Monitoring of epidermal growth factor receptor tyrosine kinase inhibitor-sensitizing and resistance mutations in the plasma DNA of patients with advanced non-small cell lung cancer during treatment with erlotinib.

BACKGROUND: The feasibility of monitoring epidermal growth factor receptor (EGFR) mutations in plasma DNA from patients with advanced non-small cell lung cancer (NSCLC) during treatment with erlotinib and its relation to disease progression was investigated. METHODS: The amount of EGFR-mutant DNA was tested in plasma DNA from patients with advanced NSCLC with allele-specific polymerase chain reaction assays. Blood samples from 23 patients with adenocarcinoma of NSCLC that carried tyrosine kinase inhibitor-sensitizing EGFR mutations were taken immediately before treatment with erlotinib. Additional blood samples were taken at timed intervals until erlotinib treatment was withdrawn. RESULTS: The amount of plasma DNA with sensitizing EGFR mutations was found to be reduced after the first cycle of erlotinib treatment in 22 of 23 patients (96%). No patients presented with the resistant T790M mutation in the pretreatment sample, but at the time of disease progression the mutation was detected in plasma from 9 patients (39%). The quantitative data from the current study demonstrated that when a T790M mutation emerged in the blood it was accompanied by an increase in the original sensitizing EGFR mutation. When T790M was detected, it was found to be present in all subsequent blood samples from that patient. Most interestingly, the results of the current study demonstrated that monitoring the EGFR mutations in the blood allows for the detection of the T790M mutation up to 344 days before disease progression is clinically evident (range, 15-344 days). CONCLUSIONS: The results of the current study demonstrated that serial monitoring of EGFR mutations in plasma DNA is feasible and may allow for the early detection of resistance mutations. These results warrant further studies to explore the clinical usefulness of such analysis.

Detection of EGFR mutations in plasma and biopsies from non-small cell lung cancer patients by allele-specific PCR assays.

BACKGROUND: Lung cancer patients with mutations in the epidermal growth factor receptor (EGFR) are primary candidates for EGFR-targeted therapy. Reliable analyses of such mutations have previously been possible only in tumour tissue. Here, we demonstrate that mutations can be detected in plasma samples with allele-specific PCR assays. METHODS: Pairs of the diagnostic biopsy and plasma obtained just prior to start of erlotinib treatment were collected from 199 patients with adenocarcinoma of non-small-cell lung cancer. DNA from both sample types was isolated and examined for the presence of mutations in exons 18-21 of the EGFR gene, employing the cobas(®) EGFR Tissue Test and cobas(®) EGFR Blood Test (in development, Roche Molecular Systems, Inc., CA, USA). RESULTS: Test results were obtained in all 199 (100%) plasma samples and 196/199 (98%) of the biopsies. EGFR-activating mutations were identified in 24/199 (12%) plasma samples and 28/196 (14%) biopsy samples, and 17/196 (9%) matched pairs contained the same mutation. Six EGFR mutations were present only in plasma samples but not in the biopsy samples. The overall concordance of the EGFR gene mutations detected in plasma and biopsy tissue was 179/196 (91%) (kappa value: 0.621). CONCLUSION: Mutational analysis of the EGFR gene in plasma samples is feasible with allele-specific PCR assays and represents a non-invasive supplement to biopsy analysis. TRIAL REGISTRATION: M-20080012 from March 10, 2008 and reported to ClinicalTrials.gov: NCT00815971.

Analytic performance studies and clinical reproducibility of a real-time PCR assay for the detection of epidermal growth factor receptor gene mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer.

BACKGROUND: Epidermal growth factor receptor (EGFR) gene mutations identify patients with non-small cell lung cancer (NSCLC) who have a high likelihood of benefiting from treatment with anti-EGFR tyrosine kinase inhibitors. Sanger sequencing is widely used for mutation detection but can be technically challenging, resulting in longer turn-around-time, with limited sensitivity for low levels of mutations. This manuscript details the technical performance verification studies and external clinical reproducibility studies of the cobas EGFR Mutation Test, a rapid multiplex real-time PCR assay designed to detect 41 mutations in exons 18, 19, 20 and 21. METHODS: The assay's limit of detection was determined using 25 formalin-fixed paraffin-embedded tissue (FFPET)-derived and plasmid DNA blends. Assay performance for a panel of 201 specimens was compared against Sanger sequencing with resolution of discordant specimens by quantitative massively parallel pyrosequencing (MPP). Internal and external reproducibility was assessed using specimens tested in duplicate by different operators, using different reagent lots, instruments and at different sites. The effects on the performance of the cobas EGFR test of endogenous substances and nine therapeutic drugs were evaluated in ten FFPET specimens. Other tests included an evaluation of the effects of necrosis, micro-organisms and homologous DNA sequences on assay performance, and the inclusivity of the assay for less frequent mutations. RESULTS: A >95% hit rate was obtained in blends with >5% mutant alleles, as determined by MPP analysis, at a total DNA input of 150 ng. The overall percent agreement between Sanger sequencing and the cobas test was 96.7% (negative percent agreement 97.5%; positive percent agreement 95.8%). Assay repeatability was 98% when tested with two operators, instruments, and reagent lots. In the external reproducibility study, the agreement was > 99% across all sites, all operators and all reagent lots for 11/12 tumors tested. Test performance was not compromised by endogenous substances, therapeutic drugs, necrosis up to 85%, and common micro-organisms. All of the assessed less common mutations except one (exon 19 deletion mutation 2236_2248 > AGAC) were detected at a similar DNA input level as that for the corresponding predominant mutation. CONCLUSION: The cobas EGFR Mutation Test is a sensitive, accurate, rapid, and reproducible assay.

Apolipoprotein B100 acts as a molecular link between lipid-induced endoplasmic reticulum stress and hepatic insulin resistance.

UNLABELLED: Accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER) results in ER stress and lipid overload-induced ER stress has been implicated in the development of insulin resistance. Here, evidence is provided for a molecular link between hepatic apolipoprotein B100 (apoB100), induction of ER stress, and attenuated insulin signaling. First, in vivo upregulation of hepatic apoB100 by a lipogenic diet was found to be closely associated with ER stress and attenuated insulin signaling in the liver. Direct in vivo overexpression of human apoB100 in a mouse transgenic model further supported the link between excessive apoB100 expression and hepatic ER stress. Human apoB100 transgenic mice exhibited hypertriglyceridemia and hyperglycemia. In vitro, accumulation of cellular apoB100 by free fatty acid (oleate) stimulation or constant expression of wild-type or N-glycosylation mutant apoB50 in hepatic cells induced ER stress. This led to perturbed activation of glycogen synthase kinase 3 and glycogen synthase by way of the activation of c-Jun N-terminal kinase and suppression of insulin signaling cascade, suggesting that dysregulation of apoB was sufficient to disturb ER homeostasis and induce hepatic insulin resistance. Small interfering (si)RNA-mediated attenuation of elevated apoB level in the apoB50-expressing cells rescued cells from lipid-induced ER stress and reversed insulin insensitivity. CONCLUSION: These findings implicate apoB100 as a molecular link between lipid-induced ER stress and hepatic insulin resistance.

Phosphatase and tensin homolog (PTEN) regulates hepatic lipogenesis, microsomal triglyceride transfer protein, and the secretion of apolipoprotein B-containing lipoproteins.

Hepatic apolipoprotein B (apoB) lipoprotein production is metabolically regulated via the phosphoinositide 3-kinase cascade; however, the role of the key negative regulator of this pathway, the tumor suppressor phosphatase with tensin homology (PTEN), is unknown. Here, we demonstrate that hepatic protein levels of apoB100 and microsomal triglyceride transfer protein (MTP) are significantly down-regulated (73% and 36%, respectively) in the liver of PTEN liver-specific knockout (KO) mice, and this is accompanied by increased triglyceride (TG) accumulation and lipogenic gene expression, and reduced hepatic apoB secretion in freshly isolated hepatocytes. MTP protein mass and lipid transfer activity were also significantly reduced in liver of PTEN KO mice. Overexpression of the dominant negative mutant PTEN C/S124 (adenovirus expressing PTEN C/S mutant [AdPTENC/S]) possessing constitutive phospoinositide 3-kinase activity in HepG2 cells led to significant reductions in both secreted apoB100 and cellular MTP mass (76% and 34%, respectively), and increased messenger RNA (mRNA) levels of sterol regulatory element binding protein 1c (SREBP-1c), fatty acid synthase (FAS), and acetyl-CoA carboxylase (ACC). Reduced apoB100 secretion induced by AdPTENC/S was associated with increased degradation of newly-synthesized cellular apoB100, in a lactacystin-sensitive manner, suggesting enhanced proteasomal degradation. AdPTENC/S also reduced apoB-lipoprotein production in McA-RH7777 and primary hamster hepatocytes. Our findings suggest a link between PTEN expression and hepatic production of apoB-containing lipoproteins. We postulate that perturbations in PTEN not only may influence hepatic insulin signaling and hepatic lipogenesis, but also may alter hepatic apoB-lipoprotein production and the MTP stability. On loss of PTEN activity, increased lipid substrate availability in the face of reduced hepatic lipoprotein production capacity can rapidly lead to hepatosteatosis and fatty liver.

MEK-ERK inhibition corrects the defect in VLDL assembly in HepG2 cells: potential role of ERK in VLDL-ApoB100 particle assembly.

OBJECTIVE: Hepatic VLDL assembly is defective in HepG2 cells, resulting in the secretion of immature triglyceride-poor LDL-sized apoB particles. We investigated the mechanisms underlying defective VLDL assembly in HepG2 and have obtained evidence implicating the MEK-ERK pathway. METHODS AND RESULTS: HepG2 cells exhibited considerably higher levels of the ERK1/2 mass and activity compared with primary hepatocytes. Inhibition of ERK1/2 using the MEK1/MEK2 inhibitor, U0126 (but not the inactive analogue) led to a significant increase in apoB secretion. In the presence of oleic acid, ERK1/2 inhibition caused a major shift in the lipoprotein distribution with a majority of particles secreted as VLDL, an effect independent of insulin. In contrast, overexpression of constitutively active MEK1 decreased apoB and large VLDL secretion. MEK1/2 inhibition significantly increased both cellular and microsomal TG mass, and mRNA levels for DGAT-1 and DGAT-2. In contrast to ERK, modulation of the PI3-K pathway or inhibition of the p38 MAP kinase, had no effect on lipoprotein density profile. Modulation of the MEK-ERK pathway in primary hamster hepatocytes led to changes in apoB secretion and altered the density profile of apoB-containing lipoproteins. CONCLUSIONS: Inhibition of the overactive ras-MEK-ERK pathway in HepG2 cells can correct the defect in VLDL assembly leading to the secretion of large, VLDL-sized particles, similar to primary hepatocytes, implicating the MEK-ERK cascade in VLDL assembly in the HepG2 model. Modulation of this pathway in primary hepatocytes also regulates apoB secretion and appears to alter the formation of VLDL-1 sized particles.

Development of an in vitro model to assess posterior capsule safety during phacoemulsification with ultrasound or AquaLase handpieces.

PURPOSE: To develop a cadaver eye model that would assess posterior capsule (PC) vulnerability when different cataract removal technologies were evaluated and use the model to evaluate the relative amplitude levels required to rupture the human PC with the AquaLase handpiece (Alcon) compared with an ultrasound (US) handpiece. SETTING: Private practice, Cincinnati, Ohio, USA. METHODS: In part 1 of the study, 26 phakic human cadaver eyes were sectioned in the anteroposterior meridian. The anterior portion of the globe was placed cornea side down, and the vitreous was gently cleared from the posterior surface of the lens capsule. Ultrasound was applied directly to the central capsule after achieving a vacuum of 100 mm Hg. The power was incremented until rupture was observed. In part 2 of the study, the same procedure was repeated with 50 eye pairs. For each pair, US was randomly applied to 1 eye and AquaLase to the other. The 50 pairs were divided into 3 groups based on vacuum level: 100, 300, or 500 mm Hg. RESULTS: Part 1 results show that at 100 mm Hg, the mean power needed to break the PC with US was 19% +/- 6% (SD). There was no correlation between time from harvest to test and rupture power (R(2) = 0.04) or between donor age and rupture power (R(2) = 0.2). When the same procedure was repeated in part 2, on average, US power ruptured the capsule at a lower power than the AquaLase magnitude at each vacuum setting. The respective means were 18.5% +/- 6.7% and 61.0% +/- 23.3% for 100 mm Hg, 15.2% +/- 5.4% and 47.1% +/- 14.5% for 300 mm Hg, and 11.8% +/- 6.5% and 20.0% +/- 9.4% for 500 mm Hg. The difference between the groups and the values within each group decreased as vacuum levels increased. CONCLUSION: This new experimental model in a cadaver eye provides a useful method for comparing factors and techniques that contribute to PC rupture.

Glucosamine-induced endoplasmic reticulum stress promotes ApoB100 degradation: evidence for Grp78-mediated targeting to proteasomal degradation.

OBJECTIVE: To investigate the role of glucosamine-mediated endoplasmic reticulum (ER) stress and Grp78 (BiP) in the intracellular degradation of apolipoprotein B100 (apoB100) in cultured hepatocytes. METHODS AND RESULTS: Glucosamine treatment (2.5 to 10 mmol/L) of HepG2 cells increased levels of the ER chaperones, 78-kDa glucose-regulated protein (Grp78) and Grp94, in a dose-dependent manner and led to significant decreases in both cellular and secreted apoB100 by up to 97% (P<0.01). In contrast, no changes were observed in ER resident (ER60, PTP-1B) or secretory (albumin, apoE) control proteins. Glucosamine-induced apoB degradation was similarly observed in primary hamster hepatocytes and McA-RH7777 cells. Glucosamine treatment led to reduced tranlocational efficiency of apoB100 in the ER and enhanced its ubiquitination and proteasomal degradation. Adenoviral overexpression of Grp78 also led to significantly decreased levels of newly synthesized apoB100 in a dose-dependent manner (P<0.01). Grp78-induced downregulation of apoB100 was sensitive to inhibition by the proteasome inhibitor, lactacystin, but not lysosomal protease inhibitors, E64 and leupeptin, suggesting that overexpression of Grp78 selectively induced proteasomal degradation of apoB100. CONCLUSIONS: These findings suggest that binding and retention by Grp78 may play a critical role in proteasomal targeting and the ER quality-control of misfolded apoB. Interaction with core lipoprotein lipids may facilitate apoB transport out of the ER by reducing Grp78-mediated ER retention.

Color perception with AcrySof natural and AcrySof single-piece intraocular lenses under photopic and mesopic conditions.

PURPOSE: To examine color perception in patients receiving bilateral implantation of an ultraviolet (UV) and blue-light filtering intraocular lens (IOL) (AcrySof Natural SN60AT, Alcon Laboratories Inc.) or a UV-only filtering IOL (AcrySof SA60AT) and to compare the results with those in a phakic group. SETTING: Cincinnati, Ohio, USA. METHODS: In this prospective study, age-matched subjects who passed the Ishihara test and had visual acuities of 20/25 or better were recruited. There were 2 pseudophakic groups (bilateral SN60AT or SA60AT IOLs) and 1 phakic group. The Farnsworth-Munsell (FM) 100-hue test was administered to each subject twice under different conditions. The phakic and AcrySof Natural SN60AT groups were tested under photopic and mesopic conditions. The SA60AT subjects were further divided into subgroups (with and without yellow clip-on lenses) and tested under photopic and mesopic conditions. RESULTS: A 1-way analysis of variance (ANOVA) of the square-root-transformed total error score showed no statistical differences (P = .637) between the treatment groups. Similarly, a 1-way ANOVA of the red-green error score (P = .729) and blue-yellow error score (P = .484) indicated no statistically significant differences between the treatment groups. The ANOVA results of the FM 100-hue test under mesopic conditions showed that the total error score in the AcrySof Natural IOL group was significantly lower (P = .046) than in the phakic group. There were no between-group differences in error scores under mesopic conditions. CONCLUSION: The FM 100-hue testing showed no difference in color perception between subjects with AcrySof Natural IOLs and those in an age-matched phakic control group or in those with a UV-only filtering AcrySof IOL with or without yellow clip-on lenses.

Incidence and prevalence of glaucoma in severe ocular surface disease.

PURPOSE: To describe the incidence and prevalence of glaucoma in a patient population with severe ocular surface disease (OSD). METHODS: A retrospective case series was compiled from all charts of patients in the Cincinnati Eye Institute/University of Cincinnati and University of Minnesota population with a diagnosis of severe OSD from 1991 to 2003. The incidence and prevalence of glaucoma in the overall patient population were identified, and stratified into disease subgroups. RESULTS: Of the 108 eyes evaluated in this study, 71 were diagnosed with glaucoma. The overall prevalence of glaucoma in patients with severe OSD is 65.7%, with a range from 42.9% to 88.4%. Analysis by subgroup shows the highest percentage of patients with concurrent glaucoma fall into the categories of aniridia and chemical injury, and the lowest was noted in those patients with autoimmune or iatrogenic OSD. Overall, the incidence of glaucoma was 20.4%, with a range of 13.6% to 60%. CONCLUSIONS: Compared with previous studies, our results show a significantly higher prevalence of glaucoma in patients with severe OSD. This information warrants increased attention to treatment and management of OSD and concurrent glaucoma.

Anterior uveitis and iris nodules that are associated with Langerhans cell histiocytosis.

PURPOSE: To describe a case of Langerhans cell histiocytosis (LCH) that involved the anterior uveal tract. DESIGN: Interventional case report. METHODS: A retrospective review was conducted on a patient with iris nodules and anterior uveitis in the setting of LCH. Visual acuity and clinical findings that were noted on slit lamp biomicroscopy were extracted. RESULTS: An 18-year-old male patient with unilateral anterior segment inflammation and iris nodules experienced visual improvement from 20/200 to 20/25 after treatment with a 5-day course of topical corticosteroids. Regression of the iris nodules and anterior segment inflammation was also noted. Bone marrow aspirate confirmed recurrent, active LCH. CONCLUSION: The clinician should include LCH in the differential diagnosis when faced with anterior segment inflammation in conjunction with iris nodules. Additionally, LCH can be treated successfully with topical corticosteroid therapy.

A progressive anterior fibrosis syndrome in patients with postsurgical congenital aniridia.

PURPOSE: To report the characteristics of a newly recognized clinical entity in congenital aniridia that we have termed aniridic fibrosis syndrome. DESIGN: Interventional case series. METHODS: Retrospective chart review of 155 eyes in 80 patients with congenital aniridia was carried out to identify and characterize eyes that had anterior chamber fibrosis. Histopathologic evaluation was performed in three eyes. RESULTS: Seven eyes in six patients were identified to have aniridic fibrosis syndrome. All eyes had undergone previous intraocular anterior segment surgery, some eyes with multiple procedures. Seven eyes had undergone cataract surgery with posterior chamber intraocular lens; six eyes had undergone previous implantation of tube shunt devices, and four eyes had undergone previous penetrating keratoplasty. Clinically, the syndrome was characterized by a progressive retrolenticular and retrocorneal membrane that caused forward displacement of intraocular lenses. Surgical findings indicated that the fibrotic membrane also can involve the ciliary body and anterior retina. Histopathologic evidence from three eyes indicated that the extensive fibrotic tissue originated from the root of the rudimentary iris and entrapped the intraocular lens haptics. Endothelial decompensation that was subsequent to the formation of the aniridic fibrosis syndrome was seen in all eyes. CONCLUSION: Aniridic fibrosis syndrome is characterized by the development of a progressive anterior chamber fibrosis. A possible mechanism that promotes the formation of this fibrotic material may be the proximity or touching of intraocular devices on immature vessels in the rudimentary iris found in aniridia. Patients with aniridia with a history of penetrating keratoplasty, intraocular lenses, and tube shunts should be monitored for aniridic fibrosis syndrome; early surgical intervention is recommended.

Detection and Dynamic Changes of EGFR Mutations from Circulating Tumor DNA as a Predictor of Survival Outcomes in NSCLC Patients Treated with First-line Intercalated Erlotinib and Chemotherapy.

PURPOSE: Blood-based circulating-free (cf) tumor DNA may be an alternative to tissue-based EGFR mutation testing in NSCLC. This exploratory analysis compares matched tumor and blood samples from the FASTACT-2 study. EXPERIMENTAL DESIGN: Patients were randomized to receive six cycles of gemcitabine/platinum plus sequential erlotinib or placebo. EGFR mutation testing was performed using the cobas tissue test and the cobas blood test (in development). Blood samples at baseline, cycle 3, and progression were assessed for blood test detection rate, sensitivity, and specificity; concordance with matched tumor analysis (n = 238), and correlation with progression-free survival (PFS) and overall survival (OS). RESULTS: Concordance between tissue and blood tests was 88%, with blood test sensitivity of 75% and a specificity of 96%. Median PFS was 13.1 versus 6.0 months for erlotinib and placebo, respectively, for those with baseline EGFR mut(+) cfDNA [HR, 0.22; 95% confidence intervals (CI), 0.14-0.33, P < 0.0001] and 6.2 versus 6.1 months, respectively, for the EGFR mut(-) cfDNA subgroup (HR, 0.83; 95% CI, 0.65-1.04, P = 0.1076). For patients with EGFR mut(+) cfDNA at baseline, median PFS was 7.2 versus 12.0 months for cycle 3 EGFR mut(+) cfDNA versus cycle 3 EGFR mut(-) patients, respectively (HR, 0.32; 95% CI, 0.21-0.48, P < 0.0001); median OS by cycle 3 status was 18.2 and 31.9 months, respectively (HR, 0.51; 95% CI, 0.31-0.84, P = 0.0066). CONCLUSIONS: Blood-based EGFR mutation analysis is relatively sensitive and highly specific. Dynamic changes in cfDNA EGFR mutation status relative to baseline may predict clinical outcomes.

Comparison of molecular testing methods for the detection of EGFR mutations in formalin-fixed paraffin-embedded tissue specimens of non-small cell lung cancer.

AIM: To conduct a methods correlation study of three different assays for the detection of mutations at EGFR gene in human formalin-fixed paraffin-embedded tumour (FFPET) specimens of non-small cell lung carcinomas (NSCLC). METHODS: We conducted a 2-site method comparison study of two european conformity (CE) in vitro diagnostic (IVD)-marked assays, the cobas EGFR Mutation Test and the Therascreen EGFR29 Mutation Kit, and 2× bidirectional Sanger sequencing. We blind-tested 124 NSCLC FFPET specimens with all three methods; the cobas test was performed at both sites. Positive (PPA) and negative percent agreements (NPA) were determined for the cobas test versus each of the other two methods. Specimens yielding discordant test results between methods were further tested using quantitative massively parallel pyrosequencing (MPP). RESULTS: PPA between cobas and Sanger was 98.8%; NPA was 79.3%. Overall there were seven discordant results. MPP confirmed an exon 19 deletion in two cases and L858R mutation in four cases. PPA between cobas and Therascreen was 98.9% and NPA was 100%. There was one discordant result. Reproducibility of the cobas test between the two sites was 99.2%. CONCLUSIONS: The invalid rates for the cobas test and Therascreen were lower than Sanger sequencing. The cobas and Therascreen assays showed a high degree of concordance, and both were more sensitive for the detection of exon 19 deletion and L858R mutations than Sanger. The cobas test was highly reproducible between the two testing sites, used the least amount of DNA input and was the only test with automated results reporting.

Inflammatory NF-kappaB activation promotes hepatic apolipoprotein B100 secretion: evidence for a link between hepatic inflammation and lipoprotein production.

Insulin-resistant states are commonly associated with chronic inflammation and hepatic overproduction of apolipoprotein B100 (apoB100), leading to hypertriglyceridemia and a metabolic dyslipidemic profile. Molecular mechanisms linking hepatic inflammatory cascades and the pathways of apoB100-lipoprotein production are, however, unknown. In the present study, we employed a diet-induced, insulin-resistant hamster model, as well as cell culture studies, to investigate the potential link between activation of hepatic inflammatory nuclear factor-kappaB (NF-kappaB) signaling cascade and the synthesis and secretion of apoB100-containing lipoproteins. Using an established insulin-resistant animal model, the fructose-fed hamster, we found that feeding fructose (previously shown to induce hepatic inflammation) for as little as 4 days reduced hepatic IkappaB (inhibitor of NF-kappaB) level, indicating activation of the inflammatory NF-kappaB cascade. Importantly, IKK (IkappaB kinase) inhibition was found to suppress apoB100 overproduction in fructose-fed hamster hepatocytes. As IKK, the upstream activator of NF-kappaB has been shown to inhibit insulin signaling, and insulin is a major regulator of apoB100, we modulated IKK activity in primary hamster hepatocytes and HepG2 cells and assessed the effects on hepatic apoB100 biosynthesis. Inhibition of the IKK-NF-kappaB pathway by BMS345541 and activation of the pathway by adenoviral-mediated IKK overexpression decreased and increased newly synthesized apoB100 levels, respectively. Pulse-chase and metabolic labeling experiments revealed that IKK activation regulates apoB100 levels at the levels of apoB100 biosynthesis and protein stability. Inhibition of the IKK-NF-kappaB pathway significantly enhanced proteasomal degradation of hepatic apoB100, while direct IKK activation led to reduced degradation and increased apoB100 mRNA translation. Together, our results reveal important links between modulation of the inflammatory IKK-NF-kappaB signaling cascade and hepatic synthesis and secretion of apoB100-containing lipoproteins. Hepatic inflammation may be an important underlying factor in hepatic apoB100 overproduction observed in insulin resistance.

Utility of lumbar puncture in diagnosis of Vogt-Koyanagi-Harada disease.

PURPOSE: To determine the significance of lumbar puncture in diagnosis of Vogt-Koyanagi-Harada disease (VKH). METHOD: A retrospective analysis was conducted on 116 consecutive patients diagnosed with VKH. Two additional patients who presented with acute VKH were included in the analysis. Demographic characteristics, including gender, age, and ethnicity, were extracted from the medical record. The stage of disease at presentation was documented. Pertinent laboratory results and diagnostic procedures such as lumbar puncture, fluorescein angiography, and echography that contributed to the diagnosis of VKH were collected. RESULTS: Lumbar puncture results for 10 patients were available. Eight of these patients presented with pleocytosis consistent with a diagnosis of VKH. Clinical features and fluorescein angiography confirmed the diagnosis in these patients. Both of the patients who did not exhibit cerebrospinal fluid (CSF) pleocytosis presented with headache, vision loss, and bilateral uveitis. Fluorescein angiography disclosed multiple foci of leakage at the retinal pigment epithelium level with accumulation of dye under the retina and disc leakage, confirming diagnosis of VKH. CONCLUSION: The utility of lumbar puncture as a diagnostic criterion for VKH should be re-evaluated given that clinical features and fluorescein angiography alone often support the diagnosis. The inherent risks and complications associated with the procedure must prompt the clinician to reserve this evaluation for atypical presentations.

Vogt-Koyanagi-Harada disease in Hispanic patients.

PURPOSE: To describe the clinical features of Vogt-Koyanagi-Harada disease (VKH) in Hispanic patients. METHODS: Retrospective review of the records of 48 Hispanic patients diagnosed with VKH. The patients were divided into two groups: patients in the early phase of VKH (n = 11) were those who presented within 1 month after the onset of symptoms; patients in the late or chronic VKH phase (n = 37) were those who presented 6 months after onset of symptoms. Demographic data, clinical features, complications and initial and final visual acuity for each patient were recorded. RESULTS: All 11 patients in early phase VKH presented with bilateral uveitis (100%). Meningismus was noted in six cases and auditory disturbances in three. Ocular findings for these 11 patients included exudative retinal detachment in ten patients (91%) and marked optic disc edema in one patient. In the late phase VKH, ocular findings included sunset glow fundus in 26 patients (70%), peripheral nummular scars in 27 (73%), and retinal pigment epithelium hyperplasia in seven (19%). Extraocular manifestations noted in this group of patients included vitiligo in four, poliosis in six, and alopecia in five; auditory disturbances were found in four patients. The visual acuity improved in 60-70% of the patients after treatment with corticosteroids alone or in combination with immunosuppressive agents. CONCLUSION: Hispanic patients with VKH often present without extraocular changes during early phase of the disease. However, once the disease evolves into the chronic phase, integumentary system involvement may become apparent in some patients.

Vogt-Koyanagi-Harada disease diagnostic criteria.

Several different sets of criteria have been proposed to establish the diagnosis of Vogt-Koyanagi-Harada disease (VKH). Various investigators have used the criteria proposed by Sugiura, those proposed by by the American Uveitis Society as well as the revised diagnostic criteria proposed by the First VKH International Workshop group. These three sets of criteria share several clinical features that are considered to be essential for establishing the diagnosis of VKH, including bilateral uveitis, meningismus, and other extraocular changes. The detection of cerebrospinal fluid pleocytosis is considered to be an absolute in the criteria proposed by Sugiura but is not required for the diagnosis of VKH by the revised diagnostic criteria. We applied the latter diagnostic criteria to 28 well-documented patients with early phase VKH and to 88 patients examined during the late phase of VKH. All of these early and late phase patients fulfilled the criteria of the revised diagnostic criteria proposed by the workshop group, indicating 100% concurrence. However, none of the above proposed criteria were prospectively validated to show the positive and negative predictive value of the proposed criteria. Such a prospective study should be undertaken to address the validity of any one or all of the above sets of VKH diagnostic criteria.

Vogt-Koyanagi-Harada disease presenting as optic neuritis.

Vogt-Koyanagi-Harada (VKH) disease is a granulomatous multisystem inflammatory disorder that classically affects the uvea, inner ear, meninges, and skin. We report three patients who presented with initial findings suggestive of bilateral optic neuritis requiring CSF analysis and brain images. None of these patients had extraocular changes. Fluorescein angiography of the retina led to the diagnosis of VKH disease in all patients. Vogt-Koyanagi-Harada disease should be included in differential diagnosis of bilateral optic neuritis, even when extraocular manifestations of the disease are absent. In such cases, fluorescein angiography will aid diagnosis.