Genetic variation at the Th2 immune gene IL13 is associated with IgE-mediated paediatric food allergy.

BACKGROUND: Food allergies pose a considerable world-wide public health burden with incidence as high as one in ten in 12-month-old infants. Few food allergy genetic risk variants have yet been identified. The Th2 immune gene IL13 is a highly plausible genetic candidate as it is central to the initiation of IgE class switching in B cells. OBJECTIVE: Here, we sought to investigate whether genetic polymorphisms at IL13 are associated with the development of challenge-proven IgE-mediated food allergy. METHOD: We genotyped nine IL13 "tag" single nucleotide polymorphisms (tag SNPs) in 367 challenge-proven food allergic cases, 199 food-sensitized tolerant cases and 156 non-food allergic controls from the HealthNuts study. 12-month-old infants were phenotyped using open oral food challenges. SNPs were tested using Cochran-Mantel-Haenszel test adjusted for ancestry strata. A replication study was conducted in an independent, co-located sample of four paediatric cohorts consisting of 203 food allergic cases and 330 non-food allergic controls. Replication sample phenotypes were defined by clinical history of reactivity, 95% PPV or challenge, and IL13 genotyping was performed. RESULTS: IL13 rs1295686 was associated with challenge-proven food allergy in the discovery sample (P=.003; OR=1.75; CI=1.20-2.53). This association was also detected in the replication sample (P=.03, OR=1.37, CI=1.03-1.82) and further supported by a meta-analysis (P=.0006, OR=1.50). However, we cannot rule out an association with food sensitization. Carriage of the rs1295686 variant A allele was also associated with elevated total plasma IgE. CONCLUSIONS AND CLINICAL RELAVANCE: We show for the first time, in two independent cohorts, that IL13 polymorphism rs1295686 (in complete linkage disequilibrium with functional variant rs20541) is associated with challenge-proven food allergy.

The skin barrier function gene SPINK5 is associated with challenge-proven IgE-mediated food allergy in infants.

BACKGROUND: A defective skin barrier is hypothesized to be an important route of sensitization to dietary antigens and may lead to food allergy in some children. Missense mutations in the serine peptidase inhibitor Kazal type 5 (SPINK5) skin barrier gene have previously been associated with allergic conditions. OBJECTIVE: To determine whether genetic variants in and around SPINK5 are associated with IgE-mediated food allergy. METHOD: We genotyped 71 "tag" single nucleotide polymorphisms (tag-SNPs) within a region spanning ~263 kb including SPINK5 (~61 kb) in n=722 (n=367 food-allergic, n=199 food-sensitized-tolerant and n=156 non-food-allergic controls) 12-month-old infants (discovery sample) phenotyped for food allergy with the gold standard oral food challenge. Transepidermal water loss (TEWL) measures were collected at 12 months from a subset (n=150) of these individuals. SNPs were tested for association with food allergy using the Cochran-Mantel-Haenszel test adjusting for ancestry strata. Association analyses were replicated in an independent sample group derived from four paediatric cohorts, total n=533 (n=203 food-allergic, n=330 non-food-allergic), mean age 2.5 years, with food allergy defined by either clinical history of reactivity, 95% positive predictive value (PPV) or challenge, corrected for ancestry by principal components. RESULTS: SPINK5 variant rs9325071 (A⟶G) was associated with challenge-proven food allergy in the discovery sample (P=.001, OR=2.95, CI=1.49-5.83). This association was further supported by replication (P=.007, OR=1.58, CI=1.13-2.20) and by meta-analysis (P=.0004, OR=1.65). Variant rs9325071 is associated with decreased SPINK5 gene expression in the skin in publicly available genotype-tissue expression data, and we generated preliminary evidence for association of this SNP with elevated TEWL also. CONCLUSIONS: We report, for the first time, association between SPINK5 variant rs9325071 and challenge-proven IgE-mediated food allergy.

The association of PTPN22 rs2476601 with juvenile idiopathic arthritis is specific to females.

A preponderance of females develop autoimmune disease, including juvenile idiopathic arthritis (JIA), yet the reason for this bias remains elusive. Evidence suggests that genetic risk of disease may be influenced by sex. PTPN22 rs2476601 is associated with JIA and numerous other autoimmune diseases, and has been reported to show female-specific association with type 1 diabetes. We performed main effect and sex-stratified association analyses to determine whether a sex-specific association exists in JIA. As expected, rs2476601 was associated with JIA in our discovery (413 cases and 690 controls) and replication (1008 cases and 9284 controls) samples. Discovery sample sex-stratified analyses demonstrated an association specifically in females (odds ratio (OR)=2.35, 95% confidence interval (CI)=1.52-3.63, P=0.00011) but not males (OR=0.91, 95% CI=0.52-1.60, P=0.75). This was similarly observed in the replication sample. There was evidence for genotype-by-sex interaction (Pinteraction=0.009). The association between rs2476601 and JIA appears restricted to females, partly accounting for the predominance of females with this disease.

Ameliorating pathogenesis by removing an exon containing a missense mutation: a potential exon-skipping therapy for laminopathies.

Exon skipping, as a therapy to restore a reading frame or switch protein isoforms, is under clinical trial. We hypothesised that removing an in-frame exon containing a mutation could also improve pathogenic phenotypes. Our model is laminopathies: incurable tissue-specific degenerative diseases associated with LMNA mutations. LMNA encodes A-type lamins, that together with B-type lamins, form the nuclear lamina. Lamins contain an alpha-helical central rod domain composed of multiple heptad repeats. Eliminating LMNA exon 3 or 5 removes six heptad repeats, so shortens, but should not otherwise significantly alter, the alpha-helix. Human Lamin A or Lamin C with a deletion corresponding to amino acids encoded by exon 5 (Lamin A/C-Δ5) localised normally in murine lmna-null cells, rescuing both nuclear shape and endogenous Lamin B1/emerin distribution. However, Lamin A carrying pathogenic mutations in exon 3 or 5, or Lamin A/C-Δ3, did not. Furthermore, Lamin A/C-Δ5 was not deleterious to wild-type cells, unlike the other Lamin A mutants including Lamin A/C-Δ3. Thus Lamin A/C-Δ5 function as effectively as wild-type Lamin A/C and better than mutant versions. Antisense oligonucleotides skipped LMNA exon 5 in human cells, demonstrating the possibility of treating certain laminopathies with this approach. This proof-of-concept is the first to report the therapeutic potential of exon skipping for diseases arising from missense mutations.

CD226 (DNAM-1) is associated with susceptibility to juvenile idiopathic arthritis.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is considered a complex genetic autoimmune disease. We investigated the association of genetic variants previously implicated in JIA, autoimmunity and/or immunoregulation, with susceptibility to JIA. METHODS: A genetic association study was performed in 639 JIA patients and 1613 healthy controls of northwest European descent. Ninety-three single nucleotide polymorphisms (SNP) were genotyped in a candidate gene approach. Results of the entire JIA patient group (all subtypes) were compared with results obtained, alternatively, with a clinically homogeneous patient group including only oligoarticular and rheumatoid factor (RF) negative polyarticular JIA patients (n=493). Meta-analyses were performed for all SNPs that have been typed in other Caucasian JIA cohorts before. RESULTS: SNPs in or near PTPN22, VTCN1, the IL2-IL21 region, ANKRD55 and TNFA were confirmed to be associated with JIA (p<0.05), strengthening the evidence for involvement of these genes in JIA. In the majority of these replicated SNPs, effect sizes were larger when analysing a homogeneous patient cohort than when analysing all subtypes. We identified two novel associations with oligoarticular and RF-negative polyarticular JIA: CD226 rs763361 (OR 1.30, 95% CI 1.12 to 1.51, p=0.0006) and CD28 rs1980422 (OR 1.29, 95% CI 1.07 to 1.55, p=0.008). Meta-analyses including reported studies confirmed the association of both SNPs with susceptibility to JIA (OR 1.16, p=0.001 and OR 1.18, p=0.001, for rs763361 and rs1980422, respectively). CONCLUSIONS: The CD226 gene has been identified as novel association with JIA, and a SNP near CD28 as a suggestive association. Both genes are probable candidate risk factors, since they are involved in costimulation of T cells.

An insight into the public acceptance of nutrigenomic-based personalised nutrition.

It is predicted that non-communicable diseases will account for over 73 % of global mortality in 2020. Given that the majority of these deaths occur in developed countries such as the UK, and that up to 80 % of chronic disease could be prevented through improvements in diet and lifestyle, it is imperative that dietary guidelines and disease prevention strategies are reviewed in order to improve their efficacy. Since the completion of the human genome project our understanding of complex interactions between environmental factors such as diet and genes has progressed considerably, as has the potential to individualise diets using dietary, phenotypic and genotypic data. Thus, there is an ambition for dietary interventions to move away from population-based guidance towards 'personalised nutrition'. The present paper reviews current evidence for the public acceptance of genetic testing and personalised nutrition in disease prevention. Health and clear consumer benefits have been identified as key motivators in the uptake of genetic testing, with individuals reporting personal experience of disease, such as those with specific symptoms, being more willing to undergo genetic testing for the purpose of personalised nutrition. This greater perceived susceptibility to disease may also improve motivation to change behaviour which is a key barrier in the success of any nutrition intervention. Several consumer concerns have been identified in the literature which should be addressed before the introduction of a nutrigenomic-based personalised nutrition service. Future research should focus on the efficacy and implementation of nutrigenomic-based personalised nutrition.

The role of genetics and environment in the rise of childhood food allergy.

Food allergy is a growing clinical and public health problem world-wide. The rising incidence is occurring more rapidly than changes to the genome sequence would allow, but it is yet to be determined whether environmental factors might act in interaction with genetic risk. That is to say, are environmental factors more likely to affect those genetically at risk? Family history is a strong risk factor for the development of food allergy as it co-aggregates with other atopic diseases and as such genetic factors do play an important role in food allergy risk. However, significant interest has now turned to the role of epigenetic modifications of the genome as the major mediator of gene-environment interaction. The consideration of the role of epigenetics in food allergy is likely to provide an insight into aetiological and biological disease mechanisms. This paper discusses the current state of knowledge regarding genetic and environmental risk factors for food allergy, and considers the potential for furthering our understanding of food allergy aetiology by examining the role of epigenetic variation.

Susceptibility variants on chromosome 7p21.1 suggest HDAC9 as a new candidate gene for male-pattern baldness.

BACKGROUND: Male-pattern baldness (androgenetic alopecia, AGA) is the most common form of hair loss among humans. Research has shown that it is caused by genetic factors. Numerous studies have unequivocally identified two major genetic risk loci for AGA: the X-chromosomal AR/EDA2R locus, and the PAX1/FOXA2 locus on chromosome 20. OBJECTIVES: To identify further candidate genes for AGA, and thus gain further insights into this phenotype. METHODS: A German sample of 581 severely affected cases and 617 controls was used to perform a genome-wide association study. The identified associated locus was further analysed by fine-mapping, and then independently replicated in an Australian sample. Expression and pathway analyses were performed to characterize the susceptibility gene identified. RESULTS: The most significant association signal was obtained for rs756853 (P = 1·64 × 10(-7) ), which is located intronically in the histone deacetylase 9 (HDAC9) gene. Fine-mapping and a family-based analysis revealed that rs756853 and the 6-kb distal rs2249817 were the most highly associated single nucleotide polymorphisms. The association finding was replicated in an independent Australian sample, when the analysis was restricted to severely affected cases and unaffected controls (P = 0·026). Analysis of rs2249817 in a combined sample of severely affected German and Australian cases and unaffected controls revealed a strong association signal (P = 9·09 × 10(-8) ). Tissue expression studies demonstrated HDAC9 expression in various tissues, including tissues of relevance to AGA. No strong genotypic effects were observed in genotype-specific expression or splice studies. Pathway analyses supported the hypothesis that HDAC9 plays a functional role in AGA via interaction with the AR gene. CONCLUSIONS: The present study suggests that HDAC9 is the third AGA susceptibility gene.

A role for the DRD4 exon III VNTR in modifying the association between nicotine dependence and neuroticism.

INTRODUCTION: Neurotic psychopathology has been extensively examined as a risk factor for nicotine dependence (ND). Genetic stratification may partially explain variability in risk estimates. Genetic variants that compromise dopaminergic neurotransmission may motivate exposure to dopamine-stimulating agents, including nicotine. The 7-repeat allele of a Variable Number Tandem Repeat (VNTR) polymorphism in DRD4 (and evolutionary derivatives 5, 6, and 8 repeats; 7R+) is associated with reduced dopamine receptor function. The purpose of this study was to examine association between both smoking initiation (SI) and progression to ND by young adulthood and (a) history of neuroticism during adolescence, (b) DRD4 7R+, and (c) interaction between neuroticism and DRD4 7R+. METHODS: Participants were drawn from the Victorian Adolescent Health Cohort Study, a longitudinal study of the health and well-being of young Australians across 8 waves (14-24 years). Neuroticism was measured at Waves 3 and 6 (mean 15.9 and 17.4 years). SI was defined as any smoking at any wave. ND was measured at Wave 8 (mean 24.1 years). Genotype data for the DRD4 VNTR were available for 839 participants. RESULTS: While adolescent neuroticism was associated with SI, evidence for association with progression to ND was weak. However, there was evidence of interaction between neuroticism and DRD4 7R+: The odds of progression to ND among those with a history of neuroticism were more than 3.5-fold higher among 7R+ carriers. CONCLUSIONS: Without considering stratification by 7R+, the association between progression to ND and neuroticism would have been assumed barely significant. However, among those carrying DRD4 7R+, risk of progression was considerably intensified.

Microbial exposure, interferon gamma gene demethylation in naïve T-cells, and the risk of allergic disease.

The period of immune programming during early life presents a critical window of opportunity for the prevention of allergic diseases. There is mounting evidence that inappropriate immune programming may involve disruption of specific epigenetic modifications (switches) at immune-related genes. This novel area of research has great potential, as epigenetic changes are known to be sensitive to environmental factors and may therefore provide a mechanistic link for the observed association between specific environmental cues, faulty immune development, and the risk of allergic disease. In addition, the dynamic and potentially reversible nature of epigenetic modifications offers potentially novel targets for therapeutic and/or preventative interventions. We review the evidence that (1) failure to up-regulate the interferon gamma (IFNgamma) response during infancy is an important determinant of the risk of allergic disease, (2) expression of the IFNgamma gene in naïve T-cells is regulated by epigenetic mechanisms, and (3) failure to up-regulate IFNgamma gene expression of naïve T-cells associated with low early life microbial exposure. Taken together, these lines of evidence suggest that low microbial exposure during early life increases the risk of allergic disease by reducing demethylation (activation) of the IFNgamma gene of naive T-cells.

Comparative efficacy of intranasal and oral vaccines against Bordetella bronchiseptica in dogs.

In order to determine the comparative efficacy of vaccines administered intranasally or orally to protect puppies from disease subsequent to experimental infection with Bordetella bronchiseptica (Bb), a randomized controlled trial was performed using 48 approximately 8-week-old specific pathogen free, Bb naive Beagle puppies. Puppies were randomized into three groups and administered vaccines containing Bb intranasally or orally, or a placebo intranasally. Twenty-one days later, all dogs were challenge exposed via aerosol administration of Bb. Clinical signs, nasal bacterial shedding and immune responses were monitored for 28 days after challenge. Intranasally vaccinated puppies had significantly lower rates of coughing, nasal discharge, retching and sneezing (i.e. were less sick clinically) than control puppies. The distinction between the orally vaccinated puppies and the control puppies was less consistent. The orally vaccinated puppies had less coughing and less retching than the control puppies, but nasal discharge and sneezing did not differ from control animals. Orally vaccinated puppies had higher rates of coughing, nasal discharge, retching and sneezing than the intranasally vaccinated puppies. Although both intranasal and oral Bb vaccines stimulated immune responses associated with disease sparing following Bb infection, the intranasal route of delivery conferred superior clinical outcomes. The observed difference in clinical efficacy suggests the need to question the rationale for the use of currently available orally administered Bb vaccines.

Association of juvenile idiopathic arthritis with PTPN22 rs2476601 is specific to females in a Greek population.

BACKGROUND: Juvenile idiopathic arthritis (JIA) is an autoimmune disease characterized by persistent chronic arthritis. Disease risk is believed to be influenced by both genetic and environmental factors. It is well established that the PTPN22 single nucleotide polymorphism (SNP) rs2476601 is associated with JIA susceptibility. It was recently reported in an Australian study that this association is restricted to females and is not observed in males. A significant source of inconsistency amongst the literature on autoimmune disease susceptibility genes stems from an inability to replicate genetic findings across different racial or ethnic groups. We therefore attempted to generate further evidence of the female-specific association of rs2476601 in a homogeneous Greek population. FINDINGS: We genotyped rs2476601 in 128 Caucasian JIA patients (70.3 % female) and 221 healthy controls (28.1 % female) from Northern Greece. Overall, PTPN22 was associated with increased risk of JIA in this Greek sample (OR = 2.3, 95 % CI 1.1 - 5.1, p = 0.038). Sex-stratified analyses showed that, once again, the risk association was restricted to females (Female: OR = 19.9, 95 % CI 1.2 - 342, p = 0.0016; Male: OR = 1.1, 95 % CI 0.3 - 3.1, p = 0.94) supporting the prior findings. CONCLUSIONS: Our data demonstrates that this sex-specific pattern of association is broadly applicable to different populations, and provides further impetus to undertake mechanistic studies to understand the impact of sex on PTPN22 in JIA.

Genetic analysis of male pattern baldness and the 5alpha-reductase genes.

Genetic predisposition and androgen dependence are important characteristics of the common patterned loss of scalp hair known as male pattern baldness. The involvement of the 5alpha-reductase enzyme in male pattern baldness has been postulated due to its role in the metabolism of testosterone to dihydrotestosterone. There are two known isozymes of 5alpha-reductase. Type I has been predominantly localized to the skin and scalp. Type II, also present on the scalp, is the target of finasteride, a promising treatment for male pattern baldness. We conducted genetic association studies of the 5alpha-reductase enzyme genes (SRD5A1 on chromosome 5 and SRD5A2 on chromosome 2) using dimorphic intragenic restriction fragment length polymorphisms. From a population survey of 828 healthy families comprising 3000 individuals, we identified 58 young bald men (aged 18-30 y) and 114 older nonbald men (aged 50-70 y) for a case control comparison. No significant differences were found between cases and controls in allele, genotype, or haplotype frequencies for restriction fragment length polymorphisms of either gene. These findings suggest that the genes encoding the two 5alpha-reductase isoenzymes are not associated with male pattern baldness. Finally, no clear inheritance pattern of male pattern baldness was observed. The relatively strong concordance for baldness between fathers and sons in this study was not consistent with a simple Mendelian autosomal dominant inheritance. A polygenic etiology should be considered.

Polymorphism of the androgen receptor gene is associated with male pattern baldness.

The common heritable loss of scalp hair known as male pattern baldness or androgenetic alopecia affects up to 80% of males by age 80. A balding scalp is characterized by high levels of the potent androgen dihydrotestosterone and increased expression of the androgen receptor gene. To determine if the androgen receptor gene is associated with male pattern baldness, we compared allele frequencies of the androgen receptor gene polymorphisms (StuI restriction fragment length polymorphism and two triplet repeat polymorphisms) in cases with cosmetically significant baldness (54 young and 392 older men) and controls (107 older men) with no indication of baldness. The androgen receptor gene StuI restriction site was found in all but one (98.1%) of the 54 young bald men (p = 0.0005) and in 92.3% of older balding men (p = 0.000004) but in only 76.6% of nonbald men. The combination of shorter CAG and GGC triplet repeat lengths was also more prevalent in bald men (p = 0.03). The ubiquity of the androgen receptor gene StuI restriction site, and higher incidence of shorter triplet repeat haplotypes in bald men suggests that these markers are very close to a functional variant that is a necessary component of the polygenic determination of male pattern baldness. Functional mutation in or near the androgen receptor gene may explain the reported high levels of expression of this gene in the balding scalp.

Significant population variation in adult male height associated with the Y chromosome and the aromatase gene.

The determination of human adult height is dependent on both environmental and genetic factors. Rare causes of abnormal stature have been identified, including mutations in the gene encoding aromatase (CYP19) and regions on the Y chromosome. However, the possible role of these loci in the genetic control of normal adult height is unknown. We have performed an association study using common biallelic polymorphisms within CYP19 and the Y chromosome to determine whether these loci are associated with variation in height in 413 adult males and 335 females drawn at random from a large population sample. An association between CYP19 and height was found (difference, 2.0 cm; 95% confidence interval, 0.16-3.8; P = 0.003), but this was more evident in men (difference, 2.3 cm; 95% confidence interval, 0.38-4.4; P = 0.05) than women (difference, 0.2 cm; 95% confidence interval, -2.1 to 1.6; P = 0.94). An association was also found with the Y chromosome (P = 0.009; difference of 1.9 cm; 95% confidence interval, 0.5-3.4). Additionally, when men were grouped according to haplotypes of the CYP19 and Y chromosome polymorphisms, a difference of 4.2 cm (95% confidence interval, 0.67-7.3) was detected (P = 0.004). These results suggest that in men, genetic variation in CYP19 and on the Y chromosome are involved in determining normal adult height, and that these loci may interact in an additive fashion.

Two distal mutations in the gene encoding emerin have profoundly different effects on emerin protein expression.

Emerin, the product of the gene responsible for X-linked Emery-Dreifuss muscular dystrophy (EDMD), has a ubiquitous tissue distribution and is localised to the nuclear envelope. We present here the relationship between emerin protein expression, nuclear localization and clinical phenotype for two distal mutations identified in unrelated EDMD patients. The first mutation predicts the replacement of the last eight amino acids of emerin with the addition of 101 amino acids, but no emerin expression is detected. The second mutation, 35 bp upstream from the first mutation, deletes six amino acids from the transmembrane region, but in this case emerin expression is seen. Emerin from this second patient is expressed at reduced levels, mistargeted and has altered biochemical properties compared to wild type emerin. In both cases the clinical phenotype was similar to patients with typical null mutations. We discuss these data in comparison with previous reports of other C-terminal mutations in the emerin gene and suggest that the efficiency of emerin's nuclear membrane localization is affected by the hydrophobicity (and possibly length) of its transmembrane region, and a longer C-terminal tail prevents nuclear localization.

Genotype-phenotype analysis in X-linked Emery-Dreifuss muscular dystrophy and identification of a missense mutation associated with a milder phenotype.

Direct sequencing of the emerin gene in 22 families with Emery-Dreifuss muscular dystrophy (EMD) revealed mutations in 21 (95%), confirming that emerin mutations can be identified in the majority of families with X-linked EMD. Most emerin mutations result in absence of the protein. In this study three mutations (a missense mutation Pro183Thr and two in-frame deletions removing residues 95-99 and 236-241, respectively) were unusual in being associated with expression of mutant protein. The phenotype in these families was compared in detail with the clinical features in cases with typical null mutations. For the in-frame deletions there were no significant differences. In the family with the missense mutation the phenotype was milder. Age at onset was later for first symptoms and for development of ankle contractures and muscle weakness. These findings have diagnostic implications as well as pointing to functionally important regions of the emerin protein.

Analyses of ovine corpora lutea for tumor necrosis factor mRNA and bioactivity during prostaglandin-induced luteolysis.

It has been suggested that tumor necrosis factor (TNF) participates in the mechanism of regression of the corpus luteum. We measured luteal expression of TNF alpha mRNA and biological activity during prostaglandin-induced luteolysis in sheep. Initiation of functional luteolysis was marked by a sharp decline in concentrations of progesterone in luteal tissue beginning 4 h after administration of luteolysin. Structural regression of corpora lutea was manifested by a reduction in glandular weight at 16 h. A luteal cytotoxic factor with TNF alpha-like bioactivity was isolated after the decrease in tissue progesterone had occurred, but before evidence of luteal resorption. We were unable to detect temporal alterations in TNF alpha mRNA in luteal samples by classical Northern blot or in situ hybridization analyses. These results imply that luteal TNF alpha is derived primarily as a preformed entity from an extraovarian source, such as infiltrating leukocytes. These results raise the possibility that this cytokine might not be involved in the early stages of luteal regression in the ewe, yet could play a secondary role, perhaps in the subsequent opsonization and removal of degenerating cells.

A comparison of in situ hybridization and immunohistochemistry for the detection of a new porcine circovirus in formalin-fixed tissues from pigs with post-weaning multisystemic wasting syndrome (PMWS).

Post-weaning multisystemic wasting syndrome (PMWS) is a recently identified condition affecting pigs in North America and Europe. Porcine circovirus antigen and nucleic acid have been demonstrated associated with lesions, and a new porcine circovirus designated PCV2 has been recovered from tissues of these animals. In this study, in situ hybridisation and immunohistochemical protocols were developed, optimized and compared for their relative sensitivity in detecting PCV2 antigens and nucleic acid in tissues from cases of PMWS that had been fixed for up to 6 months in formalin. For both immunohistochemistry and in situ hybridization, an increase in specific signal was observed following increased exposure to both protease XIV and proteinase K. Maximum signal and minimal loss of tissue morphology was seen after 40 min treatment with protease XIV (0.5 mg/ml). After optimisation, a comparison of these techniques on sequential sections demonstrated that both techniques successfully detected antigen or nucleic acid in all of the tissues examined. More positive cells, with increased signal intensity, were detected following immunohistochemistry.