RNA as a target for host defense and anti-HIV drugs.

Viruses have many strategies for negotiating entry into cells and harnessing the cellular machinery for their propagation. The diversity of strategies is however bound by the central process of translating protein from RNA. The co-evolution of cellular responses to signature viral RNA intermediates and the counter measures employed by viruses, highlight the vulnerability of this aspect of viral replication and the potential of viral RNA as a drug target. In this review we will discuss novel efforts to target the RNA intermediates of the HIV life cycle.

Gene-expressed RNA as a therapeutic: issues to consider, using ribozymes and small hairpin RNA as specific examples.

In recent years there has been a greater appreciation of both the role of RNA in intracellular gene regulation and the potential to use RNA in therapeutic modalities. In the latter case, RNA can be used as a therapeutic target or a drug. The chapters in this volume cover the varied and potent actions of RNA as antisense, ribozymes, aptamers, microRNA and small hairpin RNA in gene regulation, as well as their use as potential therapeutics for metabolic and infectious diseases. Our group has been involved in the development of anti-HIV gene expression constructs to treat HIV. In this chapter, we address the relevant scientific and some of the commercial issues in the use of RNA as a therapeutic. Specifically, the chapter discusses delivery, expression, potency, toxicity and commercial development using, as examples, hammerhead ribozymes and small hairpin RNA.

Analysis of tumor necrosis factor-alpha, lymphotoxin-alpha, tumor necrosis factor receptor II, and interleukin-6 polymorphisms in patients with idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterized by chronic inflammation that is associated with structural damage of the lung and fibrosis. Although the etiology of IPF is unknown, it is likely to involve an interaction between environmental and multiple genetic components. Animal models of pulmonary fibrosis have shown that proinflammatory mediators are critical at both the inflammatory and fibrotic stages of the disease. Genetic variants exist in genes encoding proinflammatory mediators, as well as in genes encoding their receptors, which makes these genes candidates for the pathogenesis of IPF. In the present study, we examined 12 biallelic polymorphisms in the genes for tumor necrosis factor (TNF)-alpha (+488[G/A], -238[G/A], -308[G/A]), lymphotoxin (LT)-alpha (+720[C/A], +365[C/G], and +249[A/G], determining haplotypes LT-alpha1 to LT-alpha4), tumor necrosis factor-receptor 2 (TNF-RII) (gb:M32315: 676[T/G], 1663[A/G], 1668[T/G], 1690[C/T]), and interleukin- (IL)-6 (promoter -174[G/C], intron 4[A/G]). We also examined the haplotypes determined by the three biallelic polymorphisms in each of the TNF-alpha and LT-alpha genes. As compared with a normal control population, the IPF group showed no significant deviations in genotype, allele, or haplotype frequencies. Surprisingly, in the IPF population, but not in the control population, an increased frequency of cocarriage of the IL-6 intron 4G and the TNF-RII 1690C alleles was observed, despite the location of the two genes on different chromosomes. Moreover, using impairment of carbon monoxide transfer (DL(CO)) adjusted for duration of dyspnea as a marker of rapidity of disease progression, we found that the IL-6 intron 4GG genotype was the only genotype independently associated with lower DL(CO) levels. These findings, if independently confirmed, will be the first to suggest that disease progression in IPF may be linked to a particular genetic marker or to functional polymorphisms in other genes near that marker.

Interleukin-10 gene promoter polymorphism in English and Polish healthy controls. Polymerase chain reaction haplotyping using 3' mismatches in forward and reverse primers.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system using primers with mismatches at the 3' ends was developed to determine polymorphisms in IL-10 promoter region. Three previously described biallelic polymorphisms in IL-10 were linked in a 12 reaction PCR-SSP system and the method used to provide genotype data on 233 UK and 166 Polish controls. There are eight possible polymorphic combinations in IL-10 promoter gene but only three were observed in both control groups. Population frequencies of IL-10 genotypes show, in contrast to HLA, that UK and Polish frequencies are remarkably similar.

Cytokine (TNF alpha, LT alpha and IL-10) polymorphisms in inflammatory bowel diseases and normal controls: differential effects on production and allele frequencies.

The influence of biallelic polymorphisms in the tumour necrosis factor-alpha (TNF alpha), lymphotoxin-alpha (LT alpha) and interleukin-10 (IL-10) genes on stimulated TNF alpha and IL-10 production was studied in ulcerative colitis (UC) patients, Crohn's disease (CD) patients and in healthy controls. A polymerase chain reaction sequence-specific primer (PCR-SSP) system was developed to type nine biallelic polymorphisms, three in each of the TNF alpha, LT alpha and IL-10 genes. Production of the TNF alpha and IL-10 was measured by ELISA in lipopolysaccharide (LPS) stimulated whole blood. Four haplotypes of the TNF alpha gene, three haplotypes of LT alpha and three haplotypes of IL-10 were identified. No significant differences in haplotype frequencies were found between patients and controls overall. On subgroup analysis however, haplotype TNF-2 was more frequent in women with extensive colitis compared to distal colitis (31% vs 12%; P = 0.028). This difference was even greater for the combined TNF-2-LT alpha-2 haplotype (56% vs 21%; P = 0.0007). The TNF-2 and LT alpha-2 haplotypes were associated with higher TNF alpha production in CD patients, and the TNF-4 haplotype was associated with lower TNF alpha production in UC patients. The A allele in the IL-10 promoter region at position -1082 was associated with decreased IL-10 production in CD patients and controls (P = 0.005, P = 0.015 respectively). These data provide evidence that the effect of TNF alpha, LT alpha and IL-10 gene polymorphisms on cytokine production differ in CD, UC patients and controls.

Polymorphic analysis of the high-affinity tumor necrosis factor receptor 2.

The tumor necrosis factor receptor 2 (TNF-RII, CD120b, TNF-R p75/80) gene has recently been characterised. It is located on chromosome 1p362 and consists of 10 exons and 9 introns A number of biallelic polymorphisms have been found in exons 4, 6, 9 and 10 based on differences between published sequences. In this study we have used polymerase chain reaction methodology in association with sequence-specific primers (PCR-SSP) incorporating mismatches at the 3' end to identify these polymorphisms. We were able to confirm the presence of a single biallelic polymorphism in exon 6 corresponding to a (T/G) at nucleotide 676 of TNF-RII mRNA (gb:M32315) which results in an amino acid change and three biallelic polymorphisms in exon 10 (in the3'UTR) corresponding to (A/G) at nucleotide 1663, (T/G) at nucleotide 1668 and a (C/T) at nucleotide 1690 of gb:M32315, whereas no polymorphisms were observed in exons 4 and 9. Here we report that in 192 unrelated UK Caucasian individuals the allele frequencies determined by direct counting were: 676-T (0.77), 1663-G (0.51), 1668-T (0.95), and 1690-T (0.64) and the calculated gene frequencies were; 676-T (0.52), 676-G (0.12); 1663-G (0.30), 1663-A (0.28); 1668-T (0.77), 1668-G (0.025); and 1690-T (0.40), 1690-C (0.20). Furthermore, the presence of an A allele at nucleotide position 1663 was found to be strongly associated with the presence of a C allele at nucleotide position 1690 and a G allele at nucleotide position 1668 whereas the presence of a G allele at position 1663 was associated with the absence of a C allele at nucleotide position 1690.

A rapid method of haplotyping HFE mutations and linkage disequilibrium in a Caucasoid population.

BACKGROUND: HFE mutations are associated with hereditary haemochromatosis. However, a simple method capable of demonstrating the cis/trans arrangement of alleles is lacking, and linkage disequilibrium between HFE alleles and classic HLA loci is unknown. These are important issues as the pathogenic role of the mutations is not known. AIMS: To develop a simple method of genotyping HFE mutations suitable for clinical use in addition to large disease studies. PATIENTS: A total of 330 Caucasoid cadaveric organ donor controls were examined. Ten individuals previously HLA-H genotyped by polymerase chain reaction using restriction fragment length polymorphism (PCR-RFLP) were also examined to validate the method. METHODS: A simple polymerase chain reaction using sequence specific primers (PCR-SSP) capable of haplotyping the mutations was developed. HFE allele and haplotype frequencies and linkage disequilibrium with eight HLA class I and II loci were examined in the control population. RESULTS: 27% and 19.7% of patients were positive for the 63D and 282Y alleles, respectively. No chromosome carried both 63D and 282Y. Linkage disequilibrium between 282Y and HLA-A*03 was confirmed, but was not straightforward: some A*03-associated alleles (DRB1*15, DQB1*06), but not all (B*07, Cw*0702), were associated with 282Y. CONCLUSIONS: Linkage disequilibrium data suggest that an HLA-B*07 containing haplotype contains an element affording protection from haemochromatosis and may suggest the timing of the founder 282Y mutation.

HLA associations in three mutually exclusive autoantibody subgroups in UK systemic sclerosis patients.

Systemic sclerosis (SSc) is characterized by the presence of autoantibodies, mostly IgG, which target a limited set of nuclear proteins. These antinuclear antibodies (ANA) associate with disease subgroups and specific organ involvement. Here we show that there is mutual exclusivity of individual ANA in 130 UK SSc patients, confirm clinical associations with antibody profile and extend the analysis to include genetic data. The ANA mutual exclusivity observed leads to the possibility that SSc, in these patients, is in fact three separate diseases. An alternative explanation for exclusivity relates to the fact that optimal production of IgG antibody requires T-cell help, a process restricted by the HLA class II presentation of antigen peptide. If each autoantibody has a different and tight MHC restriction, then there is a possibility that these groups arose from a common pathway and were modified by genetics into the mutually exclusive groups observed, making the separate disease theory less tenable. In order to answer this question, we have determined MHC class II restriction precisely using high-resolution HLA genotyping (SSP) coupled with an amino acid analysis program in our 130 UK SSc patients. DRB1*11 was associated with anti-topoisomerase-I antibody (ATA)-positive patients (P = 0.007) and when combined with ATA (RR = 15.82), dcSSc (RR = 11.45), or both (RR = 21.9), represented the strongest risk factor for pulmonary fibrosis. Patients with antibodies to RNA polymerases I, II and III were associated with DQB1*0201. At the amino acid level, 20 positions in DRB1 and 20 positions in DQB1 showed some significant correlation with an ANA group. Clearly, however, the linkages to MHC class II alleles are not nearly strong enough to explain the mutually exclusive nature of the autoantibody groups and our results support, but do not prove, the separate disease theory.

Rapid haplotyping of mutations in the Duffy gene using the polymerase chain reaction and sequence-specific primers.

The molecular basis for the antigenic variation and red cell expression of the Duffy antigen system has recently been elucidated. We have developed a simple one-step method for genotyping the single nucleotide polymorphisms in the promoter and exon of the Duffy gene using the polymerase chain reaction and sequence-specific primers (PCR-SSP). This method is also capable of haplotyping alleles at the two polymorphisms as being in cis or trans orientation. Twenty-four serologically typed Caucasoid and Afro-Caribbean samples were examined to validate the method, with absolute correlation between phenotype and genotype. A further 30 Gambian samples were genotyped, confirming homozygosity for the FY*null-FY*B haplotype. Allele, gene and haplotype frequencies were examined in 100 Caucasoid controls. This method permits the rapid genotyping of large numbers of samples and will prove useful as a clinical and research tool.

Polymerase chain reaction haplotyping using 3' mismatches in the forward and reverse primers: application to the biallelic polymorphisms of tumor necrosis factor and lymphotoxin alpha.

A polymerase chain reaction with sequence-specific primers (PCR-SSP) system that operates under identical conditions to HLA phototyping was devised for characterizing polymorphisms in tumor necrosis factor (TNF) and lymphotoxin alpha (LT-alpha). Mismatches at the 3' end were incorporated into the forward and reverse primers of each PCR so as to unequivocally establish the cis/trans status between the biallelic sites. Three previously described biallelic polymorphisms in TNF and three in LT-alpha were characterized in a 24-reaction PCR-SSP system. The method was used to genotype 20 cell lines and 201 HLA class I and II typed controls from the United Kingdom at the TNF and LT-alpha loci. Population frequencies of TNF haplotypes were determined as was linkage disequilibrium with HLA-A, B, Cw, DRB1 and DQB1 loci. In each gene there were 8 theoretical polymorphic combinations; 4 were observed in TNF and 4 in LT-alpha. A total of 11 TNF-LT-alpha haplotypes were determined from apparent homozygous controls and statistical analysis.

TNF and lymphotoxin-alpha polymorphisms associated with common variable immunodeficiency: role in the pathogenesis of granulomatous disease.

A subgroup of common variable immunodeficiency (CVID) patients have distinct clinical features, particularly granulomata splenomegaly, characteristic blood lymphocyte phenotype, and elevated circulating TNF levels. To investigate the genetic basis for this phenotype, 150 CVID patients and 200 controls were genotyped for six biallelic TNF and lymphotoxin-alpha (LT alpha) polymorphisms and eight class I and II HLA loci using PCR and sequence specific primers (PCR-SSP) sequence-specific primers. Clinical and immunophenotypic data were collected for 90 patients to examine associations with CVID patient subgroups. The presence of granulomata (22% of patients) was strongly associated with splenomegaly, T and B lymphopenia, reduced CD4+ CD45RA+ T cells, and CD8+ CD57+ lymphocytosis, confirming the concept of a subgroup of patients with distinct clinical and laboratory features. The uncommon TNF +488A allele was strongly associated with this subgroup (p = 0.0005). The association between "granulomatous" CVID and TNF +488A was independent of HLA class I and II associations. We postulate that the presence of the TNF +488A allele, or alleles in linkage disequilibrium with it, contributes to the high levels of TNF and granulomatous complications characteristic of this subgroup of patients.

Comprehensive, serologically equivalent DNA typing for HLA-B by PCR using sequence-specific primers (PCR-SSP).

Polymorphic products of HLA class I genes from the human major histocompatibility complex (MHC) are traditionally assigned by serology with additional heterogeneity detectable using one-dimensional isoelectric focusing (1D-IEF). With the increased availability of HLA class I DNA sequence information it has become feasible to genotype for class I by polymerase chain reaction utilising sequence-specific primers (PCR-SSP). We describe here a comprehensive HLA-B PCR-SSP typing system based on available HLA nucleotide sequences which can detect all serologically defined antigens in most heterozygous combination in 48 one-step PCR reactions. In addition, four new unsequenced variants have been identified. DNA samples from 57 International Histocompatibility Workshop reference cell lines and 160 control individuals have been typed by the HLA-B PCR-SSP technique. 3/57 cell line types and 12/160 normal control individuals types were discrepant with the reported serological types. The SSP system has been designed to be higher resolution than serology but is not a complete allele-specific PCR although many single alleles can be identified. The system is entirely complementary to previous published PCR-SSP systems for HLA-Class II and HLA-Class I in that the same PCR conditions and controls are used which allows us to do one step PCR-SSP for all relevant HLA loci in under 3 hours in a system suitable for the typing of cadaver donors.

Infiltration of the kidney by alpha beta and gamma delta T cells: effect on progression in IgA nephropathy.

We have studied renal biopsies from three groups of patients to determine if alpha beta T cells or gamma delta T cells are present, and whether their presence is correlated with disease progression in IgA nephropathy (IgAN). Group one comprised thin basement membrane disease biopsies (non-immunological control, N = 7); group two were patients with IgAN and stable renal function one year following biopsy (stable, N = 7); and group three were IgAN patients with rapidly declining renal function after one year (progressive, N = 7). Immunohistochemical staining using monoclonal antibodies (CD3, TcR beta, TcR delta) and molecular studies utilizing polymerase chain reaction amplification of cDNA transcribed from biopsy RNA, with primers specific for either the alpha beta TcR or gamma delta TcR, were undertaken. On immunohistochemistry a significant increase in CD3 + cells in progressive biopsies was seen (vs. control P = 0.002, vs. stable P = 0.002). The progressive biopsies infiltrate consisted of both alpha beta TcR (vs. control P = 0.001, vs. stable P = 0.003) and gamma delta TcR cells (vs. control P = 0.01). The RNA study demonstrated an increase in TcR C alpha transcription in the progressive (vs. control P = 0.003) biopsies. Increased TcR C delta transcription was seen in the progressive group (vs. control P = 0.01, vs. stable P = 0.02). We confirm that the presence of lymphocytes in IgAN biopsies predicts progressive disease. While alpha beta T cells are found in both stable and progressive disease, the presence of gamma delta T cells is only associated with progressive IgAN.

Predominance of T cell receptor V delta 3 in small bowel biopsies from coeliac disease patients.

Increased numbers of T cells bearing the gamma delta antigen receptor (gamma delta T cells) have been reported in small bowel biopsies of patients with latent, active or treated coeliac disease. We have studied jejunal biopsies from seven children with coeliac disease and 10 children with normal gut histology to characterize gamma delta T cell receptor (TCR) variable region gene subfamily expression in resident gamma delta T cells and compared the results with the findings in peripheral blood mononuclear cells (PBMC) obtained on the same day as the gut biopsy. Molecular analysis of RNA extracted from PBMC and biopsies was performed by reverse transcription and amplification with the polymerase chain reaction using primers specific for six TCR V delta families and four TCR V gamma families. We report, first, that a significantly increased number of gamma delta T cells expressing the TCR V delta 3 subfamily (P = 0.008) was observed in jejunal biopsies from children with coeliac disease, and second, that gamma delta T cell V region subfamily populations in gut differed from those seen in PBMC for both control and coeliac patients. Significantly reduced numbers of TCR V delta 2, V delta 3, V delta 5 (P < 0.01) and V gamma 2, V gamma 4 (P < 0.01) T cells were found in gut compared with PBMC. The difference in gamma delta T cell repertoire observed between gut and blood may reflect differences in the nature of the antigens usually encountered in these two compartments. The over-representation of TCR V delta 3 in patients with coeliac disease suggests a specific role for these cells in the induction or maintenance of the jejunal abnormality associated with this disease.