Comparison of the Frontal Plane Projection Angle and the Dynamic Valgus Index to Identify Movement Dysfunction in Females with Patellofemoral Pain.

Background: Clinicians typically measure the knee frontal plane projection angle (FPPA) during a single-leg squat to identify females with patellofemoral pain (PFP). A limitation of this measure is minimal attention to movement of the pelvis on the femur that can create knee valgus loading. The dynamic valgus index (DVI) may be a better assessment. Hypothesis/Purpose: The purpose of this study was to compare the knee FPPA and DVI between females with and without PFP and determine if the DVI better identified females with PFP than the knee FPPA. Study Design: Case-control. Methods: Sixteen females with and 16 without PFP underwent 2-dimensional motion analysis when performing five trials of a single-leg squat. The average peak knee FPPA and peak DVI were analyzed. Independent t-tests determined between-group peak knee FPPA and peak DVI differences. Receiver operating characteristic (ROC) curves determined the area under the curve (AUC) scores for sensitivity and 1 - specificity of each measure. Paired-sample area difference under the ROC curves was conducted to determine differences in the AUC for the knee FPPA and DVI. Positive likelihood ratios were calculated for each measure. The significance level was p < 0.05. Results: Females with PFP exhibited a higher knee FPPA (p = 0.001) and DVI (p = 0.015) than controls. AUC scores were .85 (p = 0.001) and .76 (p = 0.012) for the knee FPPA and DVI, respectively. Paired-sample area difference under the ROC curves showed a similar (p = 0.10) AUC for the knee FPPA and DVI. The knee FPPA had 87.5% sensitivity and 68.8% specificity; the DVI had 81.3% sensitivity and 81.0% specificity. Positive likelihood ratios for the knee FPPA and DVI were 2.8 and 4.3, respectively. Conclusion: The DVI during a single-leg squat may be another useful tool for discriminating between females with and without PFP. Level of Evidence: 3a.

Alcohol ingestion disrupts alveolar epithelial barrier function by activation of macrophage-derived transforming growth factor beta1.

BACKGROUND: Chronic alcohol abuse causes oxidative stress and impairs alveolar epithelial barrier integrity, thereby rendering the lung susceptible to acute edematous injury. Experimentally, alcohol-induced oxidative stress increases the expression of transforming growth factor ß1 (TGFß1) in the lung; however, we do not know the precise contribution of various alveolar cells in this process. In the present study, we focused on cell-cell interactions between alveolar macrophages and epithelial cells and the potential mechanisms by which TGFß1 may become activated in the alveolar space of the alcoholic lung. METHODS: Primary alveolar macrophages and epithelial cells were isolated from control- and alcohol-fed Sprague-Dawley rats. Expression of TGFß1 and the epithelial integrin αvß6 were examined by real time PCR and either immunocytochemistry or flow cytometry. Alveolar epithelial cells were cultured on transwell supports in the presence of macrophage cell lysate from control- or alcohol-fed rats or in the presence of viable macrophages ± alcohol. Epithelial barrier function was assessed by transepithelial resistance (TER) and paracellular flux of Texas Red dextran. RESULTS: TGFß1 expression was increased in alveolar macrophages from alcohol-fed rats, and TGFß1 protein was predominantly membrane-bound. Importantly, alveolar macrophage cellular lysate from alcohol-fed rats decreased TER and increased paracellular dextran flux in primary alveolar epithelial cell monolayers as compared to the lysates from control-fed rats. Alcohol-induced epithelial barrier dysfunction was prevented by anti-TGFß1 antibody treatment, indicating the presence of bioactive TGFß1 in the macrophage lysate. In addition, co-culturing macrophages and epithelial cells in the presence of alcohol decreased epithelial barrier function, which also was prevented by anti-TGFß1 and anti-αvß6 treatment. In parallel, chronic alcohol ingestion in vivo, or direct treatment with active TGFß1 in vitro, increased the expression of αvß6 integrin, which is known to activate TGFß1, in alveolar epithelial cells. CONCLUSIONS: Taken together, these data suggest that interactions between alveolar epithelial cells and macrophages contribute to the alcohol-mediated disruption of epithelial barrier function via the expression and activation of TGFß1 at points of cell-cell contact.

Chronic alcohol ingestion in rats decreases Krüppel-like factor 4 expression and intracellular zinc in the lung.

BACKGROUND: Chronic alcohol ingestion alters the dynamic balance between granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor beta1 (TGFß1) signaling within the alveolar space and, in parallel, impairs alveolar macrophage and epithelial cell function by inhibiting expression of the zinc importer ZIP4 and decreasing zinc bioavailability in the alveolar compartment. As the transcription factor Krüppel-like factor 4 (KLF4 ) binds to ZIP4 , we hypothesized that alcohol exposure and consequent perturbations in GM-CSF and TGFß1 signaling could decrease cellular KLF4 expression and/or binding as a mechanism by which it inhibits ZIP4 expression and decreases cellular zinc levels. METHODS AND RESULTS: Alcohol exposure in vitro or chronic ingestion in vivo decreased KLF4 expression in alveolar macrophages and epithelial cells. Treatment with GM-CSF or TGFß1 showed an enhancing or dampening effect on KLF4 expression and binding, respectively. Further, treatment of a rat alveolar macrophage cell line with alcohol in vitro for 4 weeks decreased the expression of the zinc transporters ZIP4 and ZNT1, and of the zinc storage protein metallothionein 1. In parallel, treating these macrophages with KLF4 siRNA decreased ZIP4 expression and decreased cellular zinc and phagocytic capacity to levels equivalent to those following alcohol exposure. In epithelial monolayers, transepithelial electrical resistance (TER) was significantly decreased by alcohol ingestion as compared with control diets, and it was restored by in vitro GM-CSF treatment. In contrast, in vitro TGFß1 treatment of the epithelial monolayers from control-fed rats significantly decreased TER as compared with untreated control monolayers. CONCLUSIONS: Taken together, these results suggest that within the alveolar space, chronic alcohol exposure decreases KLF4 and ZIP4 expression and consequently decreases zinc transport into cells, which, in turn, impairs their function. Furthermore, the dynamic decrease in the relative influence of GM-CSF versus TGFß1 could mediate the zinc deficiency and consequent cellular dysfunction that characterize the "alcoholic lung" phenotype.

Chronic ethanol consumption results in atypical liver injury in copper/zinc superoxide dismutase deficient mice.

BACKGROUND: Ethanol metabolism increases production of reactive oxygen species, including superoxide (O2(.-)) in the liver, resulting in significant oxidative stress, which causes cellular damage. Superoxide dismutase (SOD) is an antioxidant enzyme that converts superoxide to less toxic intermediates, preventing accumulation. Because the absence of SOD would confer less resistance to oxidative stress, we determined whether damage to hepatic proteolytic systems was greater in SOD(-/-) than in SOD(+/+) mice after chronic ethanol feeding. METHODS: Female wild-type (SOD(+/+)) and Cu/Zn-SOD knockout (SOD(-/-)) mice were pair-fed ethanol and control liquid diets for 24 days, after which liver injury was assessed. RESULTS: Ethanol-fed SOD(-/-) mice had 4-fold higher blood ethanol, 2.8-fold higher alanine aminotransferase levels, 20% higher liver weight, a 1.4-fold rise in hepatic protein levels, and 35 to 70% higher levels of lipid peroxides than corresponding wild-type mice. While wild-type mice exhibited fatty liver after ethanol administration, SOD(-/-) mice showed no evidence of ethanol-induced steatosis, although triglyceride levels were elevated in both groups of knockout mice. Ethanol administration caused no significant change in proteasome activity, but caused lysosomal leakage in livers of SOD(-/-) mice but not in wild-type mice. Alcohol dehydrogenase activity was reduced by 50 to 60% in ethanol-fed SOD(-/-) mice compared with all other groups. Additionally, while ethanol administration induced cytochrome P450 2E1 (CYP2E1) activity in wild-type mice, it caused no such induction in SOD(-/-) mice. Unexpectedly, ethanol feeding significantly elevated total and mitochondrial levels of glutathione in SOD knockout mice compared with wild-type mice. CONCLUSION: Ethanol-fed SOD(-/-) mice exhibited lower alcohol dehydrogenase activity and lack of CYP2E1 inducibility, thereby causing decreased ethanol metabolism compared with wild-type mice. These and other atypical responses to ethanol, including the absence of ethanol-induced steatosis and enhanced glutathione levels, appear to be linked to enhanced oxidative stress due to lack of antioxidant enzyme capacity.

Modulation of lysozyme function and degradation after nitration with peroxynitrite.

BACKGROUND: Peroxynitrite (PN) is formed from superoxide and nitric oxide, both of which are increased during hepatic ethanol metabolism. Peroxynitrite forms adducts with proteins, causing structural and functional alterations. Here, we investigated PN-induced alterations in lysozyme structure and function, and whether they altered the protein's susceptibility to proteasome-catalyzed degradation. METHODS: Hen egg lysozyme was nitrated using varying amounts of either PN or the PN donor, 3-morpholinosydnonimine (SIN-1). The activity, nitration status and the susceptibility of lysozyme to proteasome-catalyzed degradation were assessed. RESULTS: Lysozyme nitration by PN or SIN-1 caused dose-dependent formation of 3-nitrotyrosine-lysozyme adducts, causing decreased catalytic activity, and enhanced susceptibility to degradation by the 20S proteasome. Kinetic analyses revealed an increased affinity by the 20S proteasome toward nitrated lysozyme compared with the native protein. CONCLUSION: Lysozyme nitration enhances the affinity of the modified enzyme for degradation by the proteasome, thereby increasing its susceptibility to proteolysis. GENERAL SIGNIFICANCE: Increased levels of peroxynitrite have been detected in tissues of ethanol-fed animals. The damaging effects from excessive peroxynitrite in the cell increase hepatotoxicity and cellular death by protein modification due to nitration. Cellular defenses against such changes include enhanced proteolysis by the proteasome in order to maintain protein quality control.

Lysosomal leakage and lack of adaptation of hepatoprotective enzyme contribute to enhanced susceptibility to ethanol-induced liver injury in female rats.

BACKGROUND: Women exhibit greater liver damage than men after chronic alcohol consumption. Similar findings are reported in animal models. Here, we determined whether differential liver injury occurred in male and female rats after feeding these animals liquid diets containing either ethanol or isocaloric dextrose with fish oil as the sole source of lipid. METHODS: Control and ethanol liquid diets containing fish oil were pair-fed to male and female rats for 8 weeks. Liver damage was evaluated by triglyceride accumulation, lipid peroxide formation, serum transaminases, histological evaluation, and the activities of selected lysosomal and hepatoprotective enzymes. RESULTS: Fatty liver was detected after ethanol feeding in both genders, but in female rats, triglyceride levels were 60% higher, lipid peroxides were 2-fold higher, and inflammatory cells were more evident than in males. A 2-fold elevation of cathepsin B in hepatic cytosol fractions, indicating lysosomal leakage, was detected in ethanol-fed female rats but no such elevation was observed in males. The basal activity of the hepatoprotective enzyme, betaine-homocysteine methyltransferase was 4-fold higher in livers of control male rats than females, and the enzyme activity was further elevated in ethanol-fed male rats but not in females. CONCLUSIONS: Thus, female rats given ethanol in a diet containing fish oil exhibited more severe liver damage than males. We propose that this difference results, in part, from a greater tendency by females to accumulate hepatic fat, thereby enhancing the potential for oxidative stress, which in turn leads to hepatic inflammation. In addition, our findings indicate that female rats have a higher susceptibility to liver damage because of a reduced capacity for hepatoprotection.

L-Buthionine (S,R) sulfoximine depletes hepatic glutathione but protects against ethanol-induced liver injury.

BACKGROUND: L-Buthionine (S,R) sulfoximine (BSO) is an inhibitor of glutathione biosynthesis and has been used as an effective means of depleting glutathione from cells and tissues. Here we investigated whether treatment with BSO enhanced ethanol-induced liver injury in mice. METHODS: Female C57Bl/6 mice were pair fed with control and ethanol-containing liquid diets in which ethanol was 29.2% of total calories. During the final 7 days of pair feeding, groups of control-fed and ethanol-fed mice were given 0, 5 or 7.6 mM BSO in the liquid diets. RESULTS: Compared with controls, ethanol given alone decreased total liver glutathione. This effect was exacerbated in mice given ethanol with 7.6 mM BSO, causing a 72% decline in hepatic glutathione. While ethanol alone caused no decrease in mitochondrial glutathione, inclusion of 7.6 mM BSO caused a 2-fold decline compared with untreated controls. L-Buthionine (S,R) sulfoximine did not affect ethanol consumption, but serum ethanol levels in BSO-treated mice were nearly 6-fold lower than in mice given ethanol alone. The latter decline in serum ethanol was associated with a significant elevation in the specific activities of cytochrome P450 2E1 and alcohol dehydrogenase in livers of BSO-treated animals. Ethanol consumption caused a 3.5-fold elevation in serum alanine aminotransferase levels but the enzyme fell to control levels when BSO was included in the diet. L-Buthionine (S,R) sulfoximine administration also attenuated ethanol-induced steatosis, prevented the leakage of lysosomal cathepsins into the cytosol, and prevented the ethanol-elicited decline in proteasome activity. CONCLUSIONS: L-Buthionine (S,R) sulfoximine, administered with ethanol, significantly depleted hepatic glutathione, compared with controls. However, despite the decrease in hepatic antioxidant levels, liver injury by ethanol was alleviated, due, in part, to a BSO-elicited acceleration of ethanol metabolism.