Dose-dependant acute or subacute disease caused by Burkholderia pseudomallei strain NCTC 13392 in a BALB/c aerosol model of infection.

AIMS: The goal of this study was to examine, for the first time, the virulence and pathogenicity of aerosolized Burkholderia pseudomallei, strain NCTC 13392, in BALB/c mice in order to develop an animal model for testing novel medical countermeasures (MCMs) for the treatment of human acute and subacute (a disease state between acute and chronic) melioidosis. METHODS AND RESULTS: BALB/c mice were exposed to varying doses of aerosolized bacteria. Acute disease was seen in animals exposed to a very-high dose (≥103  CFU per animal) and death occurred 3-4 days postchallenge (pc). Bacteria were detected in the lungs, liver, kidney and spleen. In contrast, animals exposed to a low dose (<10 CFU per animal) survived to the end of the study (day 30 pc) but developed weight loss, a bacterial tissue burden and increasing clinical signs of infection from day 20 pc onwards, mimicking a subacute form of the disease. Pathological changes in the tissues mirrored these findings. CONCLUSIONS: This proof of concept study has shown that B. pseudomallei strain NCTC 13392 is virulent and pathogenic in BALB/c mice, when delivered by aerosol. By varying the doses of aerosolized bacteria it was possible to mimic characteristics of both human acute and subacute melioidosis, at the same time, within the same study. SIGNIFICANCE AND IMPACT OF THE STUDY: Burkholderia pseudomallei, the aetiological agent of melioidosis, causes a serious and often fatal disease in humans and animals. Novel MCMs are urgently needed for both public health and biodefense purposes. The present model provides a useful tool for the assessment and evaluation of new MCMs (e.g. therapeutics and vaccines) and offers the potential for testing new treatments for both subacute to chronic and acute melioidosis prior to human clinical trials.

Functional consequences of I56ii Dlx enhancer deletion in the developing mouse forebrain.

Dlx homeobox genes encode a group of transcription factors that play an essential role during developmental processes including maintaining the differentiation, proliferation and migration of GABAergic interneurons. The Dlx1/2 and Dlx5/6 genes are expressed in the forebrain and are arranged in convergently transcribed bigene clusters, with I12a/I12b and I56i/I56ii cis-regulatory elements (CREs) located in the intergenic region of each cluster respectively. We have characterized the phenotypic consequences of deleting I56ii on forebrain development and spatial patterning of corridor cells that are involved in guiding thalamocortical projections. Here we report that deletion of I56ii impairs expression of Dlx genes and that of potential targets including Gad2 as well as striatal markers Islet1, Meis2, and Ebf1. In addition, I56ii deletion reduces both the binding of DLX2 in the Dlx5/Dlx6 intergenic region and the presence of H3K9Ac at the Dlx5/Dlx6 locus, consistent with the reduced expression of these genes. Deletion of I56ii reduces the expression of the ISLET1 and CTIP2 in the striatum and disrupts the number of parvalbumin and calretinin expressing cells in the adult somatosensory cortex of the ΔI56ii mice. These data suggest an important regulatory role for I56ii in the developing forebrain by means of a potential regulatory mechanism which may regulate the expression of Dlx genes, notably Dlx6 as well as the spatial patterning of the ventral telencephalon, including possibly corridor cells.

A conserved MADS-box phosphorylation motif regulates differentiation and mitochondrial function in skeletal, cardiac, and smooth muscle cells.

Exposure to metabolic disease during fetal development alters cellular differentiation and perturbs metabolic homeostasis, but the underlying molecular regulators of this phenomenon in muscle cells are not completely understood. To address this, we undertook a computational approach to identify cooperating partners of the myocyte enhancer factor-2 (MEF2) family of transcription factors, known regulators of muscle differentiation and metabolic function. We demonstrate that MEF2 and the serum response factor (SRF) collaboratively regulate the expression of numerous muscle-specific genes, including microRNA-133a (miR-133a). Using tandem mass spectrometry techniques, we identify a conserved phosphorylation motif within the MEF2 and SRF Mcm1 Agamous Deficiens SRF (MADS)-box that regulates miR-133a expression and mitochondrial function in response to a lipotoxic signal. Furthermore, reconstitution of MEF2 function by expression of a neutralizing mutation in this identified phosphorylation motif restores miR-133a expression and mitochondrial membrane potential during lipotoxicity. Mechanistically, we demonstrate that miR-133a regulates mitochondrial function through translational inhibition of a mitophagy and cell death modulating protein, called Nix. Finally, we show that rodents exposed to gestational diabetes during fetal development display muscle diacylglycerol accumulation, concurrent with insulin resistance, reduced miR-133a, and elevated Nix expression, as young adult rats. Given the diverse roles of miR-133a and Nix in regulating mitochondrial function, and proliferation in certain cancers, dysregulation of this genetic pathway may have broad implications involving insulin resistance, cardiovascular disease, and cancer biology.

Sequence of pathogenic events in cynomolgus macaques infected with aerosolized monkeypox virus.

UNLABELLED: To evaluate new vaccines when human efficacy studies are not possible, the FDA's "Animal Rule" requires well-characterized models of infection. Thus, in the present study, the early pathogenic events of monkeypox infection in nonhuman primates, a surrogate for variola virus infection, were characterized. Cynomolgus macaques were exposed to aerosolized monkeypox virus (10(5) PFU). Clinical observations, viral loads, immune responses, and pathological changes were examined on days 2, 4, 6, 8, 10, and 12 postchallenge. Viral DNA (vDNA) was detected in the lungs on day 2 postchallenge, and viral antigen was detected, by immunostaining, in the epithelium of bronchi, bronchioles, and alveolar walls. Lesions comprised rare foci of dysplastic and sloughed cells in respiratory bronchioles. By day 4, vDNA was detected in the throat, tonsil, and spleen, and monkeypox antigen was detected in the lung, hilar and submandibular lymph nodes, spleen, and colon. Lung lesions comprised focal epithelial necrosis and inflammation. Body temperature peaked on day 6, pox lesions appeared on the skin, and lesions, with positive immunostaining, were present in the lung, tonsil, spleen, lymph nodes, and colon. By day 8, vDNA was present in 9/13 tissues. Blood concentrations of interleukin 1ra (IL-1ra), IL-6, and gamma interferon (IFN-γ) increased markedly. By day 10, circulating IgG antibody concentrations increased, and on day 12, animals showed early signs of recovery. These results define early events occurring in an inhalational macaque monkeypox infection model, supporting its use as a surrogate model for human smallpox. IMPORTANCE: Bioterrorism poses a major threat to public health, as the deliberate release of infectious agents, such smallpox or a related virus, monkeypox, would have catastrophic consequences. The development and testing of new medical countermeasures, e.g., vaccines, are thus priorities; however, tests for efficacy in humans cannot be performed because it would be unethical and field trials are not feasible. To overcome this, the FDA may grant marketing approval of a new product based upon the "Animal Rule," in which interventions are tested for efficacy in well-characterized animal models. Monkeypox virus infection of nonhuman primates (NHPs) presents a potential surrogate disease model for smallpox. Previously, the later stages of monkeypox infection were defined, but the early course of infection remains unstudied. Here, the early pathogenic events of inhalational monkeypox infection in NHPs were characterized, and the results support the use of this surrogate model for testing human smallpox interventions.

Efficacy of primate humoral passive transfer in a murine model of pneumonic plague is mouse strain-dependent.

New vaccines against biodefense-related and emerging pathogens are being prepared for licensure using the US Federal Drug Administration's "Animal Rule." This allows licensure of drugs and vaccines using protection data generated in animal models. A new acellular plague vaccine composed of two separate recombinant proteins (rF1 and rV) has been developed and assessed for immunogenicity in humans. Using serum obtained from human volunteers immunised with various doses of this vaccine and from immunised cynomolgus macaques, we assessed the pharmacokinetic properties of human and cynomolgus macaque IgG in BALB/c and the NIH Swiss derived Hsd:NIHS mice, respectively. Using human and cynomolgus macaque serum with known ELISA antibody titres against both vaccine components, we have shown that passive immunisation of human and nonhuman primate serum provides a reproducible delay in median time to death in mice exposed to a lethal aerosol of plague. In addition, we have shown that Hsd:NIHS mice are a better model for humoral passive transfer studies than BALB/c mice.

Efficacy of liposome-encapsulated ciprofloxacin in a murine model of Q fever.

Encapsulation of antibiotics may improve treatment of intracellular infections by prolonging antibiotic release and improving antibiotic uptake into cells. In this study, liposome-encapsulated ciprofloxacin for inhalation (CFI) was evaluated as a postexposure therapeutic for the treatment of Coxiella burnetii, the causative agent of Q fever. Intranasal treatment of male A/Jola (A/J) mice with CFI (50 mg/kg of body weight) once daily for 7 days protected mice against weight loss and clinical signs following an aerosol challenge with C. burnetii. In comparison, mice treated twice daily with oral ciprofloxacin or doxycycline (50 mg/kg) or phosphate-buffered saline (PBS) lost 15 to 20% body weight and exhibited ruffled fur, arched backs, and dehydration. Mice were culled at day 14 postchallenge. The weights and bacterial burdens of organs were determined. Mice treated with CFI exhibited reduced splenomegaly and reduced bacterial numbers in the lungs and spleen compared to mice treated with oral ciprofloxacin or doxycycline. When a single dose of CFI was administered, it provided better protection against body weight loss than 7 days of treatment with oral doxycycline, the current antibiotic of choice to treat Q fever. These data suggest that CFI has potential as a superior antibiotic to treat Q fever.

Essentials of forensic post-mortem MR imaging in adults.

Post-mortem MR (PMMR) imaging is a powerful diagnostic tool with a wide scope in forensic radiology. In the past 20 years, PMMR has been used as both an adjunct and an alternative to autopsy. The role of PMMR in forensic death investigations largely depends on the rules and habits of local jurisdictions, availability of experts, financial resources, and individual case circumstances. PMMR images are affected by post-mortem changes, including position-dependent sedimentation, variable body temperature and decomposition. Investigators must be familiar with the appearance of normal findings on PMMR to distinguish them from disease or injury. Coronal whole-body images provide a comprehensive overview. Notably, short tau inversion-recovery (STIR) images enable investigators to screen for pathological fluid accumulation, to which we refer as "forensic sentinel sign". If scan time is short, subsequent PMMR imaging may be focussed on regions with a positive forensic sentinel sign. PMMR offers excellent anatomical detail and is especially useful to visualize pathologies of the brain, heart, subcutaneous fat tissue and abdominal organs. PMMR may also be used to document skeletal injury. Cardiovascular imaging is a core area of PMMR imaging and growing evidence indicates that PMMR is able to detect ischaemic injury at an earlier stage than traditional autopsy and routine histology. The aim of this review is to present an overview of normal findings on forensic PMMR, provide general advice on the application of PMMR and summarise the current literature on PMMR imaging of the head and neck, cardiovascular system, abdomen and musculoskeletal system.

Placement of coiled catheters into the paravertebral space.

There are conflicting results with regard to the use of catheter-based techniques for continuous paravertebral block. Local anaesthetic spread within the paravertebral space is limited and the clinical effect is often variable. Discrepancies between needle tip position and final catheter position can also be problematic. The aim of this proof-of-concept study was to assess the reliability of placing a newly developed coiled catheter in human cadavers. Sixty Tuohy needles and coiled catheters were placed under ultrasound guidance, three on each side of the thoracic vertebral column in 10 human cadavers. Computed tomography was used to assess needle tip and catheter tip locations. No catheter was misplaced into the epidural, pleural or prevertebral spaces. The mean (SD) distance between catheter tips and needle tips was 8.2 (4.9) mm. The median (IQR [range]) caudo-cephalad spread of contrast dye injectate through a subset of 20 catheters was 4 (4-5[3-8]) thoracic segments. All catheters were removed without incident. Precise paravertebral catheter placement can be achieved using ultrasound-guided placement of a coiled catheter.

Survival of Mycobacterium tuberculosis during experimental aerosolization and implications for aerosol challenge models.

AIMS: We undertook a series of experiments to investigate factors that contribute to variation in Mycobacterium tuberculosis viability and infectivity, during experimental aerosolization, with an aim to optimize a strategy to enable a more reproducible delivered dose within animal models of tuberculosis. METHODS AND RESULTS: The viability and infectivity of the challenge suspension was determined in relation to aerosolization time, concentration, method of preparation and M. tuberculosis strain. Challenge stocks generated from frozen aliquots of M. tuberculosis were shown to undergo a 1 log(10) CFU ml(-1) decrease in viability during the first 10 min of aerosolization. This correlated with a decrease in surface lung lesions developing in guinea pigs challenged during this time. The phenomenon of decreased viability in vitro was not observed when using freshly grown, nonfrozen cells of M. tuberculosis. The viability of aerosolized bacilli at the point of inhalation relative to the point of aerosolization always remained constant. CONCLUSION: Based on these findings, we have developed an improved strategy by which to reproducibly deliver aerosol infection doses to individually challenged animals and separately challenged groups of animals. SIGNIFICANCE AND IMPACT OF THE STUDY: Study of the aerobiological characteristics of micro-organisms is a critical step in the validation of methodology for aerosol infection animal models, particularly where large numbers of animals and nonhuman primates are used.

Differentiation of ante-mortem and post-mortem fractures with MRI: a case report.

We describe a case of a fatal speed flying accident in which the victim was electrocuted, burned and fell from a great height. Post-mortem imaging revealed acute appearing fractures on CT, without bone marrow oedema on MRI. Based on the known clinical imaging findings of bone marrow oedema in acute fractures, we concluded that the speed flyer died from electrocution rather than the fall and that the fractures occurred post-mortem. Radiological imaging augmented the reconstruction of the peri-mortem events. Further research is needed to assess whether bone marrow oedema in acute fractures is a reliable vital sign.

Determination of lesion volume by MRI and stereology in a macaque model of tuberculosis.

Sensitive and reproducible methods are needed to measure the impact on the host following experimental challenge with Mycobacterium tuberculosis, in order to determine the degree of protection conferred by new vaccines. Here we compare how well different clinical and post-mortem measures of disease burden predict the response by the host to increasing doses of M. tuberculosis in rhesus and cynomolgus macaques. The total lung and lesion volume was quantified from magnetic resonance imaging (MRI) digital stacks obtained from lungs of M. tuberculosis infected animals that were formalin fixed and scanned ex-vivo. The total lung lesion volume relative to the fixed whole lung volume was superior at indicating disease burden when compared to thoracic radiography, pathology scores, changes in body weight and temperature, as well as erythrocyte haemoglobin concentrations and sedimentation rate. The total lesion volume accurately reflected differences in challenge doses of M. tuberculosis that ranged from 30 to 500 CFU delivered by aerosol. The determination of total lesion volume from MR images demonstrated a species-dependent difference between rhesus and cynomolgus macaques in susceptibility to M. tuberculosis infection. MR stereology provides an accurate, quantifiable and relatively simple assessment, which can be easily standardized between laboratories and should form an essential component of the clinical assessment of disease progression, or vaccine efficacy.

Recurrent posterior shoulder instability.

Recurrent posterior shoulder instability is an uncommon condition. It is often unrecognized, leading to incorrect diagnoses, delays in diagnosis, and even missed diagnoses. Posterior instability encompasses a wide spectrum of pathology, ranging from unidirectional posterior subluxation to multidirectional instability to locked posterior dislocations. Nonsurgical treatment of posterior shoulder instability is successful in most cases; however, surgical intervention is indicated when conservative treatment fails. For optimal results, the surgeon must accurately define the pattern of instability and address all soft-tissue and bony injuries present at the time of surgery. Arthroscopic treatment of posterior shoulder instability has increased application, and a variety of techniques has been described to manage posterior glenohumeral instability related to posterior capsulolabral injury.

Elevation of susceptibility to ozone-induced acute tracheobronchial injury in transgenic mice deficient in Clara cell secretory protein.

Increases in Clara cell abundance or cellular expression of Clara cell secretory protein (CCSP) may cause increased tolerance of the lung to acute oxidant injury by repeated exposure to ozone (O3). This study defines how disruption of the gene for CCSP synthesis affects the susceptibility of tracheobronchial epithelium to acute oxidant injury. Mice homozygous for a null allele of the CCSP gene (CCSP-/-) and wild type (CCSP+/+) littermates were exposed to ozone (0.2 ppm, 8 h; 1 ppm, 8 h) or filtered air. Injury was evaluated by light and scanning electron microscopy, and the abundance of necrotic, ciliated, and nonciliated cells was estimated by morphometry. Proximal and midlevel intrapulmonary airways and terminal bronchioles were evaluated. There was no difference in airway epithelial composition between CCSP+/+ and CCSP-/- mice exposed to filtered air, and exposure to 0.2 ppm ozone caused little injury to the epithelium of both CCSP+/+ and CCSP-/- mice. After exposure to 1.0 ppm ozone, CCSP-/- mice suffered from a greater degree of epithelial injury throughout the airways compared to CCSP+/+ mice. CCSP-/- mice had both ciliated and nonciliated cell injury. Furthermore, lack of CCSP was associated with a shift in airway injury to include proximal airway generations. Therefore, we conclude that CCSP modulates the susceptibility of the epithelium to oxidant-induced injury. Whether this is due to the presence of CCSP on the acellular lining layer surface and/or its intracellular distribution in the secretory cell population needs to be defined.

Biomarkers of oxidative stress study II: are oxidation products of lipids, proteins, and DNA markers of CCl4 poisoning?

Oxidation products of lipids, proteins, and DNA in the blood, plasma, and urine of rats were measured as part of a comprehensive, multilaboratory validation study searching for noninvasive biomarkers of oxidative stress. This article is the second report of the nationwide Biomarkers of Oxidative Stress Study using acute CCl4 poisoning as a rodent model for oxidative stress. The time-dependent (2, 7, and 16 h) and dose-dependent (120 and 1200 mg/kg i.p.) effects of CCl4 on concentrations of lipid hydroperoxides, TBARS, malondialdehyde (MDA), isoprostanes, protein carbonyls, methionine sulfoxidation, tyrosine products, 8-hydroxy-2'-deoxyguanosine (8-OHdG), leukocyte DNA-MDA adducts, and DNA-strand breaks were investigated to determine whether the oxidative effects of CCl4 would result in increased generation of these oxidation products. Plasma concentrations of MDA and isoprostanes (both measured by GC-MS) and urinary concentrations of isoprostanes (measured with an immunoassay or LC/MS/MS) were increased in both low-dose and high-dose CCl4-treated rats at more than one time point. The other urinary markers (MDA and 8-OHdG) showed significant elevations with treatment under three of the four conditions tested. It is concluded that measurements of MDA and isoprostanes in plasma and urine as well as 8-OHdG in urine are potential candidates for general biomarkers of oxidative stress. All other products were not changed by CCl4 or showed fewer significant effects.

Antioxidant supplementation and nasal inflammatory responses among young asthmatics exposed to high levels of ozone.

The inflammatory response to ozone in atopic asthma suggests that soluble mediators of inflammation are released in response to oxidant stress. Antioxidants may alleviate additional oxidative stress associated with photochemical oxidant pollution. This study investigates the impact of antioxidant supplementation on the nasal inflammatory response to ozone exposure in atopic asthmatic children. We conducted a randomized trial using a double-blinded design. Children with asthma (n = 117), residents of Mexico City, were given randomly a daily supplement of vitamins (50 mg/day of vitamin E and 250 mg/day of vitamin C) or placebo. Nasal lavages were performed three times during the 4-month follow-up and analysed for content of interleukin-6 (IL-6), IL-8, uric acid and glutathione (GSx). IL-6 levels in the nasal lavage were increased significantly in the placebo group after ozone exposure while no increase was observed in the supplement group. The difference in response to ozone exposure between the two groups was significant (P = 0.02). Results were similar for IL-8, but with no significant difference between the groups (P = 0.12). GSx decreased significantly in both groups. Uric acid decreased slightly in the placebo group. Our data suggest that vitamin C and E supplementation above the minimum dietary requirement in asthmatic children with a low intake of vitamin E might provide some protection against the nasal acute inflammatory response to ozone.

Effect of antioxidant supplementation on ozone-induced lung injury in human subjects.

To determine whether antioxidants can influence human susceptibility to ozone (O(3))-induced changes in lung function and airway inflammation, we placed 31 healthy nonsmoking adults (18 to 35 yr old) on a diet low in ascorbate for 3 wk. At 1 wk, subjects were exposed to filtered air for 2 h while exercising (20 L/min/m(2)), and then underwent bronchoalveolar lavage (BAL) and were randomly assigned to receive either a placebo or 250 mg of vitamin C, 50 IU of alpha-tocopherol, and 12 oz of vegetable cocktail daily for 2 wk. Subjects were then exposed to 0.4 ppm O(3) for 2 h and underwent a second BAL. On the day of the O(3) exposure, supplemented subjects were found to have significantly increased levels of plasma ascorbate, tocopherols, and carotenoids as compared with those of the placebo group. Pulmonary function testing showed that O(3)-induced reductions in FEV(1) and FVC were 30% and 24% smaller, respectively, in the supplemented cohort. In contrast, the inflammatory response to O(3) inhalation, as represented by the percent neutrophils and the concentration of interleukin-6 recovered in the BAL fluid at 1 h after O(3) exposure was not different for the two groups. These data suggest that dietary antioxidants protect against O(3)-induced pulmonary function decrements in humans.

Oxidative interactions of synthetic lung epithelial lining fluid with metal-containing particulate matter.

Epidemiology studies show association of morbidity and mortality with exposure to ambient air particulate matter (PM). Metals present in PM may catalyze oxidation of important lipids and proteins present in the lining of the respiratory tract. The present study investigated the PM-induced oxidation of human bronchoalveolar lavage (BAL) fluid (BALF) and synthetic lung epithelial lining fluid (sELF) through the measurement of oxygen incorporation and antioxidant depletion assays. Residual oil fly ash (ROFA), an emission source PM that contains approximately 10% by weight of soluble transition metals, was added (0-200 microg/ml) to BALF or sELF and exposed to 20% (18)O(2) (24 degrees C, 4 h). Oxygen incorporation was quantified as excess (18)O in the dried samples after incubation. BALF and diluted sELF yielded similar results. Oxygen incorporation was increased by ROFA addition and was enhanced by ascorbic acid (AA) and mixtures of AA and glutathione (GSH). AA depletion, but not depletion of GSH or uric acid, occurred in parallel with oxygen incorporation. AA became inhibitory to oxygen incorporation when it was present in high enough concentrations that it was not depleted by ROFA. Physiological and higher concentrations of catalase, superoxide dismutase, and glutathione peroxidase had no effect on oxygen incorporation. Both protein and lipid were found to be targets for oxygen incorporation; however, lipid appeared to be necessary for protein oxygen incorporation to occur. Based on these findings, we predict that ROFA would initiate significant oxidation of lung lining fluids after in vivo exposure and that AA, GSH, and lipid concentrations of these fluids are important determinants of this oxidation.

Modulation of cytosolic phospholipase A(2) by PPAR activators in human preadipocytes.

Cytosolic phospholipase A(2) (cPLA(2)) is responsible for the release of arachidonic acid, a precursor for eicosanoid biosynthesis, from cellular phospholipids. The objective of this study is to examine the regulation of cPLA(2) by peroxisome proliferator-activated receptor (PPAR) activators in preadipocyte SW872 (SW) cells. PPAR belong to the superfamily of nuclear hormone receptors that heterodimerize with the retinoid X receptor. In this study, the presence of both PPARalpha and PPARgamma was confirmed in SW cells by positive identification of their mRNA in the cellular homogenate. Clofibrate, a PPARalpha activator, caused an enhancement of ionophore A-23187-induced arachidonate release in SW cells. This increase resulted from an enhancement of cPLA(2) activity, which was caused by an increase in enzyme protein. Clofibrate at lower concentrations (10-200 microM) produced increases in the mRNA levels of cPLA(2) in a dose-response manner. At higher concentrations (>400 microM), clofibrate treatment resulted in the attenuation of the cPLA(2) mRNA level and protein expression. We postulate that clofibrate, acting through the PPARalpha, caused an induction in the transcription of cPLA(2) gene, which led to an increase in the cPLA(2) protein. The observed increase in arachidonate release in SW cells appeared to be a direct result of the enhanced cPLA(2) activity.

Residual oil fly ash inhalation in guinea pigs: influence of absorbate and glutathione depletion.

Inhaled urban particulate matter (PM) often contains metals that appear to contribute to its toxicity. These particles first make contact with a thin layer of epithelial lining fluid in the respiratory tract. Antioxidants present in this fluid and in cells might be important susceptibility factors in PM toxicity. We investigated the role of ascorbic acid (C) and glutathione (GSH) as determinants of susceptibility to inhaled residual oil fly ash (ROFA) in guinea pigs (male, Hartley). Guinea pigs were divided into four groups, +C+GSH, +C-GSH, -C+GSH, and -C-GSH, and exposed to clean air or ROFA (< 2.5 micron diameter, 19--25 mg/m(3) nose-only for 2.0 h). C and/or GSH were lowered by either feeding C-depleted diet (1 microg C/kg diet, 2 weeks) and/or by ip injection of a mixture of buthionine-S,R-sulfoximine (2.7 mmol/kg body weight) and diethylmaleate (1.2 mmol/kg, 2 h prior). Nasal lavage (NL) and bronchoalveolar lavage (BAL) fluid and cells were examined at 0 h and 24 h postexposure to ROFA. The C-deficient diet lowered C concentrations in BAL fluid and cells and in NL fluid by 90%, and the GSH-depletion regimen lowered both GSH and C in the BAL fluid and cells by 50%. ROFA deposition was calculated at time 0 from lung Ni levels to be 46 microg/g wet lung. In unexposed animals, the combined deficiency of C and GSH modified the cellular composition of cells recovered in lavage fluid, i.e., the increased number of eosinophils and macrophages in BAL fluid. ROFA inhalation increased lung injury in the -C-GSH group only (evidenced by increased BAL protein, LDH and neutrophils, and decreased BAL macrophages). ROFA exposure decreased C in BAL and NL at 0 h, and increased BAL C and GSH (2- to 4-fold above normal) at 24 h in nondepleted guinea pigs, but had no effect on C and GSH in depleted guinea pigs. Combined deficiency of C and GSH resulted in the highest macrophage and eosinophil counts of any group. GSH depletion was associated with increased BAL protein and LDH, increased numbers of BAL macrophages and eosinophils, and decreased rectal body temperatures. We conclude that combined deficiency of C and GSH increased susceptibility to inhaled ROFA; caused unusual BAL cellular changes; resulted in lower antioxidant concentrations in BAL than were observed with single deficiencies. Antioxidant deficiency may explain increased susceptibility to PM in elderly or diseased populations and may have important implications for extrapolating animal toxicity data to humans.