Extracellular HMGB1 exacerbates autoimmune progression and recurrence of type 1 diabetes by impairing regulatory T cell stability.

AIMS/HYPOTHESIS: High-mobility group box 1 (HMGB1), an evolutionarily conserved chromosomal protein, was rediscovered to be a 'danger signal' (alarmin) that alerts the immune system once released extracellularly. Therefore, it has been recognised contributing to the pathogenesis of autoimmune diabetes, but its exact impact on the initiation and progression of type 1 diabetes, as well as the related molecular mechanisms, are yet to be fully characterised. METHODS: In the current report, we employed NOD mice as a model to dissect the impact of blocking HMGB1 on the prevention, treatment and reversal of type 1 diabetes. To study the mechanism involved, we extensively examined the characteristics of regulatory T cells (Tregs) and their related signalling pathways upon HMGB1 stimulation. Furthermore, we investigated the relevance of our data to human autoimmune diabetes. RESULTS: Neutralising HMGB1 both delayed diabetes onset and, of particular relevance, reversed diabetes in 13 out of 20 new-onset diabetic NOD mice. Consistently, blockade of HMGB1 prevented islet isografts from autoimmune attack in diabetic NOD mice. Using transgenic reporter mice that carry a Foxp3 lineage reporter construct, we found that administration of HMGB1 impairs Treg stability and function. Mechanistic studies revealed that HMGB1 activates receptor for AGE (RAGE) and toll-like receptor (TLR)4 to enhance phosphatidylinositol 3-kinase (PI3K)-Akt-mechanistic target of rapamycin (mTOR) signalling, thereby impairing Treg stability and functionality. Indeed, high circulating levels of HMGB1 in human participants with type 1 diabetes contribute to Treg instability, suggesting that blockade of HMGB1 could be an effective therapy against type 1 diabetes in clinical settings. CONCLUSIONS/INTERPRETATION: The present data support the possibility that HMGB1 could be a viable therapeutic target to prevent the initiation, progression and recurrence of autoimmunity in the setting of type 1 diabetes.

Ubc9 deficiency selectively impairs the functionality of common lymphoid progenitors (CLPs) during bone marrow hematopoiesis.

Hematopoietic development occurs in the bone marrow, and this process begins with hematopoietic stem cells (HSCs). Ubc9 is a unique E2-conjugating enzyme required for SUMOylation, an evolutionarily conserved post-translational modification system. We herein show that a conditional Ubc9 deletion in the hematopoietic system caused decreased thymus weight and reduced lymphocyte to myeloid cell ratio. Importantly, Ubc9 deletion in the hematopoietic system only selectively impaired the development of common lymphoid progenitors (CLPs) in the bone marrow and perturbed their potential to differentiate into lymphocytes, thereby decreasing the number of T/B cells in the periphery. Ubc9 was found to be required for CLP viability, and therefore, Ubc9 deficiency rendered CLPs to undergo apoptosis and attenuated their proliferation. Thus, Ubc9 plays a critical role in the regulation of CLP function during hematopoietic development in the bone marrow.

Loss of Jak2 protects cardiac allografts from chronic rejection by attenuating Th1 response along with increased regulatory T cells.

Chronic rejection acts as the most formidable obstacle for organ transplantation in clinical settings. Herein we demonstrated in a cardiac transplantation model that blockade of Janus kinase 2 (Jak2) provides protection for cardiac allografts against chronic rejection. Specifically, loss of Jak2 almost completely abolished the production of IFN-γ+ Th1 cells, while the percentage of Foxp3+ regulatory T cells (Tregs) was significantly increased. As a result, loss of Jak2 significantly prolonged allograft survival (58 ± 30.6 days vs. 7 ± 0.3 days). Particularly, 4 out of 13 Jak2 deficient recipients (30%) showed long-term acceptance of allografts as manifested by the graft survival time > 100 days. Cellular studies revealed that Jak2 deficiency did not impact the intrinsic proliferative capability for CD4+ T cells in response to nonspecific polyclonal and allogenic stimulation. Mechanistic studies documented that the impaired Th1 development was caused by the attenuated IFN-γ/STAT1 and IL-12/STAT4 signaling along with repressed expression of Th1 transcription factors T-bet, Hlx and Runx3. However, the IL-2/STAT5 signaling remained intact, which ensured normal Treg development in Jak2-/- naïve CD4 T cells. Together, our data support that blockade of Jak2 may have therapeutic potential for prevention and treatment of allograft rejection in clinical settings.

Feasibility and Benefit of Incorporating a Multimedia Cadaver Laboratory Training Program into a Didactics Curriculum for Junior and Senior Surgical Residents.

OBJECTIVE: As operative experience in general surgery decreases and work hour limitations increase there is less exposure of surgical residents to advanced vascular and trauma exposures. Many institutions have demonstrated benefits of cadaver laboratory courses. We have incorporated a multimedia cadaver laboratory course into our general surgery residency didactics curriculum with the objective to demonstrate a benefit of the program as well as the feasibility of incorporation. STUDY DESIGN: This is a prospective study at a tertiary care institution including general surgery residents within our residency program. A curriculum was designed, requiring residents to complete multimedia learning modules before both a trauma cadaver laboratory and vascular exposure cadaver laboratory. Outcome measures included self-efficacy/confidence (precourse and postcourse 5-point Likert surveys), knowledge (net performance on precourse and postcourse multiple choice examinations), and resident perception of the curriculum (postcourse 5-point Likert survey). Data were analyzed using ANOVA paired t-tests. RESULTS: For the vascular cadaver laboratory, resident knowledge improved overall from an average of 41.2% to 50.0% of questions correct (p = 0.032) and self-efficacy/confidence improved by 0.59 from 1.52 to 2.11 out of 5 (p = 0.009). Median confidence is 1.37 out of 5 and 2.32 out of 5, before and after course, respectively. Wilcoxon nonparametric test reveals a p = 0.011. Resident's perception of the usefulness of the laboratory evaluation was 3.85 out 5. There were 85.71% agreed that the laboratory is useful and 14.29% were disagree. The Z-score is -0.1579 (means 0.1579 standard deviations a score of 3.85 below the benchmark). The percentile rank is 56.27%. The coefficient of variation is 24.68%. For the trauma cadaver laboratory, resident knowledge improved overall from an average of 55.89% to 66.17% of questions correct (p = 0.001) and self-efficacy/confidence improved by 0.75 from 1.68 out of 5 to 2.43 out of 5 (p = 0.011). Median confidence level is 1.41 out of 5 before the training course and 2.64 out of 5 after the training course. Wilcoxon signed rank test gives a p value of 0.008. Resident's perception of the usefulness of the laboratory evaluation was 3.94 out 5. There were 72.22% agreed that the laboratory is useful and 27.78% were neutral. The Z-score is -0.098 (means 0.098 standard deviations a score of 3.94 below the benchmark). The percentile rank is 53.90%. The coefficient of variation is 15.48%. CONCLUSIONS: Incorporating a multimedia cadaver laboratory into a residency education didactics curriculum was both feasible and beneficial for resident education. We demonstrate an improvement in knowledge and self efficacy/confidence following both cadaver laboratory courses.

Descriptive Analysis of Venous Thromboembolism in Georgia Trauma Centers Compared with National Trauma Centers Participating in the Trauma Quality Improvement Program.

This study was designed to compare the incidence of venous thromboembolism (VTE) in Georgia trauma centers with other national trauma centers participating in the Trauma Quality Improvement Program (TQIP). The use of chemoprophylaxis and characteristics of patients who developed VTE were also examined. We conducted a retrospective observational study of 325,703 trauma admissions to 245 trauma centers from 2013 to 2014. Patient demographics, rate of VTE, as well as the use, type, and timing of chemoprophylaxis were compared between patients admitted to Georgia and non-Georgia trauma centers. The rate of VTE in Georgia trauma centers was 1.9 per cent compared with 2.1 per cent in other national trauma centers. Overall, 49.6 per cent of Georgia patients and 45.5 per cent of patients in other trauma centers had documented chemoprophylaxis. Low molecular weight heparin was the most commonly used medication. Most patients who developed VTE did so despite receiving prophylaxis. The rate of VTE despite prophylaxis was 3.2 per cent in Georgia and 3.1 per cent in non-Georgia trauma centers. Mortality associated with VTE was higher in Georgia trauma centers compared with national TQIP benchmarks. The incidence of VTE and use of chemoprophylaxis within Georgia trauma centers were similar to national TQIP data. Interestingly, most patients who developed VTE in both populations received VTE prophylaxis. Further research is needed to develop best-practice guidelines for prevention, early detection, and treatment in high-risk populations.

Racial disparities in the use of laparoscopic surgery to treat colonic diverticulitis Are not fully explained by socioeconomics or disease complexity.

BACKGROUND: Several studies have demonstrated favorable outcomes for laparoscopic surgery over open surgery for the treatment of diverticular disease. This study was designed to analyze the relationship between race, socioeconomic status and the use of laparoscopy to address diverticulitis. METHODS: A retrospective analysis of 53,054 diverticulitis admissions was performed using data from the 2009-2013 National Inpatient Sample (NIS). The primary outcome was the use of laparoscopic versus open colectomy. Bivariate analysis and multivariable logistic regression were used to determine the raw and adjusted odds by race, insurance status, and median household income. RESULTS: Overall, 41.6% of colectomies involved the use of laparoscopy. Black patients were 19% less likely than White patients to undergo laparoscopic surgery. Hispanic patients were no more or less likely to undergo laparoscopic colectomy. Lacking private insurance was a strong predictor of undergoing open surgery. Lower income patients were 33% less likely to receive minimally invasive colectomies. CONCLUSIONS: These results demonstrate disparities in surgical treatment. Further research is warranted to understand and ameliorate treatment differences which can contribute to outcome disparities.

Repeated exposure to chlorpyrifos leads to prolonged impairments of axonal transport in the living rodent brain.

The toxicity of the class of chemicals known as the organophosphates (OP) is most commonly attributed to the inhibition of the enzyme acetylcholinesterase. However, there is significant evidence that this mechanism may not account for all of the deleterious neurologic and neurobehavioral symptoms of OP exposure, especially those associated with levels that produce no overt signs of acute toxicity. In the study described here we evaluated the effects of the commonly used OP-pesticide, chlorpyrifos (CPF) on axonal transport in the brains of living rats using manganese (Mn(2+))-enhanced magnetic resonance imaging (MEMRI) of the optic nerve (ON) projections from the retina to the superior colliculus (SC). T1-weighted MEMRI scans were evaluated at 6 and 24h after intravitreal injection of Mn(2+). As a positive control for axonal transport deficits, initial studies were conducted with the tropolone alkaloid colchicine administered by intravitreal injection. In subsequent studies both single and repeated exposures to CPF were evaluated for effects on axonal transport using MEMRI. As expected, intravitreal injection of colchicine (2.5µg) produced a robust decrease in transport of Mn(2+) along the optic nerve (ON) and to the superior colliculus (SC) (as indicated by the reduced MEMRI contrast). A single subcutaneous (s.c.) injection of CPF (18.0mg/kg) was not associated with significant alterations in the transport of Mn(2+). Conversely, 14-days of repeated s.c. exposure to CPF (18.0mg/kg/day) was associated with decreased transport of Mn(2+) along the ONs and to the SC, an effect that was also present after a 30-day (CPF-free) washout period. These results indicate that repeated exposures to a commonly used pesticide, CPF can result in persistent alterations in axonal transport in the living mammalian brain. Given the fundamental importance of axonal transport to neuronal function, these observations may (at least in part) explain some of the long term neurological deficits that have been observed in humans who have been repeatedly exposed to doses of OPs not associated with acute toxicity.

Evaluation of nicotine and cotinine analogs as potential neuroprotective agents for Alzheimer's disease.

The currently available therapies for Alzheimer's disease (AD) and related forms of dementia are limited by modest efficacy, adverse side effects, and the fact that they do not prevent the relentless progression of the illness. The purpose of the studies described here was to investigate the neuroprotective effects of the nicotine metabolite cotinine as well as a small series of cotinine and nicotine analogs (including stereoisomers) and to compare their effects to the four clinically prescribed AD therapies.

Effects of chlorpyrifos and chlorpyrifos-oxon on the dynamics and movement of mitochondria in rat cortical neurons.

Organophosphate (OP)-based pesticides have been used extensively for decades, and as a result, they have become almost ubiquitous in our environment. There is clinical and animal evidence to suggest that chronic exposures to OPs can lead to cognitive dysfunction and other neurological abnormalities, although the mechanism for these effects is unknown. We previously reported that repeated, subthreshold exposures (defined as doses not associated with signs of acute toxicity) to the commonly used OP chlorpyrifos (CPF) resulted in protracted impairments in the performance of attention and memory-related tasks in rodents as well as deficits in axonal transport ex vivo (in the sciatic nerve). Here, we investigated the effects of CPF and its active metabolite CPF oxon (CPO) on the dynamics and movement of mitochondria in rat primary cortical neurons using time-lapse imaging techniques. Exposure to CPF (1.0-20.0 µM) or CPO (5.0 nM-20.0 µM) for 1 or 24 h resulted in a concentration-dependent increase in mitochondrial length, a decrease in mitochondrial number (indicative of increased fusion events), and a decrease in their movement in axons. The changes occurred at concentrations of CPF and CPO that did not inhibit acetylcholinesterase activity (the commonly cited mechanism of acute OP toxicity), and they were not blocked by cholinergic receptor antagonists. Furthermore, the changes did not seem to be associated with direct (OP-related) effects on mitochondrial viability or function (i.e., mitochondrial membrane potential or ATP production). The results suggest that an underlying mechanism of organophosphate-based deficits in cognitive function might involve alterations in mitochondrial dynamics and/or their transport in axons.

Neuroprotective effects and mechanism of cognitive-enhancing choline analogs JWB 1-84-1 and JAY 2-22-33 in neuronal culture and Caenorhabditis elegans.

BACKGROUND: Our previous work indicated that novel analogs of choline have cytoprotective effects in vitro that might be useful in neurodegenerative conditions such as Alzheimer's disease (AD). Furthermore, two lead compounds (JWB1-84-1 and JAY2-22-33) from a library of more than 50 improved cognitive performances in a transgenic mouse model of AD. The purpose of these experiments was to more specifically investigate the neuroprotective capabilities of these lead compounds both in vitro and in vivo. RESULTS: We used N2a cells which express a Swedish mutation in the amyloid precursor protein and presenilin 1 genes to investigate the effect of JWB1-84-1 and JAY2-22-33 on ß-amyloid (Aß) levels and found that both compounds significantly reduced Aß levels. JWB1-84-1 and JAY2-22-33 also protected rat primary cortical neurons from Aß toxicity. Subsequently, we utilized the nematode Caenorhabditis elegans (C. elegans) as an in vivo model organism to identify potential molecular targets of these compounds. In the C. elegans model of Aß toxicity, human Aß is expressed intracellularly in the body wall muscle. The expression and subsequent aggregation of Aß in the muscle leads to progressive paralysis. CONCLUSION: We found that JAY2-22-33 (but not JWB1-84-1) significantly reduced Aß toxicity by delaying paralysis and this protective effect required both the insulin signaling pathway and nicotinic acetylcholine receptors (nAChRs).

Use of combination proteomic analysis to demonstrate molecular similarity of head and neck squamous cell carcinoma arising from different subsites.

OBJECTIVE: To evaluate head and neck squamous cell carcinomas (HNSCCs) for differences in protein expression between oral cavity, oropharynx, larynx, and hypopharynx subsites. DESIGN: Retrospective proteomic analysis using tissue microarray (TMA) and 2-dimensional difference gel electrophoresis (2D-DIGE). For the TMA, automated quantitative protein expression analysis was used to interrogate levels of 4 cell-cycle regulatory proteins chosen for their known roles in cancer (cyclin D1, p53, Rb, and p14). For the 2D-DIGE, lesional and normal adjacent tissues were enriched by laser capture microdissection. Total protein was extracted, analyzed by 2D-DIGE with saturation dye labeling, and evaluated for relative abundance levels of individual protein spots. SETTING: Two tertiary-care academic medical centers. PATIENTS: Seventy-one patients with HNSCC for TMA, and 14 patients with HNSCC with frozen tumor and normal tissue for 2D-DIGE. RESULTS: The automated quantitative analysis of protein expression analysis revealed no difference between subsite for cyclin D1, p53, Rb, or p14 expression. The 2D-DIGE study was based on 28 gels (14 cancer gels and 14 adjacent normal gels), and 732 spots were identified as matching across more than 90% of gels. Significance was evaluated based on false discovery rate (FDR) estimated from permuted data sets. There were no significant differences in protein expression between subsites (FDR greater than or equal to 30% in all instances). CONCLUSIONS: Observed differences in outcomes between HNSCCs from different subsites may not reflect differences in tumor biologic characteristics between subsites. Rather, it is possible that observed clinical heterogeneity among HNSCCs may be based on other factors, such as viral vs chemical carcinogenesis.

Proteomic identification of serum biomarkers for head and neck cancer surveillance.

OBJECTIVES/HYPOTHESIS: Serum protein profiling by SELDI-TOF-MS distinguishes pretreatment and post-treatment samples from patients with head and neck squamous cell cancer (HNSCC) by disease status (disease-free or recurrence) with a high degree of sensitivity and specificity. We sought to identify biomarkers for recurrence with potential utility for surveillance and incorporated 2-D DIGE and MALDI-TOF-MS techniques to overcome the limitations of SELDI-TOF-MS in determining biomarker identity. METHODS: Serum samples were collected prospectively from 143 HNSCC patients and analyzed based on disease status following treatment. RESULTS: Recurrent HNSCC occurred in 46 patients. MALDI-TOF-MS following immunodepletion of major plasma proteins followed by 2-D DIGE identified 181 proteins with differential expression between pretreatment and post-treatment samples collected 6 months or more following treatment. Classification by disease status revealed significant differential expression of 16 proteins, with recurrent HNSCC associated with underexpression of kininogen and serine protease inhibitors C-1 inhibitor, kininogen, angiotensinogen, serine/cysteine proteinase inhibitor clade G member 1, and overexpression of thiol-specific antioxidant proteins (TSA), apolipoprotein A1 and proapolipoprotein, and epidermal cytokeratin 2. CONCLUSIONS: Serum protein profiling using 2D DIGE/MALDI-TOF-MS identifies proteins with significant differential expression in HNSCC based on disease status. Recurrent HNSCC was associated with underexpression of several protease inhibitors and kininogen, which has antiangiogenic properties, and overexpression of TSA, which is a free radical scavenger, as well as several forms of apolipoprotein A1 that may serve as a carrier molecule but may also indirectly promote tumor survival through kinase activation. This profile is consistent with a more aggressive disease variant and warrants further investigation.

Stable reversal of multidrug resistance in colon cancer cells by RNA interference targeting the MDR1 gene.

Colon cancer is one of the most common cancers in the world. Overexpression of MDR1 mRNA and P-gp is associated with the classic multidrug resistance of colon cancer cells. In our previous study, we reported on the transient specific reversal of MDR1/P-gp-dependent multi-drug resistance by RNA interference (RNAi) in colon cancer cells. In this study, RNAi targeting the MDR1 gene stably reversed MDR1/P-gp-dependent multidrug resistance in colon cancer cells. The plasmid vectors pSilencer-#4029, encoding #4029 MDR1 siRNA, and pSilencer-#4123, encoding #4123 MDR1 siRNA, were constructed and then transfected into COLO 320DM, a colon cancer multidrug-resistant cell line. Clone cells were screened by G418 and identified by RT-PCR and Western blot analysis. Cellular viability was measured using the MTT assay. Cell cycle analysis and intracellular adriamycin accumulation were assessed by flow cytometry. MDR1 mRNA and P-gp expression in positive clone cells, those stably transfected with MDR1 siRNA, was inhibited. The IC50 values of the antitumor drugs were significantly decreased in the positive clones compared to the COLO 320DM parent cell line, and the PI/AI values of positive clones treated with antitumor drugs were significantly decreased compared to the parent cells. In addition, the intracellular adriamycin accumulation of positive clones treated with adriamycin was significantly increased compared with COLO 320DM. This demonstrates that the stable transfection of plasmid vectors encoding MDR1 siRNA can stably reverse the MDR1/P-gp-dependent multidrug resistance of colon cancer cells.

Specific reversal of MDR1/P-gp-dependent multidrug resistance by RNA interference in colon cancer cells.

No control cell line was available for previous RNA interference studies on reversal of multidrug resistance (MDR) in colon cancer cells. Here, human COLO 320DM, with HT-29 as the control, colon cancer cell lines were used to investigate the reversal of MDR1/P-gp-dependent MDR by siRNA (#4123 and #4029 MDR1 siRNAs) targeting to MDR1 mRNA. Both siRNAs inhibited expression of MDR1 and P-gp in COLO 320DM. The minimum inhibition concentrations were 5 nmol/l of #4123 and 25 nmol/l of #4029. #4123 MDR1 siRNA took effect in 4, 5 and 6 days at doses of 5, 25 and 100 nmol/l, respectively. Increased cytotoxicity of the antitumor drugs adriamycin and vincristine with increased intracellular adriamycin accumulation accompanied inhibition of MDR1 mRNA and P-gp expression. No such effects were found in the HT-29 control. MDR1 siRNAs specifically reversed the MDR of colon cancer cells demonstrating a possible new approach for treating MDR1/P-gp-dependent multidrug resistance.

Proteomics blood testing to distinguish chronic rhinosinusitis subtypes.

OBJECTIVES: To evaluate the potential of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) proteomic profiling of serum samples to distinguish chronic rhinosinusitis subtypes. STUDY DESIGN: Translational study of serum samples from prospectively enrolled patients undergoing sinus surgery. METHODS: Patients undergoing endoscopic sinus surgery for chronic rhinosinusitis with nasal polyposis were prospectively enrolled in an ongoing, institutional review board approved proteomics study. SELDI-TOF-MS was performed on 42 serum samples in patients with chronic rhinosinusitis with nasal polyposis (15 patients diagnosed with allergic fungal rhinosinusitis, 10 patients with Samter's triad, and 17 with chronic rhinosinusitis with nasal polyposis). Classification tree analysis on protein spectra developed from peaks detected in the 0 to 100 kD range was performed to identify disease subtypes. RESULTS: SELDI-TOF-MS correctly identified patients with allergic fungal rhinosinusitis from serum samples with 84% sensitivity and 90% specificity, and correctly identified patients with Samter's triad with 88% sensitivity and 88% specificity in two subtype comparison groups. SELDI-TOF-MS correctly identified patients with allergic fungal rhinosinusitis with 76% sensitivity and 82% specificity, and correctly identified patients with Samter's triad with 80% sensitivity and 90% specificity in three subtype comparison groups. CONCLUSION: The study provides molecular evidence that allergic fungal rhinosinusitis is a discrete subtype of chronic rhinosinusitis. SELDI-TOF-MS is a promising technology that could lead to the development of a rapid blood test, to identify severe chronic rhinosinusitis subtypes. Further investigation into the utility of this technology is warranted.

Serum inflammatory protein profiles in patients with chronic rhinosinusitis undergoing sinus surgery: a preliminary analysis.

BACKGROUND: The specific aim of this study was to evaluate surface-enhanced laser desorption/ionization time-of-flight mass spectroscopy (SELDI-TOF-MS) as a clinical screening tool in differentiating patients with chronic rhinosinusitis (CRS) from healthy control patients. METHODS: Serum samples were prospectively collected at a tertiary care academic medical center from 96 CRS patients who underwent functional endoscopic sinus surgery (FESS) and 38 control volunteers who were negatively screened according to the Rhinosinusitis Task Force guidelines on CRS. SELDI-TOF-MS was performed on serum samples to detect protein profiles in the range of 1-100 kDa. Analysis of spectral data was performed to determine the sensitivity and specificity of SELDI-TOF-MS in distinguishing patients with CRS requiring FESS from healthy controls and to determine potential markers for protein purification. RESULTS: Serum protein profiles generated from SELDI-TOF-MS in the range of 1-100 kDa were analyzed. Classification and regression tree analysis based on peak expression correctly classified patients with CRS with 77.1% sensitivity and 65.8% specificity, a positive predictive value of 88%, and a negative predictive value of 53%. Underexpression of a protein peak at 8.4 kDa was associated with CRS in 77% of cases. CONCLUSION: SELDI-TOF-MS serum protein profile analysis is able to distinguish CRS patients requiring FESS from healthy, negatively screened controls with a sensitivity of 77.1% and specificity of 65.8%. Additional investigation is required to determine if SELDI-TOF-MS will make an effective clinical tool.

Detection of papillary thyroid carcinoma with serum protein profile analysis.

OBJECTIVE: To determine the sensitivity and specificity of surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) for papillary thyroid carcinoma (PTC) detection. DESIGN: The SELDI-TOF-MS protein profiles of patients with PTC, patients with benign nodular disease (BND), and healthy controls were analyzed to determine the sensitivity and specificity of SELDI-TOF-MS assay for PTC detection. Data analysis was performed to process the spectral data and classify the disease status of the patients. SETTING: Academic tertiary care hospital. PATIENTS: Serum samples were collected prospectively from 7 patients with PTC, 8 patients with BND, and 7 healthy control volunteers. INTERVENTION: All patients diagnosed as having PTC or BND underwent thyroidectomy from October 21, 2004, to January 31, 2006. MAIN OUTCOME MEASURES: Twenty-two serum samples were analyzed. RESULTS: Most protein peaks resolved by the SELDI-TOF-MS assay were in the range of 1 to 20 kDa. Classification tree analysis based on peak expression distinguished patients with PTC from those with BND with 85.7% sensitivity and 100% specificity. Serum samples from patients with PTC differed most significantly from those of patients with BND by the underexpression of a protein peak at 11 101 Da. CONCLUSIONS: This pilot study demonstrates that proteomic analysis of serum protein profiles distinguishes patients with PTC from patients with BND with a high degree of sensitivity and specificity. Further investigation into the clinical utility of this technology in PTC biomarker detection and surveillance is warranted.

Comparative analysis of the islet proteome between NOD/Lt and ALR/Lt mice.

Although alloxan-resistant (ALR) mice share 70% of the NOD genome, ALR islets are remarkably resistant to oxidative stress and autoimmunity. Using 2D PAGE comparative analysis, we have characterized 55 proteins that are differentially expressed between the NOD and ALR islet proteome. Ten proteins were found to be highly expressed in the NOD islets. Typically, proteins associated with pancreatic inflammation and autoimmunity, such as amylase and carboxypeptidase, are highly expressed in the NOD islets. Forty-five proteins showed significantly higher expression in the ALR islets. Among these, 30 are proteins implicated in the regulation of intracellular stress including heat-shock proteins, disulfide isomerase-associated proteins, ROS detoxification enzymes, and apoptotic regulators. Our results clearly demonstrate that the 30% unique ALR genome encodes protective determinants expressed at islet levels, which render the islets of this strain of mice resistant to oxidative stress and autoimmunity.

Serum protein profile analysis following definitive treatment in patients with head and neck squamous cell carcinoma.

OBJECTIVE: To determine the sensitivity and specificity of surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) assay for head and neck squamous cell carcinoma (HNSCC) disease surveillance. DESIGN: The SELDI-TOF-MS serum protein profiles of patients with HNSCC were analyzed to determine the sensitivity and specificity of the SELDI assay for HNSCC detection following definitive treatment. SETTING: Academic research. PATIENTS: Thirty-two patients with previously untreated HNSCC. INTERVENTION: Serum samples were collected prospectively at 3-month intervals following treatment during a 24-month follow-up period. MAIN OUTCOME MEASURES: Ninety-three serum samples were analyzed. RESULTS: The SELDI-TOF-MS identified protein peaks in the range of 0 to 100 kDa. Classification tree analysis based on peak expression distinguished pretreatment from 6-month posttreatment samples with 75.0% sensitivity and 87.5% specificity. Samples collected at 3 months following treatment did not significantly differ from pretreatment samples. Serum samples from patients who were disease free at 6 months or longer following treatment differed from matched pretreatment samples by the overexpression of a protein peak at 6495 Da, while serum samples from patients with recurrence differed from matched pretreatment samples by the underexpression of a protein peak at 4493 Da. CONCLUSIONS: Proteomic analysis of serum protein profiles distinguishes pretreatment and posttreatment samples from patients with HNSCC with a high degree of sensitivity and specificity. After 6 months, serum protein profiles seem to have distinct differences in peak expression based on disease status. Further investigation of the clinical usefulness of this technology in HNSCC detection and surveillance is warranted.

Association of nuclear, cytoplasmic expression of galectin-3 with beta-catenin/Wnt-pathway activation in thyroid carcinoma.

OBJECTIVES: To characterize the localization of galectin-3 in benign and malignant thyroid neoplasms and to correlate this with alterations in beta-catenin and cyclin D1 expression. DESIGN: Immunohistochemical study of 116 paraffin-embedded archival specimens from 113 patients who had undergone thyroidectomy and tissue placed into a commercially available tissue microarray. SETTING: Tertiary care hospital. INTERVENTIONS: Thyroid tissue microarrays were stained by standard immunohistochemical protocols with monoclonal antibodies against galectin-3, beta-catenin, and cyclin D1. MAIN OUTCOME MEASURES: Nuclear and cytoplasmic expression of galectin-3 was correlated with clinical parameters, beta-catenin, and cyclin D1 expression. RESULTS: Both cytoplasmic (56%) and nuclear (42%) galectin-3 expression was observed in most malignant neoplasms but was absent in benign thyroid specimens (P<.001). Among carcinomas, cytoplasmic galectin-3 expression was observed in papillary thyroid carcinomas (82%) and follicular (33%) and medullary (9%) carcinomas but was absent in anaplastic carcinomas (P<.001). Galectin-3 nuclear expression was observed in papillary thyroid carcinomas (62%) and follicular carcinomas (33%) but was undetectable in medullary, anaplastic carcinomas (P<.001). Cytoplasmic but not nuclear galectin-3 was inversely correlated with American Joint Committee on Cancer TNM stage (P = .02). There was a strong correlation between cytoplasmic and nuclear beta-catenin expression and both nuclear (P = .04) and cytoplasmic (P = .003) galectin-3 expression. Similarly, there was a strong association between galectin-3 nuclear (P<.001) and cytoplasmic (P<.001) expression and cyclin D1 expression. CONCLUSION: Cytoplasmic and nuclear galectin-3 expression seem to be associated with activation of the Wnt-signaling pathway in well-differentiated thyroid neoplasms, suggesting that galectin-3 plays a role in thyroid carcinogenesis.