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Achievement of complete donor chimerism (CDC) after allogeneic nonmyeloablative hematopoietic stem cell transplantation (NMHSCT) is important for preventing graft rejection and for generating a graft-vs-malignancy effect. The alloreactivity of NK cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors (KIRs) with target cell HLA/KIR ligands. The influence of KIR matching on the achievement of T-cell CDC after NMHSCT has not been previously described. We analyzed 31 patients undergoing T-cell replete related donor NMHSCT following fludarabine and 200 cGy TBI. Recipient inhibitory KIR genotype and donor HLA/KIR ligand matches were used to generate an inhibitory KIR score from 1 to 4 based upon the potential number of recipient inhibitory KIRs that could be engaged with donor HLA/KIR ligands. Patients with a score of 1 were less likely to achieve T-cell CDC (P=0.016) and more likely to develop graft rejection (P=0.011) than those with scores greater than 1. Thus, patients with lower inhibitory KIR scores may have more active anti-donor immune effector cells that may reduce donor chimerism. Conversely, patients with greater inhibitory KIR scores may have less active NK cell and T-cell populations, which may make them more likely to achieve CDC.

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The reactivity of natural killer cells and some T-cell populations is regulated by killer immunoglobulin-like receptors (KIR) interactions with target cell HLA class I molecules. Such interactions have been suggested to influence outcomes after allogeneic hematopoietic stem cell transplantation, particularly for myeloid malignancies and with T-cell depletion. Donor KIR genotypes and recipient HLA KIR ligands were analyzed in 60 AML patients receiving T-cell replete, HLA-matched-related donor allogeneic bone marrow transplants. Patients were categorized according to their HLA inhibitory KIR ligand groups by determining whether or not they expressed: HLA-A3 or -A11; HLA-Bw4 and HLA-Cw groups (homozygous C1, homozygous C2 or heterozygous C1/C2). Heterozygous C1/C2 patients had significantly worse survival than those homozygous for C1 or C2 (5.8 vs 43.5 months, respectively, P=0.018) and the C1/C2 group had a higher relapse rate (47 vs 31%, respectively, P=0.048). Multivariate analysis found C1/C2 status to be an independent predictor for mortality (P=0.007, HR 2.54, confidence interval 1.29-5.00). C1/C2 heterozygosity was also associated with a delayed time to platelet engraftment, particularly for those with concurrent HLA-Bw4 expression (P=0.003). Since C1/C2 heterozygotes have a greater opportunity to engage inhibitory KIRs than do C1 or C2 homozygotes, they may more effectively inhibit KIR-positive NK- and T-cell populations involved in graft vs leukemia responses.

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We describe a new human leukocyte antigen (HLA)-Cw*07 allele that differs from Cw*0718 by a single-coding nucleotide. DNA-based genotyping identified a clinical sample from a Black African-American patient that differed from known Cw alleles. The allele was amplified independently with a haplo-specific primer and sequenced in its entirety.

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BACKGROUND: Simple anthropometric indices of body composition have particular appeal for use in children, and as such body mass index (BMI) has been used to predict percentage body fat in a number of studies. AIM: To evaluate the relationship between BMI and percentage body fat (%body fat) and a proposed, more appropriate relationship between BMI and fat mass/height(2) in a cohort of young children. SUBJECTS AND METHODS: Cross-sectional study of 109 children aged between 6 and 10 years residing in either Sydney or Brisbane, Australia. Weight and height were measured using standard methods. Body composition was measured using a stable isotope method to firstly determine total body water and subsequently fat free mass. RESULTS: The correlation between BMI and fat mass/height(2) was markedly greater than that between BMI and percentage body fat. In the entire group of children the R(2) (x100%) value for the relationship between BMI and fat mass/height(2) was 73.3% compared with 46.5% for the relationship between BMI and percentage body fat. CONCLUSIONS: We have shown that the use of BMI to predict fat mass/height(2), and consequently percentage body fat, is superior to the use of BMI to predict percentage body fat based directly upon the R(2) values of the above analysis.

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HLA-B27 is a serologic specificity that encompasses 25 different alleles that encode 23 different products (proteins): HLA-B*2701 to B*2723. These alleles are also called subtypes of HLA-B27, and they may have evolved from the most widespread subtype, B*2705. These subtypes are distinguished from changes mostly in exons 2 and 3, which encode the alpha 1 and alpha 2 domains of the B27 molecule, respectively. Occurrence of ankylosing spondylitis (AS) or related spondyloarthropathy (SpA) has thus far been documented in subjects possessing any one of the first ten (B*2701 to B*2710) subtypes studied. However, B*2706 in Southeast Asian and B*2709 in the Italian island population of Sardinia seem not to be associated with AS. The 13 most recent subtypes have not yet been studied for disease association. It is important to investigate which of them are and are not associated with AS and related SpA, and whether certain subtypes show any preferential association with some of the clinical features or forms of these diseases among the various ethnic/racial populations and geographic regions of the world. This is expected to provide clues as to the mechanism of disease association, and one of the strongest reasons to study the B27 subtypes is to learn the effects of the sequence variations on the peptide-binding specificity of the molecule. Among these peptides may be the putative arthritogenic peptide(s).

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BACKGROUND: The accurate measurement of food intake in children is important for assessing nutritional status. OBJECTIVE: We sought to both compare measurements of energy intake (EI) from diet records and of total energy expenditure (TEE) by the doubly labeled water (DLW) method and to investigate misreporting of EI. DESIGN: Forty-seven children (22 boys and 25 girls) aged 7.4 +/- 0.8 y ( +/- SD) were recruited from 25 schools in western Sydney. TEE was measured by DLW over 10 d and EI by use of 3-d food records. Misreporting was defined as [(EI - TEE)/TEE] x 100%. RESULTS: Girls had a higher (P = 0.02) percentage of body fat (28.2 +/- 7.0%) than did boys (22.9 +/- 8.0%); otherwise there were no differences among sex. Although mean (+/-SD) values for EI (7514 +/- 1260 kJ/d) and TEE (7396 +/- 1281 kJ/d) were not significantly different, there was no significant correlation between EI and TEE. EI and TEE were 9% and 11% lower, respectively, than current World Health Organization recommendations for EI. The relative bias (mean difference, EI - TEE) was low at 118 kJ/d, but the limits of agreement (bias +/- 2 SD of the difference) were wide at 118 +/- 3345 kJ/d. Although the mean percentage of misreporting was low (4 +/- 23%), the high SD indicates large intraindividual differences between EI and TEE. The most significant predictor of misreporting was dietary fat intake (r(2) = 0.45, P < 0.0001). Misreporting was not associated with sex or body composition. CONCLUSIONS: In this age group, reported EI is not representative of TEE at the individual level. However, at the population level, 3-d food records may be used for surveys of EI by 6-9-y-old children.

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BACKGROUND: The recent worldwide increase in the prevalence of childhood obesity may be due in part to a decrease in children's physical activity levels. OBJECTIVE: The current study of children in the years just before puberty aimed to 1) measure total energy expenditure (TEE) by use of the doubly labeled water (DLW) method, 2) determine the proportion of TEE related to physical activity, 3) investigate the relations between measures of physical activity and body fatness, and 4) investigate possible sex differences in these relations. DESIGN: The DLW technique was used to measure TEE over 10 d in 106 healthy children (52 boys) aged 7.8 +/- 0.9 y (x +/- SD). Fat-free mass, and hence fat mass, was derived from the (18)O dilution space. Resting energy expenditure (REE) was calculated with use of the Schofield equations. Physical activity level was calculated as TEE/REE. RESULTS: Mean TEE in both boys (7871 +/- 1135 kJ/d) and girls (7512 +/- 1195 kJ/d) was significantly different (P < 0.0001) from FAO/WHO/UNU recommendations (13% and 9% lower, respectively). There was no significant difference in physical activity level between boys (1.69 +/- 0.22) and girls (1.71 +/- 0.23). In boys but not girls, physical activity level was inversely correlated with BMI (r = -0.37, P < 0.01), fat mass (r = -0.46, P < 0.005), and percentage of body fat (r = -0.50, P < 0.0001). CONCLUSIONS: In boys but not girls, percentage of body fat is inversely associated with physical activity level. Physical activity is one factor contributing to body fatness in boys, but additional factors may influence the size of the fat stores in girls.

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With the mapping of the human genome having been completed, our ability to investigate and ideally better understand the genetic basis of rheumatic diseases is advancing at a rapid pace. Substantial evidence strongly favors a direct role for HLA-B27 in genetic susceptibility to ankylosing spondylitis and related spondyloarthropathies, although the underlying molecular basis has yet to be identified. HLA-B27 contributes only 16 to 50% of the total genetic risk for the disease, clearly indicating that other genes must be involved. However, no other putative disease genes have yet been absolutely proven. Potential genes include MHC (HLA class II, low molecular weight proteasome [LMP], transporter associated with antigen processing (TAP), tumor necrosis factor [TNF]-alpha, and major histocompatibility complex class I chain-related gene A (MICA), as well as non-MHC genes (IL-1RA, IL-6, IL-10, and CYP2D6). Genome-wide screens have identified other chromosomal areas of interest: 1p, 2q, 6p, 9q, 10q, 16q, and 19q. However, different studies have given conflicting results. HLA-B27 itself is a serologic specificity, which encompasses 25 different alleles that encode 23 different products (proteins): HLA-B*2701 to B*2723. These alleles may have evolved from the most widespread subtype, B*2705, and two of them, B*2706 in Southeast Asia and B*2709 in Sardinia, seem not to be associated with ankylosing spondylitis. The distinction between the disease associated and nonassociated subtypes may provide clues to the actual role of B27 in disease pathogenesis.

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DNA-based typing of HLA alleles occasionally results in the inability to assign a specific allele because of ambiguity in associating two or more polymorphisms to the same or to alternate homologs (cis/trans ambiguity). Since most individuals are heterozygous at a given HLA locus, the highest level of confidence in definition is obtained when the alleles are tested in isolation. By using single-strand conformation polymorphisms (SSCP) to separate heterozygous HLA-DRB1 alleles, followed by sequencing of separated conformers, we have achieved resolution of previously ambiguous assignments without the need for additional probes or primers.

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Disorders of the CYP21 gene, which is located within the major histocompatibility complex on the short arm of chromosome 6, are the leading causes of congenital adrenal hyperplasia (CAH). The coding gene and a highly homologous pseudogene are tandemly arranged with the two genes for the fourth component of complement (C4A and C4B). To analyse the prevalence rates of mutations of the CYP21 genes and the segregation of the CYP21 genes with their corresponding human leucocyte antigen (HLA)-haplotypes, 21 families with one or two children with the severe form of 21-hydroxylase deficiency were studied. Mutations of the CYP21 gene on their corresponding HLA-haplotype were detected by hybridisation of polymerase chain reaction (PCR)-amplified genomic DNA with sequence-specific oligonucleotides and solid phase direct sequencing. Our study has shown the following. (1) A single basepair mutation (A-->G or C-->G) within the second intron is the most frequent mutation leading to impaired 21-hydroxylase activity. This mutation is only detected in HLA-haplotypes associated with the salt-wasting form of CAH. (2) A large deletion of part or all of the CYP21 gene is associated with the HLA-haplotype A3, BW47, C6, DR7, DR53, DQ2 but is also observed in other HLA-haplotypes and can be detected by a simple rapid PCR restriction fragment length polymorphism method. (3) Two alleles of the coding CYP21 gene differing in a leucine codon within the first exon, (formerly described as a mutation associated with 21-hydroxylase deficiency) have been found with an equal distribution in patients with 21-hydroxylase deficiency, non-disease HLA-haplotypes and the local healthy controls.

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Previous studies suggested that peripheral blood monocytes (Mo) from HIV-infected patients were poor accessory cells (AC), although most of these studies were limited by using autologous T cells as responders. Using allogeneic T cells from uninfected volunteers as responders, the current studies demonstrate that Mo from infected individuals were comparable to Mo from uninfected volunteers as AC in Con A and pokeweed mitogen-stimulated lymphocyte proliferation assays, but were inferior to normal Mo in stimulating a mixed leukocyte reaction. This deficiency was not explained by HIV Mo-induced suppression of lymphoproliferation or by death of responding CD4 lymphocytes induced by HIV transmission from infected Mo in 6-day MLR cultures. Mo from HIV-infected patients retained the ability to stimulate mumps-specific T cell lines in response to antigen, demonstrating that Mo from these individuals could process and display antigen on their cell surface in association with a functional DR molecule. Taken together these results suggest that Mo from HIV-infected patients (i) retain the ability to act as AC in T cell responses to mitogenic signals or to stimulate already activated antigen-specific T cells, but (ii) fail to stimulate resting and/or unprimed T cells in response to alloantigen and perhaps de novo antigen exposure. It is possible this Mo defect may have an adverse effect on the immune responsiveness of HIV-infected individuals.

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Staphylococcal enterotoxin A (SEA) induced the production of interferon-gamma (IFN-gamma) by spleen cells from ICR Swiss mice during the first 24 h of culture. Splenocytes from females produced higher levels of IFN-gamma than did those from males at 8, 12, and 16 h. By 20 h after SEA stimulation, IFN-gamma production by spleen cells from males was similar to that of females. The cell types involved in IFN-gamma production in this SEA/spleen cell system were analyzed by depletion studies. Removal of Thy-1+ cells by panning prevented production of IFN-gamma in the 24 h after SEA stimulation. In vivo depletion of asialo GM1+ (AGM1+) cells prevented production of IFN-gamma through 16 h of culture with SEA, but permitted a modest IFN-gamma response at 20 h that was similar in magnitude in both sexes. Following removal of L3T4+ and Lyt-2+ cells by panning, IFN-gamma production was detected at 12 h after SEA stimulation and maintained through 24 h of culture with cells from females producing higher levels of IFN-gamma. These data suggest that male ICR Swiss mice are deficient in the activity of Thy-1+, AGM1+, L3T4-, and Lyt-2- cells in the early (8-16 h) production of IFN-gamma following SEA stimulation of spleen cells.

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Human spleen cells were fractionated by percoll density gradient centrifugation and by sorting in the FACS with mixtures of fluorescent antibodies against T cells, B cells, monocytes, and Sig-bearing cells. Cells responsible for powerful MLR stimulation were class II HLA antigen-positive and were concentrated in preparations depleted of all the markers listed above. These cells represented 1-2% of the initial spleen cells. They were remarkably more active than other HLA class II antigen-positive cells. The procedure described allows rapid enrichment for the responsible cells. It should be useful for further characterization of these cells and for performing studies on their function.

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Human leukocyte antigen (HLA) typing was performed in 174 patients with rheumatoid arthritis and 222 white control subjects. Increases in HLA-DR4 and HLA-DR1 were observed as in previous studies. Each of these appeared to be inherited as dominant risk factors. Southern blotting with a DR-beta probe after digestion of genomic DNA with the restriction enzyme Bam HI showed seven bands. Three of them correlated with DR4 and were increased in rheumatoid arthritis patients. Subsets of DR4 were determined by polymerase chain reaction amplification followed by hybridization with oligonucleotide probes. Dw4 was increased in rheumatoid arthritis patients, and the frequency of the other subsets appeared to be similar in rheumatoid arthritis patients and control subjects. A polymorphism associated with the T cell receptor V-beta-8 gene family was significantly increased in rheumatoid arthritis patients.

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In a new series of RA patients the association with HLA-DR4 was again found to be highly significant. Also increased were the DR4 associated antigens DRw53 and DQw3. Hybridization of a DQ-beta probe to Bg1 II-digested DNA showed that a variant of DQw3, characterized by a 4.3 kb fragment and related to the serological specificity TA10, was markedly increased among DR4 positive RA patients. HLA-DR1 was also increased in RA and appeared to be independent of the increase of DR4. Although the DR1 association was weaker, it was observed in both patients with and without RF. In contrast, DR4 was found to be increased significantly only in the RF positive group of patients, as previously observed. Development of RA in multiple-case families was found to be linked to the inheritance of DR4 positive haplotypes. The possible role of risk factors associated with polymorphic markers of the T-cell receptor genes was investigated. An allelic marker associated with one of the variable gene subfamilies of the beta-chain was found to be associated with RA. The results suggest that susceptibility for development of RA is influenced by alleles of the T-cell receptor in conjunction with the class II HLA factors with which the T-cell receptor interacts in the course of the immune response.

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The HLA-D region antigens DR5 (w11,w12), DRw6 (w13,w14), DRw8, DRw52, and DQw1 have previously been shown to be increased in frequency in subsets of patients with juvenile arthritis. Since the HLA-D region is complex (composed of at least 3 subregions encoding multiple molecules, each in turn presenting multiple alloantigenic epitopes), we sought to clarify whether one strongly associated factor might explain the previous findings. To search for the pertinent HLA-D region stimulatory epitopes, alloreactive T cells were primed against DR5 and DRw6 haplotypes and cloned by limiting dilution. Three T cell clones and 1 alloantiserum met the criteria for significant association with juvenile arthritis on patient testing, including DR5, DRw6, and DRw8 haplotypes. Monoclonal antibody blocking revealed that all 4 recognized epitopes on DR subregion products. For 2 of the clones, the relative risks for JA (10.5 and 9.4) were higher than the risks with any other previously described typing reagents.

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The ability of cells with different amounts of HLA-DQ or -DR to support T-cell proliferation in response to foreign antigens was investigated. Adherent cells were stained with monoclonal antibodies and sorted in the fluorescence-activated cell sorter (FACS) into (a) DQ-positive and DQ-negative subsets, with monoclonal anti-DQ; or (b) subsets expressing different density of DR determinants. Expression of HLA-DQ correlated with increased density of DR. The subset of cells expressing detectable DQ and increased density of DR was found to be more efficient in presenting mumps or tetanus toxoid antigen to T cells than were the DQ-negative, low-DR density adherent cells. Similar results were obtained with primary cultures of T cells from blood and with cloned antigen-specific T-cell lines, restricted by a single DR-subregion specificity. Our results suggest that quantitative variation in DR/DQ molecules expressed on monocytes correlates with their ability to support T-cell responses to nominal antigens. It is not clear whether this is due to only class II antigen density on the surface of the accessory cells or whether other factors are involved.

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Previous studies have demonstrated restriction fragment length polymorphisms (RFLP) in the vicinity of the alpha and beta genes of the human T-cell receptor. In the course of experiments designed to discover additional polymorphic restriction sites, we found a new RFLP of the T-cell alpha gene recognized by the restriction enzyme Taq I. The site was localized to the interval between the most 3' joining (J) exon and the most 5' constant (C) region exon, about 7 kb distant from the previously described Bgl II polymorphic site which mapped to the vicinity of the 3' untranslated exon. With the use of these two polymorphic markers, four Ti-alpha alleles could be identified, allowing unambiguous assignment of all Ti-alpha genes in some families. These markers may be useful in identifying possible immune response genes or disease predisposition genes associated with the genes of the T-cell receptor for antigen.

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