Site-specific immunophenotyping of keloid disease demonstrates immune upregulation and the presence of lymphoid aggregates.

BACKGROUND: Keloid disease (KD) is a common fibroproliferative disorder of unknown aetiology. T cells and macrophages are increased in KD and are thought to contribute to its pathogenesis. However, while a link between inflammation and fibrotic disorders is well known for other disorders, it remains undetermined in KD. OBJECTIVES: Systematically to immunophenotype the inflammatory infiltrate of KD in situ in a site-specific manner, and to compare this with normal skin and scar tissue. METHODS: Sixty-eight keloid cases were screened for the presence of all three (intralesional, perilesional and extralesional) keloid-associated specific tissue sites. Subsequently, a complete set of 25 keloid biopsies (from different patients) was compared with normal skin (n = 11) and normal scar (n = 11) samples and subjected to systematic, site-specific quantitative immunohistomorphometry and histochemistry, using a range of immunological markers of B cells, T cells, macrophages, mast cells (MCs) and Langerhans cells. RESULTS: T cells, B cells, degranulated and mature MCs (coexpressing OX40 ligand) and alternative macrophages (M2) were all significantly increased in intralesional and perilesional KD sites compared with normal skin and scar tissue (P < 0·05). Additionally, 10 of 68 KD cases (15%) showed the presence of distinctive lymphoid aggregates, which resembled mucosa-associated lymphoid tissue (MALT). CONCLUSIONS: The increased number and activity of MCs and M2 may implicate inflammation in the fibrotic process in KD. The distinct KD-associated lymphoid aggregate resembles MALT, for which we propose the term 'keloid-associated lymphoid tissue' (KALT). It may perpetuate inflammatory stimuli that promote KD growth. KALT, MCs and M2 are promising novel targets for future KD therapy.

Poly(A) RT-PCR measurement of diagnostic genes in pancreatic juice in pancreatic cancer.

BACKGROUND: The last decade has seen significant progress in understanding the molecular biology of pancreatic adenocarcinoma. There is now an urgent need to translate these molecular techniques to clinical practice in order to improve diagnosis and prediction of response to treatment. The objectives of this study are to utilise poly(A) RT-PCR to measure expression levels of diagnostic Indicator genes, selected from microarray studies, of RNA from intraoperatively sampled pancreatic ductal juice and to correlate these expression levels with those in matched pancreatic tissue resection samples. METHODS: Intraoperative sampling of pancreatic juice and collection of matched tissue samples was undertaken in patients undergoing pancreaticoduodenectomy for suspected tumour. RNA was isolated and poly(A) PCR and real-time PCR used to measure expression levels of 30 genes. Spearman's rank correlation test was used to examine the relationship of gene expression between pancreatic juice and tissue. RESULTS: Of the 30 Indicator genes measured, just one, ANXA1, showed a significant correlation of expression level between pancreatic juice and tissue samples, whereas three genes, IGFBP3 (P0.035), PSCA (P0.001) and SPINK1 (P0.05), showed significantly different expression between cancerous and benign pancreatic tissue samples. CONCLUSIONS: These results demonstrate that RNA analysis of pancreatic juice is feasible using the poly(A) cDNA technique, that correlation of gene expression exists between pancreatic juice and tissue for very few genes and that gene expression profiling can distinguish between benign and malignant pancreatic tissue. This indicates possible use of the technique for measurement of Indicator genes in pancreatic tissue for diagnosis of pancreatic cancer from very small tissue samples.

Quantum dots light up pathology.

Quantum dots (QDs) are novel nanocrystal fluorophores with extremely high fluorescence efficiency and minimal photobleaching. They also possess a constant excitation wavelength together with sharp and symmetrical tunable emission spectra. These unique optical properties make them near-perfect fluorescent markers and there has recently been rapid development of their use for bioimaging. QDs can be conjugated to a wide range of biological targets, including proteins, antibodies, and nucleic acid probes, rendering them of particular interest to pathology researchers. They have been used in multiplex immunohistochemistry and in situ hybridization, which when combined with multispectral imaging, has enabled quantitative measurement of gene expression in situ. QDs have also been used for live in vivo animal imaging and are now being applied to an ever-increasing range of biological problems. These are detailed in this review, which also acts to outline the important advances that have been made in their range of applications. The relative novelty of QDs can present problems in their practical use and guidelines for their application are given.

Comparison of gene-expression profiles in parallel bone marrow and peripheral blood samples in acute myeloid leukaemia by real-time polymerase chain reaction.

BACKGROUND: Gene signatures (Indicator genes) in bone marrow that provide more precise prognostication in haematological malignancy have been identified by microarray expression studies. It would be beneficial to measure these diagnostic signatures in peripheral blood. AIMS: To determine the degree of correspondence of gene expression for a set of Indicator genes between bone marrow and peripheral blood in acute myeloid leukaemia (AML). METHODS: Parallel bone marrow aspirate and peripheral blood samples were obtained from 19 patients diagnosed with AML and mononuclear cells isolated from both sample types. mRNA was globally amplified by polyadenylated real-time polymerase chain reaction (polyA RT-PCR); the expression of 15 AML Indicator genes, identified from previous microarray studies, was measured by RT-PCR. All values were normalised to the mean expression of three housekeeping genes (IF2-beta, GAP and RbS9) and were statistically compared using SPSS software. RESULTS: No significant difference in expression between bone marrow and peripheral blood was observed for 10 of the genes (leptin receptor, CD33, adipsin, proteoglycan 1, MB-1, cyclin D3, hSNF2b, proteasome iota, HkrT-1 and E2A), indicating its possible use in monitoring disease activity in peripheral blood samples, whereas c-myb, HOXA9, LYN, cystatin c and LTC4s showed significantly different expression between bone marrow and peripheral blood samples. CONCLUSION: These results indicate a possible use for the method in monitoring AML in peripheral blood by RT-PCR measurement of Indicator genes. In addition, the initial use of polyA PCR facilitates translation to very small clinical samples, including fractionated cell populations, of particular importance for monitoring haematological malignancy.

Adipocytic proportion of bone marrow is inversely related to bone formation in osteoporosis.

AIMS: Preliminary studies have suggested that there is an increase in adipocytic tissue in osteoporotic (OP) bone, supporting in vitro evidence for a switch in differentiation of stromal cells from the osteoblastic to the adipocytic lineage. To investigate this the variation of the ratio of adipose tissue to haemopoietic/stromal tissue in OP bone was measured. METHODS: The ratio of adipocytic to haemopoietic/stromal tissue (A/H) was measured by semi-automated image analysis in iliac crest biopsies from 127 patients with osteoporosis (84 female patients, 48 male patients; mean age, 55 years; range, 5-80). Fourteen patients with normal histomorphometric data (nine women; five men; mean age, 48 years; range 21-70) acted as controls. RESULTS: The ratio of A/H was higher in OP bone than in the normal controls (OP mean 43.06% v normal mean 22.4%; p < 0.001). Multiple regression analysis showed that 98.5% of the variability in the A/H ratio was the result of age and several measures of bone formation, including cancellous wall thickness, osteoid volume, cancellous thickness, cortical wall thickness, cancellous apposition rate, and bone formation rate, together with cancellous separation (each significant at p < 0.001). Those with the greatest effect on the A/H ratio (in decreasing order) were cancellous apposition rate, osteoid volume, and age. CONCLUSIONS: Cancellous apposition rate, osteoid volume, and age were associated with the increase in the proportion of adipose tissue present in OP bone. Of these, cancellous apposition rate reflects osteoblast activity, indicating that the increase in the volume of adipose tissue in osteoporosis is associated with reduced bone formation, supporting the postulated switch in differentiation of stromal cells from the osteoblastic to the adipocytic pathway in osteoporosis.

A study of four cases of extra-orbital giant cell angiofibroma with documentation of some unusual features.

AIMS: To document the clinical, light microscopic, immunohistochemical and ultrastructural features of four cases of extra-orbital giant cell angiofibromas. METHODS AND RESULTS: Sections of formalin-fixed paraffin-embedded specimens were studied by haematoxylin and eosin, reticulin and immunohistochemical stains. Electron microscopy was carried out in two cases on tissue fixed in formalin. The age of the patients ranged from 30 to 41 years. Two patients presented with a soft tissue swelling in the left groin, one patient had a left axillary soft tissue lump and one patient presented with a parotid lump. All lesions were well circumscribed and contained variably cellular and vascularized tissue composed of round to spindle cells with a patternless arrangement, scattered multinucleate giant cells and pseudovascular spaces conforming to the description of giant cell angiofibroma. Mononuclear and multinucleate tumour cells were both positive for vimentin and CD34; one tumour exhibited focal S100 protein and GFAP positivity. Both of the tumours examined by electron microscopy showed fibroblastic features, but in addition one contained cells having Schwannian features. All four patients were well without recurrent disease on follow-up (average 25 months). CONCLUSION: Giant cell angiofibroma shares many features with solitary fibrous tumour and giant cell fibroblastoma and shows a wider distribution than initially recognized. Rarely, Schwannian differentiation may be observed in these tumours.

Dual colour FISH in paraffin wax embedded bone trephines for identification of numerical and structural chromosomal abnormalities in acute myeloid leukaemia and myelodysplasia.

AIMS/BACKGROUND: The advent of new treatments for haematological malignancies has led to the need for a correlation between cytogenetic and morphological abnormalities. This study aimed to achieve this by the application of interphase cytogenetics to marrow trephine sections, a technique not previously reported for formalin fixed, paraffin wax embedded trephine biopsies. METHODS: Dual colour fluorescence in situ hybridisation (FISH) was used to detect numerical and structural abnormalities in routinely processed paraffin wax embedded trephine biopsies. Three cases with t(8;21) and three with t(15;17) were analysed, together with a case of trisomy 8. Chromosome specific probes were hybridised with sections and disclosed by fluorescein isothiocyanate and rhodamine/Texas red labelled antidigoxigenin and antibiotin amplification; translocations were identified by colocalisation of probes using a double wavelength bypass filter. RESULTS: A translocation signal was present in 12% and 11.5% of the cells counted in the t(8;21) and t(15;17) cases, respectively, but in none of the normal controls (p < 0.001). In the case of trisomy 8, 9% of the cells counted contained three hybridisation signals for chromosome 8, whereas no cell contained more than two in the normal control (p < 0.001). CONCLUSIONS: This technique is useful for archived routinely processed material, enabling it to be used as a research tool but also, and perhaps more importantly, in clinical practice.

Osteoporosis in men: a cellular endocrine perspective of an increasingly common clinical problem.

Although it has been accepted that osteoporosis is common in women, only recently have we become aware that it is also widespread in men; one in twelve men in the UK have osteoporosis. In many cases, there are recognisable causes for their osteoporosis, but a significant proportion (approximately one third) of these men have idiopathic disease. A major problem is that these cases are difficult to treat. An important therapeutic strategy would be to identify men at risk from osteoporosis sufficiently early, so that they can begin preventative measures. Moreover, development of novel means of treating these men would be an important clinical advance. With the emphasis on osteoporosis in women, however, the cellular and molecular basis for male idiopathic osteoporosis (MIO) is still poorly understood. Nevertheless, there are some aspects of skeletal regulation which may be specific for men and which could form the basis for addressing these problems. Thus, the importance of oestrogen in maintaining the adult skeleton in men as well as women implies that bone cells in men can respond to low levels of the hormone. Both oestrogen receptor (ER) alpha and beta are expressed in bone in vivo, which may be important for oestrogen action on bone in men. Furthermore, in osteoporosis generally, there is increasing evidence for defective osteoblast differentiation such that there is a surfeit of adipocytes over osteoblasts. A low peak bone mass is a powerful risk factor for osteoporosis in later life; bone formation and, by implication, osteoblast differentiation, is key to the mechanism by which it is accrued. GH and IGFs are important for regulating osteoblast differentiation. Evidence now suggests that they are associated with bone mineral density, particularly in men. The genes for ERs, GH and IGF-I might be useful candidates with which we can begin to detect men at risk from osteoporosis. Furthermore, the mechanisms by which oestrogen, GH and IGF-I regulate the male skeleton could provide the basis for developing novel means of treating MIO.

Subtractive hybridization--genetic takeaways and the search for meaning.

Gene expression profiling relies on mRNA extraction from defined cell systems, which in the case of pathological processes necessarily results in the use of small quantities of tissues, sometimes as little as a few cells. This obviates the use of many systems of gene expression profiling and is best carried out using cDNA amplified by poly(A) reverse transcription polymerase chain reaction, which is capable of generating material representative of all the expressed genes in samples as small as one cell. Analysis of this material using subtractive hybridization compares the genes expressed at different stages of a biological/pathological process allowing identification of the all the genes upregulated during the process. The identification of the genes present is not dependent on their prior description or on the choice of genes used in a screen and as such the method is ideal for identifying novel genes or unsuspected genes. We have used the method to identify genes involved in normal osteoblastic differentiation and in Paget's disease of bone and it has been widely used to study normal differentiation and pathological processes in a number of systems. The method, its applications and its relationship with the other methods of gene expression profiling are reviewed.

Dopexamine reduces the incidence of acute inflammation in the gut mucosa after abdominal surgery in high-risk patients.

OBJECTIVE: To evaluate the effect of dopexamine on the incidence of acute inflammation in the stomach/duodenum in patients undergoing abdominal surgery > or =1.5 hrs with a minimum of one high-risk criterion. DESIGN: Prospective, randomized, double-blind, placebo-controlled study. This study was conducted as a side arm to a multicenter, multinational study. SETTING: University hospital in an adult intensive care unit. PATIENTS: Thirty-eight patients. INTERVENTIONS: Patients were stabilized with fluid, blood products, and supplementary oxygen to achieve predetermined goals: cardiac index > 2.5 L/min/m2, mean arterial blood pressure of 70 mm Hg, pulmonary arterial occlusion pressure of 10 mm Hg, hemoglobin of 100 g/L, and arterial saturation of 94%. After stabilization, the study drug (either placebo [group A], dopexamine 0.5 microg/kg/min [group B], or dopexamine 2.0 microg/kg/min [group C]) was commenced. The study drug infusion was started 2 to 12 hrs before surgery and infused for 24 hrs after surgery. Estimation of upper gut blood flow was assessed using a gastric tonometer, and gastroscopy with biopsy was performed before surgery (after induction of anesthesia) and 72 hrs after surgery. Comparisons were made between endoscopic findings and histologic proof of acute inflammatory changes. In addition, biopsies were assessed for the presence in the mucosa of mast cells, myeloperoxidase activity, and inducible nitric oxide synthase. MEASUREMENTS AND MAIN RESULTS: Intramucosal pH decreased significantly with time in all three groups (p < .001), reaching the lowest point at the end of surgery. There was no difference among the groups. Endoscopy visualized acute inflammatory changes in 58.3% of group A patients, 46.2% of group B patients, and 53.90% of group C patients after hemodynamic optimization. At 72 hrs, dopexamine-treated patients compared with placebo-treated patients had a significantly lower incidence of gastric and duodenal acute inflammatory changes, as defined by myeloperoxidase activity (37.5% in groups B and C vs. 86% in group A; p < .05). CONCLUSION: Dopexamine in doses of 0.5 and 2.0 microg/kg/min affords significant histologic protection to the upper gastrointestinal tract mucosa 72 hrs after operation in high-risk surgical patients undergoing abdominal surgery.

Osteoblastic differentiation and mRNA analysis of STRO-1-positive human bone marrow stromal cells using primary in vitro culture and poly (A) PCR.

Investigation of osteoblast dysfunction in osteoporosis has been hampered by a poor understanding of normal early osteoblast differentiation, due to a relative lack of markers for the earliest cells in the lineage. Attempts to identify such markers have used cultures of animal or immortalized human cells, of uncertain relevance to human biology, or heterogeneous cultures in which genetic variability precludes the isolation of stage-specific genotypic markers. Primary in vitro generation of clonal populations of human bone marrow stromal cells was used in order to overcome these problems. Fibroblast-like stromal cells were isolated from human sternal bone marrow. They showed differentiation to an osteoblastic phenotype when stimulated with dexamethasone (10(-7) M) and fluorescence activated cell analysis demonstrated immunopositivity for STRO-1 (an antibody that recognizes osteoprogenitor stem cells of the colony-forming unit-fibroblastic) in from 8 to 40 per cent of the cells, dependent on time post-harvest. Cells positive for STRO-1 were immunoselected using magnetic activated cell sorting and seeded at low density (10 cells/cm2) to produce clones. Each clone was subpassaged, osteoblastic differentiation stimulated with dexamethasone, and mRMA-extracted at time points post-stimulation (0 h and 1-14 days). A novel poly (A) reverse transcriptase-polymerase chain reaction (RT-PCR) was used to amplify cDNA representative of all transcripts expressed at each time point. Differential gene expression within the amplified cDNA was assessed using 3' end cDNA probes to osteocalcin, osteopontin, and collagen type I (positive), demonstrating the acquisition of an osteoblastic phenotype. Time-specific gene products for early osteoblast differentiation have been generated from primary human cultures, utilizing very low density seeding and poly (A) RT-PCR. These products overcome the problems associated with animal, immortalized or heterogeneous culture and can be used to study normal and altered early osteoblast differentiation, indicating the possibility of using the same system to study other disease states.

Differential patterns of altered bone formation in different bone compartments in established osteoporosis.

AIM: To investigate the level of bone formation in the different bone compartments in cases of established osteoporosis, as previous work has concentrated on trabecular bone alone. METHODS: Bone formation rates were measured histomorphometrically, in the periosteal (P), cortical (C), subcortical (SC), and trabecular (T) compartments in iliac crest biopsies from 159 patients with established osteoporosis. The values were standardised using age and sex matched control data and patterns of differential change determined by analysis of parametric status (increased, normal, reduced). RESULTS: Mean bone formation was reduced in all four compartments. This was more marked (4.4/4.1 standard deviations below the mean in C/T, v 2.3/0.9 in P/SC) and more frequent (reduced in 81.5%/78.3% in T/C, v 43.3%/44% in P/SC) in the trabecular and cortical compartments than in the periosteal or subcortical bone. Parametric status was equal in trabecular and cortical bone in 85.4% of cases, and in periosteal and subcortical bone in 65.7%, but in all four compartments in only 35.1%, indicating differential alteration of bone formation in the two sets of compartments (T/C v P/SC). CONCLUSIONS: Altered trabecular bone formation is important in osteoporosis, but there are differential patterns of alteration in the other three compartments, emphasising the presence of different microenvironments in bone; thus the effect on the cortical compartment was similar to that on the trabecular, while the subcortical and periosteal compartments also showed linkage. The linkage between the two pairs was divergent, indicating different control of bone formation, with resultant different patterns of perturbation in osteoporosis.

In situ zymographic localisation of type II collagen degrading activity in osteoarthritic human articular cartilage.

OBJECTIVES: Chondrocytic matrix metalloproteinases (MMPs) are believed to be important in osteoarthritic cartilage degradation. The cartilage lesion of osteoarthritis (OA) is focal and often progressive. During its development chondrocytes differentially up and down regulate production of mRNA for individual MMPs. This observation has potential implications for understanding the disease processes that lead to progressive cartilage loss in OA and designing appropriate targeted treatment. The complex regulation of MMP mediated effects means there is a pressing need to establish whether visualisation of MMP mRNA or protein equates to enzyme activity. The technique of in situ zymography (ISZ) offers a way of examining diseased human tissue for in vivo production of an excess of degrading enzyme over inhibitor. The primary objective of this study was to assess, and if positive follow, collagen II degrading activity in cartilage during development of the OA lesion. A secondary objective was to assess whether there was any correlation between sites of collagen II degrading activity and expression of the collagenase (MMP-13), recently implicated in type II collagen degredation in this lesion. METHODS: Biopsied human normal and osteoarthritic cartilage, showing various degrees of damage, was examined by in situ zymography, with and without enzyme inhibitors, to establish sites of type II collagenase activity. Paired samples were probed for MMP-13 mRNA using 35S-labelled oligonucleotide probes. Comparative analyses were performed. RESULTS: In situ zymography showed collagen II degrading activity over chondrocytes only in osteoarthritic cartilage. Distribution and amount varied with the extent of cartilage damage and position of chondrocytes, being greatest in deep cartilage and in cartilage lesions where fissuring was occurring. The enzyme causing the degradation behaved as a matrix metalloproteinase. MMP-13 mRNA expression codistributed with the type II collagenase activity. CONCLUSION: In OA, chondrocytes can degrade type II collagen. The type II collagen degrading activity varies in site and amount as the cartilage lesion progresses and throughout codistributes with MMP-13 mRNA expression.

Differential patterns of osteoblast dysfunction in trabecular bone in patients with established osteoporosis.

AIMS: To analyse osteoblast function in 153 cases of established osteoporosis as previous work has indicated that osteoporosis is a heterogeneous condition characterised by different patterns of osteoclast and osteoblast dysfunction. METHODS: Histomorphometric data from 153 cases with established osteoporosis was used to analyse osteoblast function, using the following parameters: osteoblast number was assessed using the ratio of osteoblast surface to bone surface (ObS:BS); the percentage of active osteoblasts was assessed by using mineralising surface as a proportion of osteoid surface (sLS + dLS/OS); and the efficiency of active osteoblasts was assessed using the ratio of double to total labelled surface (dLS:tLS). The values of each parameter were standardised using age and sex matched control data and a three dimensional matrix was used to identify groups of patients with similar patterns of altered function. RESULTS: The largest group (60 cases) showed a reduction in all three parameters, while a small group (9 cases) had normal osteoblast function. However, one group showed reduction in osteoblast number only (23 cases), while another group showed a normal number of osteoblasts but both reduced percentage and efficiency of activity (14 cases). The results also suggest that efficiency of activity falls first and that this eventually leads to exit from the active pool. CONCLUSIONS: These results demonstrate the presence of heterogeneity of osteoblast dysfunction in osteoporosis, indicating that the disease is caused by interference at a variety of target sites along the pathway of osteoblast proliferation, differentiation, and activation. Greater understanding of this pathway and of the variety of alterations in the pathway that can occur in osteoporosis may allow more focused therapy for different patient groups identified on the basis of histomorphometric analysis.

Pericardial involvement in systemic sclerosis.

OBJECTIVE: To determine the frequency and histological characteristics of pericardial involvement in systemic sclerosis. METHOD: Necropsy sections of pericardium from 44 patients with systemic sclerosis were studied, together with sections from 19 age/sex matched controls. Sections were stained with haematoxylin and eosin, acid toluidine blue, and elastic van Gieson. Mast cells were counted in 10 random high power fields and the degree of fibrosis was quantified using a Chalkley count. RESULTS: Chronic pericarditis was seen in 31 (77.5%) of the systemic sclerosis cases, but in only one of the controls. The characteristic changes of uraemic pericarditis were not seen. The degree of fibrosis was greater in those with systemic sclerosis, though numbers of mast cells, thought to be important in fibrogenesis, were similar in both groups. Myocardial fibrosis was seen in 15 (37.5%) of systemic sclerosis cases but in none of the controls. CONCLUSION: The incidence of pericarditis and myocardial fibrosis is much greater than in controls. The results indicate that pericarditis is a primary disease (rather than secondary to uraemia).

Melanosis coli is associated with an increase in colonic epithelial apoptosis and not with laxative use.

We have investigated the clinical presentation, laxative use and histopathology of 38 patients with a histological diagnosis of melanosis coli and measured the colonic epithelial apoptosis in these cases. The presence of lipofuscin was confirmed in all cases. Fifteen of the cases had constipation, whilst eight had diarrhoea. Neither constipation nor diarrhoea was present in 13 cases and both were present, at different times, in two. Laxatives had been used in all those with constipation, in only one with diarrhoea and in none of the others. The mean apoptotic count was significantly increased in those with melanosis coli compared with the controls. In the majority of cases with constipation there was no other abnormality, whilst an additional diagnosis was present in the majority of the remainder. Colonic epithelial apoptosis was increased in melanosis coli and the majority of cases were not associated with laxative use. These results support the proposed role of apoptosis in melanosis coli, but indicate that melanosis coli is a non-specific marker of increased apoptosis with many possible causes, of which the use of laxatives is only one.

Pulmonary herpes simplex in burns patients.

In this study we aimed to determine the incidence of herpes simplex virus (HSV) in the lungs of burns patients, and its association with the presence of adult respiratory distress syndrome (ARDS) and pneumonia. Haematoxylin and eosin (H&E), and immunohistochemical (IHC) staining for HSV was performed on lung tissue from 54 patients who had died following burn injury and from nine control cases. Polymerase chain reaction (PCR) for HSV deoxyribonucleic acid (DNA) was performed on a subset both of burns cases and controls. No viral inclusions were detected in H&E sections, but 50% of the burns cases were positive for HSV by IHC staining; no control cases were positive. Nuclear and cytoplasmic immunopositivity for HSV was seen in macrophages and epithelial lining cells. HSV was strongly associated with ARDS (p=0.007), but not with pneumonia (p=0.577). The relative risk of HSV infection was higher for cases with ARDS (2.21) than for those with pneumonia (1.26). PCR for HSV DNA was positive in three out of five burns cases, and in one out of five control cases. Immunohistochemical staining is more sensitive for the detection of herpes simplex virus than haematoxylin and eosin staining for detection of viral inclusions. Burns cases have a high incidence of pulmonary herpes simplex virus infection. Polymerase chain reaction results may not be fully representative due to problems of tissue necrosis postmortem. Pulmonary herpes simplex virus is strongly associated with adult respiratory distress syndrome and the two may be causally linked. Early detection and treatment of pulmonary herpes simplex virus in burns patients may reduce pulmonary complications and mortality.