Treatment of stable slipped capital femoral epiphysis: systematic review and exploratory patient level analysis.

BACKGROUND: Several aspects of slipped capital femoral epiphysis (SCFE) treatment remain controversial. Loder's work has been instrumental in changing our understanding and approach to the management of the condition when he introduced the concept of "slip instability" and showed that avascular necrosis (AVN) developed in 47% of unstable slips but none of the stable slips. As the two types of SCFE behave differently in terms of presentation, progress and complications, we approached them as two different conditions to highlight these differences. This paper focuses on treatments of stable SCFE. MATERIALS AND METHODS: An extensive literature search was carried out from multiple databases. One thousand six hundred and twenty-three citations were screened. Three hundred and sixteen full publications were obtained for further scrutiny. Fifty-eight studies (2262 hips) were included in the review. These studies evaluated 6 interventions. AVN was chosen as a surrogate for bad outcome. Secondary outcomes were chondrolysis (CL), femoro-acetabular impingement (FAI), osteoarthritis (OA) and patients' reported outcomes. The latter were pooled when they met our predefined criteria. RESULTS: The type of surgical intervention was an important risk factor. Pinning in situ (PIS) was associated with the lowest AVN rate (1.4%). Moreover, the CL, FAI and OA rates were relatively low in patients who underwent PIS. These were not translated into high patient satisfaction rates among these patients, with only 47% reporting an "excellent" outcome. In contrast, 87% of patients who underwent Ganz surgical dislocation reported an "excellent" outcome. The Ganz surgical dislocation was associated with an AVN rate of 3.3%; double that observed in pinning in situ. CONCLUSION: Pinning in situ is the best treatment for mild and moderate stable slip. Ganz surgical dislocation gives higher patient satisfaction for severe stable slip but the risk of AVN is doubled compared with pinning in situ. Devices that allow continued growth may be better than standard screws. LEVEL OF EVIDENCE: Level III.

Revisiting the target-masker linguistic similarity hypothesis.

The linguistic similarity hypothesis states that it is more difficult to segregate target and masker speech when they are linguistically similar. For example, recognition of English target speech should be more impaired by the presence of Dutch masking speech than Mandarin masking speech because Dutch and English are more linguistically similar than Mandarin and English. Across four experiments, English target speech was consistently recognized more poorly when presented in English masking speech than in silence, speech-shaped noise, or an unintelligible masker (i.e., Dutch or Mandarin). However, we found no evidence for graded masking effects-Dutch did not impair performance more than Mandarin in any experiment, despite 650 participants being tested. This general pattern was consistent when using both a cross-modal paradigm (in which target speech was lipread and maskers were presented aurally; Experiments 1a and 1b) and an auditory-only paradigm (in which both the targets and maskers were presented aurally; Experiments 2a and 2b). These findings suggest that the linguistic similarity hypothesis should be refined to reflect the existing evidence: There is greater release from masking when the masker language differs from the target speech than when it is the same as the target speech. However, evidence that unintelligible maskers impair speech identification to a greater extent when they are more linguistically similar to the target language remains elusive.

Topical metal chelation therapy ameliorates oxidation-induced toxicity in diabetic cataract.

Oxidative stress plays a critical role in cataractogenesis, the leading cause of blindness worldwide. Since transition metals generate reactive oxygen species (ROS) formation, metal chelation therapy has been proposed for treatment of cataracts. However, the effectiveness of most chelators is limited by low tissue penetrability. This study is the first to demonstrate that the topically applied divalent metal chelator ethylenediamine tetraacetic acid (EDTA) combined with the carrier and permeability enhancer methyl sulfonyl methane (MSM) ameliorates both oxidation-induced lens opacification and the associated toxic accumulation of protein-4-hydroxynonenal (HNE) adducts. Both in vitro (rat lens culture) and in vivo (diabetic rats), EDTA-MSM (1) significantly reduced lens opacification by about 40-50%, (2) significantly diminished lens epithelial cell proliferation and fiber cell swelling in early stages of cataract formation in vivo, and (3) notably decreased the levels of protein-HNE adducts. These findings have important implications specifically for the treatment of cataract and generally for other diseases in which oxidative stress plays a key pathogenic role.

Metal chelation attenuates oxidative stress, inflammation, and vertical burn progression in a porcine brass comb burn model.

Oxidative stress and inflammation may mediate cellular damage and tissue destruction as the burn wound continues to progress after the abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of a metal chelator proprietary lotion (Livionex Formulation (LF) lotion), that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent progression or reduce burn wound severity in a porcine model. We have reported earlier that in a rat burn model, LF lotion reduces thermal injury progression. Here, we used the porcine brass comb burn model that closely mimics the human condition for contact burns and applied LF lotion every 8 h starting 15 min after the injury. We found that LF lotion reduces the depth of cell death as assessed by TUNEL staining and blood vessel blockage in the treated burn sites and interspaces. The protein expression of pro-inflammatory markers IL-6, TNF-a, and TNFα Converting Enzyme (TACE), and lipid aldehyde production (protein-HNE) was reduced with LF treatment. LF lotion reversed the burn-induced decrease in the aldehyde dehydrogenase (ALDH-1) expression in the burn sites and interspaces. These data show that a topically applied EDTA-containing lotion protects both vertical and horizontal burn progression when applied after thermal injury. Curbing burn wound conversion and halting the progression of second partial burn to third-degree full-thickness burn remains challenging when it comes to burn treatment strategies during the acute phase. Burn wound conversion can be reduced with targeted treatments to attenuate the oxidative and inflammatory response in the immediate aftermath of the injury. Our studies suggest that LF lotion could be such a targeted treatment.

Thermal injury induces early blood vessel occlusion in a porcine model of brass comb burn.

Burn wound progression is an important determinant of patient morbidity and mortality after injury. In this study, we used the brass comb contact burn to determine burn wound vertical injury progression with a focus on blood vessel occlusion and endothelial cell death. Class A 3-month-old Yorkshire pigs received a brass comb contact burn. Burn wounds were sampled at 0, 30 min, 1, 2, 4, and 24 h. Hematoxylin Phloxin Saffron staining and vimentin immunostaining were performed to determine the depth of blood vessel occlusion and endothelial cell death, respectively. The depth of blood vessel occlusion increased by 30 min (p < 0.005) and peaked by 1 to 4 h (p > 0.05). The depth of endothelial cell death risen to a plateau at 30 min (p < 0.005) to 2 h and then peaked at 24 h (p < 0.03). We observed a progression of blood vessel occlusion and vascular endothelial cell death from the middle of the dermis to the hypodermis within 2 h to 4 h after the initial injury, namely a progression from a second-degree (partial thickness) to third-degree (full thickness) burn. These data suggest that therapeutic interventions during this time window may provide a better outcome by reducing or preventing vertical progression of blood vascular occlusion or endothelial cell death.

Understanding Speech Amid the Jingle and Jangle: Recommendations for Improving Measurement Practices in Listening Effort Research.

The latent constructs psychologists study are typically not directly accessible, so researchers must design measurement instruments that are intended to provide insights about those constructs. Construct validation-assessing whether instruments measure what they intend to-is therefore critical for ensuring that the conclusions we draw actually reflect the intended phenomena. Insufficient construct validation can lead to the jingle fallacy-falsely assuming two instruments measure the same construct because the instruments share a name (Thorndike, 1904)-and the jangle fallacy-falsely assuming two instruments measure different constructs because the instruments have different names (Kelley, 1927). In this paper, we examine construct validation practices in research on listening effort and identify patterns that strongly suggest the presence of jingle and jangle in the literature. We argue that the lack of construct validation for listening effort measures has led to inconsistent findings and hindered our understanding of the construct. We also provide specific recommendations for improving construct validation of listening effort instruments, drawing on the framework laid out in a recent paper on improving measurement practices (Flake & Fried, 2020). Although this paper addresses listening effort, the issues raised and recommendations presented are widely applicable to tasks used in research on auditory perception and cognitive psychology.

Metal chelation reduces skin epithelial inflammation and rescues epithelial cells from toxicity due to thermal injury in a rat model.

BACKGROUND: One of the most pervasive complications of burn injury is wound progression, characterized by continuous tissue destruction in untreated wounds, which leads to wound infection, inflammation, oxidative stress and excessive scar formation. We determined whether additional tissue destruction could be attenuated with Livionex formulation (LF) lotion, which contains a metal-chelating agent and reduces inflammation in burn wounds. METHODS: We subjected male Sprague Dawley rats to a 2% total body surface area (TBSA) burn using a brass comb model and topically applied LF lotion (containing ethylenediaminetetraacetic acid and methyl sulfonyl methane) to the affected area every 8 hours over 3 days. Inflammatory cytokine levels, cell apoptosis and wound healing were compared in LF lotion-treated and untreated rats. Statistical analysis was performed using a one-way analysis of variance in conjunction with Tukey's post-hoc test. RESULTS: Serum inflammatory cytokines were not detectable after 3 days, suggesting that small burn wounds induce only an immediate, localized inflammatory response. Microscopy revealed that LF lotion improved burn site pathology. Deoxynucleotidyl transferase biotin-d-UTP nick-end labeling staining showed reduced cell death in the LF-treated samples. LF lotion prevented the spread of tissue damage, as seen by increased amounts of Ki-67-positive nuclei in the adjacent epidermis and hair follicles. Tumor necrosis factor-alpha, interleukin-6 and inducible nitric oxide synthase levels in LF-treated skin sections from burned rats were comparable to the levels observed in unburned control sections, indicating that LF lotion reduces inflammation in and around the burn site. CONCLUSIONS: These results establish LF lotion as a therapeutic agent for reducing inflammatory stress, cell death and tissue destruction when applied immediately after a burn injury. Further studies of LF lotion on large TBSA burns will determine its efficacy as an emergency treatment for reducing long-term morbidity and scarring.

Molecular cloning and oxidative modification of human lens ALDH1A1: implication in impaired detoxification of lipid aldehydes.

Earlier studies showed that human lens ALDH1A1 plays a critical role in protection against oxidative stress-induced cytotoxicity in human lens epithelial cells (HLEC), and opacification of rat and mouse lens. The complete coding sequence of ALDH1A1 was cloned from human lens cDNA library by using PCR methods and expressed it in Escherichia coli. The cloned human lens ALDH1A1 cDNA encodes a 501-amino-acid protein (molecular mass = 54.8 kD) that is 100% identical to human liver ALDH1A1 and shares significant identity with the same isozyme from other tissues and species. The purified recombinant human lens ALDH1A1 exhibited optimal catalytic activity at pH 8 and preferred NAD(+) as cofactor and specifically catalyzed the oxidation of toxic lipid aldehydes such as 4-hydroxynonenal (HNE; K(m) = 4.8 microM) and malonaldehyde (K(m) MDA = 3.5 microM). Citral, disulfiram, and cyanamide were found to inhibit human lens ALDH1A1 at IC50 values of 55, 101, and 22610 microM, respectively, whereas diethylstilbestrol (DES) was found to be an activator (EC(50), 1.3 microM). Further, modification of recombinant human lens ALDH1A1 with nitric oxide donors such as S-nitroso-N-acetylpenicillamine (SNAP) and S-nitrosoglutathione (GSNO) significantly inhibited the enzyme activity. It therefore appears that activation of ALDH1A1, which efficiently catalyzes the detoxification of lipid-derived toxic aldehydes, and/or prevention of its oxidative modification may be novel therapeutic interventions against oxidative stress-induced lens pathologies.

[Metabolism and detoxification of the lipid derived aldehyde, 4-Hydroxynonenal in diabetic cataractogenesis in rat].

OBJECTIVE: To study the metabolism of 4-hydroxynonenal (HNE), one of lipid derived aldehydes (LDAs), in diabetic rat lens and its role in diabetic cataract formation. METHODS: Experimental research. A factor design was used to set up the experiment statistically upon two factors: diabetic and normal control as treatment factors; day 30, 45 and 70 as the time factors. Normal and diabetic rats' lenses were incubated with HNE for 2 hours. HNE metabolites in the culture media were studied by high performance liquid chromatography (HPLC). Aldehyde dehydrogenase (ALDH) activity in normal and diabetic rat lens (30, 45 and 70 d after inducing of cataract) was detected by a spectrophotometer, ALDH protein and HNE-protein were detected by Western Blot. All data were analyzed by the Bonferroni test using SAS 8.0 software. RESULTS: The major pathway for HNE metabolism in normal lens was conjugation with glutathione (GSH) to form GS-HNE (45%), followed by HNE's oxidation to 4-hydroxy-2-nonenoic acid (HNA) by ALDH, which accounted for approximately 9.1% of HNE. The conjugation of HNE with GSH in diabetic lens was decreased approximately 64% at day 30 compared with the controls (F = 49.59, P < 0.001). The pathway of HNE oxidation by ALDH in the diabetic lens was enhanced approximately 1.7 times at day 70 compared to day 30 (F = 11.51, P = 0.0442). A higher ALDH activity, greater amount of ALDH protein, and less amount of HNE-protein adduct were presented in diabetic rat lens. CONCLUSIONS: The pathway of conjugation of HNE with GSH is inhibited in diabetic lens which may play a role in the formation of diabetic cataract. The oxidation of HNE by ALDH is a compensation process for protecting the lens against diabetic damage.

Endoscopic management of a prolapsed posterior costal cartilage graft in laryngotracheal reconstruction.

Laryngotracheal reconstruction is the standard of care for management of high grade pediatric laryngotracheal stenosis. One of the complications of a reconstruction with a posterior costal cartilage graft is graft prolapse. Typically, a revision laryngotracheal reconstruction will be needed to correct this complication. We describe a case in which a non-invasive endoscopic approach using laser was undertaken for a prolapsed posterior costal cartilage graft in a child who had undergone an anterior-posterior costal cartilage laryngotracheal reconstruction for Grade 3 stenosis. This, according to our knowledge, has not been previously described and provides an alternative to revision surgery.

The PTPN22*C1858T functional polymorphism is associated with susceptibility to inflammatory polyarthritis but neither this nor other variants spanning the gene is associated with disease outcome.

BACKGROUND: The PTPN22 gene has been widely confirmed as a susceptibility gene for rheumatoid arthritis (RA) in populations of Northern European descent. The aim of the current study was to explore the role of variants spanning the PTPN22 gene in determining susceptibility to and outcome of inflammatory polyarthritis (IP). PATIENTS AND METHODS: Single nucleotide polymorphism (SNP) variants spanning the gene were genotyped using the Sequenom MassArray platform and tested, firstly for their association with susceptibility to IP. Genotype frequencies were compared between new onset IP cases (n = 843) and population controls (n = 471). Secondly, a within-cohort analysis was performed testing each variant for association with a number of clinical outcome measures reflecting disease severity including radiological erosions, physical function, measured using the Health Assessment Questionnaire (HAQ) score, and disease activity at defined time-points following disease presentation. RESULTS: A significant association between carriage of the PTPN22*1858T allele and IP (odds ratio (OR) = 1.4 (95% CI 1.1-1.9), p = 0.02) was observed. The strength of the effect was similar in the RA subgroup (OR = 1.4 (95% CI 1.0-1.9), p = 0.05). No association between IP susceptibility and any of the other SNPs was detected. No association was detected for any of the SNPs tested, including the PTPN22*C1858T polymorphism, for either erosive status, Larsen score by 5 years or other markers of clinical outcome. CONCLUSION: The PTPN22*C1858T polymorphism is associated with susceptibility to IP, but we have found no evidence for association of this or other variants spanning the gene with clinical outcome measures.

An assessment of pelvic binder placement at a UK major trauma centre.

Introduction Pelvic binders are used to reduce the haemorrhage associated with pelvic ring injuries. Application at the level of the greater trochanters is required. We assessed the frequency of their use in patients with pelvic ring injuries and their positioning in patients presenting to a single major trauma centre. Methods A retrospective review of our trauma database was performed to randomly select 1000 patients for study from April 2012 to December 2016. Patients with a pelvic binder or a pelvic ring injury defined by the Young and Burgess classification were included. Computed tomography was used to identify and measure pelvic binder placement. Results 140 patients were identified: 110/140 had a binder placed. Of the total, 54 (49.1%) patients had satisfactory placement and 56 (50.9%) had unsatisfactory placement; 30/67 (44.8%) patients with a pelvic ring injury had no binder applied, of whom 6 (20%) had an unstable injury; 9/67 patients died. Discussion This is the first study assessing pelvic binder placement in patients at a UK major trauma centre. Unsatisfactory positioning of the pelvic binder is a common problem and it was not used in a large proportion of patients with pelvic ring injuries. This demonstrates that there is a need for continuing education for teams dealing with major trauma.

Laparoscopic-assisted transversus abdominis plane block as an effective analgesic in total extraperitoneal inguinal hernia repair: a double-blind, randomized controlled trial.

PURPOSE: Laparoscopic inguinal hernia repair has facilitated early mobilization. Management of post-operative pain is paramount in these day case procedures. The aim of this study was to compare laparoscopic-assisted transversus abdominis plane (TAP) block with periportal local anaesthetic infiltration in managing post-operative pain. METHODS: A double-blind, randomized controlled trial was conducted with patients undergoing elective laparoscopic inguinal hernia repair (January 2016-October 2017). The intervention group received laparoscopic-assisted TAP block with 30 ml 0.25% Bupivacaine. The control group received 15ml of 0.5% Bupivacaine at the periportal sites. Primary outcome measure was assessment of post-operative pain scores using numerical rating on visual analogue scale (VAS) at rest and on coughing at 3 h. Efficacy of TAP block was assessed as reduction in mean pain scores in the order of 2 points using the VAS. RESULTS: 60 (57 males and 3 females) were enrolled; 30 patients were randomized to each group. Patient demographics, anaesthetic and surgical times were similar in both groups. Mean pain scores were significantly reduced in the intervention group at 3 (3.1 vs 1.1 p < 0.001) and 6 h (4.1 vs 1.7 p < 0.001) at rest and on coughing at 3 (4.8 vs 2.1 p < 0.001) and 6 h (5.4 vs 3.0 p < 0.001). Patient satisfaction was higher (8.0 vs 6.8 p < 0.001) and rescue analgesic requirements (169.4vs 71.3 p < 0.001) lower in the intervention group. CONCLUSIONS: This analysis has demonstrated the therapeutic benefit of laparoscopic-assisted TAP block in initial post-operative pain management for patients undergoing elective laparoscopic inguinal hernia repair.

Amelioration of Endotoxin-Induced Inflammatory Toxic Response by a Metal Chelator in Rat Eyes.

Purpose: Metal ions play a key role in exacerbating toxicity associated with oxidative stress and inflammation. This study examines the effects of a formulation containing the metal chelator ethylenediaminetetraacetic acid (EDTA) and permeability enhancer methyl sulfonyl methane (MSM) on the early course of inflammation in endotoxin-induced uveitis (EIU). The proprietary MSM/EDTA formulation of Livionex, Inc., which was used for this study, is covered by several patents and pending patent applications. Methods: EIU was induced by using subcutaneous injection of lipopolysaccharide (LPS) into the thighs of Lewis rats. Treatment consisted of topical application to the eyes of either PBS or eye drops designated as ME that contain EDTA and MSM. Clinical signs of uveitis were monitored at 6 and 24 hours postinjection. Oxidative and inflammatory markers were evaluated by ELISA or immunohistochemistry. Results: Rats treated with ME showed fewer clinical signs of uveitis including reduced miosis, fibrinous exudates, and dilated blood vessels. The aqueous humor of treated rats contained fewer leukocytes, lower protein levels, and less PGE2. Formation of protein adducts with the lipid peroxidation end-product, 4-hydroxynonenal, expression of NF-κB, TNF-α, and MMP-9 were all reduced in rats treated with ME. Conclusions: Our results indicate that ME eye drops downregulate the ocular inflammatory response in LPS treated rats, suggesting that induction of EIU involves metal ions and chelation therapy with ME is a potential treatment for uveitis.

Correction to: Laparoscopic-assisted transversus abdominis plane block as an effective analgesic in total extraperitoneal inguinal hernia repair: a double-blind, randomized controlled trial.

In the original publication, one of the co-author 'M. Riaz' details were missed to include in the author group. The complete author group should read as A. Mughal, A. Khan, J. Rehman, H. Naseem, M. Riaz, R. Waldron, M. Duggan, W. Khan, K. Barry, I. Z. Khan.

Role of aldehyde dehydrogenase isozymes in the defense of rat lens and human lens epithelial cells against oxidative stress.

PURPOSE: 4-Hydroxynonenal (HNE), a metastable lipid peroxidation product, is highly toxic to various cell types if not detoxified. Because of its constant exposure to light, the ocular lens continuously generates reactive oxygen species which, under conditions of oxidative stress, may lead to excessive lipid peroxidation and consequent formation of lipid-derived aldehydes (LDAs) such as HNE. The contribution of various isozymes of aldehyde dehydrogenase (ALDH) to the oxidation of LDAs has never been systematically investigated in the lens. The present study was undertaken to ascertain the role of ALDH1A1 and -3A1 in HNE metabolism and HNE-induced toxicity in cultured human lens epithelial cells (HLECs) and in rat and mouse lenses. METHODS: The metabolism of 3H-HNE was studied in ALDH3A1-knockout mouse lens and in HLECs transfected with ALDH1A1- or -3A1-specific antisense RNA and short interfering (Si)RNA. Appropriate controls were used, including wild-type mouse lens, scrambled oligonucleotides, and a transfection reagent. Transfected HLECs were exposed to oxidative stress (Fenton reaction) or HNE (30 microM) for 3 hours. Toxicity parameters, such as cell viability, apoptosis, and protein-HNE adducts and oxidation of exogenously added 3H-HNE were measured. Rat lenses were transfected with the SiRNA specific to ALDH1A1, and oxidation of 3H-HNE and the susceptibility of the transfected lenses to oxidation-induced opacification were measured. RESULTS: Rat lenses transfected with ALDH1A1-specific SiRNA, or cultured in the presence of the ALDH inhibitor cyanamide/disulfiram and subjected to oxidative stress displayed accelerated loss of transparency and a diminished capacity to oxidize HNE. Similarly, inhibition of ALDH1A1 in HLECs by ALDH1A1-specific antisense RNA or SiRNA was associated with decreased oxidation of 3H-HNE and increased susceptibility of the cells to oxidative damage, including apoptosis. Furthermore, 3H-HNE metabolism and HNE-induced toxicity were not affected in ALDH3A1-specific SiRNA- or antisense RNA-treated rat lenses, HLECs, or ALDH3A1-null mouse lenses. CONCLUSIONS: The results suggest that, under oxidative stress, HNE produced in the lens epithelium can cause toxicity and thus contribute to oxidation-induced cataractogenesis. Furthermore, the studies indicate that ALDH1A1 is a critical isozyme for maintaining clarity in human, rat, and mouse lenses.

Attenuation of p38α MAPK stress response signaling delays the in vivo aging of skeletal muscle myofibers and progenitor cells.

Functional competence and self-renewal of mammalian skeletal muscle myofibers and progenitor cells declines with age. Progression of the muscle aging phenotype involves the decline of juvenile protective factorsi.e., proteins whose beneficial functions translate directly to the quality of life, and self-renewal of progenitor cells. These characteristics occur simultaneously with the age-associated increase of p38α stress response signaling. This suggests that the maintenance of low levels of p38α activity of juvenile tissues may delay or attenuate aging. We used the dominant negative haploinsufficient p38α mouse (DN-p38α(AF/+)) to demonstrate that in vivo attenuation of p38α activity in the gastrocnemius of the aged mutant delays age-associated processes that include: a) the decline of the juvenile protective factors, BubR1, aldehyde dehydrogenase 1A (ALDH1A1), and aldehyde dehydrogenase 2 (ALDH2); b) attenuated expression of p16(Ink4a) and p19(Arf) tumor suppressor genes of the Cdkn2a locus; c) decreased levels of hydroxynonenal protein adducts, expression of COX2 and iNOS; d) decline of the senescent progenitor cell pool level and d) the loss of gastrocnemius muscle mass. We propose that elevated P-p38α activity promotes skeletal muscle aging and that the homeostasis of p38α impacts the maintenance of a beneficial healthspan.

Topically applied metal chelator reduces thermal injury progression in a rat model of brass comb burn.

UNLABELLED: Oxidative stress may be involved in the cellular damage and tissue destruction as burn wounds continues to progress after abatement of the initial insult. Since iron and calcium ions play key roles in oxidative stress, this study tested whether topical application of Livionex formulation (LF) lotion, that contains disodium EDTA as a metal chelator and methyl sulfonyl methane (MSM) as a permeability enhancer, would prevent or reduce burns. METHODS: We used an established brass comb burn model with some modifications. Topical application of LF lotion was started 5 min post-burn, and repeated every 8 h for 3 consecutive days. Rats were euthanized and skin harvested for histochemistry and immunohistochemistry. Formation of protein adducts of 4-hydroxynonenal (HNE), malonadialdehyde (MDA) and acrolein (ACR) and expression of aldehyde dehydrogenase (ALDH) isozymes, ALDH1 and ALDH2 were assessed. RESULTS: LF lotion-treated burn sites and interspaces showed mild morphological improvement compared to untreated burn sites. Furthermore, the lotion significantly decreased the immunostaining of lipid aldehyde-protein adducts including protein -HNE, -MDA and -ACR adducts, and restored the expression of aldehyde dehydrogenase isozymes in the unburned interspaces. CONCLUSION: This data, for the first time, demonstrates that a topically applied EDTA-containing lotion protects burns progression with a concomitant decrease in the accumulation of reactive lipid aldehydes and protection of aldehyde dehydrogenase isozymes. Present studies are suggestive of therapeutic intervention of burns by this novel lotion.

Metabolism of lipid derived aldehyde, 4-hydroxynonenal in human lens epithelial cells and rat lens.

PURPOSE: An earlier study showed that 4-hydroxynonenal (HNE), formed as a result of increased lipid peroxidation in oxidative stress, causes loss of lens transparency. To determine how HNE is detoxified in ocular tissues, its metabolism in cultured human lens epithelial cells (HLECs) as well as rat lens was investigated. METHODS: Rat lens or HLECs were incubated with 30 nmol (5 x 10(5) cpm/ micromol) of HNE in 2 mL Krebs-Hansleit buffer for 1 hour at 37 degrees C. The medium, after ultrafiltration was analyzed by high performance liquid chromatography (HPLC), using a C-18 reversed-phase column. The metabolites were separated by using a gradient consisting of solvent A (0.1% aqueous trifluoroacetic acid) and solvent B (100% acetonitrile) at a flow rate of 1 mL/min. Fractions containing radioactivity were pooled and analyzed using electrospray ionization mass spectroscopy (ESI-MS) or gas chromatography-chemical ionization mass spectroscopy (GC/CI-MS). RESULTS: On HPLC, the incubation media from cultured lens and HLECs separated into three major radioactive peaks. Peak I of the HLECs and lens treated with HNE was identified to be a mixture of glutathione (GS) conjugates of HNE and 1,4-dihydroxy-2-nonene (DHN). The identity of the conjugates was confirmed by ESI-MS. Based on the retention times, peaks II, and III were assigned to 4-hydroxy-2-nonenoic acid (HNA) and unmetabolized HNE, respectively. The identities of HNA and HNE were confirmed by spiking the tissue extracts with synthetic metabolites and finally by GC/CI-MS. Sorbinil, an aldose reductase (AR) inhibitor, attenuated GS-DHN levels and cyanamide, an aldehyde dehydrogenase inhibitor, decreased formation of HNA. CONCLUSIONS: The results show that the major metabolic transformation of HNE in rat lens and HLECs involves conjugation with GS and oxidation to HNA. The GS-HNE conjugate is reduced to GS-DHN by AR. Thus, under normal physiological conditions, the lens has multiple routes to detoxify HNE. However, oxidative stress may overwhelm the metabolic capacity of the lens to detoxify HNE and lead to formation of cataract.

Protection of HLE B-3 cells against hydrogen peroxide- and naphthalene-induced lipid peroxidation and apoptosis by transfection with hGSTA1 and hGSTA2.

PURPOSE: To investigate the physiological role of two major alpha-class glutathione S-transferases (GSTs), hGSTA1-1 and hGSTA2-2 in protection against oxidative stress and lipid peroxidation (LPO) in human lens epithelial (HLE B-3) cells. METHODS: Total GSTs were purified from HLE B-3 cells by glutathione (GSH)-affinity chromatography and characterized by Western blot analysis, isoelectric focusing, and kinetic studies. The relative contributions of the alpha-class GSTs and the Se-dependent glutathione peroxidase (GPx)-1 in GSH-dependent reduction of phospholipid hydroperoxide (PL-OOH) were quantitated through immunoprecipitation studies using separately the specific polyclonal antibodies against human alpha-class GSTs and GPx-1. HLE B-3 cell membranes were prepared, peroxidized, and used to examine whether hGSTA1-1 and hGSTA2-2 catalyzes the reduction of membrane PL-OOH in situ using the microiodometric and spectrophotometric assays. The protective effects of the alpha-class GSTs against H2O2- and naphthalene-induced LPO and apoptosis were examined by transfecting HLE B-3 cells with cDNAs of hGSTA1 and hGSTA2. RESULTS. HLE B-3 cells expressed only the alpha and pi class GSTs. The Michaelis-Menten constant (k(m)) and turnover number (k(cat)) of purified total GSTs toward phosphatidylcholine hydroperoxide (PC-OOH) were found to be 30 +/- 4 microM and 1.95 +/- 0.26 seconds, respectively. The alpha-class GSTs accounted for approximately 65% of the total GPx activity of HLE B-3 cells toward PC-OOH. Our results demonstrate for the first time that hGSTA1-1 and hGSTA2-2 effectively catalyzed GSH-dependent reduction of membrane PL-OOH in situ in HLE B-3 cells. Transfection with hGSTA1 or hGSTA2 protected these cells from H2O2- and naphthalene-induced LPO and attenuated H2O2- and naphthalene-induced apoptosis through inhibiting caspase 3 activation. CONCLUSIONS: These results demonstrate that the alpha-class GSTs hGSTA1-1 and hGSTA2-2 play a major role as antioxidant enzymes and are the main determinants of the levels of LPO caused by oxidative stress in human lens epithelial cells.