also whether the C4 genes are long (6-7-kb intron present) or short (6-7-kb intron absent). Haplotypes that have either two long C4 genes or one long and one short C4 gene generate BssH II fragments of approximately 115 or approximately 105 kb, respectively. Haplotypes that have either a single long or a single short C4 gene generate BssH II fragments of approximately 80 or approximately 70 kb, respectively. This technique has been used to analyze the DNA isolated from PBMC and allows the complete definition of the C4 gene organization of an individual without the need for family studies.

Extensive deletions and insertions in different MHC supratypes detected by pusled field gel electrophoresis.

The genomic organization of the human MHC was examined in multiple examples of six different supratypes using pulsed field electrophoresis (PFGE) after digestion of genomic DNA with infrequency cutting restriction endonucleases. Differences in restriction fragment length and band intensity were shown to be specific for each supratype. Mapping of the MHC revealed that each supratype contains previously undescribed deletions and insertions between HLA B and DQ regions.

Differences in gene copy number carried by different MHC ancestral haplotypes. Quantitation after physical separation of haplotypes by pulsed field gel electrophoresis.

We have examined the hypothesis that MHC ancestral haplotypes have a specific content of genes regulating the extent of autoimmune reactions. Gene copy number was quantitated by objective densitometry after PFGE was used to separate heterozygous AHs of different lengths. Initially we analyzed examples of known gene copy number at the C4 and 21 hydroxylase loci and showed that the approach provides predictable results. We then studied heterozygotes containing one characterized and one uncharacterized AH with particular attention to the gene copy number at the C4, Cyp21, and DRB loci. Each AH studied has a characteristic gene copy number at each locus studied. The same may be true of TNF, but other possibilities must be considered. AHs are markers for extensive chromosomal segments including particular numbers of several functional genes. Since AHs mark susceptibility to autoimmune disease, differences in gene copy number may be implicated.

Human natural killer (NK) alloreactivity and its association with the major histocompatibility complex: ancestral haplotypes encode particular NK-defined haplotypes.

As ancestral haplotypes of the major histocompatibility complex (MHC) appear to define identical MHC haplotypes in unrelated individuals, unrelated individuals sharing the same ancestral haplotype should also share the same NK-defined allospecificities that have recently been shown to map to the human MHC. To test this prediction, multiple cell lines from unrelated individuals sharing the same ancestral haplotypes were tested for the NK-defined allospecificities. It was found that cells sharing the same ancestral haplotypes do have the same NK-defined specificities. Furthermore, the NK-defined phenotype of cells that possess two different ancestral haplotypes can be predicted from the NK-defined phenotypes of unrelated cells that are homozygous for the ancestral haplotypes concerned. Although the group 1 and 2 NK-defined allospecificities can be explained to some extent by HLA-C alleles, evidence is presented that additional genes may modify the phenotype conferred by HLA-C.

Adipose invasion of muscle in Wagyu cattle: Monitoring by histology and melting temperature.

Remarkably, Wagyu cattle progressively desaturate intramuscular and subcutaneous fat leading to melting temperatures (Tm) well below 38°C. In parallel, the adipose tissue expands, arborises and invades the muscle. The process is aggressive in that it leads to loss of myofibres resulting in much smaller fascicles and therefore fine marbling or snowflaking. The "Microscopic score" appears to be an excellent measure of marbling especially for lesser and greater degrees which are not quantified reliably by others methods. By comparing muscle groups, we conclude that the tailhead is a suitable site for sequential monitoring. Melting temperatures of intramuscular and subcutaneous tissue are also useful.

Epistasis between the MHC and the RCA alpha block in primary Sjögren syndrome.

OBJECTIVE: The RCA alpha block (Regulators of Complement Activation, 1q32) contains critical complement regulatory genes such as CR1 and MCP. This study examined RCA alpha block haplotype associations with both disease susceptibility and diversification of the anti-Ro/La autoantibody response in primary Sjögren syndrome (pSS). METHODS: 115 patients with pSS and 98 controls were included in the study. 93 of 109 (85%) of the patients with pSS were seropositive for Ro/La autoantibodies. The Genomic Matching Technique (GMT) was used to define RCA alpha block ancestral haplotypes (AH). RESULTS: RCA alpha block haplotypes, AH1 and AH3, were both associated with autoantibody-positive pSS (p = 0.0003). Autoantibody associations with both HLA DR3 and DR15 have been previously defined. There was an epistatic interaction (p = 0.023) between RCA alpha AH1 and HLA DR3, and this genotypic combination was present in 48% of autoantibody-positive patients with pSS compared with 8% of controls. This epistasis is most simply attributable to an interaction between C4 and its receptor, CR1, encoded within the RCA alpha block. Both DR3 and a relative C4 deficiency are carried on the major histocompatibility complex 8.1 ancestral haplotype. Only four of 92 (4%) autoantibody-positive patients with pSS did not carry any risk RCA alpha or HLA haplotype, compared with 36 of 96 (38%) controls, and there were differences in haplotype frequencies within autoantibody subsets of pSS. CONCLUSIONS: Normal population variation in the RCA alpha block, in addition to the major histocompatibility complex, contributes genetic susceptibility to systemic autoimmune disease and the autoantibody response. This finding provides evidence for the role of regulation of complement activation in disease pathogenesis.

The implications of intergenic polymorphism for major histocompatibility complex evolution.

A systematic survey of six intergenic regions flanking the human HLA-B locus in eight haplotypes reveals the regions to be up to 20 times more polymorphic than the reported average degree of human neutral polymorphism. Furthermore, the extent of polymorphism is directly related to the proximity to the HLA-B locus. Apparently linkage to HLA-B locus alleles, which are under balancing selection, maintains the neutral polymorphism of adjacent regions. For these linked polymorphisms to persist, recombination in the 200-kb interval from HLA-B to TNF must occur at a low frequency. The high degree of polymorphism found distal to HLA-B suggests that recombination is uncommon on both sides of the HLA-B locus. The least-squares estimate is 0.15% per megabase with an estimated range from 0.02 to 0.54%. These findings place strong restrictions on possible recombinational mechanisms for the generation of diversity at the HLA-B.

Indels and imperfect duplication have driven the evolution of human Complement Receptor 1 (CR1) and CR1-like from their precursor CR1 alpha: importance of functional sets.

This study examines the effects of duplication and insertions-deletions (indels) by comparing human complement receptor 1 (CR1) and human CR1-like (CR1L) with syntenic genes from four other vertebrates (chimpanzee, baboon, rat, and mouse). By phylogenetic analysis, the domains of these genes can be classified into 10 distinct subfamilies (a, b, c, d, e, f, g(-like), h, j, and k), which have been largely conserved throughout vertebrate and invertebrate evolution. In spite of many complex and diverse duplications and indels, the subfamily order of domains (a, j, e, f, b, k, d, g(-like)) has been maintained. The number of domain sets has increased progressively, thereby expanding the functional repertoire.

Use of the genomic matching technique to complement multiplex STR profiling reduces DNA profiling costs in high volume crimes and intelligence led screens.

The genomic matching technique (GMT) targets duplicated polymorphic sequences within genomic blocks in the human major histocompatibility complex (MHC), differentiating between individuals at the DNA level using a single primer pair per block. The GMT is currently used to supplement human leukocyte antigen (HLA) typing to match donor and recipient pairs for bone marrow transplantation and has the potential to be employed as a powerful exclusion tool in forensic biology. The GMT is highly reproducible, produces DNA profiles from less than 1 ng of DNA and was successfully employed to profile a range of forensic samples including buccal swabs, handled objects and fingerprints. Furthermore, GMT profiles from a single genomic block in the MHC are likely to be more discriminatory than known highly polymorphic short tandem repeat (STR) loci such as ACTBP2. As such, the GMT can reduce the cost of investigations that require profiling of multiple suspects or samples from one or more crime scenes and could be extended to profile genomic blocks in other polymorphic genetic systems in the human genome.

The identification of MHC identical siblings without HLA typing.

By comparing genomic sequences of different MHC haplotypes, we defined highly polymorphic markers. After amplification, electrophoresis, and scanning with a laser, we have identified profiles that serve as signatures of the haplotype and its component alleles. One set of markers can be used to define the block that includes HLA-B and HLA-C, among other loci. Another set provides signatures for the entire HLA-DR and -DQ multigene cluster. By profile overlay, it is possible to identify siblings who share both haplotypes from HLA-C to HLA-DQ. Here we demonstrate the value of genomic analysis ("block matching") in selecting genotypically identical siblings prior to transplantation. Forty-six siblings from 10 families were genotyped by family analysis after meticulous HLA, C4, and Bf typing including molecular methods for HLA-DRB1. In 43 siblings, the haplotype assignments were unequivocal. Twenty-two identical sibling pairs could then be compared with 77 nonidentical pairs. Independent genomic analysis yielded entirely concordant results. In three siblings, the possibility of parental recombination was considered but could not be defined by the conventional typing. By genomic analysis, however, it was clear that recombination had indeed occurred in one case. In the remaining two cases, additional, more telomeric markers will be necessary to resolve the issue. This simple, cost-effective method has immediate application to the identification of matched pairs (HLA-C to HLA-DQ) for bone marrow and renal transplantation.

Zidovudine-induced restoration of cell-mediated immunity to mycobacteria in immunodeficient HIV-infected patients.

OBJECTIVE: To describe a localized form of Mycobacterium avium intracellulare (MAI) infection occurring concurrently with the restoration of cutaneous delayed-type hypersensitivity (DTH) responses to mycobacterial antigens after commencement of zidovudine therapy in immunodeficient HIV-infected patients. PATIENTS: The first 108 Western Australian patients with a CD4+ T-cell count of < 200 x 10(6)/l and/or symptomatic disease to be given zidovudine (ZDV), of whom 72 had adequate DTH data. METHODS: DTH responses to seven antigens were measured by the 'Multitest' method before and on at least two occasions during the 6 months after commencing ZDV. All patients were reviewed at regular intervals and clinical events recorded. RESULTS: Of the 64 patients who were anergic to tuberculin before commencing ZDV, 27 (42%) developed a DTH response to tuberculin after ZDV. Four of the nine patients with a 'Multitest' tuberculin response of > or = 8 mm and one patient who developed a positive Mantoux test to M. avium purified protein derivative developed an illness characterized by localized MAI infection, lymphadenopathy and/or severe fevers after 1-2 weeks. CONCLUSIONS: The development of localized MAI infection and/or fevers shortly after commencing ZDV in immunodeficient HIV-infected patients may reflect restoration of cellular immunity to mycobacterial antigens in some patients rather than early failure of therapy or hypersensitivity to ZDV.

Extensive nucleotide variability within a 370 kb sequence from the central region of the major histocompatibility complex.

The recent availability of the genomic sequence spanning the central and telomeric end of the major histocompatibility complex (MHC) has allowed a detailed study of its organisation, gene content and level of nucleotide variability. Previous analyses of nucleotide variability in the MHC have focused on the coding regions of the human leukocyte antigen (HLA) Class I and II genes. Non-coding nucleotide variability has been considered a by-product of exonic diversity. However, with the advent of genomic sequencing, the extent of non-coding nucleotide variability within the MHC has just begun to be appreciated. In this study, we compared different human haplotypes in 370 kb of sequence in the central region of the MHC to show the following: 1. unusually high levels of non-coding nucleotide variability, up to 80 times greater than elsewhere in the genome; 2. non-coding nucleotide variability greater than 1% at nucleotide sites distant to the Class I genes; 3. nucleotide variability greater than 1% maintained over regions containing highly linked loci; and 4. distinct troughs and peaks in the level of nucleotide variability. We will discuss these observations in relation to a possible role of nucleotide variability in the organisation of the MHC.

Severity and outcome of Pneumocystis carinii pneumonia (PCP) in patients of known and unknown HIV status.

Of 170 Western Australian patients who had their first AIDS-defining illness between 1 January 1983 and 31 December 1991, 61 (36%) were of unknown HIV antibody status (AIDS presenters), while 109 (64%) were of known HIV antibody status (HIV presenters). Pneumocystis carinii pneumonia (PCP) was less common as the AIDS-defining illness in HIV presenters (41% versus 62%, p = 0.005). In this study of 70 patients with PCP as the index AIDS diagnosis, 36 were HIV presenters and 34 were AIDS presenters. Ten HIV presenters were taking prophylaxis at the time PCP manifested. The duration of symptoms of cough or dyspnea before the diagnosis of PCP was shorter, and the arterial PO2 measurement on admission was higher in those on prophylaxis, and a lower proportion of patients on prophylaxis required hospital admission (p < or = 0.05 for all comparisons). Furthermore, the CD4 counts at diagnosis of PCP were lower in patients taking PCP prophylaxis (mean 26 x 10(6)/L) than in patients who were not (mean 94 x 10(6)/L, p = 0.007). Of seven patients who died of PCP, none were receiving treatment for HIV disease before AIDS presentation. These findings suggest that PCP is prevented or deferred in patients receiving care for HIV disease and is less severe as a result of early diagnosis and treatment.

Genetics of human complement component C4 and evolution the central MHC.

The two classes of human complement component C4 proteins C4A and C4B manifest differential chemical reactivities and binding affinities towards target surfaces and complement receptor CR1. There are multiple, polymorphic allotypes of C4A and C4B proteins. A complex multiplication pattern of C4A and C4B genes with variations in gene size, gene dosage and flanking genes exists in the population. This is probably driven by the selection pressure to respond to a great variety of parasites efficiently and effectively, which the bony fish achieved through the multiplication and diversification of the related complement C3 proteins. Complement C4, C3 and C5 belong to the alpha2 macroglobulin protein family but acquired specific features that include an anaphylatoxin domain, a netrin (NTR) domain, and stretches of basic residues for proteolytic processings to form multiple chain structures. Complement C3 and C4 are important in the innate immune response as they opsonize parasites for phagocytosis. The emergence of complement C3 predates proteins involved in the adaptive immune response as C3 is present in deuterostome invertebrates such as echinoderms. The human C4 genes are located in the central MHC at chromosome 6p21.3. C3 and C5 are located at chromosome 19 and 9, respectively, with representatives of the other groups of genes paralogous to the MHC at 19p13.1-p13.3, 1q21-25, and 9q33-34. The central MHC also contains genes for complement components C2 and Bf. These genes appear to have similar evolutionary histories to C3/C4/C5 and are used here to illustrate stepwise processes resulting in co-location of diverse domains, chromosomal duplication, local segmental duplication and divergence of sequence and function. This model of evolution is useful in the investigation of innate and acquired immunity and in seeking explanations for diseases associated with MHC ancestral haplotypes.

Serum exchange and use of dilutions have improved precision of measurement of islet cell antibodies.

In an attempt to improve the diagnostic value of measuring antibodies to islet cell cytoplasmic antigen, coded sera were distributed to 38 laboratories and results were returned for analysis. Comparison between laboratories revealed that results for some individual sera differed by up to nine doubling dilutions and even within laboratories duplicate samples could differ by six doubling dilutions. By including dilutions of sera it was possible to draw a standard curve for each laboratory and this revealed major variations in shape, slope and intercept. However, using each laboratory's standard curve and converting results to units, a substantial improvement was obtained. The approach described improves standardisation and will permit laboratories to identify poor precision and/or any systematic change in assay performance.

Antinuclear antibody. Precise and accurate quantitation without serial dilution.

A new method for measuring homogeneous pattern antinuclear antibody by immunofluorescence has been validated. This method exploits the differential sensitivity of rat heart muscle, kidney tubules and liver parenchyma as substrates for ANA, and employs calibrating sera. It provides quantitative measurement in the range 2.5-10 WHO IU/ml and semi-quantitative measurement up to 30 WHO IU/ml, without the need for serial dilution. The routine use of standards together with this method improves precision and provides conversion of measurements to IU/ml. These modifications make ANA a precise screening test for the exclusion of SLE.

An enzyme-linked immunosorbent assay for antistriational antibodies associated with myasthenia gravis and thymoma: comparison with indirect immunofluorescence.

Autoantibodies to the striations of skeletal muscle (AStrA) detected by immunofluorescence are useful in the diagnosis of a thymoma associated with myasthenia gravis (MG). With the intention of developing a better method, an enzyme-linked immunosorbent assay (ELISA) has been evaluated in 147 MG patients and 200 healthy controls. An additional 107 patients with various autoimmune diseases and autoantibodies were also tested. With a crude actomyosin preparation, the ELISA gave similar results to immunofluorescence, viz. positives in 42% of MG patients, but in all with a histologically proven thymoma. Less than 1% of the healthy controls were positive but false positives were found in patients with liver disease and anti-smooth muscle antibodies. After treatment of rheumatoid arthritis with D-penicillamine the titre of AStrA may rise. The ELISA was shown to be sensitive and reproducible, but immunofluorescence is a more practical method of distinguishing between the different categories of anti-muscle antibodies. ELISA should prove particularly useful for quantitation and sequential monitoring.

Amyloidosis associated with dermatomyositis and features of multiple myeloma. The progression of amyloidosis associated with corticosteroid and cytotoxic drug therapy.

Described here is a 59 year old man with dermatomyositis and hypogammaglobulinemia. His muscle power improved after corticosteroid therapy, but extensive amyloidosis and repeated infections appeared. Bone marrow morphology suggested multiple myeloma, but treatment with cytotoxic drugs had no beneficial effect on the amyloidosis. Because of rapid progression of the amyloidosis and further infections, cytotoxic drug therapy was stopped, corticosteroid dosage was decreased, and supplementary immunoglobulin therapy was instituted. The infections occurred less frequently and the amyloidosis appeared to regress. This case suggests that immunosuppressive therapy may exacerbate amyloidosis. The literature is reviewed, and the possible role of humoral immunodeficiency in the pathogenesis of amyloidosis is discussed. It is suggested that supplementary immunoglobulin may be beneficial in amyloidosis.

Penicillamine-associated myasthenia gravis, antiacetylcholine receptor and antistriational antibodies.

Myasthenia gravis with thymic hyperplasia developed in a patient with Wilson's disease after eight years of penicillamine treatment. Four months prior to the onset of myasthenia, penicillin hypersensitivity was observed. Immunofluorescence on the excised thymus revealed immunoglobulin and complement deposition, but the myasthenia persisted after thymectomy and continuation of penicillamine therapy. Increased antiacetylcholine receptor antibody was demonstrable throughout. This patient subsequently became pregnant, enabling studies to be performed on the transplacental transfer of the immunoglobulin G (IgG) class antiacetylcholine receptor antibody. Eleven cases of rheumatoid arthritis with penicillamine-associated antistriational antibodies have also been observed; in three of these cases there was evidence of myasthenia gravis. These observations extend earlier reports of the association of penicillamine with myasthenia gravis and suggest that antistriational antibody, antiacetylcholine receptor antibody and thymic hyperplasia may be independent effects of penicillamine therapy.

Arthritis associated with a crystallizing cryoprecipitable IgG paraprotein.

A crystallizing IgG-lambda cryoprotein was found in the synovial fluid of a patient with peripheral erosive arthritis and tenosynovitis. The same crystallizing paraprotein could be demonstrated in serum incubated at 4 degrees C, but its formation could be inhibited by the in vitro addition of D-penicillamine. Crystals were present in synovial tissue and appeared to be initiating an inflammatory reaction via complement activation. Slit lamp examination showed crystals in Bowman's membrane. Plasmapheresis led to temporary improvement in the synovitis and tenosynovitis.