The families of 29 patients with systemic lupus erythematosus and 42 normal subjects were studied to determine the inheritance of the HLA-A, B, C, and DR antigens and also the complement polymorphisms for C2, C4A, C4B, and Bf, which are encoded in the same region of the sixth chromosome. Null (silent) alleles for C4A, C4B, or C2 were found in 24 of the 29 (83%) patients compared with 18 of the 42 (43%) normal controls. HLA-DR3 was present in 20 (69%) of the patients and seven out of 39 (18%) of the normal controls. There was strong linkage disequilibrium between DR3 and the null alleles for C4A and C4B.The data did not permit the relative contributions of DR3 and null factors of C4A and C4B as genetic risk factors to be distinguished. The Al, B5, B7, B8, DR2, and DR3. The importance of these associations remains uncertain, but one possibility is that they reflect linkage disequilibrium with other loci that determine risk factors. In this context the HLA region encoded complement polymorphisms C2, C4A, C4B, and Bf may be relevant candidates. The polymorphism is particularly complex and extensive for the C4A and B loci.5 In addition to the expressed polymorphic variations null (silent) alleles for C2 and both C4 loci have been described," 8 and these are associated with no detectable product. Furthermore, variation in haemolytic activity between the C4 gene products has been observed, and one, C4A6, is nonhaemolytic when inherited in certain haplotypes. 9 Proved complement deficiency states account for only a small minority of cases of systemic lupus erythematosus, but no

Background-CD4ϩ CD28 null T cells are present in increased numbers in the peripheral blood of patients with acute coronary syndrome (ACS) compared with patients with chronic stable angina (CSA). The triggers of activation and expansion of these cells to date remain unclear. Methods and Results-Twenty-one patients with ACS and 12 CSA patients with angiographically confirmed coronary artery disease and 9 healthy volunteers were investigated. Peripheral blood leukocytes were stimulated with human cytomegalovirus (HCMV), Chlamydia pneumoniae, human heat-shock protein 60 (hHSP60), or oxidized LDL (ox-LDL). CD4 ϩ CD28 null cells were separated by flow cytometry and assessed for antigen recognition using upregulation of interferon-␥ and perforin mRNA transcription as criteria for activation. CD4ϩ CD28 null cells from 12 of 21 patients with ACS reacted with hHSP60. No response was detected to HCMV, C pneumoniae, or ox-LDL. Incubation of the cells with anti-MHC class II and anti-CD4 antibodies but not anti-class I antibodies blocked antigen presentation, confirming recognition of the hHSP60 to be via the MHC class II pathway. Patients with CSA had low numbers of CD4 ϩ CD28 null cells. These cells were nonreactive to any of the antigens used. Circulating CD4 ϩ CD28 null cells were present in 5 of the 9 healthy controls. None reacted with hHSP60. Conclusions-We have shown that hHSP60 is an antigen recognized by CD4ϩ CD28 null T cells of ACS patients. Endothelial cells express hHSP60 either constitutively or under stress conditions. Circulating hHSP60-specific CD4 ϩ CD28 null cells may, along other inflammatory mechanisms, contribute to vascular damage in these patients.

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The immune suppression inherent in allogeneic stem cell transplantation (SCT) offers a favorable environment for infection by opportunistic agents, such as human cytomegalovirus (CMV). Despite the application of potent antiviral prophylaxis, patients remain at risk for CMV infection until adequate immunity is restored. CMV-specific CD8(+) T cell counts were monitored, using HLA-A2 tetrameric complexes, to establish the level of immune response to the viral phosphoprotein UL83 in patients after allogeneic SCT. Correlating this with viral replication and clinical status shows that the level of tetramer-positive T cells provides an assessment of CMV immune reconstitution after stem cell transplantation. Most patients with seropositive donors did reconstitute long-term CMV immunity, unless prolonged immunosuppression to control graft-versus-host disease was induced. Together with polymerase chain reaction testing, this technique provides measurable parameters that can be a guide to therapeutic decision making and can form the basis of CMV immunotherapy.

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Although biuret based protein assays are theoretically applicable to peptide measurement, there is a high level of inter-peptide variation, determined largely by peptide hydrophobicity. This variation in peptide reactivity can be significantly reduced by heat-denaturation of peptides at 95 °C for 5 minutes in the presence of 0.1 M NaOH containing 1% (w/v) SDS, prior to incubation for 30 min at 37 °C in BCA standard working reagent. This modification to the standard bicinchoninic acid (BCA) assay protocol allows for an accurate, rapid and economical estimation of the peptide concentration within an unknown sample.Key words: Assay; BCA; bicinchoninic acid; estimation; measurement; peptide.There are a wide range of methods for estimating protein concentration, driven by the rise in proteomics/peptidomics over the last few years, but fewer approaches are available for quantifying the peptide content of a sample.Measurement of UV absorbance is often recommended however absorbance at 280 nm is influenced by frequency of tyrosine and tryptophan (as well as nucleotide and nucleic acid contamination), whilst absorbance at 210 nm is complicated by other organic compounds [1]. A number of fluorescent and luminescent approaches to peptide quantification are in use [2,3,4], however, the majority of these methods either react with the peptide amino-terminus or are biased towards certain amino acids within the sequence, and are frequently sensitive to photo-bleaching. Colorimetric protein assays, including the biuretbased Lowry [5] and bicinchoninic acid (BCA) [6] assays are occasionally utilised

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This study reports the frequencies of HLA antigens and the polymorphic variants of C4, C2, and Bf for 200 patients with multiple sclerosis (MS) living in the Grampian region of Scotland, an area of high disease prevalence. A group of 128 normal subjects from the same region were typed for comparison. Although the frequencies of HLA-B7 and DR2 in the patient group (43.3% and 49.4%, respectively) were found to be similar to those reported for other Northern European HLA studies on patients with MS, high frequencies of these antigens were also observed in the group of normal Grampian subjects (38.3% and 40.6%) the differences were not statistically significant. However, a significant association was found between the recently defined Class II HLA antigen, DQw1, and MS (P less than 0.006) when compared with controls. There were no significant differences in frequency of the polymorphisms of C4, C2, and Bf when the group of patients with MS was compared with the control group of normal subjects. The patients were subdivided according to disease severity, remittent versus progressive clinical course, age of onset of the disease and initial symptoms. The frequencies of the HLA and complement polymorphisms (C4, C2, and Bf) were analysed in these subdivisions. DQw1 was found with similar frequency in severe and benign disease (78% and 80%, respectively) but DR2 was most frequent in the group of patients with remittent disease (54%). There were no significant differences in frequency of the polymorphisms of C4, C2, and Bf between the above subgroups of patients and overall no significant HLA associations were found with age of onset of disease or initial symptoms. The findings suggest that in an area of high prevalence of MS, the disease is more closely associated with DQw1 than DR2. Furthermore, there was no evidence to support the hypothesis that the HLA region complement gene polymorphisms show significant association with a putative HLA-linked MS susceptibility gene.