Perceived stress and coping strategies among candidates for heart transplantation during the organ waiting period.

The effectiveness of the transplantation team in diffusing stress during the pretransplantation period is increasingly important the longer the patient remains on the transplant waiting list. This study describes the stressors and coping strategies of heart transplant candidates during the waiting period. Thirty-nine candidates on the active list for heart transplantation from four mid-East Coast transplantation centers participated. With a possible stress score of 0 to 243, the mean score for this sample was a low 72.84 (standard deviation = 37.47). The three most common stressors were (1) requiring a heart transplant, (2) having terminal heart disease, and (3) worrying family members. The three most common coping strategies were (1) thinking positively, (2) using humor, and (3) trying to keep life as normal as possible. The finding of low stress levels was surprising but may reflect the presence of hope or the patient's desire to spare family members worry--a concern commonly cited by patients. Another explanation is that patients desiring to be perceived as ideal transplant recipients may have underreported their stress. This suggests that the transplantation team should support positive coping strategies when possible and that both patient and family coping should be closely monitored throughout the waiting period.

The chemical structure of the C4d fragment of the human complement component C4.

The complete amino acid sequence of the C4d fragment (380 residues long) of the human complement component C4 is presented. Most of the sequence was determined by analysis of CNBr peptides and tryptic peptides obtained from S-carboxymethylated protein. The sequence of the amino terminal 88 residues [Campbell R. D., Gagnon J. and Porter R. R. (1981) Biochem. J. 199, 359-370] and a 106 residue polymorphic segment of C4d [Chakravarti D. N., Campbell R. D. and Gagon J. (1983) FEBS Lett. 154, 387-390] was extended. Some overlaps not provided by the protein sequence analysis were obtained from the amino acid sequence predicted by the nucleotide sequence [Belt K. T., Carroll M. C. and Porter R. R. (1984) Cell 36, 907-914]. The present protein sequence data provide information for the isolation of all the CNBr and succinylated tryptic peptides of C4d. In addition to the polymorphism previously described, two other sets of polymorphic amino acid residues at positions 153 (Ile/Ser) and 154 (Gln/Ala) have been identified. The major site of glycosylation has been shown to be an asparagine residue located in the sequence -Asn-Val-Thr- in the carboxy terminal end of C4d. A remarkable difference in the predicted secondary structure of C4d arising from one set of four polymorphic residues in a stretch of six residues and another single polymorphic residue suggests a structural basis for the origin of the different chemical reactivities of the C4 isotypes (C4A and C4B) and their serological difference in the expression of Rodgers or Chido blood group antigens. Possible non-covalent membrane attachment sites have been suggested from the hydropathy profile. Comparison of the C4d sequence with human C3, C5 and alpha 2-macroglobulin revealed extended stretches of sequence similarity (between 19 and 38% homology) with the corresponding regions of these proteins.

Molecular characterization of the HLA-linked steroid 21-hydroxylase B gene from an individual with congenital adrenal hyperplasia.

21-Hydroxylase deficiency which causes congenital adrenal hyperplasia is one of the most common defects of adrenal steroidogenesis. There are two 21-hydroxylase genes in man, A and B, and these have been mapped to the HLA class III region. Only the 21-hydroxylase B gene is thought to be active. To understand the molecular basis of congenital adrenal hyperplasia in a patient with the salt-wasting form of the disease, we cloned and characterized his single 21-hydroxylase B gene. The nucleotide sequence of this gene and a 21-hydroxylase B gene from a normal individual have been determined. Comparison of the two sequences has revealed 11 nucleotide alterations, of which two are in the 5' flanking region, four are in introns, one is in the 3' untranslated region and four are in exons. Two of the differences in exons cause codon changes, with Ser-269 and Asn-494 in the normal 21-hydroxylase B gene being converted to Thr and Ser, respectively. These amino acid substitutions may give an insight into those residues necessary for 21-hydroxylase enzymatic activity. We have also confirmed that the 21-hydroxylase A gene is a pseudogene due to three deleterious mutations in the exons. In addition, comparison of the 21-hydroxylase B gene sequence with other published sequences indicates that this microsomal cytochrome P-450 may be polymorphic.

Structural basis of the polymorphism of human complement components C4A and C4B: gene size, reactivity and antigenicity.

The human complement components C4A and C4B are highly homologous proteins, but they show markedly different, class-specific, chemical reactivities. They also differ serologically in that C4A generally expresses the Rodgers (Rg) blood group antigens while C4B generally expresses the Chido (Ch) blood group antigens. C4A 1 and C4B 5 are exceptional variants which possess their class-specific chemical reactivities, but express essentially the reversed antigenicities. The genes encoding the typical Rg-positive C4A 3a and Ch-positive C4B 3 allotypes and the interesting variants C4A 1 and C4B 5 have been cloned. Characterization of the cloned DNA has revealed that the genes encoding the A 3a, A 1 and B 3 allotypes are 22 kb long, but that encoding B 5 is only 16 kb long. Comparison of derived amino acid sequences of the polymorphic C4d fragment has shown that C4A and C4B can be defined by only four isotypic amino acid differences at position 1101-1106. Over this region C4A has the sequence PCPVLD while C4B has the sequence LSPVIH, and this presumably is the cause of their different chemical reactivities. Moreover, the probable locations of the two Rg and the six Ch antigenic determinants have been deduced. Our structural data on the C4A and C4B polymorphism pattern suggests a gene conversion-like mechanism is operating in mixing the generally discrete serological phenotypes between C4A and C4B.

Deletion of the steroid 21-hydroxylase and complement C4 genes in congenital adrenal hyperplasia.

DNA was analysed from 20 patients with congenital adrenal hyperplasia due to cytochrome P-450 steroid 21-hydroxylase deficiency. Using probes recognising sequences in both the 21-hydroxylase gene and the adjacent fourth component of complement (C4), one patient was found to have a homozygous deletion of DNA which encompassed the C4B and 21-hydroxylase B genes. Evidence is presented for this deletion arising by recombination between homologous regions of 21-hydroxylase A and B. Seven patients appeared to be heterozygous for the same deletion, but no detectable alteration in the 21-hydroxylase gene could be demonstrated in others.

The molecular genetics of components of complement.

Rapid progress has been made in establishing linkages and in chromosome allocation of the genes of some 9 complement components. In the MHC, C2, Factor B, and two C4 or C4 related genes have been placed in some detail in both man and mouse. The gene coding for the cytochrome P-450 21-hydroxylase has been shown to be duplicated and immediately 3' to the two C4 genes, though it appears to be functionally and structurally unrelated to the complement components. Thus six genes have been mapped to this region where particular haplotypes are associated with increased susceptibility to a number of diseases, some of which are autoimmune in character. The complete gene structure of Factor B has been solved in man and rapid progress is being made with the C2 and C4 genes. The structural basis of the polymorphisms of these genes is being established. In C4, the polymorphism is exceptionally complex with varying numbers of loci and probably more than 50 allotypes occurring in man. A structural basis has also been found for the big differences in the biological activity of some of the C4 allotypes in man. Apart from the genes in the MHC, linkage has been found between the genes coding for C4bp, CR1, and Factor H. Remarkably there are sequence homologies between these proteins and C2 and Factor B, probably related to the ability to bind to one or other of the structurally similar proteins C3b and C4b. The complete cDNA sequences of C3 and C4 in mouse and man have given much information on the many posttranslational modifications of these proteins. A partial structure has been obtained for the C3 gene and the homology shown between C3, C4, C5, alpha 2-macroglobulin, and pregnancy zone protein. Although the amount of detailed information in the molecular genetics of complement components is accumulating rapidly, there appears to be a reasonable prospect that linkages and homologies will classify the data into a comprehensible form.

The purification and properties of some less common allotypes of the fourth component of human complement.

Human complement component C4 is coded by two genes situated between HLA-D and HLA-B. Both genes are highly polymorphic; C4-A gene products normally carry the blood group antigen Rodgers and C4-B proteins usually carry the Chido antigen. Using a monoclonal antibody which binds Rodgers-positive and Chido-positive proteins with different affinities, we have purified a number of less common C4 allotypes and compared their properties. All C4-B allotypes tested have similar specific hemolytic activities and binding efficiencies to small molecules. All C4-A proteins tested had similar binding to small molecules and hemolytic activities except for the C4-A6 proteins from two individuals with different extended haplotypes, both of which had identical hemolytic activities and much lower ones than other C4-A allotypes. Two allotypes, C4-A1, Rodgers-negative but Chido-positive, and C4-B5, Chido-negative but probably Rodgers-positive, were found to behave as typical C4-A and C4-B proteins, respectively, apart from the switch in their antigenic properties.

The complement components coded in the major histocompatibility complexes and their biological activities.

The complement system has two pathways of activity, both dependent on the sequential conversion of proteolytic zymogens to active proteases leading to a common lytic complex and both with control proteins which inhibit or inactivate different steps in the cascade. Three of the components--C2, factor B and C4--are coded by closely linked genes in the MHC of man and mouse and have been placed relative to each other. The genes are polymorphic, particularly C4, with variable numbers of loci as well as many mutant forms. Some alleles of C4 show strikingly different reactivities in their haemolytic activity and this may be relevant to the association of susceptibility to autoimmune diseases with particular haplotypes in this section of HLA.

Deletion of complement C4 and steroid 21-hydroxylase genes in the HLA class III region.

Molecular maps have been prepared of the HLA region on human chromosome 6 that includes the complement C4 and steroid 21-hydroxylase genes (21-OH), using DNA of individuals deficient (QO) in either of the two forms C4A or C4B. In all, 18 haplotypes with C4A QO were examined by Southern analysis and two had deletions of 28-30 kb that included both the C4A and 21-OHA genes. Of six C4B QO haplotypes, one had a deletion that included both the C4B and 21-OHA genes. Thus, some of the C4 null alleles are due to deletion of the gene but the majority in this sample are not. Deletion occurred in two common haplotypes suggesting that in the population as a whole, C4A deficiency is due to deletion in about one-half the C4A QO haplotypes. As duplication of C4A or C4B genes does occur, the possibility that unequal cross-over could explain the C4 deletion was examined by preparing cosmid clones from the DNA of an individual typed C4A QO. A cloned genomic fragment containing the single C4B gene was isolated and found to be similar to the homologous region of a cosmid from a normal individual carrying a C4A gene. This suggests that if a cross-over has occurred it is in a region where the two genes are identical. The biological significance of the rather frequent occurrence in the population of haplotypes with C4A or C4B deletion together with the accompanying deletion of the 21-OHA gene is discussed.

The origin of the very variable haemolytic activities of the common human complement component C4 allotypes including C4-A6.

The human complement component C4 occurs in many different forms which show big differences in their haemolytic activities. This phenomenon seems likely to be of considerable importance both physiologically and pathologically. C4 is coded by duplicated genes between HLA-D and HLA-B loci in the major histocompatibility complex in man. Several fold differences in haemolytic activity between products of the two loci C4-A and C4-B have been correlated with changes of six amino acid residues in this large protein of 1722 residues and with differences of several fold in the covalent binding of C4 to antibody-antigen aggregates. Some allotypes of one locus also differ markedly, notably C4-A6 which has 1/10th the haemolytic activity of other C4-A allotypes. A monoclonal antibody affinity column has been prepared which is able to separate C4-A from C4-B proteins and, using serum from an individual expressing only the C4-A6 allele at the C4-A locus, C4-A6 protein has been prepared. Investigation has shown C4-A6 to have the same reactivity as other C4-A allotypes except in the formation of the complex protease, C5 convertase. This protease is formed from C4, C2 and C3 and if C4-A6 is used it has approximately 1/5th the catalytic activity compared with other C4-A allotype. Allelic differences in sequence identified in C4 proteins so far are few and it is probable that the big difference in catalytic activity of C5 convertase is caused by very small changes in structure.

The polymorphism of the complement genes in HLA.

Genes coding for the complement proteins C2, C4A, C4B and factor B lie between HLA-D and HLA-B in HLA, the major histocompatibility complex in man. All the complement components are polymorphic, particularly C4, which has many alleles at each locus. The genetic complexity of C4 extends to the number of loci each of which may be deleted or duplicated on the chromosome. The different forms of C4 showed markedly different reactivities with small molecules and on haemolytic activity in the complement system. Surprisingly, amino acid sequences of the several allelic forms of C4 appear to be very similar, with less than 1% of residue positions being changed between alleles of C4A and C4B. These results may be relevant to the increased susceptibility to autoimmune disease which is associated with particular haplotypes of the HLA complex.

Mapping of steroid 21-hydroxylase genes adjacent to complement component C4 genes in HLA, the major histocompatibility complex in man.

The genes for four components (C) of complement in the human major histocompatibility complex (HLA) have been aligned previously in a series of overlapping cosmid cloned inserts. Those inserts, which contained the two C4 genes C4A and C4B, hybridized with human adrenal mRNA, indicating that they contain a gene expressed in the adrenal. The mRNA fraction of 2.4 kilobases (kb) hybridizes with genomic DNA of 4.5 kb, which is duplicated and lies about 1.5 kb 3' of both the C4A and the C4B complement genes. Sequencing of a 430-base section and comparison with the published cDNA sequence of bovine cytochrome P-450 21-hydroxylase, peptide sequences of porcine 21-hydroxylase, and a cDNA sequence of a rat liver cytochrome P-450 identified the gene as coding for human steroid 21-hydroxylase [steroid,hydrogen-donor:oxygen oxidoreductase (21-hydroxylating), EC 1.14.99.10]. Mapping of the gene was helped by use of a synthetic oligonucleotide based on the bovine cDNA sequence.

Polymorphism of human complement component C4.

An assessment has been made of the polymorphism of human complement component C4 by comparing derived amino acid sequences of cDNA and genomic DNA with limited amino acid sequences. In all, one complete and six partial sequences have been obtained from material from three individuals and include two C4A and two C4B alleles. Differences were found between the 4 alleles from 2 loci in only 15 of the 1722 amino acid residues, and 12 lie within one section of 230 residues, which in 1 allele also contains a 3-residue deletion. In three variable positions, an allelic difference in one C4 type was common to the other types. Three nucleotide differences were found in four introns. In spite of marked differences in their chemical reactivity, the many allelic forms appear to differ in less than 1% of their amino acid residue positions. This unusual pattern of polymorphism may be due to recent duplication of the C4 gene, or may have arisen by selection as a result of the biological role of C4, which interacts in the complement sequence with nine other proteins necessitating conservation of much of the surface structure.

Structure and organization of the C4 genes.

This 200 000 Mr serum protein is coded for by at least two separate loci, C4A and C4B, which map in the HLA Class III region on chromosome 6 in man. Both loci are highly polymorphic with more than 30 alleles, including null alleles assigned to the two loci. The complete nucleotide sequence of a full length C4A cDNA clone and a substantial part of a C4b cDNA clone has shown class differences which can be used to synthesize nucleotide probes specific for C4A and C4B. Three C4 loci of approximately 16 kilobases each spaced by 10 kilobases have been identified in DNA from one individual and aligned 30 kilobases from the factor B gene by overlapping cloned genomic fragments from a cosmid library. Characterization of these genes by restriction mapping, nucleotide sequence analysis and hybridization with C4A and C4B specific synthetic oligonucleotides show that these genes are very similar.

A comparison of the properties of two classes, C4A and C4B, of the human complement component C4.

A remarkable difference has been observed between the reactivity of the two forms of human complement component C4. C4B binds twice as effectively as C4A to antibody-coated red cells, but the reverse occurs with protein-antigen complexes. C4B reacts much more effectively with hydroxyl groups than C4A and this is reversed for reaction with amino groups in spite of the very small difference in amino acid sequence between the two forms of C4. No other differences in stability, activation or inactivation were observed. These findings emphasise the biological advantage of the duplication of the C4 gene in its reaction with a wide range of antigenic structures. The correlation of the presence of different forms of C4 with susceptibility to autoimmune diseases may be explicable by these big differences in binding reactivity.