A computerised decision support system for cardiovascular risk management 'live' in the electronic health record environment: development, validation and implementation-the Utrecht Cardiovascular Cohort Initiative.

PURPOSE: We set out to develop a real-time computerised decision support system (CDSS) embedded in the electronic health record (EHR) with information on risk factors, estimated risk, and guideline-based advice on treatment strategy in order to improve adherence to cardiovascular risk management (CVRM) guidelines with the ultimate aim of improving patient healthcare. METHODS: We defined a project plan including the scope and requirements, infrastructure and interface, data quality and study population, validation and evaluation of the CDSS. RESULTS: In collaboration with clinicians, data scientists, epidemiologists, ICT architects, and user experience and interface designers we developed a CDSS that provides 'live' information on CVRM within the environment of the EHR. The CDSS provides information on cardiovascular risk factors (age, sex, medical and family history, smoking, blood pressure, lipids, kidney function, and glucose intolerance measurements), estimated 10-year cardiovascular risk, guideline-compliant suggestions for both pharmacological and non-pharmacological treatment to optimise risk factors, and an estimate on the change in 10-year risk of cardiovascular disease if treatment goals are adhered to. Our pilot study identified a number of issues that needed to be addressed, such as missing data, rules and regulations, privacy, and patient participation. CONCLUSION: Development of a CDSS is complex and requires a multidisciplinary approach. We identified opportunities and challenges in our project developing a CDSS aimed at improving adherence to CVRM guidelines. The regulatory environment, including guidance on scientific evaluation, legislation, and privacy issues needs to evolve within this emerging field of eHealth.

The effect of contact load on CoCrMo wear and the formation and retention of tribofilms.

Tribochemical reactions in a protein lubricated metal-on-metal (MoM) sliding contact may play a significant role for its wear performance. Such reactions lead to the formation of a carbonaceous 'tribofilm', which can act as a protective layer against corrosion and wear. The purpose of this study was to determine the effect of contact load on wear and the formation and retention of tribofilms. Wear tests were performed in a custom-made ball-on-flat testing apparatus that incorporated an electrochemical cell. A ceramic ball was used to articulate against low-carbon wrought CoCrMo alloy pins in bovine serum. Using a range of contact loads at a single potentiostatic condition (close to free potential), weight loss and changes in surface properties were evaluated. We determined that wear was influenced by the loading condition. As expected, wear increased with load, but the association between applied load and measured weight loss was not linear. In the intermediate load region, in the range of 32-48 N (~58-80 MPa), there was more than an order of magnitude drop in the wear per unit load, and the wear versus load data suggested an inflexion point at 49 N. Regression analyses yielded a cubic model (R2=0.991; p=0.0002), where the cubic term, which represents the inflexion, was highly significant (p=0.0021). This model is supported by the observations that the minimum in the friction versus load curve is at 52 N and the highest relative increase in polarization resistance occurred at 49 N. Scanning electron microscopy and Raman spectroscopy indicated the absence of a tribofilm for the low and within the contact area of the high load cases. Synergistic interactions of wear and corrosion seem to play an important role.

Fretting-corrosion in Hip Implant Modular Junctions: New Experimental Set-up and Initial Outcome.

Modern hip prostheses feature a modular implant design with at least one tapered junction. This design can lead to several complications due to the introduction of additional interfaces, which are subjected to various loading conditions and micromotion. The main objective of current study is to develop a fretting corrosion apparatus, which is able characterize the mechanical and electrochemical behaviour of various existing metal alloy couples during fretting motion. This study describes the design and the main considerations during the development of a novel fretting corrosion apparatus, as well as determination of the machine compliance and the initial testing results. Machine compliance considerations and frictional interactions of the couples are discussed in detail. For the preliminary tests, metal alloy pins, made of Ti6Al4V and wrought high-carbon CoCrMo were mechanically polished to a surface roughness of less than 20nm. 2 pins (Diameter = 11mm) of either Ti6Al4V or CoCrMo were loaded onto a Ti6Al4V alloy rod at a normal force of 200N. The interface types included: Ti6Al4V-Ti6Al4V-Ti6Al4V, Ti6Al4V-Ti6Al4V-CoCrMo, and CoCrMo-Ti6Al4V-CoCrMo. The Ti6Al4V rod articulated against the metal alloy pins in a sinusoidal fretting motion with a displacement amplitude of ±50µm. Bovine calf serum (30g/L of protein content) was selected as a lubricant and tested at 2 different pH levels (pH 3.0 and 7.6). In all cases, current and friction energy were monitored during the fretting process. The results indicated distinct, material-specific current evolutions and friction energies. No significant differences were observed in electrochemical or mechanical behaviour in response to pH change. In general, Ti6Al4V-Ti6Al4V-Ti6Al4V couples displayed the earliest passivation and superior electrochemical behaviour compared to Ti6Al4V-Ti6Al4V-CoCrMo and CoCrMo-Ti6Al4V-CoCrMo under fretting conditions. In addition, fluctuations in current were observed in specific regions at all instances where Ti6Al4V was coupled with Ti6Al4V. These fluctuations were not observed in instances where Ti6Al4V was coupled with CoCrMo. These findings suggest transitions in the degradation mechanisms at the modular junction as a function of material couples/contacts. The findings may assist in improving the current hip modular junctions.

What do we know about taper corrosion in total hip arthroplasty?

Mechanically assisted crevice corrosion (MACC) at metal/metal modular junctions in which at least one of the components is fabricated from cobalt-chromium alloy, has reemerged as a potential clinically significant complication in total hip arthroplasty. The clinical manifestation of MACC may include the development of an adverse local tissue reaction (ALTR), similar to what has been described in association with metal-on-metal bearing total hip and resurfacing arthroplasty. The clinical presentation of MACC-associated ALTRs may include pain and possibly late recurrent dislocations. Abnormal metal artifact reduction sequence magnetic resonance images and elevated serum metal levels (cobalt elevations out of proportion to chromium elevations) can be helpful in the diagnosis of these MACC-associated ALTRs.

Prospective study on serum metal levels in patients with metal-on-metal lumbar disc arthroplasty.

PURPOSE: Metal-on-metal total disc replacement is a recent alternative treatment for degenerative disc disease. Wear and corrosion of these implants can lead to local and systemic transport of metal debris. This prospective longitudinal study examined the serum chromium and cobalt levels in 24 patients with cobalt-chromium alloy metal-on-metal lumbar disc replacements. METHODS: Serum was assayed for chromium (Cr) and cobalt (Co) using high-resolution inductively-coupled plasma-mass spectrometry. Detection limits were 0.015 ng/mL for Cr and 0.04 ng/mL for Co. RESULTS: Median serum Co levels at pre-op, 3, 6, 12, 24, and 36-months post-op were 0.10, 1.03, 0.96, 0.98, 0.67, and 0.52 ng/mL, respectively. Median serum Cr levels were 0.06, 0.49, 0.65, 0.43, 0.52, and 0.50 ng/mL, respectively. CONCLUSION: In general, these results indicated that serum Co and Cr levels are elevated at all postoperative time points and are of the same order of magnitude as those observed in well-functioning metal-on-metal surface replacements of the hip and in metal-on-metal total hip replacements at similar postoperative time points.

Senescence bypass screen identifies TBX2, which represses Cdkn2a (p19(ARF)) and is amplified in a subset of human breast cancers.

To identify new immortalizing genes with potential roles in tumorigenesis, we performed a genetic screen aimed to bypass the rapid and tight senescence arrest of primary fibroblasts deficient for the oncogene Bmi1. We identified the T-box member TBX2 as a potent immortalizing gene that acts by downregulating Cdkn2a (p19(ARF)). TBX2 represses the Cdkn2a (p19(ARF)) promoter and attenuates E2F1, Myc or HRAS-mediated induction of Cdkn2a (p19(ARF)). We found TBX2 to be amplified in a subset of primary human breast cancers, indicating that it might contribute to breast cancer development.

Tribocorrosion behavior of CoCrMo alloy for hip prosthesis as a function of loads: a comparison between two testing systems.

Metal-on-metal (MOM) hip prosthesis bearings have enjoyed renewed popularity, but concerns remain with wear debris and metal ion release causing a negative response in the surrounding tissues. Further understanding into the wear and corrosion mechanisms occurring in MOM hips is therefore essential.The purpose of this study was to evaluate the tribocorrosion behaviour, or interplay between corrosion and wear, of a low-carbon CoCrMo alloy as a function of loading. The tribocorrosion tests were performed using two tribometer configurations. In the first configuration, "System A", a linearly reciprocating alumina ball slid against the flat metal immersed in a phosphate buffer solution (PBS). In the second configuration, "System B", the flat end of a cylindrical metal pin was pressed against an alumina ball that oscillated rotationally, using bovine calf serum (BCS) as the lubricant and electrolyte. System B was custom-built to emulate in vivo conditions. The tribocorrosion tests were performed under potentiostatic conditions at -0.345V, with a sliding duration of 1800 seconds and a frequency of 1Hz. In System A the applied loads were 0.05, 0.5, and 1N (138, 296 and 373MPa, respectively) and in System B were 16, 32, and 64N (474, 597, and 752MPa, respectively). Electrochemical impedance spectroscopy (EIS) and polarization resistance were estimated. The total mass loss (K(wc)) in the CoCrMo was determined. The mass loss due to wear (K(w)) and that due to corrosion (K(c)) were determined. The dominant wear regime for the CoCrMo alloy subjected to sliding changes from wear-corrosion to mechanical wear as the contact stress increases. An attempt was made to compare both system, in their tribochemical responses and formulate some insights in the total degradation processes. Our results also suggest that the proteins in the serum lubricant assist in the generation of a protective layer against corrosion during sliding. The study highlights the need of adequate methodology/guidelines to compare the results from different test systems and translating in solving the practical problems.

Neutralizing antibodies mediate virus-immune pathology of COVID-19.

SARS-CoV-2 is a novel beta-coronavirus causing over 200.000 lethal cases within six months of first infecting humans. SARS-CoV-2 causes COVID-19, a form of severe acute respiratory syndrome (SARS). COVID-19 is characterized by two phases: the first resembles the flu with pneumonia, but after about seven or eight days the disease suddenly worsens to a sepsis-like syndrome. It is difficult to explain this virus-immune-pathology sequence from virology or immunology only. This paper hypothesizes that host-produced anti-spike protein antibodies are responsible for immune-induced viral dissemination. Subsequently, systemic distribution of virus-antibodies complexes activates the immune pathology observed in severe COVID-19. This hypothesis may be counterintuitive to immunologist that consider many anti-spike antibodies to be virus-neutralizing antibodies. Although anti-spike antibodies may hinder infection of epithelial cells, antibody binding to the spike protein may facilitate virus infection of myeloid leukocytes. If myeloid leukocytes reenter the circulation, they could spread the virus from a locoregional infection to a systemic disease. Disseminated virus in combination with antibodies results in dispersed virus-antibody complexes that overstimulate the immune system. The hypothesis aligns with the sequences of virus, immune and pathological events in COVID-19. The delay in onset from both syndromes results from an immune system still naïve to the non-cross-reactive spike protein. Details of this hypothesis are in concordance with many clinical characteristics of COVID-19, including its predominant lethality for the elderly, and the mostly asymptomatic course of disease in children. It predicts putative detrimental effects of vaccines that induce virus-neutralizing antibodies against the spike protein, as has been shown for other coronaviruses. This hypothesis has consequences for treatment of patients, evaluation of personal and herd immunity and vaccine development. In patients, cellular immunity should be stimulated. Neutralizing antibodies might not be indicative for immunity. Vaccines should aim to stimulate cellular immunity COVID-19 and/or stimulate humoral immunity against viral proteins except for the immunodominant spike protein.

[Small risk of developing Lyme borreliosis following a tick bite on Ameland: research in a general practice]. / Kleine kans op lymeborreliose na een tekenbeet op Ameland: onderzoek in een huisartsenpraktijk.

OBJECTIVE: To investigate the percentage of ticks infected with Borrelia burgdorferi on the Dutch North Sea island of Ameland, and the risk of developing Lyme disease following tick bite on the island. DESIGN: Prospective, observational. METHOD: Ticks were collected from patients who visited a general practitioner and were tested for the DNA of B. burgdorferi. After 6 months the patients were interviewed by phone using a standardised questionnaire. RESULTS: From 2004-2006, 216 ticks were collected from 167 persons. Most ticks were removed within 24 hours. In 44 ticks (20.4%) B. burgdorferi DNA was detected. Follow up information was available on 146 persons, 41 (28.1%) of whom had been bitten by a Borrelia-positive tick. None of the persons developed a typical erythema migrans. From the 13 persons (9%) reporting a non-specific redness of the skin (diameter less than 5 cm) at the site of the tick bite, 5 had been bitten by a positive tick and 8 by a negative tick. One patient bitten by a positive tick reported systemic symptoms related to Lyme borreliosis, namely fatigue, perspiration and joint ache, without local redness. CONCLUSION: The probability of developing Lyme borreliosis was low even though a relatively large percentage of the ticks collected were positive for B. burgdorferi. This is probably connected to the fact that in the majority of cases the tick had been removed within 24 hours.

Treatment of ocular squamous cell carcinomas in cattle with interleukin-2.

In total, 174 bovine ocular squamous cell carcinomas of varying sizes (20 to 2800 mm(2) in area) were treated daily with peritumoural injections of solvent, or solvent containing 5000 U, 20,000 U, 200,000 U, 500,000 U, 1 million U or 2 million U interleukin-2 (IL-2) for 10 days. The tumours were measured and clinically staged before treatment and at one, three, four, nine and 20 months after treatment. After 20 months, 14 per cent of the tumours treated with the solvent had regressed completely, a significantly smaller proportion than the 55 per cent treated with 5000 U IL-2, 52 per cent treated with 20,000 U IL-2, 58 per cent treated with 200,000 U IL-2, 50 per cent treated with 500,000 U IL-2, 69 per cent of tumours treated with 1 million U IL-2, 52 per cent treated with 2 million U IL-2. The tumours on the third eyelid and limbus were the most responsive.

The state of methamphetamine ('tik') use among youth in the Western Cape, South Africa.

BACKGROUND: Methamphetamine use among youth in the Western Cape Province of South Africa has increased at alarming rates over the past decade. Although current estimates of youth use exist, they range from 2 - 12%. OBJECTIVES: To identify (i) the prevalence of methamphetamine use in Western Cape youth and (ii) the association between use and known risk factors for methamphetamine use. METHODS: Data were obtained from 10 000 Western Cape Province Grade 8 learners in 54 secondary schools (mean age 14.0 years). Prevalence was descriptively reported while risk factors for past-month use were modelled in a hierarchical logistic regression with demographic, socioeconomic status, substance use, sexual activity and relationship predictors. RESULTS: Approximately 5% (n=496) of learners had used methamphetamine within their lifetime. Of these users, 65% (n=322) had used in the past month or week. Compared to never users, past-month users were more likely to be male, less likely to have a present or partially present mother, less likely to live in an apartment/flat/brick house, more likely to have used alcohol and tobacco and more likely to report having a same-sex partner. CONCLUSION: Results replicate previously known methamphetamine risk factors and highlight the need to address methamphetamine use in comprehensive prevention initiatives.

Systemic cobalt toxicity from total hip arthroplasties: review of a rare condition Part 1 - history, mechanism, measurements, and pathophysiology.

UNLABELLED: Recently, the use of metal-on-metal articulations in total hip arthroplasty (THA) has led to an increase in adverse events owing to local soft-tissue reactions from metal ions and wear debris. While the majority of these implants perform well, it has been increasingly recognised that a small proportion of patients may develop complications secondary to systemic cobalt toxicity when these implants fail. However, distinguishing true toxicity from benign elevations in cobalt ion levels can be challenging. The purpose of this two part series is to review the use of cobalt alloys in THA and to highlight the following related topics of interest: mechanisms of cobalt ion release and their measurement, definitions of pathological cobalt ion levels, and the pathophysiology, risk factors and treatment of cobalt toxicity. Historically, these metal-on-metal arthroplasties are composed of a chromium-cobalt articulation. The release of cobalt is due to the mechanical and oxidative stresses placed on the prosthetic joint. It exerts its pathological effects through direct cellular toxicity. This manuscript will highlight the pathophysiology of cobalt toxicity in patients with metal-on-metal hip arthroplasties. TAKE HOME MESSAGE: Patients with new or evolving hip symptoms with a prior history of THA warrant orthopaedic surgical evaluation. Increased awareness of the range of systemic symptoms associated with cobalt toxicity, coupled with prompt orthopaedic intervention, may forestall the development of further complications.

Systemic cobalt toxicity from total hip arthroplasties: review of a rare condition Part 2. measurement, risk factors, and step-wise approach to treatment.

UNLABELLED: As adverse events related to metal on metal hip arthroplasty have been better understood, there has been increased interest in toxicity related to the high circulating levels of cobalt ions. However, distinguishing true toxicity from benign elevations in cobalt levels can be challenging. The purpose of this review is to examine the use of cobalt alloys in total hip arthroplasty, to review the methods of measuring circulating cobalt levels, to define a level of cobalt which is considered pathological and to review the pathophysiology, risk factors and treatment of cobalt toxicity. To the best of our knowledge, there are 18 published cases where cobalt metal ion toxicity has been attributed to the use of cobalt-chromium alloys in hip arthroplasty. Of these cases, the great majority reported systemic toxic reactions at serum cobalt levels more than 100 µg/L. This review highlights some of the clinical features of cobalt toxicity, with the goal that early awareness may decrease the risk factors for the development of cobalt toxicity and/or reduce its severity. TAKE HOME MESSAGE: Severe adverse events can arise from the release of cobalt from metal-on-metal arthroplasties, and as such, orthopaedic surgeons should not only be aware of the presenting problems, but also have the knowledge to treat appropriately.

Specific expression in mouse mesoderm- and neural crest-derived tissues of a human PDGFRA promoter/lacZ transgene.

The platelet-derived growth factor alpha-receptor (PDGFR-alpha) displays a lineage-specific expression pattern in the mouse embryo and is required for normal development of mesoderm and cephalic neural crest derivatives. The purpose of the present study was to demonstrate the in vivo promoter function of genomic DNA fragments representing the 5'-flanking part of the human PDGFRA gene. 2.2, 0.9 and 0.4 kb PDGFRA promoter fragments, ligated to a lacZ reporter gene, were microinjected into fertilized mouse eggs and transgenic mouse lines were established. The expression patterns were basically similar in the 2.2 and 0.9 kb lines and overlapped grossly the endogenous Pdgfra gene expression pattern. The transgenic line with the highest expression level was chosen for detailed analysis. Expression was, as expected, mainly confined to tissues of mesodermal and neural crest origin. No expression was found in epithelial tissues of endo- or ectodermal origin. The promoter fragments were also active in neuroepithelium and in certain neuronal cell types that did not faithfully express PDGFR-alpha mRNA, while they failed to specify reporter expression in PDGFR-alpha expressing O-2A progenitor cells and other glial elements of the central nervous system. Thus, the isolated human PDGFRA promoter contains most but not all of the regulatory elements that are necessary to establish tissue specific gene expression during development.

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta.

Two mutants of Tagetes erecta displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M2 population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C13 monothiophene 2-(but-3-en-1-ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecta and also high amounts of two C13 bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagetes erecta. These experiments also showed that one mutant is deficient in demethylation of the monothiophene.

Particulate wear debris activates protein tyrosine kinases and nuclear factor kappaB, which down-regulates type I collagen synthesis in human osteoblasts.

Particulate wear debris generated mechanically from prosthetic materials is phagocytosed by a variety of cell types within the periprosthetic space including osteoblasts, which cells with an altered function may contribute to periprosthetic osteolysis. Exposure of osteoblast-like osteosarcoma cells or bone marrow-derived primary osteoblasts to either metallic or polymeric particles of phagocytosable sizes resulted in a marked decrease in the steady-state messenger RNA (mRNA) levels of procollagen alpha1[I] and procollagen alpha1[III]. In contrast, no significant effect was observed for the osteoblast-specific genes, such as osteonectin and osteocalcin (OC). In kinetic studies, particles once phagocytosed, maintained a significant suppressive effect on collagen gene expression and type I collagen synthesis for up to five passages. Large particles of a size that cannot be phagocytosed also down-regulated collagen gene expression suggesting that an initial contact between cells and particles can generate gene responsive signals independently of the phagocytosis process. Concerning such signaling, titanium particles rapidly increased protein tyrosine phosphorylation and nuclear transcription factor kappaB (NF-kappaB) binding activity before the phagocytosis of particles. Protein tyrosine kinase (PTK) inhibitors such as genistein and the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) significantly reduced the suppressive effect of titanium on collagen gene expression suggesting particles suppress collagen gene expression through the NF-kappaB signaling pathway. These results provide a mechanism by which particulate wear debris can antagonize the transcription of the procollagen alpha1[I] gene in osteoblasts, which may contribute to reduced bone formation and progressive periprosthetic osteolysis.

Bone resorption activity of particulate-stimulated macrophages.

Particulate wear debris from bone cement or prosthetic components can stimulate macrophages to cause bone resorption in a dose-dependent manner. This bone resorption activity of particulate-stimulated macrophages is associated with increased levels of both prostaglandin E2 (PGE2) and interleukin-1 (IL-1). In this study we compared the effect of particulate size, concentration, and composition on the secretion of IL-1 and PGE2 by peritoneal macrophages and on the bone-resorbing activity of conditioned medium (CM) harvested from particulate-challenged macrophages. Particulates (titanium, Ti; polymethylmethacrylate, PMMA; and polystyrene, PS) only with phagocytosable size stimulated peritoneal macrophages to secrete IL-1 and PGE2 in a dose- and time-dependent manner. Ti particles (1-3 microns) exhibited significantly enhanced bone-resorbing activity measured as 45Ca release. The maximum bone-resorbing response was observed at a concentration of 0.1% Ti (approximately 10-15 Ti particulates per cell), which also corresponded with the highest IL-1 levels measured in particulate-challenged CM. This was measured using either conditioned media from Ti-stimulated macrophages or in cocultures of calvarial bone and macrophages in the presence of Ti. Exogenous PGE2 and recombinant human IL-1 could significantly increase the 45Ca release; indomethacin (IM) significantly reduced both the spontaneous calcium efflux and active 45Ca release from in vivo labeled calvarial bones. However, IM and/or anti-IL-1 antibodies could suppress only partly the macrophage-mediated bone resorption, indicating that, in a macrophage-bone coculture system, factors other than PGE2 and IL-1 also may regulate particulate-induced bone resorption, probably involving multiple cell types.

The potential role of fibroblasts in periprosthetic osteolysis: fibroblast response to titanium particles.

Periprosthetic osteolysis with or without aseptic loosening is a major clinical problem in total hip arthroplasty. While the macrophage response to prosthetic wear debris and its role in periprosthetic osteolysis has been extensively studied, information regarding other cell types (fibroblasts, osteoblasts) is limited. This study explored the response of fibroblasts to particulate wear debris. Fibroblasts isolated from interfacial membranes of patients with failed total hip replacements and normal synovial tissue, when challenged with small-sized ( < 3 microns) titanium (Ti) particles, responded with significantly enhanced expressions of collagenase, stromelysin and, to a much lesser extent, their tissue inhibitor of metalloproteinases (TIMP). These "regulated" expressions at both mRNA and protein levels were correlated with the size and composition of particles. De novo protein synthesis was required for the regulation of these mRNAs. A similar effect could be induced by the treatment of the cells with particle-free conditioned medium from Ti particle-stimulated fibroblasts. Furthermore, this conditioned medium significantly suppressed the mRNA levels of procollagen alpha 1 (I) and alpha 1 (III) in osteoblast-like MG-63 cells. It is concluded that fibroblasts stimulated with certain particle debris may play an important role in periprosthetic osteolysis by releasing bone-resorbing metalloproteinases and mediator(s) which resulted in suppressed collagen synthesis in osteoblasts.

On the mutagenic action of some enzyme immunoassay substrates.

The mutagenic action of six compounds used in ELISA, EMIT and EIA assays, was investigated by means of the fluctuation test, with Klebsiella pneumoniae as a test organism, and the Ames' plate incorporation test using Salmonella typhimurium TA 98, TA 100 and TA 1537. It appears that 2,2'-azino-di(3-ethyl-benzthiazoline sulphonic acid (6)) or ABTS exerts mutagenic action on Klebsiella pneumoniae at a concentration of 11 g/l, on Salmonella typhimurium TA 100 at a top agar concentration of 0.1 g/l, on Salmonella typhimurium TA 98 at 0.2 g/l and on Salmonella typhimurium TA 1537 at 10 g/l. With umbelliferone, mutagenic action was found only with Klebsiella pneumoniae at a concentration of 0.8 g/l or higher. With o-phenylenediamine, strong mutagenic activity was found only with strain TA 98 and metabolic activation at a top agar concentration of 0.001 g/l. With 5-aminosalicylic acid, beta-methylumbelliferone and p-nitrophenyl-phosphate, no mutagenic action was observed.

An automated method for the quantification of immunostained human Langerhans cells.

Allergic contact dermatitis is a frequent and increasing health problem. For ethical reasons, the current animal tests used to screen for contact sensitizers should be replaced by in vitro alternatives. Contact sensitizers have been shown to accelerate Langerhans cell (LC) migration from human organotypic skin explant cultures (hOSECs) more rapidly than non-sensitizers and it has been proposed that the hOSEC model could be used to screen for sensitizers. However, chemically induced decreases in epidermal LC numbers need to be accurately quantified if the alterations in epidermal LC numbers are to form the basis of an alternative system for screening contact sensitizers in vitro. As manual counting of LCs is labour intensive and subject to intra- and inter-personal variation we developed an image analysis routine, using the Leica QWin image analysis software, to quantify LCs in situ using immunohistochemically stained skin sections. LCs can be identified using antibodies against the membrane molecule CD1a or the Lag antibody, which recognises cytoplasmic Birbeck granules. Quantification of epidermal LC number using the image analysis software had a much lower inter-person variation than when the same specimens were counted manually, using both the anti-Lag and CD1a antibodies. The software-aided quantification of epidermal LCs provides an accurate method for measuring chemically-induced changes in LC numbers.