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.

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.

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.

A mixture model approach to the tests of concordance and discordance between two large-scale experiments with two-sample groups.

MOTIVATION: Due to advances in experimental technologies, such as microarray, mass spectrometry and nuclear magnetic resonance, it is feasible to obtain large-scale data sets, in which measurements for a large number of features can be simultaneously collected. However, the sample sizes of these data sets are usually small due to their relatively high costs, which leads to the issue of concordance among different data sets collected for the same study: features should have consistent behavior in different data sets. There is a lack of rigorous statistical methods for evaluating this concordance or discordance. METHODS: Based on a three-component normal-mixture model, we propose two likelihood ratio tests for evaluating the concordance and discordance between two large-scale data sets with two sample groups. The parameter estimation is achieved through the expectation-maximization (E-M) algorithm. A normal-distribution-quantile-based method is used for data transformation. RESULTS: To evaluate the proposed tests, we conducted some simulation studies, which suggested their satisfactory performances. As applications, the proposed tests were applied to three SELDI-MS data sets with replicates. One data set has replicates from different platforms and the other two have replicates from the same platform. We found that data generated by SELDI-MS showed satisfactory concordance between replicates from the same platform but unsatisfactory concordance between replicates from different platforms. AVAILABILITY: The R codes are freely available at http://home.gwu.edu/~ylai/research/Concordance.

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.

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.

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.

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.

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.

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.

Serum levels of an isoform of apolipoprotein A-II as a potential marker for prostate cancer.

PURPOSE: We recently showed that protein expression profiling of serum using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) has potential as a diagnostic approach for detection of prostate cancer. As a parallel effort, we have been pursuing the identification of the protein(s) comprising the individual discriminatory "peaks" and evaluating their utility as potential biomarkers for prostate disease. EXPERIMENTAL DESIGN: We employed liquid chromatography, gel electrophoresis and tandem mass spectroscopy to isolate and identify a protein that correlates with observed SELDI-TOF MS mass/charge (m/z) values. Immunodepletion, immunoassay, and Western analysis were used to verify that the identified protein generated the observed SELDI peak. Subsequent immunohistochemistry was used to examine the expression of the proteins in prostate tumors. RESULTS: An 8,946 m/z SELDI-TOF MS peak was found to retain discriminatory value in each of two separate data sets with an increased expression in the diseased state. Sequence identification by liquid chromatography-MS/MS and subsequent immunoassays verified that an isoform of apolipoprotein A-II (ApoA-II) is the observed 8,946 m/z SELDI peak. Immunohistochemistry revealed that ApoA-II is overexpressed in prostate tumors. SELDI-based immunoassay revealed that an 8.9-kDa isoform of ApoA-II is specifically overexpressed in serum from individuals with prostate cancer. ApoA-II was also overexpressed in the serum of individuals with prostate cancer who have normal prostate-specific antigen (0-4.0 ng/mL). CONCLUSIONS: We have identified an isoform of ApoA-II giving rise to an 8.9K m/z SELDI "peak" that is specifically overexpressed in prostate disease. The ability of ApoA-II to detect disease in patients with normal prostate-specific antigen suggests potential utility of the marker in identifying indolent disease.

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

OBJECTIVES: To analyze surface-enhanced laser desorption and ionization time-of-flight mass spectrometry (SELDI-TOF-MS) protein profiles of patients with head and neck squamous cell carcinoma (HNSCC) and healthy controls and to determine the sensitivity and specificity of SELDI assay for HNSCC detection before and after treatment. DESIGN: Proteomic analysis and comparison of serum samples. SETTING: Tertiary care academic medical center. SUBJECTS: Seventy-eight patients with HNSCC and 68 healthy controls. MAIN OUTCOME MEASURES: Serum samples were prospectively collected from 78 patients with HNSCC and 68 healthy control volunteers. SELDI-TOF-MS was performed on serum samples to identify protein peaks in the range of 0 to 100 kDa. Classification analysis of the spectral data was performed and used to classify the disease status of the patients. RESULTS: The SELDI-TOF-MS assay generated serum protein profiles ranging from 0 to 100 kDa. After background subtraction, mass calibration, and normalization, 545 protein peaks were identified. Classification tree analysis based on peak expression correctly classified patients with HNSCC with 82% sensitivity and 76% specificity. Subgroup analysis correctly classified 83% of oral cavity tumors, 81% of oropharyngeal tumors, and 88% of laryngeal tumors. Pretreatment and posttreatment samples were available from 12 patients, and the posttreatment samples were correctly classified in 86% of the patients at 3 months and 75% of the patients at 6 months. CONCLUSIONS: Proteomic SELDI-TOF-MS analysis of serum protein profiles distinguishes patients with HNSCC from controls with a high degree of sensitivity and specificity. Further investigation into the clinical utility of this technology in HNSCC detection and surveillance is warranted.

Serum protein fingerprinting coupled with a pattern-matching algorithm distinguishes prostate cancer from benign prostate hyperplasia and healthy men.

The prostate-specific antigen test has been a major factor in increasing awareness and better patient management of prostate cancer (PCA), but its lack of specificity limits its use in diagnosis and makes for poor early detection of PCA. The objective of our studies is to identify better biomarkers for early detection of PCA using protein profiling technologies that can simultaneously resolve and analyze multiple proteins. Evaluating multiple proteins will be essential to establishing signature proteomic patterns that distinguish cancer from noncancer as well as identify all genetic subtypes of the cancer and their biological activity. In this study, we used a protein biochip surface enhanced laser desorption/ionization mass spectrometry approach coupled with an artificial intelligence learning algorithm to differentiate PCA from noncancer cohorts. Surface enhanced laser desorption/ionization mass spectrometry protein profiles of serum from 167 PCA patients, 77 patients with benign prostate hyperplasia, and 82 age-matched unaffected healthy men were used to train and develop a decision tree classification algorithm that used a nine-protein mass pattern that correctly classified 96% of the samples. A blinded test set, separated from the training set by a stratified random sampling before the analysis, was used to determine the sensitivity and specificity of the classification system. A sensitivity of 83%, a specificity of 97%, and a positive predictive value of 96% for the study population and 91% for the general population were obtained when comparing the PCA versus noncancer (benign prostate hyperplasia/healthy men) groups. This high-throughput proteomic classification system will provide a highly accurate and innovative approach for the early detection/diagnosis of PCA.

Serum protein profiles to identify head and neck cancer.

PURPOSE: New and more consistent biomarkers of head and neck squamous cell carcinoma (HNSCC) are needed to improve early detection of disease and to monitor successful patient management. The purpose of this study was to determine whether a new proteomic technology could correctly identify protein expression profiles for cancer in patient serum samples. EXPERIMENTAL DESIGN: Surface-enhanced laser desorption/ionization-time of flight-mass spectrometry ProteinChip system was used to screen for differentially expressed proteins in serum from 99 patients with HNSCC and 102 normal controls. Protein peak clustering and classification analyses of the surface-enhanced laser desorption/ionization spectral data were performed using the Biomarker Wizard and Biomarker Patterns software (version 3.0), respectively (Ciphergen Biosystems, Fremont, CA). RESULTS: Several proteins, with masses ranging from 2778 to 20800 Da, were differentially expressed between HNSCC and the healthy controls. The serum protein expression profiles were used to develop and train a classification and regression tree algorithm, which reliably achieved a sensitivity of 83.3% and a specificity of 100% in discriminating HNSCC from normal controls. CONCLUSIONS: We propose that this technique has potential for the development of a screening test for the detection of HNSCC.

Normal, benign, preneoplastic, and malignant prostate cells have distinct protein expression profiles resolved by surface enhanced laser desorption/ionization mass spectrometry.

PURPOSE: The objective of this study was to discover protein biomarkers that differentiate malignant from nonmalignant cell populations, especially early protein alterations that signal the initiation of a developing cancer. We hypothesized that Surface Enhanced Laser Desorption/Ionization-time of flight-mass spectrometry-assisted protein profiling could detect these protein alterations. EXPERIMENTAL DESIGN: Epithelial cell populations [benign prostatic hyperplasia (BPH), prostate intraepithelial neoplasia (PIN), and prostate cancer (PCA)] were procured from nine prostatectomy specimens using laser capture microdissection. Surface Enhanced Laser Desorption/Ionization-time of flight-mass spectrometry analysis was performed on cell lysates, and the relative intensity levels of each protein or peptide in the mass spectra was calculated and compared for each cell type. RESULTS: Several small molecular mass peptides or proteins (3000-5000 Da) were found in greater abundance in PIN and PCA cell lysates. Another peak, with an average mass of 5666 Da, was observed to be up-regulated in 86% of the BPH cell lysates. Higher levels of this same peak were found in only 22% of the PIN lysates and none of the PCA lysates. Expression differences were also found for intracellular levels of prostate-specific antigen, which were reduced in PIN and PCA cells when compared with matched normals. Although no single protein alteration was observed in all PIN/PCA samples, combining two or more of the markers was effective in distinguishing the benign cell types (normal/BPH) from diseased cell types (PIN/PCA). Logistic regression analysis using seven differentially expressed proteins resulted in a predictive equation that correctly distinguished the diseased lysates with a sensitivity and specificity of 93.3 and 93.8%, respectively. CONCLUSIONS: We have shown that the protein profiles from prostate cells with different disease states have discriminating differences. These differentially regulated proteins are potential markers for early detection and/or risk factors for development of prostate cancer. Studies are under way to identify these protein/peptides, with the goal of developing a diagnostic test for the early detection of prostate cancer.

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.

Computational protein biomarker prediction: a case study for prostate cancer.

BACKGROUND: Recent technological advances in mass spectrometry pose challenges in computational mathematics and statistics to process the mass spectral data into predictive models with clinical and biological significance. We discuss several classification-based approaches to finding protein biomarker candidates using protein profiles obtained via mass spectrometry, and we assess their statistical significance. Our overall goal is to implicate peaks that have a high likelihood of being biologically linked to a given disease state, and thus to narrow the search for biomarker candidates. RESULTS: Thorough cross-validation studies and randomization tests are performed on a prostate cancer dataset with over 300 patients, obtained at the Eastern Virginia Medical School using SELDI-TOF mass spectrometry. We obtain average classification accuracies of 87% on a four-group classification problem using a two-stage linear SVM-based procedure and just 13 peaks, with other methods performing comparably. CONCLUSIONS: Modern feature selection and classification methods are powerful techniques for both the identification of biomarker candidates and the related problem of building predictive models from protein mass spectrometric profiles. Cross-validation and randomization are essential tools that must be performed carefully in order not to bias the results unfairly. However, only a biological validation and identification of the underlying proteins will ultimately confirm the actual value and power of any computational predictions.