Clinical laboratory verification of thyroglobulin concentrations in the presence of autoantibodies to thyroglobulin: comparison of EIA, radioimmunoassay and LC MS/MS measurements in an Urban Hospital.

OBJECTIVE: Thyroglobulin (Tg) measurements assess recurrence in post-thyroidectomy thyroid cancer patients. Tg measurements by enzyme immunoassays (EIA) can be falsely elevated by interference from Tg autoantibodies (TgAb). Radioimmunoassay (RIA) is less susceptible to TgAb interference and has been the standard-of-care test for TgAb positive patients. Recently developed liquid chromatography tandem mass spectrometry (LC-MS/MS) methods may eliminate TgAb interference. We assessed the performance of Tg measurements by EIA, RIA and LC-MS/MS to evaluate TgAb interference differences. RESULTS: We measured TgAb and Tg in 50 plasma samples from 40 patients in whom Tg measurement was part of their routine follow-up and 10 healthy volunteers. Discrepancy between EIA and both LC-MS/MS and RIA was observed at low Tg concentrations (≤ 7.55 ng/mL) in TgAb positive specimens (LC-MS/MS = 1.9 * EIA - 0.03, r = 0.68). RIA and LC-MS/MS Tg measurements in TgAb positive specimens with low Tg concentrations had improved correlation but demonstrated bias (LC MS/MS = 0.6 * RIA - 1.4, r = 0.90). Disagreement between methods may be attributed to LC-MS/MS reported Tg concentrations as undetectable compared to RIA. It seems likely that most discrepant cases are falsely elevated in RIA due to TgAb interference, however, some cases appear below the detection limit of LC-MS/MS; implementation of LC-MS/MS by clinicians will require lower detection limits.

Focused evolution of HIV-1 neutralizing antibodies revealed by structures and deep sequencing.

Antibody VRC01 is a human immunoglobulin that neutralizes about 90% of HIV-1 isolates. To understand how such broadly neutralizing antibodies develop, we used x-ray crystallography and 454 pyrosequencing to characterize additional VRC01-like antibodies from HIV-1-infected individuals. Crystal structures revealed a convergent mode of binding for diverse antibodies to the same CD4-binding-site epitope. A functional genomics analysis of expressed heavy and light chains revealed common pathways of antibody-heavy chain maturation, confined to the IGHV1-2*02 lineage, involving dozens of somatic changes, and capable of pairing with different light chains. Broadly neutralizing HIV-1 immunity associated with VRC01-like antibodies thus involves the evolution of antibodies to a highly affinity-matured state required to recognize an invariant viral structure, with lineages defined from thousands of sequences providing a genetic roadmap of their development.

Analysis of somatic hypermutation in X-linked hyper-IgM syndrome shows specific deficiencies in mutational targeting.

Subjects with X-linked hyper-IgM syndrome (X-HIgM) have a markedly reduced frequency of CD27(+) memory B cells, and their Ig genes have a low level of somatic hypermutation (SHM). To analyze the nature of SHM in X-HIgM, we sequenced 209 nonproductive and 926 productive Ig heavy chain genes. In nonproductive rearrangements that were not subjected to selection, as well as productive rearrangements, most of the mutations were within targeted RGYW, WRCY, WA, or TW motifs (R = purine, Y = pyrimidine, and W = A or T). However, there was significantly decreased targeting of the hypermutable G in RGYW motifs. Moreover, the ratio of transitions to transversions was markedly increased compared with normal. Microarray analysis documented that specific genes involved in SHM, including activation-induced cytidine deaminase (AICDA) and uracil-DNA glycosylase (UNG2), were up-regulated in normal germinal center (GC) B cells, but not induced by CD40 ligation. Similar results were obtained from light chain rearrangements. These results indicate that in the absence of CD40-CD154 interactions, there is a marked reduction in SHM and, specifically, mutations of AICDA-targeted G residues in RGYW motifs along with a decrease in transversions normally related to UNG2 activity.

Effective therapy for nephritis in (NZB x NZW)F1 mice with triptolide and tripdiolide, the principal active components of the Chinese herbal remedy Tripterygium wilfordii Hook F.

OBJECTIVE: Triptolide and tripdiolide are thought to be active components of the Chinese antirheumatic herbal remedy Tripterygium wilfordii Hook F, which has been shown to be effective in treating murine lupus nephritis. This study was undertaken to examine the therapeutic effect of triptolide and tripdiolide on established lupus nephritis in (NZB x NZW)F1 mice. METHODS: (NZB x NZW)F1 mice were treated with vehicle, triptolide, or tripdiolide for 15 weeks beginning at the age of 29 weeks (after the development of lupus nephritis). Body weight, proteinuria, and anti-double-stranded DNA (anti-dsDNA) antibodies were monitored, and the kidney and spleen were assessed histologically. Culture supernatants of spleen mononuclear cells were assayed for cytokines. RESULTS: By 28 weeks, most (NZB x NZW)F1 mice had developed lupus nephritis. Vehicle-treated mice exhibited progressive proteinuria, hypoalbuminemia, elevated blood urea nitrogen (BUN) levels, and evidence of severe nephritis. In contrast, proteinuria and BUN levels were significantly reduced in mice treated with either triptolide or tripdiolide as compared with those treated with vehicle. There was no hypoalbuminemia or apparent evidence of lupus nephritis in mice treated with either of the 2 diterpenoids. At 44 weeks of age, the survival rate in mice treated with vehicle (35.7%) was markedly lower than that in mice treated with either triptolide (87.5%) or tripdiolide (88.2%). The mean level of anti-dsDNA antibody in mice treated with tripdiolide was lower than that in the vehicle-treated mice upon completion of the treatment course. Production of tumor necrosis factor, interleukin-6, and monocyte chemoattractant protein 1 by spleen cells was also decreased after diterpenoid therapy. CONCLUSION: Therapy with triptolide or tripdiolide significantly ameliorated lupus nephritis in (NZB x NZW)F1 mice, reduced cytokine and chemokine production, and prolonged survival.

The NF-kappaB canonical pathway is involved in the control of the exonucleolytic processing of coding ends during V(D)J recombination.

V(D)J recombination is essential to produce an Ig repertoire with a large range of Ag specificities. Although NF-kappaB-binding sites are present in the human and mouse IgH, Igkappa, and Iglambda enhancer modules and RAG expression is controlled by NF-kappaB, it is not known whether NF-kappaB regulates V(D)J recombination mechanisms after RAG-mediated dsDNA breaks. To clarify the involvement of NF-kappaB in human V(D)J recombination, we amplified Ig gene rearrangements from individual peripheral B cells of patients with X-linked anhidrotic ectodermal dysplasia with hyper-IgM syndrome (HED-ID) who have deficient expression of the NF-kappaB essential modulator (NEMO/Ikkgamma). The amplification of nonproductive Ig gene rearrangements from HED-ID B cells reflects the influence of the Ikkgamma-mediated canonical NF-kappaB pathway on specific molecular mechanisms involved in V(D)J recombination. We found that the CDR3(H) from HED-ID B cells were abnormally long, as a result of a marked reduction in the exonuclease activity on the V, D, and J germline coding ends, whereas random N-nucleotide addition and palindromic overhangs (P nucleotides) were comparable to controls. This suggests that an intact canonical NF-kappaB pathway is essential for normal exonucleolytic activity during human V(D)J recombination, whereas terminal deoxynucleotide transferase, Artemis, and DNA-dependent protein kinase catalytic subunit activity are not affected. The generation of memory B cells and somatic hypermutation were markedly deficient confirming a role for NF-kappaB in these events of B cell maturation. However, selection of the primary B cell repertoire appeared to be intact and was partially able to correct the defects generated by abnormal V(D)J recombination.

B-cell repertoire and clonal analysis in unaffected first degree relatives in familial chronic lymphocytic leukaemia kindred.

Monoclonal B cell lymphocytosis (MBL) was detected in four unaffected first-degree relatives (FDR) in a familial chronic lymphocytic leukaemia (CLL) kindred. The proband remains untreated and two male siblings have died. The four unaffected siblings have been followed for a five-year period. All four FDR developed a kappa(+)CD5(+) MBL detected by flow cytometry. Poymerase chain reaction (PCR) for IGHV rearrangement showed evidence of oligoclonality in three of these individuals. Single cell PCR of flow cytometric sorted kappa(+) cells combined with Ig kappa light chain gene sequencing revealed further evidence of monoclonality in two of these individuals. Three of these individuals all showed evidence of hyper-somatic mutations. The B-cell repertoire in unaffected FDR in familial CLL offers a new area to investigate the interface between the immune system and lymphoid neoplasm.

Staphylococcal protein a deletes B-1a and marginal zone B lymphocytes expressing human immunoglobulins: an immune evasion mechanism.

Protein A (SpA) of Staphylococcus aureus is endowed with the capacity to interact with the H chain variable region (V(H)) of human Abs and to target >40% of B lymphocytes. To investigate whether this property represents a virulence factor and to determine the in vivo consequences of the confrontation of SpA with B lymphocytes, we used transgenic mice expressing fully human Abs. We found that administration of soluble SpA reduces B-1a lymphocytes of the peritoneal cavity and marginal zone B lymphocytes of the spleen, resulting in a markedly deficient type 2 humoral response. Single-cell PCR analysis and sequencing of the Ab V(H) gene repertoire revealed a significant reduction of V(H)3+ marginal zone B cells. Since the two B lymphocyte subsets targeted are involved in innate immune functions, our data suggest that crippling of humoral immunity by S. aureus represents an immune evasion mechanism that may aggravate recurrent infections.

Specific in vivo deletion of B-cell subpopulations expressing human immunoglobulins by the B-cell superantigen protein L.

Some pathogens have evolved to produce proteins, called B-cell superantigens, that can interact with human immunoglobulin variable regions, independently of the combining site, and activate B lymphocytes that express the target immunoglobulins. However, the in vivo consequences of these interactions on human B-cell numbers and function are largely unknown. Using transgenic mice expressing fully human immunoglobulins, we studied the consequences of in vivo exposure of protein L of Peptostreptococcus magnus with human immunoglobulins. In the mature pool of B cells, protein L exposure resulted in a specific reduction of splenic marginal-zone B cells and peritoneal B-1 cells. Splenic B cells exhibited a skewed light-chain repertoire consistent with the capacity of protein L to bind specific kappa gene products. Remarkably, these two B-cell subsets are implicated in innate B-cell immunity, allowing rapid clearance of pathogens. Thus, the present study reveals a novel mechanism that may be used by some infectious agents to subvert a first line of the host's immune defense.

Different patterns of bcl-6 and p53 gene mutations in tonsillar B cells indicate separate mutational mechanisms.

Mutations within the 5'-non-coding region of the bcl-6 gene can occur in lymphomas that originate from germinal centers (GCs), as well as in normal memory and GC B cells. Mutations in the p53 gene occur in 50% of human cancers. Since both bcl-6 and p53 can be mutated in certain circumstances, we investigated the accumulation of mutations in these genes in individual tonsillar B and T cells to determine whether the mutations exhibited a pattern anticipated from the B-cell hypermutation machinery. In tonsillar GC B cells, the overall mutational frequencies in the 5'-non-coding region of the bcl-6 gene was 0.85 x 10(-3)/bp. In contrast, there were no mutations in a region 2.8 kb downstream of the promoter. RGYW (purine, guanine, pyrimidine, A/T) targeting and a significantly lower mutational frequency in nai;ve B and GC founder B cells compared with GC B cells suggested that a similar mutator mechanism was active on Ig genes and this non-Ig gene. The mutational frequency in the exon-7-region of p53 was similar in the GC, memory and nai;ve B-cell subsets (1.02 x 10(-3) to 1.25 x 10(-3)/bp). RGYW/WRCY motifs were not targeted preferentially in the p53 gene. Moreover, a comparable mutational frequency of p53 was noted in tonsillar B and T cells. Hence, mutations in p53 do not appear to be the result of the B-cell hypermutational mechanism.