Structure of an autoimmune T cell receptor complexed with class II peptide-MHC: insights into MHC bias and antigen specificity

T cell receptor crossreactivity with different peptide ligands and biased recognition of MHC are coupled features of antigen recognition that are necessary for the T cell's diverse functional repertoire. In the crystal structure between an autoreactive, EAE T cell clone 172.10 and myelin basic protein (1-11) presented by class II MHC I-Au, recognition of the MHC is dominated by the Vbeta domain of the TCR, which interacts with the MHC alpha chain in a manner suggestive of a germline-encoded TCR/MHC "anchor point." Strikingly, there are few specific contacts between the TCR CDR3 loops and the MBP peptide. We also find that over 1,000,000 different peptides derived from combinatorial libraries can activate 172.10, yet the TCR strongly prefers the native MBP contact residues. We suggest that while TCR scanning of pMHC may be degenerate due to the TCR germline bias for MHC, recognition of structurally distinct agonist peptides is not indicative of TCR promiscuity, but rather highly specific alternative solutions to TCR engagement.     

Relevance of HIV-1-specific CD4+ helper T-cell responses during structured treatment interruptions in patients with CD4+ T-cell nadir above 400/mm3

OBJECTIVES: To analyze the dynamics of both HIV-1-specific CD4 and CD8 T-cell responses during structured treatment interruptions (STIs) in chronically HIV-1-infected (CHI) patients and to correlate them with the viral set point achieved. METHODS: Forty-five early-stage CHI patients who were on highly active antiretroviral therapy (HAART) for at least 1 year and underwent STI were included. Plasma viral load (VL), peripheral blood mononuclear cell (PBMC) lymphoproliferative (LPR) response to HIV p24 protein, and HIV-1 epitope-specific interferon-gammarelease from CD8 T cells were measured over a minimum study period of 2 years. RESULTS: VL set point during final STI was both significantly lower than, and positively correlated to, baseline VL (P < 0.0001: mean VL reduction 0.77 log10, and r = 0.42, P = 0.004, respectively). CD4 LPRs to p24 increased significantly (P = 0.001) between day 0 of the first STI cycle and 4th STI but decreased thereafter. VL set point during final STI was significantly and negatively correlated with LPRs to p24 at both 2nd STI and 4th STI. Nevertheless, at week 52, 12 weeks after the end of the last STI, LPRs were weak and transient in all patients and were not correlated with VL set point. Moreover, the magnitude and breadth of HIV-1-specific CD8 T-cell responses increased significantly (P < 0.0001) between day 0 and week 52. The largest increases occurred during the final STI. Even though VL reached set point by week 12 of the final STI, HIV-1-specific CD8 T-cell responses did not stabilize but rather increased until the end of the follow-up and did not correlate with plasma VL (r = 0.01, P = 0.88). CONCLUSIONS: STIs do not lead to control of viral replication in CHI patients, probably due to the fact that boosted CTL responses lack strong and durable helper T-cell responses. To reset the VL set point, new approaches that effectively augment and preserve helper T-cell responses should be investigated.     

Clinical and molecular study in congenital muscular dystrophy with partial laminin alpha 2 (LAMA2) deficiency

Complete laminin alpha2 (LAMA2) deficiency causes approximately half of congenital muscular dystrophy (CMD) cases. Many loss-of-function mutations have been reported in these severe, neonatal-onset patients, but only single missense mutations have been found in milder CMD with partial laminin alpha2 deficiency. Here, we studied nine patients diagnosed with CMD who showed abnormal white-matter signal at brain MRI and partial deficiency of laminin alpha2 on immunofluorescence of muscle biopsy. We screened the entire 9.5 kb laminin alpha2 mRNA from patient muscle biopsy by direct capillary automated sequencing, single strand conformational polymorphism (SSCP), or denaturing high performance liquid chromatography (DHPLC) of overlapping RT-PCR products followed by direct sequencing of heteroduplexes. We identified laminin alpha2 sequence changes in six of nine CMD patients. Each of the gene changes identified, except one, was novel, including three missense changes and two splice-site mutations. The finding of partial laminin alpha2 deficiency by immunostaining is not specific for laminin alpha2 gene mutation carriers, with only two patients (22%) showing clear causative mutations, and an additional three patients (33%) showing possible mutations. The clinical presentation and disease progression was homogeneous in the laminin alpha2-mutation positive and negative CMD patients.     

Comparison of serum hepatitis C virus RNA and core antigen concentrations and determination of whether levels are associated with liver histology or affected by specimen storage time

An enzyme immunoassay has recently been developed for the hepatitis C virus (HCV) core antigen. To evaluate the possible association between core antigen and HCV RNA levels with regards to the change in liver histology over time as well as study the effect of duration of storage on viral load results, sequential sera were analyzed from 45 patients with chronic HCV infection who had undergone two or more liver biopsies. A relatively strong association was found between the core antigen and HCV RNA concentrations (r(s) = 0.8), with a core antigen level of 1 pg/ml corresponding to approximately 1,000 IU/ml. All 42 sera with detectable HCV RNA at the time of the second biopsy had core antigen concentrations above 1 pg/ml, and the three sera without detectable HCV RNA had concentrations below 1 pg/ml. No association was found between HCV RNA or core antigen levels and the stage of fibrosis in biopsy samples, progression of fibrosis, necro-inflammatory grade, steatosis, genotype, alanine aminotransferase level, or alcohol consumption. A significant association was demonstrated between the storage time of the samples and both the HCV RNA and core antigen concentrations. The median log HCV RNA concentrations (international units/milliliter) were 3.92 for the sera obtained at the time of the first biopsy (median storage time, 13.0 years) and 4.41 for the sera obtained at the time of the second biopsy (median storage time, 6.6 years) compared to 5.96, the median for 102 different routine clinical patient samples.     

Amyloidosis and the serum amyloid A protein response to muramyl dipeptide analogs and different mycobacterial species.

Serum amyloid A protein (SAA) elevation accompanies induction of secondary amyloidosis in mice given Mycobacterium butyricum in Freund adjuvant. The synthesis of SAA by cultured hepatocytes is induced by a macrophage-derived mediator, which has been identified as interleukin 1. In these studies, SAA synthesis has been used as an index of macrophage activation to examine the in vivo response of mice to challenge with seven different mycobacteria and with synthetic analogs of the immunoadjuvant N-acetylmuramyl-L-alanyl-D-isoglutamine [MDP(L-D)]. SAA synthesis was stimulated by administration (by the intraperitoneal route) of the mycobacteria dissolved in saline, with Mycobacterium vaccae being the most active and Mycobacterium leprae being the least stimulatory. MDP(L-D), which is the minimal structure (molecular weight, 492) able to substitute for mycobacteria in Freund adjuvant, stimulated SAA synthesis, whereas the MDP(D-D) isomer was inactive. The butyl ester of MDP, which induces no detectable pyrogenicity but retains adjuvanticity, required a 100-fold greater dosage than MDP(L-D) in stimulating SAA synthesis. Amyloidosis was detected histologically only when active SAA inducers MDP(L-D), M. vaccae, and M. butyricum, were administered in incomplete Freund adjuvant, with amyloid-enhancing factor. These studies demonstrated that SAA elevation was a sensitive in vivo marker of the capacity of antigens to stimulate macrophages to produce interleukin 1. A point of considerable relevance to the human use of MDP was the observation that repeated injections of the adjuvant MDP in saline did not induce secondary amyloidosis.     

Short-circuiting autoimmune disease by target-tissue-derived nitric oxide

A previous report from this laboratory suggested that expression of skeletal-muscle-derived, inducible nitric oxide synthase (iNOS), is associated with resistance to the autoimmune model of myasthenia gravis (MG) demonstrated by Wistar Furth rats following the passive transfer of antibody reactive with the nicotinic acetylcholine receptor (AChR). The study reported below demonstrates an association between increased expression of iNOS/NO in Wistar Furth rats and the induction of programmed cell death (apoptosis) in both macrophages and CD4+ T cells that attempt to traffic through targeted muscles. It is concluded that production of muscle-derived NO is protective in experimental MG, and in part, dictates the severity of eventual immunopathology.     

Characterization of plasmid-borne afa-3 gene clusters encoding afimbrial adhesins expressed by Escherichia coli strains associated with intestinal or urinary tract infections.

The afa gene clusters encode afimbrial adhesins (AFA) that are expressed by uropathogenic and diarrhea-associated Escherichia coli strains and belong to a family of hemagglutinins recognizing the Dr blood group antigen as a receptor. This family so far includes AFA-I and AFA-III as well as the Dr and F1845 adhesins (B. Nowicki, A. Labigne, S. Moseley, R. Hull, S. Hull, and J. Moulds, Infect. Immun. 58:279-281, 1990). Reported in this work is the genetic organization of the afa-3 gene cluster cloned from a uropathogenic E. coli strain (A30) which expressed a subtype of AFA designated AFA-III. The amino acid sequence of AFA-III was deduced from the nucleotide sequence of the afaE3 gene and was found to be highly homologous to that of the Dr adhesin (98.1% identity). A polymerase chain reaction assay was developed to detect the presence of afa-3 gene clusters in E. coli strains. Study of the genetic support of the afa-3 gene clusters in the strains which showed positive amplification revealed that they were always located on large, 100-kb plasmids whether the strains originated from patients with cystitis or with diarrhea. Moreover, the cloned afa-3 gene clusters from A30 and from the diarrhea-associated strain AL845 appeared to be carried by 9-kb plasmid regions which displayed a similar genetic organization. Chloramphenicol was reported to be a potent inhibitor of receptor binding by the Dr adhesin (Nowicki et al., Infect. Immun. 58:279-281, 1990). AFA-III expressed by strains AL845 and AL847 appeared to mediate, like the Dr adhesin, chloramphenicol-sensitive hemagglutination, whereas AFA-III produced by A30 conferred chloramphenicol-resistant adherence. A comparison of the sequences of these four proteins indicated that the amino acid at position 52 of the processed AFA could be part of the receptor-binding domain.     

Transplant aspirative cytology: Analysis of morphological and immunocytochemical parameters in renal allograft dysfunction

We have analyzed 245 transplant aspirative cytologies (TACs) from 96 renal allograft patients. TACs were divided in two chronological groups: Early (TACs performed during the first 3-mo post-transplantation) and late (TACs performed after the third month post-transplantation), in order to assess the effect of allograft tolerance on TAC features. Both morphological and immunocytochemical aspects were evaluated, including CD4, CD8, IL2-R, and HLA-DR immunolabeling. A final diagnosis for each case of allograft dysfunction was achieved by other independent diagnostic means. Four diagnostic groups were considered in the present study: acute rejection (AR), chronic rejection (CR), acute tubular necrosis (ATN), and Cyclosporin A toxicity (CsA-T). In addition, a control group (C) was established from patients with stable allograft function. We found that immunocytochemical analysis of TACs is particularly helpful in the diagnosis of late allograft dysfunction, a time period when the simple cytological study of renal infiltrate is not informative enough to help take therapeutic decisions.     

Effects of enzyme replacement therapy on pain and health related quality of life in patients with Fabry disease: data from FOS (Fabry Outcome Survey)

Background: Fabry disease is an X linked lysosomal storage disease caused by deficiency of the lysosomal enzyme α-galactosidase A. This leads to accumulation of globotriaosylceramide in nearly all tissues, including the blood vessels, kidney, myocardium, and nervous system. Symptoms often begin in childhood and include acroparaesthesia, with burning or tingling pain that spreads from the extremities to more proximal sites.

Aims: This study set out to evaluate pain and its influence on quality of life in patients with Fabry disease receiving enzyme replacement therapy (ERT) with agalsidase alfa.

Methods: Data were obtained from the Fabry Outcome Survey. Pain was measured using the Brief Pain Inventory (BPI), and health-related quality of life (HRQoL) was documented with the European Quality of Life Questionnaire (EQ-5D).

Results: The mean (SD) score for "pain at its worst" on the BPI prior to ERT was 5.1 (2.7). One year after commencement of ERT, this had improved by 0.5, and improved by a further 0.6 after 2 years (p<0.05). Similar statistically significant improvements were seen for "pain on average" and "pain now" after 2 years of ERT. The mean HRQoL utility score prior to ERT was 0.66 (0.32). After 12 months of treatment with agalsidase alfa, this had improved to 0.74 (0.26; p<0.05); this improvement was maintained after 2 years.

Conclusions: ERT with agalsidase alfa significantly reduces pain and improves quality of life in patients with Fabry disease.

     

Vitamin C-induced loss of redox-dependent viability in lung microvascular endothelial cells

Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.