Detection of adrenomedullin and nitric oxide in different forms of periodontal disease.

BACKGROUND AND OBJECTIVE: The multifunctional molecules adrenomedullin (AM) and nitric oxide (NO) are both involved in the host response to microbial challenge during periodontal disease. Whether they coexist in periodontal inflammation and if equally produced in the different forms of periodontal disease has not previously been investigated. The aims of this study were to describe the locations of AM and NO in healthy and inflamed gingival tissues and to determine and compare their levels in the gingival crevicular fluid and saliva of patients with gingivitis, chronic periodontitis and aggressive periodontitis. MATERIAL AND METHODS: AM and inducible nitric oxide synthase (iNOS) were immunolocalized in clinically healthy and inflamed gingival tissue sections. The cells expressing AM and iNOS were characterized using immunocytochemistry with different markers for macrophages [cluster differentiation (CD)68 and CD14)], dendritic cells (CD83), neutrophils [neutrophil gelatinase-associated lipocalin (nGAL)] and natural killer cells (CD56). In an initial study, the levels of AM and NO were also measured in samples of gingival crevicular fluid and saliva obtained from patients with a diagnosis of gingivitis (n = 9), chronic periodontitis (n = 9) and aggressive periodontitis (n = 9) using an ELISA and the nitrate/nitrite (NO metabolites) Griess assay, respectively. RESULTS: Low levels of AM- and iNOS-expressing cells were detected in healthy gingival tissues in comparison with three-fold higher levels of these cells in inflamed tissues. These cells were localized mainly in the epithelial layer but were also present in deeper connective tissue. AM and iNOS were co-localized in particular cells within inflamed tissues, namely CD68(+) (52%) and CD14(+) (36%) macrophages, but also in nGAL(+) neutrophils (16%) and CD83(+) dendritic cells (14%). Interestingly, AM and NO levels in saliva were both found to be higher (p < 0.01) in patients with aggressive periodontitis than in patients with chronic periodontitis or gingivitis. In contrast, in gingival crevicular fluid, the levels of NO showed marked differences among patients with chronic periodontitis, aggressive periodontitis and gingivitis (p < 0.01), and the levels of AM were higher (p < 0.01) in both chronic and aggressive periodontitis compared with gingivitis alone. CONCLUSION: The data presented demonstrate a functional linkage between AM and NO in periodontal disease, with salivary and gingival crevicular fluid levels possibly associated with different forms and severities of periodontal disease. Exacerbated production of both AM and NO in saliva suggests their potential use as salivary markers of aggressive periodontitis.

Regulation of adrenomedullin and nitric oxide production by periodontal bacteria.

OBJECTIVES: In periodontitis the host response to bacterial challenge includes activity of the multifunctional molecules adrenomedullin (AM) and nitric oxide (NO). The aim of this study was to investigate the role of periodontal bacteria in regulating the production of these molecules from cultured cells. MATERIAL AND METHODS: Regulation of AM and NO production from oral keratinocytes when challenged with culture supernatants from Aggregatibacter actinomycetemcomitans, Campylobacter rectus, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia, Veillonella atypica, Streptococcus salivarius and Candida albicans was examined. AM and NO were measured in cell culture supernatants using an enzyme-linked immunosorbent assay and the nitrate/nitrite (NO metabolites) Griess assay respectively. Cellular production of AM and inducible NO synthase was also analysed in target cells by immunofluorescence and Western blot analysis. The inter-relationship of AM and NO production were further investigated with macrophages. RESULTS: A. actinomycetemcomitans and C. rectus induced maximal levels of both AM and NO after 6 and 48 h respectively from oral keratinocytes. AM production in macrophages was upregulated in response to the NO donor S-nitrosoglutathione and partially blocked by the inducible NO synthase inhibitor, N(ω) -Nitro-l-arginine methyl ester hydrochloride. Likewise, NO production was increased upon exposure to AM, while the AM receptor antagonist AM 22-52 reduced the release of NO. CONCLUSIONS: Pathogens associated with aggressive periodontitis, A. actinomycetemcomitans and C. rectus, were more effective than those associated with chronic periodontitis, P. gingivalis and Prev. intermedia, and commensals, S. salivarius and V. atypica, as regards the upregulation of AM and NO production from oral keratinocytes. Interaction between these molecules was also demonstrated with macrophages. Understanding the coordinated regulation of AM and NO production in response to periodontal bacteria may identify ways to promote their protective effects and minimize destructive potential.

Effects of hydroxyapatite and PDGF concentrations on osteoblast growth in a nanohydroxyapatite-polylactic acid composite for guided tissue regeneration.

The technique of guided tissue regeneration (GTR) has evolved over recent years in an attempt to achieve periodontal tissue regeneration by the use of a barrier membrane. However, there are significant limitations in the currently available membranes and overall outcomes may be limited. A degradable composite material was investigated as a potential GTR membrane material. Polylactic acid (PLA) and nanohydroxyapatite (nHA) composite was analysed, its bioactive potential and suitability as a carrier system for growth factors were assessed. The effect of nHA concentrations and the addition of platelet derived growth factor (PDGF) on osteoblast proliferation and differentiation was investigated. The bioactivity was dependent on the nHA concentration in the films, with more apatite deposited on films containing higher nHA content. Osteoblasts proliferated well on samples containing low nHA content and differentiated on films with higher nHA content. The composite films were able to deliver PDGF and cell proliferation increased on samples that were pre-absorbed with the growth factor. nHA-PLA composite films are able to deliver active PDGF. In addition the bioactivity and cell differentiation was higher on films containing more nHA. The use of a nHA-PLA composite material containing a high concentration of nHA may be a useful material for GTR membrane as it will not only act as a barrier, but may also be able to enhance bone regeneration by delivery of biologically active molecules.

Effects of low-dose doxycycline on cytokine secretion in human monocytes stimulated with Aggregatibacter actinomycetemcomitans.

Doxycycline is an antibiotic used in the treatment of a variety of inflammatory conditions, including periodontitis. Apart from its antimicrobial properties, this drug also has independent anti-inflammatory effects at sub-antimicrobial doses. The present study aimed to investigate the effects of low-doses of doxycycline (LDD) on cytokine production by human monocytic cells challenged with the periodontal pathogen Aggregatibacter actinomycetemcomitans, for up to 6 h. The simultaneous regulation of 12 cytokines were measured by a Human Cytokine Array Kit. To validate the array findings, selected cytokines were also measured by enzyme-linked immunosorbant assay (ELISA). A. actinomycetemcomitans stimulated the production of tumor necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1ß, IL-6 and IL-8 by the cells after 6 h of challenge, and doxycycline significantly inhibited this effect. The kinetics of this regulation demonstrated an early (within 2 h) and significant (P<0.05) inhibition of pro-inflammatory cytokines, with a mild (0.5-fold) up-regulation of the anti-inflammatory cytokine IL-10. The results indicate that LDD acts as an anti-inflammatory agent in human monocytic cells stimulated with A. actinomycetemcomitans. This model provides clear evidence that some of the clinically proven benefits of LDD may be related to its ability to regulate inflammatory mediator release by monocytic cells. This property may contribute to the clinically proven benefits of this antibiotic as an adjunctive treatment for periodontitis.

Absorption and release of protein from hydroxyapatite-polylactic acid (HA-PLA) membranes.

OBJECTIVES: The aim of this study was to investigate the kinetics of protein interactions with a novel hydroxyapatite-polylactide (HA-PLA) composite membrane material. METHODS: Trilayer PLA and HA-PLA composite membranes reinforced with PLA fibres were used to absorb and release protein which was measured by a BioRad assay. The proteins used were fetal calf serum and bovine serum albumin. PLA and HA-PLA composite films were manufactured to test permeability. RESULTS: Maximal protein absorption was seen within 5min of treating materials; a nearly 8-fold increase in total absorption was seen with HA-containing composites compared to those without HA. These also exhibited a more gradual and sustained release of protein for periods of up to 96h, for example at 24h protein concentrations released were 2.20+/-2.80 and 0.49+/-5.38microg/ml for membranes with and without HA respectively. In addition low pressure and temperature used during production of membranes also allowed greater and more sustained protein release. HA-PLA composite films also showed marked increased permeability compared to plain PLA films, for example after 24h PLA only films 3.64+/-1.01microg/ml, PLA film with 25% HA: 44.99+/-35.61microg/ml, PLA film with 75% HA: 153.12+/-65.57microg/ml. CONCLUSIONS: The results demonstrate that these composite membranes rapidly absorb protein and that the absorbed protein is released slowly for periods of up to 96h, dependent on constituents of the material and the manufacturing conditions. Incorporation of HA into these membranes was the key factor for improved protein kinetics and membrane permeability.

The T1799A point mutation is present in posterior uveal melanoma.

An activating mutation in exon 15 of the BRAF gene is present in a high proportion of cutaneous pigmented lesions. Until recently this mutation had however only been identified in one case of posterior uveal melanoma. Despite this apparent lack of the BRAF mutation, inappropriate downstream activation of the Ras/Raf/MAPK pathway has been described in posterior uveal melanoma. Based on the already recognised morphological and cytogenetic heterogeneity in uveal melanoma, we hypothesised that the BRAF mutation may be present in uveal melanoma but only in some of the tumour cells. In this study, we analysed 20 ciliary body and 30 choroidal melanomas using a nested PCR-based technique resulting in the amplification of a nested product only if the mutation was present. This sensitive technique can identify mutated DNA in the presence of wild-type DNA. The mutation was identified in 4 of 20 (20%) ciliary body and 11 of 30 (40%) choroidal melanomas. Further analysis of separate areas within the same choroidal melanoma demonstrated that the mutation was not present in the entire tumour. In conclusion, the T1799A BRAF mutation is present in a proportion of posterior uveal melanomas but within these tumours the distribution of the mutation is heterogeneous.

Porphyromonas gingivalis antagonises Campylobacter rectus induced cytokine production by human monocytes.

Porphyromonas gingivalis and Campylobacter rectus are two major bacterial species implicated in the pathogenesis of periodontitis. P. gingivalis can antagonise the inflammatory response to other periodontal pathogens, a property commonly attributed to its lipopolysaccharide (LPS). The aim of this study was to investigate the capacity of P. gingivalis to antagonise C. rectus induced cytokine stimulation from human monocytes, and to investigate the involvement of its LPS. Primary human monocytes and Monomac-6 cells were challenged with culture supernatants from P. gingivalis and C. rectus, and levels of IL-1beta, IL-6 and IL-8 produced were measured by ELISA after 6h incubation. Purified P. gingivalis LPS was also added alone or in combination with C. rectus culture supernatant. Both species significantly stimulated the production of all three cytokines from the two cell lines, but P. gingivalis was considerably weaker inducer. Co-stimulation of the cells with P. gingivalis and C. rectus suppressed the cytokine-stimulatory capacity of the latter. P. gingivalis LPS alone was sufficient to antagonise IL-6 and IL-8, but not IL-1beta stimulation by C. rectus. In conclusion, mixed infections may impair host immune responses by reducing pro-inflammatory cytokine levels, which may be of relevance to the pathogenesis of periodontitis.

Differential expression of receptor activator of nuclear factor-kappaB ligand and osteoprotegerin mRNA in periodontal diseases.

BACKGROUND AND OBJECTIVE: Receptor activator of nuclear factor-kappaB ligand (RANKL) is responsible for the induction of osteoclastogenesis and bone resorption, whereas its decoy receptor, osteoprotegerin, can directly block this action. Because this dyad of cytokines is crucial for regulating the bone remodelling process, imbalances in their expression may cause a switch from the physiological state to enhanced bone resorption or formation. This study investigated the mRNA expression of RANKL and osteoprotegerin, as well as their relative ratio, in the gingival tissues of patients with various forms of periodontal diseases. MATERIAL AND METHODS: Gingival tissue was obtained from nine healthy subjects and 41 patients, who had gingivitis, chronic periodontitis, generalized aggressive periodontitis, and chronic periodontitis and were receiving immunosuppressant therapy. Quantitative real-time polymerase chain reaction was employed to evaluate the mRNA expression of RANKL and osteoprotegerin in these tissues. RESULTS: Compared with healthy individuals, patients in all periodontitis groups, but not those with gingivitis, exhibited stronger RANKL expression and a higher relative RANKL/osteoprotegerin ratio. In addition, osteoprotegerin expression was weaker in patients with chronic periodontitis. When patients with generalized aggressive periodontitis and chronic periodontitis were compared, the former exhibited stronger RANKL expression, whereas the latter exhibited weaker osteoprotegerin expression, and there was no difference in their relative ratio. When chronic periodontitis patients were compared with chronic periodontitis patients receiving immunosuppressant therapy, osteoprotegerin, but not RANKL, expression was stronger in the latter. CONCLUSION: This study demonstrates that RANKL and osteoprotegerin expression are differentially regulated in various forms of periodontitis, and the relative RANKL/osteoprotegerin ratio appears to be indicative of disease occurrence. This information may confer diagnostic and therapeutic value in periodontitis.

Interleukin-1alpha stimulation in monocytes by periodontal bacteria: antagonistic effects of Porphyromonas gingivalis.

Periodontal pathogenic bacteria are associated with elevated levels of interleukin-1alpha (IL-1alpha) but it is unclear if all species can induce cytokine production equally. Porphyromonas gingivalis may be able antagonize IL-1alpha induced by other species through the activity of its proteases or lipopolysaccharide (LPS). Monomac-6 cells and primary human monocytes were treated with culture supernatants from Porphyromonas gingivalis, Fusobacterium nucleatum, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedius, Veillonella atypical and Prevotella nigrescens. IL-1alpha protein levels were measured after 6 h of incubation. In addition, monocytes were co-stimulated with supernatants from P. gingivalis and other bacteria. The role of P. gingivalis proteases was tested using Arg-X and Lys-X mutant strains. The role of LPS was investigated using purified P. gingivalis LPS and polymixin depletion. All species tested induced significant IL-1alpha production, but P. gingivalis was the weakest. Co-stimulation of monocytes with P. gingivalis antagonized the ability of other bacterial species to induce IL-1alpha production. This effect was at its greatest with C. rectus (resulting in a 70% reduction). Gingipain mutant strains and chemical inhibition of protease activity did not reduce antagonistic activity. However, 100 ng/ml of P. gingivalis LPS can reproduce the antagonistic activity of P. gingivalis culture supernatants. Periodontitis-associated bacterial species stimulate IL-1alpha production by monocytes. P. gingivalis can antagonize this effect, and its LPS appears to be the crucial component. This study highlights the importance of mixed infections in the pathogenesis of periodontal disease because reduction of pro-inflammatory cytokine levels may impair the ability of the host to tackle infection.

Parasites of domestic and wild animals in South Africa. XLVII. Ticks of tortoises and other reptiles.

A total of 586 reptiles, belonging to 35 species and five subspecies, were examined in surveys aimed at determining the species spectrum and geographic distribution of ticks that infest them. Of these reptiles 509 were tortoises, 28 monitor or other lizards, and 49 snakes. Nine ixodid tick species, of which seven belonged to the genus Amblyomma, and one argasid tick, Ornithodoros compactus were recovered. Seven of the ten tick species are parasites of reptiles. Amongst these seven species Amblyomma marmoreum was most prevalent and numerous on leopard tortoises, Geochelone pardalis; Amblyomma nuttalliwas present only on Bell's hinged tortoises, Kinixys belliana; and most Amblyomma sylvaticum were collected from angulate tortoises, Chersina angulata. Amblyomma exornatum (formerly Aponomma exornatum) was only recovered from monitor lizards, Varanus spp.; most Amblyomma latum (formerly Aponomma latum) were from snakes; and a single nymph of Amblyomma transversale (formerly Aponomma transversale) was collected from a southern African python, Python natalensis. All 30 Namaqualand speckled padloper tortoises, Homopus signatus signatus, examined were infested with O. compactus. The seasonal occurrence of A. sylvaticum and the geographic distribution of this tick and of A. marmoreum, A. nuttalli, A. exornatum, A. latum and O. compactus are illustrated.

Hosts, seasonality and geographic distribution of the South African tortoise tick, Amblyomma marmoreum.

The tortoise tick Amblyomma marmoreum was collected from large numbers of reptiles and other animals during the course of numerous surveys conducted in South Africa. A total of 1229 ticks, of which 550 were adults, were recovered from 309 reptiles belonging to 13 species, with leopard tortoises, Geochelone pardalis being the most heavily infested. The 269 birds sampled harboured 4901 larvae, 217 nymphs and no adult ticks, and the prevalence of infestation was greatest on helmeted guineafowls, Numida meleagris. Only two larvae were recovered from 610 rodents, including 31 spring hares, Pedetes capensis, whereas 1144 other small mammals yielded 1835 immature ticks, of which 1655 were collected from 623 scrub hares, Lepus saxatilis. The 213 carnivores examined harboured 2459 ticks of which none were adult. A single adult tick and 6684 larvae and 62 nymphs were recovered from 656 large herbivores, and a total of 4081 immature ticks and three adults were collected from 1543 domestic animals and 194 humans. Adult male and female A. marmoreum were most numerous on reptiles during January and February, and larvae during March. The largest numbers of larvae were present on domestic cattle and helmeted guineafowls in the Eastern Cape Province during March or April respectively, whereas larvae were most numerous on helmeted guineafowls, scrub hares and the vegetation in north-eastern Mpumalanga Province during May. In both provinces nymphs were most numerous between October and December. Amblyomma marmoreum appears to be most prevalent in the western regions of the Western and Eastern Cape and Free State provinces, and the north-eastern regions of the Northern Cape, KwaZulu-Natal, Mpumulanga and Limpopo provinces.

Is carotid duplex scanning sufficient as the sole investigation prior to carotid endarterectomy?

Carotid endarterectomy (CEA) is the accepted treatment for certain patients who have had, or who are at risk of having, a stroke if they have a significant narrowing of the internal carotid artery. Rapid and accurate classification of the degree of stenosis is important as the benefit of surgery is highly dependent on this. The aim of this study was to assess whether the addition of angiography to duplex scanning resulted in a change in patient management in a unit where duplex scanning was used as the sole imaging investigation prior to CEA. The study population consisted of 64 patients with significant internal carotid artery stenosis on duplex scanning who were suitable for, and wished to be considered for, CEA. All patients underwent an angiogram. In this study 9 (14%) patients did not proceed to surgery on the basis of angiography and in a further 11 (17%) patients insufficient views of the distal vessel were obtained on duplex scanning. Three of these patients had extensive disease which excluded surgery. One patient experienced a transient ischaemic attack (TIA) at the time of angiography. In conclusion, this audit has highlighted the limitations in performing duplex scanning alone, and the costs that this can incur on the patient who may undergo an unnecessary operation. We cannot recommend duplex scanning as the sole investigation prior to CEA. There is need to evaluate the role of additional non-invasive carotid imaging such as magnetic resonance angiography or CT angiography in the assessment of these patients.

Assessing the utility of the stop dialysate flow method in patients receiving haemodiafiltration.

BACKGROUND: The stop dialysate flow (SDF) method of post-dialysis urea sampling is the most commonly used method in the UK. It can also be used with a published formula to predict 30 min equilibrated urea accurately. The method has not been validated in patients undergoing haemodiafiltration (HDF). Given the increased use of HDF across Europe, we felt it prudent to assess the utility of the SDF method and prediction equation in this modality. METHODS: Fourteen patients from two renal units were studied. Blood samples were taken at 1 min intervals from the arterial side of the dialysis circuit in the first 5 min after HDF had ceased whilst blood circulation continued. A peripheral sample was taken from the contralateral arm immediately after HDF had ceased and a 30 min sample was taken from the arterial needle. These samples were used to assess the utility of 5 min arterial blood urea and the 30 min prediction formula, respectively. RESULTS: Blood urea measured from the arterial circuit at 5 min correlated closely with the contralateral sample taken immediately post-HDF, with no significant difference (6.45+/-2.11 vs 6.52+/-2.19 mmol/l, P = 0.39). The use of 5 min arterial blood urea and prediction formula allowed an accurate prediction of 30 min urea (R2 = 0.96). CONCLUSIONS: The use of the SDF method with a 5 min post-HDF arterial sample is valid in patients receiving HDF. The previously published prediction formula for estimating 30 min urea is also valid using the 5 min post-HDF sample.

Alphagan allergy may increase the propensity for multiple eye-drop allergy.

AIMS: Since its introduction in 1996, brimonidine tartrate 0.2% ophthalmic solution (Alphagan, Allergan) twice daily has become established as an effective intra ocular pressure-lowering treatment. While the efficacy of Alphagan cannot be questioned, we gained the clinical impression that the drug has an unacceptably high rate of allergy. Of greater concern, we suspected that patients suffering from local Alphagan allergy had a higher rate of allergy to subsequently used topical preparations. We analysed data from a large scale study of glaucoma patients to establish whether our suspicions were correct. SUBJECTS AND METHODS: We have created a database of the entire glaucoma treatment histories for consecutive patients attending a single consultant's clinics (DMIM) at Glasgow Royal Infirmary between May 1999 and September 2001. All have undergone medical treatment for primary open angle glaucoma, ocular hypertension, or normal tension glaucoma. Patients with any other form of glaucoma, and patients in whom a full record of treatment was not available were excluded from the study. RESULTS: Alphagan was discontinued due to allergy on 73 per 100,000 patient treatment days. This was a far higher frequency than for other preparations. In patients allergic to both Alphagan and another preparation (Timoptol, Trusopt and Xalatan), the mean interval between the first and second allergy was shorter when Alphagan allergy occurred first. This was statistically significant in Timoptol and Trusopt cross-reactivity. CONCLUSIONS: Alphagan has high allergenicity, and may increase the likelihood of allergy to subsequently used preparations.

Bilateral uveal melanoma: a series of four cases.

AIM: To describe the occurrence of bilateral primary choroidal melanoma in four patients. METHODS: All patients attending the Liverpool Ocular Oncology Centre with uveal melanoma between January 1993 and February 2002 were identified, and those with bilateral primary choroidal melanoma were reviewed. Their presentation and management are described. RESULTS: Four patients, all female, were identified. Patient 1 presented with a right juxtapapillary melanoma at the age of 64, which was treated with krypton laser and endoresection, and then when aged 73 required proton beam radiotherapy for a melanoma in her left eye. Patient 2 presented at the age of 82 with bilateral choroidal melanomas and underwent simultaneous bilateral plaque radiotherapy. Patient 3 presented with bilateral choroidal melanomas at the age of 75 and was treated initially with bilateral proton beam radiotherapy. Patient 4 was treated at the age of 54 with right plaque radiotherapy for a choroidal melanoma, and 3 years later needed plaque radiotherapy for a melanoma in her other eye. CONCLUSION: Bilateral choroidal melanoma is possible and should be a consideration in the continuing management of patients with choroidal melanoma.

Indirect cytotoxicity evaluation of pseudowollastonite.

This study aimed to evaluate the cytotoxicity of substances leached by pseudowollastonite (CaSiO(3)). It has been previously shown that calcium (Ca(2+)) and silicate (SiO(3)(-)) ions are released from pseudowollastonite into biological solutions. Both of these ions are known to influence the biological metabolism of osteoblastic cells essential in the mineralization process and bone-bonding mechanism. The indirect toxicity evaluation was performed by extraction method, according to International Standard Organization (ISO). Pseudowollastonite pellets obtained by solid-state reaction were incubated, in culture medium, during 24, 48, 72 or 168 h at different concentrations (5, 10, 15, 50, 100, 200 mg/ml). The cytotoxicity of each extract in presence of human osteoblastic cell line (SaOS-2) was quantitatively assessed by measuring the viability (succinate dehydrogenase activity, MTT), the membrane integrity (the uptake of the neutral red by viable cells, NR) as well as the cell necrosis by measuring the lactate dehydrogenase (LDH) released in the culture medium. No significant alteration of membrane integrity or cell suffering was detectable. However, increased cell metabolism was observed for cells exposed to pseudowollastonite extract with longest extraction time (168 h). In conclusion, mineral elements leached by pseudowollastonite do not significantly affect the metabolism of osteoblastic cells.

Validation of a simple, rapid, and economical technique for distinguishing type 1 and 2 fibres in fixed and frozen skeletal muscle.

AIMS: To produce a method of distinguishing between type 1 and 2 skeletal muscle fibres that would be more economical and reproducible than the standard ATPase method and be applicable to both fixed and frozen tissue. Because the ATPase method has been accepted as the basis for fibre identification for the past 50 years, the new method should not give significantly different results. METHODS: Isoforms of myosin correlate with isoforms of myofibrillar ATPase and an immunohistochemical (IHC) double labelling protocol was devised using monoclonal antibodies to fast and slow myosin. This required one tissue section rather than four. The results of the two methods were compared by means of morphometric analysis of skeletal muscle biopsies from 20 normal healthy volunteers. RESULTS: There were no significant differences (p = 0.57) in the percentages of type 1 (46% using the IHC method v 48% using ATPase) or type 2 fibres (54% v 52%, respectively). The 2a and 2b subtypes were distinguished easily. Analysis of variance revealed that cross sectional area (mu m(2)), diameter (mu m), form factor, and density of fibre staining (a measure of substrate-enzyme or protein) were all similar. The method worked equally well on fixed material. CONCLUSION: An IHC method based on the fast and slow isoforms of myosin shows no significant differences in fibre type analysis from the standard ATPase method although it provides important advantages because it is applicable to fixed (including archival) material, is economical and reproducible, and yields a permanent preparation.

Antiviral pathway activation in patients with chronic fatigue syndrome and acute infection.

Gene expression of key enzymes in 2 antiviral pathways (ribonuclease latent [RNase L] and RNA-regulated protein kinase [PKR]) was compared in 22 patients with chronic fatigue syndrome (CFS), 10 patients with acute gastroenteritis, and 21 healthy volunteers. Pathway activation in the group of patients with infections differed significantly from that of the other 2 groups, in whom there was no evidence of upregulation. Therefore, assay of activation is unlikely to provide the basis for a diagnostic test for CFS.

Human beta defensin-1 and -2 expression in human pilosebaceous units: upregulation in acne vulgaris lesions.

A rich residential microflora is harboured by the distal outer root sheath of the hair follicle and the hair canal - normally without causing skin diseases. Although the basic mechanisms involved in the development of inflammation during acne vulgaris remain unclear, microbial agents might play an important role in this process. In this study we have analyzed by in situ hybridization and immunohistochemistry the expression patterns of two antimicrobial peptides, human beta defensin-1 and human beta defensin-2, in healthy human hair follicles as well as in perilesional and intralesional skin of acne vulgaris lesions such as comedones, papules, and pustules. Strong defensin-1 and defensin-2 immunoreactivity was found in all suprabasal layers of the epidermis, the distal outer root sheath of the hair follicle, and the pilosebaceous duct. Marked defensin-1 and defensin-2 immunoreactivity was also found in the sebaceous gland and in the basal layer of the central outer root sheath including the bulge region. The majority of acne biopsies displayed a marked upregulation of defensin-2 immunoreactivity in the lesional and perilesional epithelium - in particular in pustules - and a less marked upregulation of defensin-1 immunoreactivity. The upregulation of beta-defensin expression in acne vulgaris lesions compared to controls suggests that beta-defensins may be involved in the pathogenesis of acne vulgaris.