Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding.

AIMS/HYPOTHESIS: While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD). METHODS: C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle. RESULTS: Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic-euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance. CONCLUSIONS/INTERPRETATION: HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.

AMP-activated protein kinase (AMPK)α2 plays a role in determining the cellular fate of glucose in insulin-resistant mouse skeletal muscle.

AIMS/HYPOTHESIS: We determined whether: (1) an acute lipid infusion impairs skeletal muscle AMP-activated protein kinase (AMPK)α2 activity, increases inducible nitric oxide synthase (iNOS) and causes peripheral insulin resistance in conscious, unstressed, lean mice; and (2) restoration of AMPKα2 activity during the lipid infusion attenuates the increase in iNOS and reverses the defect in insulin sensitivity in vivo. METHODS: Chow-fed, 18-week-old C57BL/6J male mice were surgically catheterised. After 5 days they received: (1) a 5 h infusion of 5 ml kg(-1) h(-1) Intralipid + 6 U/h heparin (Lipid treatment) or saline (Control); (2) Lipid treatment or Control, followed by a 2 h hyperinsulinaemic-euglycaemic clamp (insulin clamp; 4 mU kg(-1) min(-1)); and (3) infusion of the AMPK activator, 5-aminoimidazole-4-carboxamide 1-ß-D-ribofuranoside (AICAR) (1 mg kg(-1) min(-1)), or saline during Lipid treatment, followed by a 2 h insulin clamp. In a separate protocol, mice producing a muscle-specific kinase-dead AMPKα2 subunit (α2-KD) underwent an insulin clamp to determine the role of AMPKα2 in insulin-mediated muscle glucose metabolism. RESULTS: Lipid treatment decreased AMPKα2 activity, increased iNOS abundance/activation and reduced whole-body insulin sensitivity in vivo. AICAR increased AMPKα2 activity twofold; this did not suppress iNOS or improve whole-body or tissue-specific rates of glucose uptake during Lipid treatment. AICAR caused a marked increase in insulin-mediated glycogen synthesis in skeletal muscle. Consistent with this latter result, lean α2-KD mice exhibited impaired insulin-stimulated glycogen synthesis even though muscle glucose uptake was not affected. CONCLUSIONS/INTERPRETATION: Acute induction of insulin resistance via lipid infusion in healthy mice impairs AMPKα2, increases iNOS and causes insulin resistance in vivo. However, these changes do not appear to be interrelated. Rather, a functionally active AMPKα2 subunit is required for insulin-stimulated muscle glycogen synthesis.

Systematic review: the prognostic role of alpha-fetoprotein following liver transplantation for hepatocellular carcinoma.

BACKGROUND: Liver transplantation (LT) offers a possible cure for carefully selected patients with hepatocellular carcinoma (HCC). Studies report that preoperative alpha-fetoprotein (AFP) is a prognostic indicator that can predict survival and recurrence in these patients. AIM: To undertake a systematic review of available literature on preoperative AFP as a predictor of survival and recurrence following LT for HCC. METHODS: A literature search was performed using Medline, Embase, Cochrane Library, CINAHL and Google scholar databases to identify studies reporting AFP as a prognostic marker in LT for HCC. Primary outcomes of interest were overall survival and recurrence. Secondary outcomes were correlation of pre-LT AFP with vascular invasion and grade of tumour differentiation. RESULTS: A total of 13 studies met the inclusion criteria (12,159 patients). The majority were male (9603, 78.9%). All were observational studies and only one prospective. Methodological quality was rated as poor for all studies, with selection and observer bias apparent for most cohorts. Reported survival rates and recurrence rates varied widely between the studies although overall demonstrated better outcomes for those with lower (<1000 ng/mL) pre-LT AFP levels. Similarly, rates of vascular invasion and poor tumour differentiation were higher in those with high pre-LT AFP levels. CONCLUSIONS: A quantity of AFP >1000 ng/mL is associated with poorer outcomes from liver transplantation for hepatocellular carcinoma. The quality of studies was generally poor and precluded valid statistical meta-analysis. There is a need to improve the performance and reporting of primary prognostic studies to facilitate high quality systematic review and meta-analysis.

Obesity impairs skeletal muscle AMPK signaling during exercise: role of AMPKα2 in the regulation of exercise capacity in vivo.

OBJECTIVE: Skeletal muscle AMP-activated protein kinase (AMPK)α2 activity is impaired in obese, insulin-resistant individuals during exercise. We determined whether this defect contributes to the metabolic dysregulation and reduced exercise capacity observed in the obese state. DESIGN: C57BL/6J wild-type (WT) mice and/or mice expressing a kinase dead AMPKα2 subunit in skeletal muscle (α2-KD) were fed chow or high-fat (HF) diets from 3 to 16 weeks of age. At 15 weeks, mice performed an exercise stress test to determine exercise capacity. In WT mice, muscle glucose uptake and skeletal muscle AMPKα2 activity was assessed in chronically catheterized mice (carotid artery/jugular vein) at 16 weeks. In a separate study, HF-fed WT and α2-KD mice performed 5 weeks of exercise training (from 15 to 20 weeks of age) to test whether AMPKα2 is necessary to restore work tolerance. RESULTS: HF-fed WT mice had reduced exercise tolerance during an exercise stress test, and an attenuation in muscle glucose uptake and AMPKα2 activity during a single bout of exercise (P<0.05 versus chow). In chow-fed α2-KD mice, running speed and time were impaired ∼45 and ∼55%, respectively (P<0.05 versus WT chow); HF feeding further reduced running time ∼25% (P<0.05 versus α2-KD chow). In response to 5 weeks of exercise training, HF-fed WT and α2-KD mice increased maximum running speed ∼35% (P<0.05 versus pre-training) and maintained body weight at pre-training levels, whereas body weight increased in untrained HF WT and α2-KD mice. Exercise training restored running speed to levels seen in healthy, chow-fed mice. CONCLUSION: HF feeding impairs AMPKα2 activity in skeletal muscle during exercise in vivo. Although this defect directly contributes to reduced exercise capacity, findings in HF-fed α2-KD mice show that AMPKα2-independent mechanisms are also involved. Importantly, α2-KD mice on a HF-fed diet adapt to regular exercise by increasing exercise tolerance, demonstrating that this adaptation is independent of skeletal muscle AMPKα2 activity.

Impact of macrophage toll-like receptor 4 deficiency on macrophage infiltration into adipose tissue and the artery wall in mice.

AIMS/HYPOTHESIS: Toll-like receptor 4 (TLR4) is a receptor for saturated fatty acids (SFAs), global deficiency of which has been shown to protect against inflammation, insulin resistance and atherosclerotic lesion formation. Because macrophages express Tlr4 and are important in insulin resistance and atherosclerotic lesion formation due to their infiltration of white adipose tissue (WAT) and the artery wall, respectively, we hypothesised that deficiency of macrophage TLR4 could protect against these disorders. METHODS: Bone marrow transplantation of agouti, LDL-receptor deficient (A(y)/a; Ldlr (-/-)) mice with marrow from either C57BL/6 or Tlr4 (-/-) mice was performed. Recipient mice with Tlr4 (+/+) marrow (MthetaTLR4(+/+)) or with Tlr4 (-/-) marrow (MthetaTLR4(-/-)) were then placed on one of four diets: (1) low fat; (2) high fat; (3) high fat rich in SFAs (HF(SFA)); and (4) HF(SFA) supplemented with fish oil. RESULTS: There were no differences in body composition or plasma lipids between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice on any of the diets. However, we observed a decrease in some macrophage and inflammatory markers in WAT of female low fat-fed MthetaTLR4(-/-) mice compared with MthetaTLR4(+/+) mice. MthetaTLR4(-/-) mice fed low-fat diet also displayed decreased atherosclerotic lesion area. There were no differences in macrophage accrual in WAT or atherosclerosis between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed any of the high-fat diets. Finally, no difference was seen in insulin sensitivity between MthetaTLR4(+/+) and MthetaTLR4(-/-) mice fed the HF(SFA) diet. CONCLUSIONS/INTERPRETATION: These data suggest that under certain dietary conditions, macrophage expression of Tlr4 can be an important mediator of macrophage accumulation in WAT and the artery wall.

Differential attenuation of AMPK activation during acute exercise following exercise training or AICAR treatment.

Short-term exercise training in humans attenuates AMP-activated protein kinase (AMPK) activation during subsequent exercise conducted at the same absolute workload. Short-term 5-aminoimidazole-4-carboxyamide-ribonucleoside (AICAR) administration in rats mimics exercise training on skeletal muscle in terms of increasing insulin sensitivity, mitochondrial enzymes, and GLUT4 content, but it is not known whether these adaptations are accompanied by reduced AMPK activation during subsequent exercise. We compared the effect of 10 days of treadmill training (60 min/day) with 10 days of AICAR administration (0.5 mg/g body weight ip) on subsequent AMPK activation during 45 min of treadmill exercise in male Sprague-Dawley rats. Compared with nonexercised control rats, acute exercise significantly (P < 0.05) increased AMPKalpha Thr172 phosphorylation (p-AMPKalpha; 1.6-fold) and ACCbeta Ser218 phosphorylation (p-ACCbeta; 4.9-fold) in the soleus and p-ACCbeta 2.2-fold in the extensor digitorum longus. Ten days of exercise training abolished the increase in soleus p-AMPKalpha and attenuated the increase in p-ACCbeta (nonsignificant 2-fold increase). Ten days of AICAR administration also attenuated the exercise-induced increases in AMPK signaling in the soleus although not as effectively as 10 days of exercise training (nonsignificant 1.3-fold increase in p-AMPKalpha; significant 3-fold increase in p-ACCbeta). The increase in skeletal muscle 2-deoxyglucose uptake during exercise was greater after either 10 days of exercise training or AICAR administration. In conclusion, 10 days of AICAR administration substantially mimics the effect of 10 days training on attenuating skeletal muscle AMPK activation in response to subsequent exercise.

L-Arginine infusion increases glucose clearance during prolonged exercise in humans.

Nitric oxide synthase (NOS) inhibition has been shown in humans to attenuate exercise-induced increases in muscle glucose uptake. We examined the effect of infusing the NO precursor L-arginine (L-Arg) on glucose kinetics during exercise in humans. Nine endurance-trained males cycled for 120 min at 72+/-1% Vo(2 peak) followed immediately by a 15-min "all-out" cycling performance bout. A [6,6-(2)H]glucose tracer was infused throughout exercise, and either saline alone (Control, CON) or saline containing L-Arg HCL (L-Arg, 30 g at 0.5 g/min) was confused in a double-blind, randomized order during the last 60 min of exercise. L-Arg augmented the increases in glucose rate of appearance, glucose rate of disappearance, and glucose clearance rate (L-Arg: 16.1+/-1.8 ml.min(-1).kg(-1); CON: 11.9+/- 0.7 ml.min(-1).kg(-1) at 120 min, P<0.05) during exercise, with a net effect of reducing plasma glucose concentration during exercise. L-Arg infusion had no significant effect on plasma insulin concentration but attenuated the increase in nonesterified fatty acid and glycerol concentrations during exercise. L-Arg infusion had no effect on cycling exercise performance. In conclusion, L-Arg infusion during exercise significantly increases skeletal muscle glucose clearance in humans. Because plasma insulin concentration was unaffected by L-Arg infusion, greater NO production may have been responsible for this effect.

Effect of exercise intensity and hypoxia on skeletal muscle AMPK signaling and substrate metabolism in humans.

We compared in human skeletal muscle the effect of absolute vs. relative exercise intensity on AMP-activated protein kinase (AMPK) signaling and substrate metabolism under normoxic and hypoxic conditions. Eight untrained males cycled for 30 min under hypoxic conditions (11.5% O(2), 111 +/- 12 W, 72 +/- 3% hypoxia Vo(2 peak); 72% Hypoxia) or under normoxic conditions (20.9% O(2)) matched to the same absolute (111 +/- 12 W, 51 +/- 1% normoxia Vo(2 peak); 51% Normoxia) or relative (to Vo(2 peak)) intensity (171 +/- 18 W, 73 +/- 1% normoxia Vo(2 peak); 73% Normoxia). Increases (P < 0.05) in AMPK activity, AMPKalpha Thr(172) phosphorylation, ACCbeta Ser(221) phosphorylation, free AMP content, and glucose clearance were more influenced by the absolute than by the relative exercise intensity, being greatest in 73% Normoxia with no difference between 51% Normoxia and 72% Hypoxia. In contrast to this, increases in muscle glycogen use, muscle lactate content, and plasma catecholamine concentration were more influenced by the relative than by the absolute exercise intensity, being similar in 72% Hypoxia and 73% Normoxia, with both trials higher than in 51% Normoxia. In conclusion, increases in muscle AMPK signaling, free AMP content, and glucose disposal during exercise are largely determined by the absolute exercise intensity, whereas increases in plasma catecholamine levels, muscle glycogen use, and muscle lactate levels are more closely associated with the relative exercise intensity.

Incidence of histoplasmosis following allogeneic bone marrow transplant or solid organ transplant in a hyperendemic area.

Questions have arisen regarding the risk of developing symptomatic Histoplasma capsulatum infection among patients who undergo transplant-related immunosuppression in areas endemic for histoplasmosis. Our medical center is located in a hyperendemic area for histoplasmosis, where three large outbreaks occurred since 1978. We undertook a retrospective chart review of 137 patients who received allogeneic bone marrow transplant and of 449 patients who received solid organ transplant from January 1994 to December 1996 in order to assess the incidence of active histoplasmosis. Charts were reviewed before and after transplantation for clinical outcomes, H. capsulatum serologies and antigen results, and microbiological and radiological results. After a mean follow-up duration exceeding 16 months, no patient was diagnosed with histoplasmosis. In the absence of an outbreak, histoplasmosis is a rare infection following the immunosuppression of allogeneic bone marrow or solid organ transplantation even in a hyperendemic area. Pre-transplant serologies or chest radiographs consistent with prior infection were not associated with post-transplant histoplasmosis.

Addressing psychosocial issues and problems of co-morbidity for Native American clients with substance abuse problems: a conference proceedings.

This proceedings consists of five papers presented at a conference titled: Psychosocial Issues and Problems of Co-Morbidity for Native American Clients with Substance Abuse Problems. The conference was held June 2-4, 1999, in Albuquerque New Mexico, and was hosted by the Native American Research and Training Center (NARTC) and the National Center for American Indian and Alaska Native Mental Health Research.

Effects of aerosol-vapor JP-8 jet fuel on the functional observational battery, and learning and memory in the rat.

To determine whether JP-8 jet fuel affects parameters of the Functional Observational Battery (FOB), visual discrimination, or spatial learning and memory, the authors exposed groups of male Fischer Brown Norway hybrid rats for 28 d to aerosol/vapor-delivered JP-8, or to JP-8 followed by 15 min of aerosolized substance P analogue, or to sham-confined fresh room air. Behavioral testing was accomplished with the U.S. Environmental Protection Agency's Functional Observational Battery. The authors used the Morris swim task to test visual and spatial learning and memory testing. The spatial test included examination of memory for the original target location following 15 d of JP-8 exposure, as well as a 3-d new target location learning paradigm implemented the day that followed the final day of exposure. Only JP-8 exposed animals had significant weight loss by the 2nd week of exposure compared with JP-8 with substance P and control rats; this finding compares with those of prior studies of JP-8 jet fuel. Rats exposed to JP-8 with or without substance P exhibited significantly greater rearing and less grooming behavior over time than did controls during Functional Observational Battery open-field testing. Exposed rats also swam significantly faster than controls during the new target location training and testing, thus supporting the increased activity noted during Functional Observational Battery testing. There were no significant differences between the exposed and control groups' performances during acquisition, retention, or learning of the new platform location in either the visual discrimination or spatial version of the Morris swim task. The data suggest that although visual discrimination and spatial learning and memory were not disrupted by JP-8 exposure, arousal indices and activity measures were distinctly different in these animals.

Cytoplasmic signals mediate apical early endosomal targeting of endotubin in MDCK cells.

Endotubin is an integral membrane protein that targets into apical endosomes in polarized epithelial cells. Although the role of cytoplasmic targeting signals as mediators of basolateral targeting and endocytosis is well established, it has been suggested that apical targeting requires either N-glycosylation of the ectoplasmic domains or partitioning of macromolecules into glycolipid-rich rafts. However, we have previously shown that the cytoplasmic portion of endotubin possesses signals that are necessary for its proper sorting into the apical early endosomes. To further define the targeting signals involved in this apically directed event, as well as to determine if the cytoplasmic domain was sufficient to mediate apical endosomal targeting, we generated a panel of endotubin and Tac-antigen chimeras and expressed them in Madin-Darby canine kidney cells. We show that both the apically targeting wild-type endotubin and a basolaterally targeted cytoplasmic domain mutant do not associate with rafts and are TX-100 soluble. The cytoplasmic tail of endotubin is sufficient for apical endosomal targeting, as chimeras with the endotubin cytoplasmic domain and Tac transmembrane and extracellular domains are efficiently targeted to the apical endosomal compartment. Furthermore, we show that overexpression of these chimeras results in their missorting to the basolateral membrane, indicating that the apical sorting process is a saturable event. These results show that cells contain machinery in both the biosynthetic and endosomal compartments that recognize cytoplasmic apical sorting signals.

Haemophilus ducreyi lipooligosaccharide mutant defective in expression of beta-1,4-glucosyltransferase is virulent in humans.

The lipooligosaccharide (LOS) of Haemophilus ducreyi contains a major glycoform that is immunochemically identical to paragloboside, a glycosphingolipid precursor of major human blood group antigens. We recently identified the gene responsible for the glucosyltransferase activity and constructed an isogenic mutant (35000glu-) deficient in this activity. 35000glu- makes an LOS that consists only of the heptose trisaccharide core and 2-keto-deoxyoctulosonic acid (KDO). For this study, the mutant was reconstructed in the 35000HP (human passaged [HP]) background. Five human subjects were inoculated with 35000HP and 35000HPglu- in a dose-response trial. The pustule formation rates were 40% (95% confidence interval [CI], 13.7 to 72.6%) at 10 sites for 35000HP and 46.7% (95% CI, 24.8 to 69.9%) at 15 sites for 35000HPglu-. The histopathology and recovery rates of H. ducreyi from surface cultures and biopsies obtained from mutant and parent sites were similar. These results indicate that the expression of glycoforms with sugar moieties extending beyond the heptose trisaccharide core is not required for pustule formation by H. ducreyi in humans.

Expression of cytolethal distending toxin and hemolysin is not required for pustule formation by Haemophilus ducreyi in human volunteers.

Haemophilus ducreyi makes cytolethal distending toxin (CDT) and hemolysin. In a previous human challenge trial, an isogenic hemolysin-deficient mutant caused pustules with a rate similar to that of its parent. To test whether CDT was required for pustule formation, six human subjects were inoculated with a CDT mutant and parent at multiple sites. The pustule formation rates were similar at both parent and mutant sites. A CDT and hemolysin double mutant was constructed and tested in five additional subjects. The pustule formation rates were similar for the parent and double mutant. These results indicate that neither the expression of CDT, nor that of hemolysin, nor both are required for pustule formation by H. ducreyi in humans.

Age-related differences in pulmonary inflammatory responses to JP-8 jet fuel aerosol inhalation.

Our previous studies have demonstrated that JP-8 jet fuel aerosol inhalation induced lung injury and dysfunction. To further examine JP-8 jet fuel-induced inflammatory mechanisms, a total of 40 male C57BL/6 mice (young, 3.5 months; adult, 12 months; half in each age group) were randomly assigned to the exposure or control groups. Mice were nose-only exposed to room air or atmospheres of 1000 mg/m3 JP-8 jet fuel for 1 h/day for 7 days. Lung injury was assessed by pulmonary mechanics, respiratory permeability, lavaged cell profile, and chemical mediators in bronchoalveolar lavage fluid (BALF). The young and adult mice exposed to JP-8 jet fuel had similar values with regards to increased lung dynamic compliance, lung permeability, BALF cell count, and decreased PGE2. However, there were several different responses between the young-versus-adult mice with respect to BALF cell differential, TNF-alpha, and 8-iso-PGF2,, levels after exposure to JP-8 jet fuel. These data suggest that JP-8 jet fuel may have different inflammatory mechanisms leading to lung injury and dysfunction in the younger-versus-adult mice.

Mutations in NYX, encoding the leucine-rich proteoglycan nyctalopin, cause X-linked complete congenital stationary night blindness.

During development, visual photoreceptors, bipolar cells and other neurons establish connections within the retina enabling the eye to process visual images over approximately 7 log units of illumination. Within the retina, cells that respond to light increment and light decrement are separated into ON- and OFF-pathways. Hereditary diseases are known to disturb these retinal pathways, causing either progressive degeneration or stationary deficits. Congenital stationary night blindness (CSNB) is a group of stable retinal disorders that are characterized by abnormal night vision. Genetic subtypes of CSNB have been defined and different disease actions have been postulated. The molecular bases have been elucidated in several subtypes, providing a better understanding of the disease mechanisms and developmental retinal neurobiology. Here we have studied 22 families with 'complete' X-linked CSNB (CSNB1; MIM 310500; ref. 4) in which affected males have night blindness, some photopic vision loss and a defect of the ON-pathway. We have found 14 different mutations, including 1 founder mutation in 7 families from the United States, in a novel candidate gene, NYX. NYX, which encodes a glycosylphosphatidyl (GPI)-anchored protein called nyctalopin, is a new and unique member of the small leucine-rich proteoglycan (SLRP) family. The role of other SLRP proteins suggests that mutant nyctalopin disrupts developing retinal interconnections involving the ON-bipolar cells, leading to the visual losses seen in patients with complete CSNB.

Short-term pulmonary response to inhaled JP-8 jet fuel aerosol in mice.

B6.A.D. (Ahr(d)/Nat(s)) mice were utilized to investigate the short-term pulmonary response to JP-8 jet fuel (JP-8) aerosol inhalation. Mice were nose-only exposed to atmospheres of 0 to 118 mg/m3 for 1 h/d over a period of 7 days to further test the hypothesis that JP-8 concentrations below the permissible exposure level (PEL) of 350 mg/m3 will induce lung injury. At 24 to 30 hours after the final exposure, pulmonary function and respiratory permeability were measured on anesthetized mice and then randomly assigned for bronchoalveolar lavage or histopathology. Bronchoalveolar lavage fluid (BALF) was analyzed for total protein, lactic dehydrogenase (LDH), N-acetyl-beta-D-glucosaminidase (NAG), and cytology. Respiratory permeability increases were observed following doses of 48 and 118 mg/m3 and were supported by concomitant BALF increases in total protein and LDH. Conversely, NAG and alveolar macrophage levels decreased following the same exposure concentrations. Morphological lung injury was characterized by the targeting of bronchiolar epithelium and consisted of perivascular edema, Clara cell vacuolization, and necrosis. Alveolar injury included sporadic pulmonary edema, intra-alveolar hemorrhage, and alterations in type II epithelial cells. These results indicate that repeated inhalation of aerosolized JP-8 induces physiological, biochemical, cellular, and morphological lung injury. This study also provides evidence for the reevaluation of the 350 mg/m3 PEL for more volatile petroleum distillates with regard to respirable aerosols.

Expression of peptidoglycan-associated lipoprotein is required for virulence in the human model of Haemophilus ducreyi infection.

Haemophilus ducreyi expresses a peptidoglycan-associated lipoprotein (PAL) that exhibits extensive homology to Haemophilus influenzae protein 6. We constructed an isogenic PAL mutant (35000HP-SMS4) by the use of a suicide vector that contains lacZ as a counterselectable marker. H. ducreyi 35000HP-SMS4 and its parent, 35000HP, had similar growth rates in broth and similar lipooligosaccharide profiles. 35000HP-SMS4 formed smaller, more transparent colonies than 35000HP and, unlike its parent, was hypersensitive to antibiotics. Complementation of the mutant in trans restored the parental phenotypes. To test whether expression of PAL is required for virulence, nine human volunteers were experimentally infected. Each subject was inoculated with two doses (41 to 89 CFU) of live 35000HP and one dose of heat-killed bacteria on one arm and with three doses (ranging from 28 to 800 CFU) of live 35000HP-SMS4 on the other arm. Papules developed at similar rates at sites inoculated with the mutant or parent but were significantly smaller at mutant-inoculated sites than at parent-inoculated sites. The pustule formation rate was 72% (95% confidence interval [CI], 46.5 to 90.3%) at 18 parent sites and 11% (95% CI, 2.4 to 29.2%) at 27 mutant sites (P < 0.0001). The rates of recovery of H. ducreyi from surface cultures were 8% (n = 130; 95% CI, 4.3 to 14.6%) for parent-inoculated sites and 0% (n = 120; 95% CI, 0.0 to 2.5%) for mutant-inoculated sites (P < 0.001). H. ducreyi was recovered from six of seven biopsied parent-inoculated sites and from one of three biopsied mutant-inoculated sites. Confocal microscopy confirmed that the bacteria present in a mutant inoculation site pustule lacked a PAL-specific epitope. Although biosafety regulations precluded our testing the complemented mutant in humans, these results suggest that expression of PAL facilitates the ability of H. ducreyi to progress to the pustular stage of disease.

Effects of short-term JP-8 jet fuel exposure on cell-mediated immunity.

The U.S. Air Force has implemented the widespread use of JP-8 jet fuel in its operations, although a thorough understanding of its potential effects upon exposed personnel is unclear. Exposure to environmental toxicants such as JP-8 may have significant effects on host physiology. Jet fuel exposure has been shown to cause human liver dysfunction, abnormal electroencephalograms, shortened attention spans, and decreased sensorimotor speed. Previous studies have shown that short-term, low-concentration JP-8 exposure had significant effects on the immune system; e.g., decreased viable immune cell numbers, decreased immune organ weights, and loss of immune function that persisted for extended periods of time (i.e., up to 4 weeks post-exposure). In the current study, an in-depth analysis of the effects of JP-8 exposure on cellular immunity was performed. Short-term (7 days, 1 h/day), low-concentration (1000 mg/m3) exposures were conducted in mice, and T cell and natural killer (NK) cell functions were analyzed 24 h after the last exposure. The exposure regimen was found to almost completely ablate NK cell function, as well as significantly suppress the generation of lymphokine-activated killer (LAK) cell activity. Furthermore, JP-8 exposure suppressed the generation of cytotoxic T lymphocyte (CTL) cells from precursor T cells, and inhibited helper T cell activity. These findings demonstrate that JP-8 jet fuel exposure has significant detrimental effects on immune functions of exposed individuals. JP-8 jet fuel should be considered a potential and significant immunotoxicant. Chronic exposure to JP-8 may have serious implications to the long-term health of exposed individuals.