The inhibitory effect of copper, zinc, and manganese on Legionella longbeachae and other Legionella spp. in vitro.

Legionella longbeachae is an important cause of Legionnaires' disease in Australasia and is associated with exposure to potting soils. Our aim was to identify ways to reduce the load of L. longbeachae in potting soils. Inductively-coupled plasma optical emission spectrometry (ICP-OES) of an all-purpose potting mix showed copper (Cu) concentrations (mg/kg) range from 15.8 to 23.6. Zinc (Zn) and manganese (Mn) were significantly higher than Cu ranging from 88.6-106 to 171-203, respectively. Minimal inhibitory and bactericidal concentrations of 10 salts used in the horticultural industry were determined for Legionella species in buffered yeast extract (BYE) broth. For L. longbeachae (n = 9) the median (range) minimum inhibitory concentration (MIC) (mg/L) of copper sulfate was 31.25 (15.6-31.25), zinc sulfate 31.25 (7.81-31.25), and manganese sulfate 31.25 (7.81-62.5). The MIC and minimum bactericidal concentration (MBC) were within one dilution of each other. Susceptibility to Cu and Zn salts increased as the concentration of pyrophosphate iron in the media decreased. The MIC values for these three metals against Legionella pneumophila (n = 3) and Legionella micdadei (n = 4) were similar. Combinations of Cu, Zn, and Mn were additive. Legionella longbeachae has similar susceptibility to Cu and other metal ions in comparison to L. pneumophila.

Monoclonal Antibodies and Invasive Aspergillosis: Diagnostic and Therapeutic Perspectives.

Invasive aspergillosis (IA) is a life-threatening fungal disease that causes high morbidity and mortality in immunosuppressed patients. Early and accurate diagnosis and treatment of IA remain challenging. Given the broad range of non-specific clinical symptoms and the shortcomings of current diagnostic techniques, most patients are either diagnosed as "possible" or "probable" cases but not "proven". Moreover, because of the lack of sensitive and specific tests, many high-risk patients receive an empirical therapy or a prolonged treatment of high-priced antifungal agents, leading to unnecessary adverse effects and a high risk of drug resistance. More precise diagnostic techniques alongside a targeted antifungal treatment are fundamental requirements for reducing the morbidity and mortality of IA. Monoclonal antibodies (mAbs) with high specificity in targeting the corresponding antigen(s) may have the potential to improve diagnostic tests and form the basis for novel IA treatments. This review summarizes the up-to-date application of mAb-based approaches in assisting IA diagnosis and therapy.

Hydrogen Sulfide and Substance P Levels in Patients with Escherichia coli and Klebsiella pneumoniae Bacteraemia.

Hydrogen sulfide (H2S) and substance P (SP) are known from animal models and in vitro studies as proinflammatory mediators. In this study, peripheral blood concentrations of H2S and SP were measured in patients with Escherichia coli or Klebsiella pneumoniae bacteraemia. Fifty patients were recruited from general wards at Christchurch Hospital, during 2020-2021. Samples from age- and sex-matched healthy subjects previously recruited as controls for studies of cardiovascular disease were used as controls. The concentrations of H2S were higher than controls on day 0, day 1, and day 2, and SP was higher than controls on all 4 days. The concentrations of H2S were highest on day 0, whereas SP concentrations were higher on day 2 than other days. Interleukin-6 and C-reactive protein were significantly higher on day 0 and day 1, respectively. The concentrations of H2S and SP did not differ between 15 non-septic (SIRS 0-1) and the 35 septic subjects (SIRS ≥ 2). Substance P concentrations were higher in subjects with abdominal infection than urinary tract infections on day 0 (p = 0.0002) and day 1 (p = 0.0091). In conclusion, the peak H2S concentrations precede the SP peak in patients with Gram-negative bacteraemia, but this response varies with the site of infection.

Two Monoclonal Antibodies That Specifically Recognize Aspergillus Cell Wall Antigens and Can Detect Circulating Antigens in Infected Mice.

Invasive aspergillosis (IA) is a life-threatening disease mainly caused by Aspergillus fumigatus and Aspergillus flavus. Early diagnosis of this condition is crucial for patient treatment and survival. As current diagnostic techniques for IA lack sufficient accuracy, we have raised two monoclonal antibodies (1D2 and 4E4) against A. fumigatus cell wall fragments that may provide a platform for a new diagnostic approach. The immunoreactivity of these antibodies was tested by immunofluorescence and ELISA against various Aspergillus and Candida species in vitro and by immunohistochemistry in A. fumigatus infected mouse tissues. Both monoclonal antibodies (mAbs) showed intensive fluorescence with the hyphae wall of A. fumigatus and A. flavus, but there was no staining with other Aspergillus species or Candida species. Both mAbs also showed strong immunoreactivity to the cell wall of A. fumigatus hyphae in the infected liver, spleen and kidney of mice with IA. The antigens identified by 1D2 and 4E4 might be glycoproteins and the epitopes are most likely a protein or peptide rather than a carbohydrate. An antibody-based antigen capture ELISA detected the extracellular antigens released by A. fumigatus, A. flavus, A. niger and A. terreus, but not in Candida species. The antigen could be detected in the plasma of mice after 48 h of infection by double-sandwich ELISA. In conclusion, both 1D2 and 4E4 mAbs are potentially promising diagnostic tools to investigate invasive aspergillosis.

Enhancement of Culture of Legionella longbeachae from Respiratory Samples by Use of Immunomagnetic Separation and Antimicrobial Decontamination.

Legionella longbeachae is the commonest Legionella species identified in patients with community-acquired pneumonia in New Zealand. Isolation of the organism on culture is the gold standard for the diagnosis of Legionnaires disease, but it has poor sensitivity (40%) compared with quantitative PCR (qPCR). We have developed a selective decontamination process using glycine, vancomycin, polymyxin, and cycloheximide (GVPC) with immunomagnetic separation (IMS) for culturing L. longbeachae A polyclonal antibody specific for L. longbeachae was produced from New Zealand White rabbits and coupled to tosyl-activated magnetic beads. Stored L. longbeachae qPCR-positive respiratory samples were retrieved from -80°C storage for testing. One portion of test samples was mixed with GVPC and the antibody bead complex, separated, washed, and cultured on modified Wadowsky and Yee agar (MWY) agar. Another portion was exposed to HCl-KCl acidic buffer (pH 2.2) before incubation on MWY agar. qPCR used probes specific for the ITS (internal transcribed spacer) region of the L. longbeachae genome. Cultures were positive in 10/53 (19%) samples after acid wash and 26/53 (49%) after GVPC-IMS (P = 0.001). Growth of contaminants was rare. The mean qPCR threshold cycle values were lower in culture-positive samples after acid wash than in the culture-negative samples (mean, 29.9 versus 34.8; difference, 4.9; 95% confidence interval [CI], ±2.9; P = 0.001) but not after GVPC-IMS (mean, 33.0 versus 34.7; difference, 1.7; 95% CI, ±2.48; P = 0.16). The sensitivity of culture for L. longbeachae in respiratory specimens may be improved by using GVPC-IMS rather than acid wash for decontamination, but this should be confirmed in a prospective study of fresh specimens.

Low Vitamin C Concentrations in Patients with Community-Acquired Pneumonia Resolved with Pragmatic Administration of Intravenous and Oral Vitamin C.

Community-acquired pneumonia (CAP) is characterized by elevated markers of inflammation and oxidative stress and depleted circulating concentrations of the antioxidant nutrient vitamin C. A feasibility trial of intravenous and oral vitamin C supplementation, matched to the timing of intravenous and oral antibiotic formulations, was carried out and changes in vitamin C status were monitored to determine whether saturating status could be achieved throughout the administration period. Patients with moderate and severe CAP (CURB-65 ≥ 2; n = 75) who were receiving intravenous antimicrobial therapy were randomized to placebo (n = 39) or intravenous vitamin C (2.5 g per 8 h; n = 36) before moving to oral vitamin C (1 g three times daily) when prescribed oral antimicrobials. Blood samples were collected at baseline and then daily whilst in the hospital. Vitamin C concentrations were determined by high-performance liquid chromatography. The inflammatory and infection biomarkers C-reactive protein and procalcitonin were elevated at baseline (158 (61, 277) mg/L and 414 (155, 1708) ng/L, respectively), and vitamin C concentrations were depleted (15 (7, 25) µmol/L). There was an inverse association between vitamin C and C-reactive protein concentrations (r = -0.312, p = 0.01). Within one day of intervention initiation, plasma vitamin C concentrations in the vitamin C group reached median concentrations of 227 (109, 422) µmol/L, and circulating concentrations remained at ≥150 µmol/L for the duration of the intervention, whilst median vitamin C concentrations in the placebo group remained low (≤35 µmol/L). There was a trend toward decreased duration of hospital stay (p = 0.07) and time to clinical stability (p = 0.08) in the vitamin C group. In conclusion, patients with moderate to severe CAP have inadequate plasma vitamin C concentrations for the duration of their hospital stay. The administration of intravenous or oral vitamin C, titrated to match the antimicrobial formulation, provided saturating plasma vitamin C concentrations whilst in the hospital. There were trends toward shorter duration of hospital stay and time to clinical stability. Thus, larger trials assessing the impact of intravenous and oral vitamin C intervention on CAP clinical outcomes are indicated.

Adjunctive intravenous then oral vitamin C for moderate and severe community-acquired pneumonia in hospitalized adults: feasibility of randomized controlled trial.

Patients hospitalised with community acquired pneumonia (CAP) have low peripheral blood vitamin C concentrations and limited antioxidant capacity. The feasibility of a trial of vitamin C supplementation to improve patient outcomes was assessed. Participants with moderate and severe CAP (CURB-65 ≥ 2) on intravenous antimicrobial treatment were randomised to either intravenous vitamin C (2.5 g 8 hourly) or placebo before switching to oral intervention (1 g tds) for 7 days when they were prescribed oral antimicrobial therapy. Of 344 patients screened 75 (22%) were randomised and analysed. The median age was 76 years, and 43 (57%) were male. In each group, one serious adverse event that was potentially intervention related occurred, and one subject discontinued treatment. Vitamin C concentrations were 226 µmol/L in the vitamin C group and 19 µmol/L in the placebo group (p < 0.001) after 3 intravneous doses. There were no signficant differences between the vitamin C and placebo groups for death within 28 days (0 vs. 2; p = 0.49), median length of stay (69 vs. 121 h; p = 0.07), time to clinical stability (22 vs. 49 h; p = 0.08), or readmission within 30 days (1 vs. 4; p = 0.22). The vitamin C doses given were safe, well tolerated and saturating. A randomised controlled trial to assess the efficacy of vitamin C in patients with CAP would require 932 participants (CURB-65 ≥ 2) to observe a difference in mortality and 200 participants to observe a difference with a composite endpoint such as mortality plus discharge after 7 days in hospital. These studies are feasible in a multicentre setting.

Developments in novel breath tests for bacterial and fungal pulmonary infection.

PURPOSE OF REVIEW: Breath testing has developed over the last 20 years. New techniques that can identify fingerprints for specific diseases and specific markers of respiratory pathogens have been applied to breath analysis. This review discusses the recent advances in breath analysis for the diagnosis of bacterial and fungal lower respiratory tract infections. RECENT FINDINGS: The current techniques continue to develop rapidly, but preconcentration techniques are needed to analyse many target volatile organic compounds for most systems. Breath testing with an electronic nose is promising for the diagnosis of tuberculosis (TB), and specific volatiles identifiable by gas chromatography with mass spectrometry have been identified in breath for Mycobacterium tuberculosis, Pseudomonas aeruginosa and Aspergillus fumigatus, but are found at very low concentrations in breath. Contamination from the environment is an ongoing confounding influence. Exhaled breath condensate (EBC) is disappointing as a diagnostic sample. SUMMARY: Careful attention needs to be paid to the sensitivity and specificity of a technique and confounding from the environment. The role of technologies such as selected ion flow tube-mass spectrometry is emerging. The electronic nose requires further validation for TB. The identification of specific microbial biomarkers aids the quest for improved accuracy. EBC is currently of limited value.

A Novel Monoclonal Antibody 1D2 That Broadly Inhibits Clinically Important Aspergillus Species.

Aspergillus fumigatus is a ubiquitous airborne fungus, is the predominant cause (>90%) of invasive aspergillosis (IA) in immunosuppressed patients and has a high mortality. New approaches to prevention and treatment are needed because of the poor efficacy, toxicity and side effects of the current anti-Aspergillus drugs on patients. Thus, we aim to explore a new avenue to combat Aspergillus infection by using a novel monoclonal antibody (mAb) 1D2 against a glycoprotein on the cell wall of Aspergillus. The ability of this mAb to inhibit attachment, germination, and growth of Aspergillus conidia and hyphae in vitro were examined. A dose-dependent growth inhibition of Aspergillus conidia in the presence of mAb 1D2 was found. The mAb 1D2 inhibited attachment of Aspergillus conidia to an untreated slide surface and fibronectin-treated surface compared to an unrelated mAb 6B10. When conidia were exposed to 1D2 concomitantly with inoculation into culture media, the mAb prevented the swelling and germination of conidia. This inhibitory ability of 1D2 was less apparent if it was added two hours after inoculation. Damage to hyphae was also observed when 1D2 was added to Aspergillus hyphae that had been incubated in media overnight. These in vitro results indicate that mAb 1D2 broadly inhibits clinically important Aspergillus species and has a promising therapeutic effect both as prophylaxis to inhibit an Aspergillus infection as well as a treatment.

Novel diagnostics: progress toward a breath test for invasive Aspergillus fumigatus.

In this paper we will briefly review some of the possible techniques for the development of a breath test for aspergillosis and describe progress made toward validating 2-Pentyl furan (2PF) as a marker of Aspergillus infection. Breath testing to diagnose pulmonary aspergillosis is attractive because of the proximity of the lesion to the sample and the simplicity of obtaining diagnostic specimens. Techniques to detect volatile organic compounds (VOCs) in breath discussed include the electronic nose, selective ion flow mass spectrometry (SIFT), ion mobility spectrometry (IMS) and gas chromatography with mass spectrometry (GC/MS). We have used GC/MS to identify Aspergillus-derived VOC's in the head space of cultures. A promising diagnostic marker molecule is 2PF, which was not detectable from cultures of bacterial respiratory pathogens with the possible exception of Streptococcus pneumoniae. 2-Pentylfuran is not known to be produced by mammalian metabolism and was not detectable in the breath of healthy controls, or neutropenic subjects. In contrast, 2PF was found in the breath of patients with lung disease who were colonized or infected with A. fumigatus. Case reports are presented of two severely immune compromised patients with invasive aspergillosis from whom 2PF was detected in multiple breath samples but became undetectable with effective treatment. A well conducted prospective trial is needed to validate the clinical usefulness of this marker for diagnosis and monitoring of invasive aspergillosis. Unanswered questions remaining include how much 2PF, if any, is produced by extensive lung inflammation, and whether food containing high levels of 2PF can cause false positive breath tests either from contamination in the mouth or gastrointestinal absorption and subsequent excretion in the breath.

Legionellosis Caused by Non-Legionella pneumophila Species, with a Focus on Legionella longbeachae.

Although known as causes of community-acquired pneumonia and Pontiac fever, the global burden of infection caused by Legionella species other than Legionella pneumophila is under-recognised. Non-L. pneumophila legionellae have a worldwide distribution, although common testing strategies for legionellosis favour detection of L. pneumophila over other Legionella species, leading to an inherent diagnostic bias and under-detection of cases. When systematically tested for in Australia and New Zealand, L. longbeachae was shown to be a leading cause of community-acquired pneumonia. Exposure to potting soils and compost is a particular risk for infection from L. longbeachae, and L. longbeachae may be better adapted to soil and composting plant material than other Legionella species. It is possible that the high rate of L. longbeachae reported in Australia and New Zealand is related to the composition of commercial potting soils which, unlike European products, contain pine bark and sawdust. Genetic studies have demonstrated that the Legionella genomes are highly plastic, with areas of the chromosome showing high levels of recombination as well as horizontal gene transfer both within and between species via plasmids. This, combined with various secretion systems and extensive effector repertoires that enable the bacterium to hijack host cell functions and resources, is instrumental in shaping its pathogenesis, survival and growth. Prevention of legionellosis is hampered by surveillance systems that are compromised by ascertainment bias, which limits commitment to an effective public health response. Current prevention strategies in Australia and New Zealand are directed at individual gardeners who use potting soils and compost. This consists of advice to avoid aerosols generated by the use of potting soils and use masks and gloves, but there is little evidence that this is effective. There is a need to better understand the epidemiology of L. longbeachae and other Legionella species in order to develop effective treatment and preventative strategies globally.

2-Aminoacetophenone as a potential breath biomarker for Pseudomonas aeruginosa in the cystic fibrosis lung.

BACKGROUND: Pseudomonas aeruginosa infections are associated with progressive life threatening decline of lung function in cystic fibrosis sufferers. Growth of Ps. aeruginosa releases a "grape-like" odour that has been identified as the microbial volatile organic compound 2-aminoacetophenone (2-AA). METHODS: We investigated 2-AA for its specificity to Ps. aeruginosa and its suitability as a potential breath biomarker of colonisation or infection by Solid Phase Micro Extraction and Gas Chromatography-Mass Spectrometry (GC/MS). RESULTS: Cultures of 20 clinical strains of Ps. aeruginosa but not other respiratory pathogens had high concentrations of 2-AA in the head space of in vitro cultures when analysed by GC/MS. 2-AA was stable for 6 hours in deactivated glass sampling bulbs but was not stable in Tedlar® bags. Optimisation of GC/MS allowed detection levels of 2-AA to low pico mol/mol range in breath. The 2-AA was detected in a significantly higher proportion of subjects colonised with Ps. aeruginosa 15/16 (93.7%) than both the healthy controls 5/17 (29%) (p < 0.0002) and CF patients not colonised with Ps. aeruginosa 4/13(30.7%) (p < 0.001). The sensitivity and specificity of the 2-AA breath test compared to isolation of Ps. aeruginosa in sputum and/or BALF was 93.8% (95% CI, 67-99) and 69.2% (95% CI, 38-89) respectively. The peak integration values for 2-AA analysis in the breath samples were significantly higher in Ps. aeruginosa colonised subjects (median 242, range 0-1243) than the healthy controls (median 0, range 0-161; p < 0.001) and CF subjects not colonised with Ps. aeruginosa (median 0, range 0-287; p < 0.003). CONCLUSIONS: Our results report 2-AA as a promising breath biomarker for the detection of Ps. aeruginosa infections in the cystic fibrosis lung.

Validating a breath collection and analysis system for the new tuberculosis breath test.

Mycobacterium tuberculosis releases four volatile compounds, methyl phenyl-acetate, methyl nicotinate, methyl p-anisate and o-phenylanisole, some of which we have previously been reported to be detected in the breath of tuberculosis patients (Syhre et al 2009 Tuberculosis 89 263-6). These volatiles have the potential to offer a non-invasive and sensitive breath test for the detection of tuberculosis infection. To determine the best sample collection and pre-concentration system a number of variables were examined. The four markers were most stable when breath was collected in a salinized glass sampling bulb compared to either Tedlar® TA, Supel(TM) Inert Foil or Supel(TM) Inert Gas bags. Concentration of breath onto thermal desorption cartridges indicated that Tenax® TA was the most universal sorbent for the collection of all four volatiles. Increasing the number of breath exhalations captured and analysed actively increased the detectable level of volatiles. The most important discovery was samples of methyl nicotinate, methyl p-anisate and o-phenylanisole remained stable on Tenax® TA cartridges for over two months at various altitudes.

Quantification of hydrogen cyanide (HCN) in breath using selected ion flow tube mass spectrometry--HCN is not a biomarker of Pseudomonas in chronic suppurative lung disease.

Hydrogen cyanide (HCN) in exhaled breath has been proposed as a biomarker for airway inflammation, and also a marker of the presence in the airways of specific organisms, especially Pseudomonas aeruginosa. However the production of HCN by salivary peroxidase in the oral cavity increases orally exhaled concentrations, and may not reflect the condition of the lower airways. Using SIFT-MS we aimed to determine an appropriate single-exhalation breathing maneuver which avoids the interference of HCN produced in the oral cavity. We have established that the SIFT-MS Voice200™ is suitable for the online measurement of HCN in exhaled breath. In healthy volunteers a significantly higher end exhaled HCN concentration was measured in oral exhalations compared to nasal exhalations (mean ± SD) 4.5 ± 0.6 ppb versus 2.4 ± 0.3 ppb, p < 0.01. For the accurate and reproducible quantification of end exhaled HCN in breath a nasal inhalation to full vital capacity and nasal exhalation at controlled flow is recommended. This technique was subsequently used to measure exhaled HCN in a group of patients with chronic suppurative lung disease (CSLD) and known microbiological colonization status to determine utility of HCN measurement to detect P. aeruginosa. Median nasal end exhaled HCN concentrations were higher in patients with CSLD (3.7 ppb) than normal subjects (2.0 ppb). However no differences between exhaled HCN concentrations of subjects colonized with P. aeruginosa and other organisms were identified, indicating that breath HCN is not a suitable biomarker of P. aeruginosa colonization.

Assessment of potential causes of falsely positive Mycobacterium tuberculosis breath test.

A suite of volatiles have previously been identified as specific markers of Mycobacterium tuberculosis metabolism in vitro. These markers - methyl phenylacetate, methyl p-anisate, methyl nicotinate, o-phenylanisole with the addition of methyl salicylate, may also be derived from other sources and confound development of a breath test for tuberculosis. To identify potential sources of these potential biomarkers food products, cosmetics, TB medication, environmental air and cigarette smoke were analysed for these markers using solid phase microextraction coupled with Gas Chromatography/Mass Spectrometry. Breath from healthy subjects, including smokers was also tested. Methyl salicylate was commonly detected, making this unsuitable as a specific marker for M. tuberculosis. Methyl nicotinate was detected repeatedly in cigarettes. Methyl phenylacetate was detected in 1.7% of healthy subjects and o-phenylanisole in just 1% of healthy breath indicating these may be more suitable for inclusion in the tuberculosis breath test due to their low "background" level. These results justify further clinical studies to further explore these markers as specific indicators of M. tuberculosis infection.

Potential sources of 2-aminoacetophenone to confound the Pseudomonas aeruginosa breath test, including analysis of a food challenge study.

2-Aminoacetophenone can be detected in the breath of Pseudomonas aeruginosa colonized cystic fibrosis patients; however, low levels were also detected in a small proportion of healthy subjects. It was hypothesized that food, beverages, cosmetics or medications could be a source of contamination of 2-aminoacetophenone in breath. To determine the potential confounding of these products on 2-aminoacetophenone breath analysis, screening for this volatile was performed in the laboratory by gas chromatography/mass spectrometry and a food challenge study carried out. 2-Aminoacetophenone was detected in four of the 78 samples tested in vitro: corn chips and canned tuna (high pmol mol(-1)) and egg white and one of the three beers (low pmol mol(-1)). No 2-aminoacetophenone was detected in the CF medication or cosmetics tested. Twenty-eight out of 30 environmental air samples were negative for 2-aminoacetophenone (below 50 pmol mol(-1)). A challenge study with ten healthy subjects was performed to determine if 2-aminoacetophenone from corn chips was detectable on the breath after consumption. Analysis of mixed breath samples reported that the levels of 2-aminoacetophenone were immediately elevated after corn chip consumption, but after 2 h the level of 2-aminoacetophenone had reduced back to the 'baseline' for each subject.

Determining the limits and confounders for the 2-pentyl furan breath test by gas chromatography/mass spectrometry.

Aspergillus fumigatus produces 2-pentyl furan (2-PF), a volatile compound not produced by many other pathogens or normal human metabolism. 2-Pentyl furan has been detected in the breath of patients with invasive aspergillosis (IA) by SPME pre-concentration coupled with CG/MS providing the possibility of an attractive diagnostic test. The limit of detection (LOD) and quantification (LOQ) for peak integration were assessed both statistically and empirically respectively. 2-Pentyl furan was detected from 10 of 45 food stuffs tested. Levels were highest from soymilk (3 of 3 brands), lower from pumpkin, peanuts, rolled oats 2, Ensure Plus, tinned asparagus, tinned beans and a vegetable exact (Marmite). No 2-PF was detectable in anti-fungal medications used to treat IA or commonly used cosmetics tested. There was no difference in 2-PF breath levels between morning and afternoon or fasting and non fasting samples taken from healthy subjects eating a diet without 2-PF rich foods. 2-Pentyl furan levels were present in breath samples immediately after a mouth rinse with soy milk (P<0.001), and in some subjects after ingesting soy milk and rinsing their mouth with water. The breath test for 2-PF can be conducted without an overnight fast or at a specified time provided the mouth has been rinsed 30 min or more from when 2-PF containing products have been ingested.

Hypothiocyanous acid is a potent inhibitor of apoptosis and caspase 3 activation in endothelial cells.

Hypothiocyanous acid (HOSCN) is a common, thiol-specific oxidant with strong antibacterial activity. It is thought to be nontoxic to mammalian cells, although its ability to specifically target intracellular thiols may potentially cause cellular dysfunction. In this study we demonstrate specific effects of HOSCN on human endothelial cells, with exposure to high concentrations resulting in morphology changes unlike those seen with other oxidants. Effects were time- and dose-dependent and were accompanied by loss of total cell thiols and GSH and by inactivation of glyceraldehyde-3-phosphate dehydrogenase. High-dose exposure was cytotoxic, but lesser doses did not cause cell death, and apoptosis was not initiated by any concentration of HOSCN. In fact, initiation of apoptosis was blocked by minimal HOSCN exposure, with activation of caspase 3 and cleavage of the proenzyme being prevented. This was unlikely to be due to direct oxidation of the caspase 3 active-site cysteine and suggests alternative targeting of the caspase pathway. The survival of endothelial cells when HOSCN is present together with an inducer of apoptosis suggests that HOSCN differs from most other oxidants and could affect endothelial cell survival pathways in a way that may have an impact on vascular function.