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Aim: To describe the impact of paediatric myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD) on academic and cognitive outcomes., Method: This was an observational, retrospective, and descriptive single-centre study, carried out on a paediatric case series of children with MOGAD., Results: A total of 51 patients were included (22 females); their median age was 8 years and the median follow-up duration was 31.1 months (interquartile range 23.5). The most frequent clinical presentation was acute disseminated encephalomyelitis (54.9%), followed by optic neuritis (35.5%). At the last follow-up, regardless of the clinical phenotype at disease onset, 39.5% of patients with MOGAD received academic and educational interventions (p < 0.05 compared to before disease onset), including academic accommodations (p < 0.05) or the need for a learning support assistant (p < 0.05). Ten patients were evaluated with the Wechsler Intelligence Scale for Children, Fifth Edition (WISC-V). The overall IQ was calculated for six patients (mean = 92); two of these patients had an IQ lower than 85. No difference was found regarding prenatal and neonatal neurodevelopmental characteristics between this cohort and the general population., Interpretation: MOGAD was associated with a need for academic support; lower scores were found on the WISC-V. Patients with MOGAD should receive cognitive and academic assessments to inform educational planning and support academic success., (© 2024 The Author(s). Developmental Medicine & Child Neurology published by John Wiley & Sons Ltd on behalf of Mac Keith Press.)

Recruitment and retention are challenges for prospective pediatric cohort studies, particularly those involving serial venipunctures. We investigated factors underlying enrollment and retention in the Pandemic Response Repository through Microbial and Immune Surveillance and Epidemiology (PREMISE) Enterovirus D68 (EV-D68) Pilot Study, a multicenter prospective longitudinal cohort study assessing the utility of immunologic surveillance for pandemic preparedness. This study enrolls children ≤10 years for two blood draws, pre- and post-EV-D68 season, separated by 6-18 months. Overall, 174 children were enrolled in Cohort 1 of the study and 120 (69 %) of children completed the study, with follow-up blood samples obtained from 101 (58 %) of participants. Families were primarily motivated to participate by a desire to help other children, advance science, and better prepare for the next pandemic. Adding research blood draws to clinically indicated blood draws improved enrollment, and multiple study touch points facilitated retention. These findings can be applied to improve recruitment and retention in future pandemic preparedness efforts and longitudinal pediatric cohort studies., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: HN-T received funding from the Centers for Disease Control and Prevention for her involvement in the “Duration of Enterovirus D68 Shedding from the Upper Respiratory Tract” study and received support from the Congenital and Perinatal Infections Consortium (CPIC) and Cambridge Innovation Institute for conference attendance at IDWeek (2022, 2023) and Next Generation Dx Summit (2023), respectively. AS and AM receive support from the NIH, NIAID Vaccine Research Center. MRV receives support from the following grants/contracts: K08AI156125, 75N91019D00024, R01AI169461; received honorariums for lectures from NIAID, Uniformed Services University, University of Maryland-Baltimore, Duke University, Baylor College of Medicine; and has a patent application pending (PCT/US2020/043,415). MLH receives support from the NIH, NIAID Vaccine Research Center; reports the following grants: NHLBI-R01, NCATS-CTSA K12; NCATS-UM1; NSF-SCH; participates in the GSK Advisory Board for asthma; and is the AAAAI EORD Interest Section Vice Chair., (© 2024 The Authors. Published by Elsevier Inc.)

Purpose: The objective of this study is to explore the impact of artificial intelligence (AI) development on the sports industry labor market, the ways in which AI has influenced the demand for labor, created new job opportunities, and impacted existing job roles., Methodology: It refers to the inductive approach in the spirit technological determinism theory. It is based on the literature review and written qualitative, semi-structured interviews ( N  = 14) with sports human resources, management, and technology professionals (purposive sampling). Analysis involved inductive coding and line-by-line analytics of the data., Findings: The labor market implications of AI in the sports industry are multifaceted. New job roles are likely to emerge, demanding a blend of AI expertise, data-analysis skills, and sports domain knowledge. Professionals in roles such as sports data analysts and marketing experts may find increasing opportunities. However, certain jobs undergo transformation as AI automates routine tasks. It requires individuals to upskill or transition to roles that require a deeper understanding of AI. This necessitates the creation of responsibilities focused on ethical AI governance and oversight., Originality: It is important to research the impact of AI dissemination on the sports industry labor market in a holistic manner because the effects of AI are complex and far-reaching. While there are potential benefits to the implementation of AI, there are also potential risks and challenges that need to be addressed, the implementation of AI in the sports industry could have broader social and ethical implications that need to be considered., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Glebova, Madsen, Mihaľová, Géczi, Mittelman and Jorgič.)

Background: Night shift work is associated with adverse pathophysiologic effects on maternal and fetal well-being. Although emergency medicine (EM) residents work frequent night shifts, there is no existing guidance for residency program directors (PDs) regarding scheduling pregnant residents. Our study assessed scheduling practices for pregnant EM residents, differences based on program and PD characteristics, barriers and attitudes toward implementing a formal scheduling policy, and PDs' awareness of literature describing adverse effects of night shifts on maternal-fetal outcomes., Methods: We conducted an anonymous, web-based survey of U.S. EM residencies ( N  = 276). Quantitative data were summarized; chi-square analysis and logistic regression were used to assess relationships between program and PD characteristics and schedule accommodations. Qualitative description was used to analyze an open-ended question, organizing findings into major and minor themes., Results: Of the 167 completed surveys (response rate 61%), 67% of programs reported no formal policy for scheduling pregnant residents but made adjustments on an individual basis including block changes (85%), decreased (46%) or no night shifts (34%), and working shifts earlier in pregnancy to cover later shifts (20%). Barriers to adjustments included staffing constraints (60%), equity concerns (45%), or impact on wellness (41%) among all residents and privacy (28%). PDs endorsed scheduling adjustments as important (mean 8.1, 0-10 scale) and reported guidance from graduate medical education governance would be useful (60%). Larger program size, but not PD gender or proportion of female residents, was associated with an increased likelihood of scheduling modifications. Twenty-five percent of PDs reported little knowledge of literature regarding night shift work and pregnancy. Qualitative themes supported quantitative findings., Conclusions: Most EM residency programs do not have formal scheduling policies for pregnant residents, but most PDs support making adjustments and do so informally. More education and guidance for PDs are needed to promote the development of formal policies., Competing Interests: The authors declare no potential conflict of interest., (© 2022 Society for Academic Emergency Medicine.)

Introduction: Despite the burdens that resident attrition places upon programs and fellow trainees, emergency medicine (EM) as a specialty has only begun to explore the issue. Our primary objectives were to quantify attrition in EM residency programs and elucidate the reasons behind it. Our secondary objectives were to describe demographic characteristics of residents undergoing attrition, personal factors associated with attrition, and methods of resident replacement., Methods: We conducted a national survey study of all EM program directors (PDs) during the 2018-2019 academic year. PDs were asked to identify all residents who had left their program prior to completion of training within the last four academic years (2015-2016 to 2018-2019), provide relevant demographic information, select perceived reasons for attrition, and report any resident replacements. Frequencies, percentages, proportions, and 95% confidence intervals were obtained for program- and resident-specific demographics. We performed Fisher's exact tests to compare reasons for attrition between age groups., Results: Of 217 PDs successfully contacted, 118 completed the questionnaire (response rate of 54%). A third of programs (39 of 118) reported at least one resident attrition. A total of 52 residents underwent attrition. Attrition was most likely to occur prior to completion of two years of training. Gender and underrepresented minority status were not associated with attrition. Older residents were more likely to leave due to academic challenges. The most common reported reason for attrition was to switch specialties. Resident replacement was found in 42% of cases., Conclusion: One-third of programs were affected by resident attrition. Gender and underrepresented minority status were not associated with attrition.

Artemisia annua L. and artemisinin, have been used for millennia to treat malaria. We used human liver microsomes (HLM) and rats to compare hepatic metabolism, tissue distribution, and inflammation attenuation by dried leaves of A. annua (DLA) and pure artemisinin. For HLM assays, extracts, teas, and phytochemicals from DLA were tested and IC 50 values for CYP2B6 and CYP3A4 were measured. For tissue distribution studies, artemisinin or DLA was orally delivered to rats, tissues harvested at 1 h, and blood, urine and feces over 8 h; all were analyzed for artemisinin and deoxyartemisinin by GC-MS. For inflammation, rats received an intraperitoneal injection of water or lipopolysaccharide (LPS) and 70 mg/kg oral artemisinin as pure drug or DLA. Serum was collected over 8 h and analyzed by ELISA for TNF-α, IL-6, and IL-10. DLA-delivered artemisinin distributed to tissues in higher concentrations in vivo, but elimination remained mostly unchanged. This seemed to be due to inhibition of first-pass metabolism by DLA phytochemicals, as demonstrated by HLM assays of DLA extracts, teas and phytochemicals. DLA was more effective than artemisinin in males at attenuating proinflammatory cytokine production; the data were less conclusive in females. These results suggest that the oral consumption of artemisinin as DLA enhances the bioavailability and anti-inflammatory potency of artemisinin., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Background: Resident attrition negatively affects residents and programs. The incidence of attrition in emergency medicine (EM) and program-specific factors associated with attrition remain unclear., Objective: We quantified the percentage of EM residencies affected by attrition between 2007 and 2016 and identified program-specific factors associated with attrition., Methods: We performed a retrospective analysis of data derived from the American Medical Association National Graduate Medical Education Census. We defined attrition as any postgraduate who left their residency training program prior to completion. We calculated the percentage of residency programs that experienced attrition and the overall incidence of attrition. We used Fisher's exact tests, Wilcoxon rank sum tests, and t tests, as well as multivariable logistic regression, to identify program-specific factors associated with attrition., Results: Between 2007 and 2016, 139 EM residency programs (82%) experienced attrition of at least 1 resident. An average of 23% of EM training programs experienced attrition annually. The incidence of EM resident attrition averaged 0.85% per year. Program-specific factors associated with attrition include 4-year residencies ( P = .031), programs with medium class size ( P = .0003), more female residents ( P = .002), and more female faculty ( P = .003). After analysis, only medium class size (compared to small) was associated with attrition (odds ratio = 4.96, 95% confidence interval 1.65-14.91)., Conclusions: Between 2007 and 2016, while the incidence of resident attrition in EM was low (< 1%), the majority of programs experienced resident attrition. Medium class size (7 to 12 residents) was the only program-specific factor associated with increased attrition., Competing Interests: Conflict of interest: The authors declare they have no competing interests., (Accreditation Council for Graduate Medical Education 2019.)

The majority of breast cancers (90-95%) arise due to mediators distinct from inherited genetic mutations. One major mediator of breast cancer involves chronic inflammation. M1 macrophages are an integral component of chronic inflammation and the breast cancer tumor microenvironment (TME). Previous studies have demonstrated that up to 50% of the breast tumor comprise of tumor-associated macrophages (TAMs) and increased TAM infiltration has been associated with poor patient prognosis. Furthermore, breast cancer associated deaths are predominantly attributed to invasive cancers and metastasis with epithelial-mesenchymal transition (EMT) being implicated. In this study, we investigated the effects of cellular crosstalk between TAMs and breast cancer using an in vitro model system. M1 polarized THP-1 macrophage conditioned media (CM) was generated and used to evaluate cellular and functional changes of breast cancer lines T47D and MCF-7. We observed that T47D and MCF-7 exhibited a partial EMT phenotype in the presence of activated THP-1 CM. Additionally, MCF-7 displayed a significant increase in migratory and invasive properties. We conclude that M1 secretory factors can promote a partial EMT of epithelial-like breast cancer cells. The targeting of M1 macrophages or their secretory components may inhibit EMT and limit the invasive potential of breast cancer., Competing Interests: CONFLICTS OF INTEREST No potential conflicts of interest were disclosed.

Objectives: The small body of neuropsychological research in paediatric obsessive-compulsive disorder (OCD) yields inconsistent results. A recent meta-analysis found small effect sizes, concluding that paediatric OCD may not be associated with cognitive impairments, stressing the need for more research. We investigated neuropsychological performance in a large sample of youths with OCD, while assessing potential moderators., Methods: Participants with OCD (n = 102) and matched controls (n = 161) were thoroughly screened and blindly evaluated for comorbidities, and completed a neuropsychological battery assessing processing speed, visuospatial abilities (VSA), working memory (WM), non-verbal memory (NVM), and executive functions (EF)., Results: Compared to controls, youths with OCD exhibited underperformance on tasks assessing processing speed. On tests of VSA and WM, underperformance was found only on timed tasks. There were no differences on NVM and EF tasks. Notably, the OCD group's standardised scores were in the normative range. Test performance was not associated with demographic or clinical variables., Conclusions: Youths with OCD exhibited intact performance on memory and EF tests, but slower processing speed, and underperformance only on timed VSA and WM tasks. While the OCD group performed in the normative range, these findings reveal relative weaknesses that may be overlooked. Such an oversight may be of particular importance in clinical and school settings.

Background: The available research on the relationship between neuropsychological functioning and the therapeutic outcome of obsessive-compulsive disorder (OCD) has yielded inconsistent results. In this study, our aim was twofold. First, we sought to evaluate the effects of cognitive behavioral group therapy (CBGT) on neurocognitive functions in OCD patients. Second, we assessed the viability of neuropsychological test performance as a predictor of treatment response to CBGT., Methods: One hundred fifty carefully screened OCD patients were randomized to receive either 12-week CBGT (n = 75) or to remain on a waiting list (WL; n = 75) for the corresponding time. Forty-seven participants dropped out of the study, leaving 103 participants that were included in the analysis (CBGT, n = 61; WL, n = 42). Participants had several neuropsychological domains evaluated both at baseline and at end-point., Results: A significant difference in obsessive-compulsive, anxiety, and depression symptoms was observed between treated patients and controls favoring the CBGT group, but no significant differences were found on neuropsychological measures after 3 months of CBGT. In addition, there were no differences between treatment responders and nonresponders on all neuropsychological outcome measures. Employing a conservative alpha, neuropsychological test performance did not predict CBGT treatment response., Conclusions: Although the CBGT group demonstrated significant improvement in OCD symptoms, no significant difference was found on all neuropsychological domains, and test performance did not predict treatment response., (© 2016 Wiley Periodicals, Inc.)

Background: Research into the neuropsychology of pediatric obsessive-compulsive disorder (OCD) reveals inconsistent results, limiting the ability to draw conclusions about possible neurocognitive deficits in youth with OCD. The aim of this study was to conduct a meta-analysis of the available literature., Methods: We identified 36 studies, of which 11 studies met inclusion criteria. Results were categorized into nine functional subdomains: planning, response inhibition/interference control, set shifting/cognitive flexibility, verbal memory, nonverbal memory, processing speed, working memory, visuospatial functions, and attention. For each domain, weighted pooled Hedges' g effect size was calculated using random model analyses., Results: Small effect sizes were found across all subdomains, none of which were found to be statistically significant., Discussion: Results indicate that youth with OCD do not exhibit noteworthy neuropsychological deficits. This is in line with recent suggestions that OCD may not be characterized by clinically meaningful neuropsychological impairments. However, the small number of available controlled studies highlights the urgent need for more neuropsychological research in this population, as well as for further exploration of the neurodevelopmental hypothesis in pediatric OCD. Finally, the relatively low persistence rates of OCD into adulthood should be taken under consideration, especially in the context of the putative neuropsychological performance differences between adult and pediatric OCD populations., (© 2015 Association for Child and Adolescent Mental Health.)

The inconsistent nature of the neuropsychology literature pertaining to obsessive-compulsive disorder (OCD) has long been recognized. However, individual studies, systematic reviews, and recent meta-analytic reviews were unsuccessful in establishing a consensus regarding a disorder-specific neuropsychological profile. In an attempt to identify methodological factors that may contribute to the inconsistency that is characteristic of this body of research, a systematic review of methodological factors in studies comparing OCD patients and non-psychiatric controls on neuropsychological tests was conducted. This review covered 115 studies that included nearly 3500 patients. Results revealed a range of methodological weaknesses. Some of these weaknesses have been previously noted in the broader neuropsychological literature, while some are more specific to psychiatric disorders, and to OCD. These methodological shortcomings have the potential to hinder the identification of a specific neuropsychological profile associated with OCD as well as to obscure the association between neurocognitive dysfunctions and contemporary neurobiological models. Rectifying these weaknesses may facilitate replicability, and promote our ability to extract cogent, meaningful, and more unified inferences regarding the neuropsychology of OCD. To that end, we present a set of methodological recommendations to facilitate future neuropsychology research in psychiatric disorders in general, and in OCD in particular., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

The concept of comorbidity between attention deficit/hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD) has been discussed for two decades. No review, however, has examined this question in light of the stark contrast in disorder-specific phenomenology and neurobiology. We review reported prevalence rates and the methodological, phenomenological, and theoretical issues concerning concomitant ADHD-OCD. Reported co-occurrence rates are highly inconsistent in the literature. Studies aimed at examining the potential for comorbidity have suffered from various methodological problems, including the existence of very few community samples, highly variable exclusionary criteria, and possible clinical misinterpretation of symptoms. Despite numerous studies suggesting an ADHD-OCD comorbidity, thus far etiological (i.e., genetic) backing has been provided only for a pediatric comorbidity. Additionally, inflated rates of ADHD-OCD co-occurrence may be mediated by the presence of tic disorders, and evidence of impaired neuronal maturational processes in pediatric OCD may lead to possibly transient phenotypical expressions that resemble ADHD symptomatology. Thus, clinicians are encouraged to consider the possibility that ADHD-like symptoms resulting from OCD-specific symptomatology may be misdiagnosed as ADHD. This suggestion may account for the lower co-occurrence rates reported in adolescents and adults and for the lack of a theoretical account for comorbidity in these age groups. Existing literature is summarized and critically reviewed, and recommendations are made for future research.

Variants of exposure therapy are effective for treating obsessive-compulsive and related disorders (OCRDs). However, significant numbers of patients do not respond adequately to exposure therapy resulting in continued distress and functional impairment. Therefore, novel approaches to augmenting exposure therapy are needed to adequately treat non- and partial-responders. Emerging research suggests that interventions that augment learning and memory processes associated with exposure therapy (i.e., extinction training) may display promise in enhancing treatment response in OCRDs. As the most studied example, d-cycloserine (DCS) is a relatively safe cognitive enhancer that appears to accelerate treatment gains associated with exposure therapy. This article reviews research on the use of DCS and other putative cognitive modifiers as they relate to the treatment (or prospective treatment) of obsessive-compulsive disorder and other OCRDs.

A vast and heterogeneous body of literature on the neuropsychology of obsessive-compulsive disorder (OCD) has accumulated in recent decades, yielding inconsistent results. In an attempt to quantitatively summarize the literature, we conducted a meta-analysis of 115 studies (including 3452 patients), comparing adult OCD patients with healthy controls on tests of 10 neuropsychological domains. Across studies, medium mean effect sizes were found for all executive function subdomains, processing speed, and sustained attention. Small effect sizes were found for visuospatial abilities and working memory. A large effect size was found for non-verbal memory whereas a small effect size was found for verbal memory, where only the former was found to be associated with impairments in executive functions. Moderators of effect sizes were also investigated. Results are discussed in terms of their clinical significance as well as their implications for current neurobiological models of OCD and methodological caveats., (© 2013.)

A one-dimensional mathematical model is developed and implemented to describe the coupled transport of citrate-stabilized silver nanoparticles (nAg) and dissolved silver ions in porous media. This hybrid numerical simulator employs an Eulerian finite difference (FD) method to model the reactive transport of dissolved constituents and a Lagrangian (random-walk particle-tracking (RWPT)) approach to capture the transport and differential aging of nanoparticles. Model performance is demonstrated by comparison of simulations with data obtained from a series of nAg transport and dissolution column experiments. A three pore volume pulse of a citrate-stabilized nAg suspension (ca. 3 mg/L) was introduced into a 12 or 16 cm long column packed with water-saturated quartz sand at a pore-water velocity of ca. 7.6 m/day and pH 4 or 7. While low retention levels (ca.17%) and no dissolution were observed for the pH 7 column, analysis of column effluent samples for pH 4 conditions indicated that ca. 88% of the injected silver mass was retained in the column, while 6% was eluted as particles (nAg) and 6% as dissolved ions (Ag(+)). Hybrid model simulations, employing a lumped nAg dissolution coefficient of (3.45 ± 0.35) × 10(-2)/h, are shown to accurately capture measured nAg transport and Ag(+) release behavior. A model sensitivity analysis explores the influence of flow velocity and particle size on nAg transport and fate, indicating that as velocity and particle size decrease, nAg dissolution and Ag(+) transport processes increasingly dominate silver mobility.

Exposure-based cognitive-behavioral therapy and serotonin reuptake inhibitor medications are efficacious treatment options for the management of pediatric obsessive-compulsive disorder. Despite established efficacy, many youths receiving either therapy remain symptomatic after acute treatment. Regardless of the rationale for persistent symptoms, a clear need emerges for treatment options that restore functioning efficiently to symptomatic youths. One innovative approach builds upon the identified role of NMDA receptors in the fear extinction process. Instead of breaking existing connections during fear extinction, new associations develop that eventually predominate over prior associations. Recent investigations have explored augmenting exposure-based cognitive-behavioral therapy with the NMDA partial agonist d-cycloserine, with preliminary results demonstrating expedited treatment gains and moderately larger effects above exposure and response prevention therapy alone. A large randomized clinical trial is underway to evaluate the efficacy and efficiency of this therapeutic combination in pediatric obsessive-compulsive disorder. Results from this trial may translate into improved management practices.

Background: Adjuvants serve as catalysts of the innate immune response by initiating a localized site of inflammation that is mitigated by the interactions between antigens and toll like receptor (TLR) proteins. Currently, the majority of vaccines are formulated with aluminum based adjuvants, which are associated with various side effects. In an effort to develop a new class of adjuvants, agonists of TLR proteins, such as bacterial products, would be natural candidates. Lipopolysaccharide (LPS), a major structural component of gram negative bacteria cell walls, induces the systemic inflammation observed in septic shock by interacting with TLR-4. The use of synthetic peptides of LPS or TLR-4 agonists, which mimic the interaction between TLR-4 and LPS, can potentially regulate cellular signal transduction pathways such that a localized inflammatory response is achieved similar to that generated by adjuvants., Methodology/principal Findings: We report the identification and activity of several peptides isolated using phage display combinatorial peptide technology, which functionally mimicked LPS. The activity of the LPS-TLR-4 interaction was assessed by NF-κB nuclear translocation analyses in HEK-BLUE™-4 cells, a cell culture model that expresses only TLR-4, and the murine macrophage cell line, RAW264.7. Furthermore, the LPS peptide mimics were capable of inducing inflammatory cytokine secretion from RAW264.7 cells. Lastly, ELISA analysis of serum from vaccinated BALB/c mice revealed that the LPS peptide mimics act as a functional adjuvant., Conclusions/significance: Our data demonstrate the identification of synthetic peptides that mimic LPS by interacting with TLR-4. This LPS mimotope-TLR-4 interaction will allow for the development and use of these peptides as a new class of adjuvants, namely TLR-4 agonists.

Prostate cancer (PCa) is one of the most common types of cancer in men in the United States and is the second leading cause of cancer related death in men. Clinically, secreted prostate specific antigen (PSA) has gained recognition because of its proteolytic activity being directly linked to PCa cell proliferation leading to disease initiation and progression. Using phage display technology, we identified four distinct cyclical peptides. These peptides apart from differences in their amino acid sequence, elicited minimal cross reactive antibody responses against each other. One of the four peptides analyzed produced an antibody response that recognizes the PSA protein. We demonstrate that the synthetic PSA peptide mimics identified in our study are immunologically active and produce neutralizing activity and this has relevance and utility for prostate cancer disease progression., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Endothelial progenitor cells are increasingly being studied in various diseases ranging from ischemia, diabetic retinopathy, and in cancer. The discovery that these cells can be mobilized from their bone marrow niche to sites of inflammation and tumor to induce neovasculogenesis has afforded a novel opportunity to understand the tissue microenvironment and specific cell-cell interactive pathways. This review provides a comprehensive up-to-date understanding of the physiological function and therapeutic utility of these cells. The emphasis is on the systemic factors that modulate their differentiation/mobilization and survival and presents the challenges of its potential therapeutic clinical utility as a diagnostic and prognostic reagent.

Heat shock proteins such as gp96 are immunogenic and are widely used as vaccines in immunotherapy of cancers. The present study focuses on the use of peptide mimotopes as immunotherapeutic vaccines for prostate cancer. To this end, we developed a 15-mer gp96 peptide mimotope specifically reactive to MAT-LyLu gp96-peptide complex using combinatorial single-chain antibody and peptide phage display library. The immunogenicity of the synthesized gp96 mimotope was analyzed initially in normal BALB/c mice in combination with various adjuvants such as complete Freund's adjuvant (CFA), aluminum salts (ALUM), granulocyte-macrophage colony-stimulating factor (GM-CSF), and liposome, of which CFA served as a positive control. The antibody response was determined and found that the gp96 mimotope with ALUM showed a significant increase in antibody titer, followed by GM-CSF and liposomes. Further, the T cell (CD4(+) and CD8(+)) populations from splenocytes, as well as IgG isotypes, interleukin-4, and interleukin-5 of gp96 mimotope with ALUM-immunized animals, were analyzed. The results suggest that the gp96 mimotope may elicit a potent and effective antitumor antibody response. Further, the study identifies ALUM and GM-CSF as adjuvant options to drive an appropriate protective immune response as these adjuvants have prior use in humans.

To explore the role of the HLH subdomain in bHLHZ proteins, we designed sets of minimalist proteins based on bHLHZ protein Max, bHLH/PAS protein Arnt and bZIP protein C/EBP. In the first, the Max bHLH and C/EBP leucine zipper were fused such that the leucine heptad repeats were not in register; therefore, the protein dimerization interface was disrupted. Max1bHLH-C/EBP showed little ability to activate transcription from the E-box (5'-CACGTG) in the yeast one-hybrid assay, and no E-box binding by quantitative fluorescence anisotropy. Max1bHLH-C/EBP's activity was significantly improved after library selection (three amino acids randomized between HLH and leucine zipper), despite the Max bHLH and C/EBP zipper still being out of register: a representative mutant gave a high nanomolar K(d) value for E-box binding. Thus, selection proved to be a powerful tool for salvaging the flawed Max1bHLH-C/EBP, although the out-of-register mutants still did not achieve the strong DNA-binding affinities displayed by their in-register counterparts. ArntbHLH-C/EBP hybrids further demonstrated the importance of maintaining register, as out-of-register mutants showed no E-box-responsive activity, whereas the in-register hybrid showed moderate activity. In another design, we eliminated the HLH altogether and fused the Max basic region to the C/EBP zipper to generate bZIP-like hybrids. Despite numerous designs and selections, these hybrids possessed no E-box-responsive activity. Finally, we tested the importance of the loop sequence in MaxbHLHZ by fluorescence and circular dichroism. In one mutant, the loop was shortened by two residues; in the other, the Lys57:DNA-backbone interaction was abolished by mutation to Gly57. Both showed markedly decreased E-box-binding relative to MaxbHLHZ. Our results suggest that, in contrast to the more rigid bZIP, the HLH is capable of significant conformational adaptation to enable gene-regulatory function and is required for protein dimerization and positioning the basic region for DNA recognition.

Cancer-derived heat shock protein gp96 induces a tumor-specific protective immune response primarily mediated by cytotoxic T lymphocytes (CTL) directed toward cancer-associated peptides associated with gp96. Both innate and adaptive immune responses have been demonstrated using a cell culture-based signaling mechanism. When used as an extraneous vaccine, one critical interaction which must occur for an immune response to be generated is the interaction between gp96 and the antigen presenting cell (APC) surface receptors (CD91, SR-A, TLR-2, and TLR-4). Our previous study concluded that gp96 purified from various rat and human prostate cancers is differentially glycosylated based on the amino and neutral monosaccharide content, and it was postulated that the monosaccharides may play a role in its biological activity. In this report, we report differences in the cancer-specific sialic acid content of gp96 purified from normal rat prostate compared to two rat prostate cancers, MAT-LyLu and Dunning G, as well as between two human prostate cancer cells, LnCaP and DU145. We also examined the modulatory effect of sialic acid residues on the binding of gp96 to APCs and its subsequent activation. Our results supported the contention that significant differences in the sialic acid content exist between Dunning G, MAT-LyLu, and normal rat prostate gp96, which affected its binding and biochemical activity to APCs. We therefore postulate that varied glycans of HPS96, a hitherto neglected structural component, may play a pivotal role in its anticancer activity. We suggest that construction of the glycan tree is a key to identification of the necessary and sufficient elements in the structure-function activity of HSP96.

The concept of cancer immunotherapy provides a fresh perspective as it is not associated with many of the drawbacks of conventional therapies such as chemotherapy, radiotherapy and surgery. When fully activated the immune system has immense potential as is evident from mis-matched transplanted organs undergoing rapid immunological attack and rejection. However, the development of immune strategies for cancer therapy has been associated with challenges of their own. Early attempts at cancer vaccination were carried out in an empirical manner that did not always lead to reproducibility. This led to a search of tumor associated antigens with the belief that specific targeting of these antigens would lead to successful tumor elimination. Active vaccination with TAA peptides or passive vaccination with specific lymphocytes against these TAAs did not however demonstrate encouraging results in clinical trials. This was mainly because of the lack of an activating immune response which is required for continuous stimulation of lymphocytes and also because of the selection of tumor escape variants that did not express the particular TAA. On the positive side, attempts at characterizing TAAs illuminated the molecular changes that attribute a malignant phenotype to cancer cells. Attempts at cytokine therapy were also met with challenges of high systemic toxicity and a lack of specific lymphocyte activation. It was therefore realized that an ideal vaccinating agent should be able to combine the effects of both these therapeutic strategies, i.e., it should be able to induce an innate immune response which can be tailored to a tumor specific adaptive immune response. By this, the immunosuppressive tumor environment can be altered to become immune activating, thus facilitating the infiltration of myeloid and lymphoid cells that can act in concert leading to tumor regression. In this regard, immunotherapeutic approaches such as DNA vaccines, dendritic cell based vaccines, HSP based vaccines and gene transfer technology, are being developed and further refined to overcome their inherent limitations. Animal experiments with these therapeutic modalities have demonstrated exciting results, although their evaluation in clinical trials has not indicated exceptional tumor protection in a large percentage of the patients. These observations only further underscore the multivariate and dynamic nature of the immune system and the many ways in which tumor cells modulate themselves and their surroundings to escape immune surveillance. Assessment of successful therapeutic intervention will require periodic evaluations of the suppressive nature of the tumor microenvironment accompanied by qualitative and quantitative measurements of lymphocyte responses in patients. With the development of advanced genetic technologies and continuous identification of tumor antigens, the field of cancer immunotherapy is progressing at an exciting pace giving us hope for the advent of effective treatment modalities that will prolong tumor free survival and enhance the quality of life in patients with malignant disease.

Our labs are focused on identifying amino acid sequences having the ability to react specifically with the functional binding site of a complementary antibody. Our epitopic definition is based on the analysis of the similarity level of antigenic amino acid sequences to the host proteome. Here, the similarity profile to the human proteome of an HCV E1 immunodominant epitope, i.e. the HCV E1(315-328)HRMAWDMMMNWSPT sequence, led to i) characterizing the immunoreactive HCV E1 315-328 region as a sequence endowed with a low level of similarity to human proteins; ii) defining 2 contiguous immunodominant linear determinants respectively located at the NH(2) and COOH terminus of the conserved viral antigenic sequence. This study supports the hypothesis that low sequence similarity to the host's proteome modulates the pool of epitopic amino acid sequences in a viral antigen, and appears of potential value in defining immunogenic viral peptide sequences to be used in immunotherapeutic approaches for HCV treatment.

Experimental analyses and literature survey reveal low-redundancy to the host proteins as a common denominator of immunogenic sequences mapped along tumor-, autoimmune-, and infectious disease-associated-proteins. The hypothesis that immunogenicity of peptide sequences is linked to proteomic redundancy is discussed.

In order to identify small regions of the genome whose specific copy number alteration is associated with high genomic instability in the form of overall genome-wide copy number aberrations, we have analyzed array-based comparative genomic hybridization (aCGH) data from 33 sporadic colorectal carcinomas. Copy number changes of a small number of specific regions were significantly correlated with elevated overall amplifications and deletions scattered throughout the entire genome. One significant region at 9q34 includes the c-ABL gene. Another region spanning 22q11-q13 includes the breakpoint cluster region (BCR) of the Philadelphia chromosome. Coordinate 22q11-q13 alterations were observed in 9 of 11 tumors with the 9q34 alteration. Additional regions on 1q and 14q were associated with overall genome-wide copy number changes, while copy number aberrations on chromosome 7p, 7q, and 13q21.1-q31.3 were found associated with this instability only in tumors from patients with a smoking history. Our analysis demonstrates there are a small number of regions of the genome where gain or loss is commonly associated with a tumor's overall level of copy number aberrations. Our finding BCR and ABL located within two of the instability-associated regions, and the involvement of these two regions occurring coordinately, suggests a system akin to the BCR-ABL translocation of CML may be involved in genomic instability in about one-third of human colorectal carcinomas.

Recently we defined the tyrosinase233-247IPYWDWRDAEKCDIC peptide as the pentadecamer sequence hosting the linear determinant of the anti-tyrosinase MAb T311. In order to understand the molecular features that render epitopic the amino acid tyrosinase233-247 sequence, we 1) analyzed the MHC II affinity of the tyrosinase233-247IPYWDWRDAEKCDIC peptide using RankPep as a binding prediction program, and 2) experimentally verified the actual peptide-MHC II interaction by carrying out peptide electrophoretic mobility shift assays. Nine random tyrosinase peptides were used as controls. Under the conditions used in this study, it was found that all of the ten tyrosinase peptides were capable of binding to Ad/Ed molecules in the gel-shift binding assay. Moreover, there was no correlation between the theoretical predicted Ad/Ed affinity and the experimental actual peptide binding. We conclude that Ad/Ed binding potential appears influential in the defining of tyrosinase233-247IPYWDWRDAEKCDIC peptide epitopicity.

Background: Although described by Hippocrates in 400 B.C., pemphigus disease still needs a safe therapeutical approach, given that the currently used therapies (i.e. corticosteroids and immunosuppressive drugs) often provoke collateral effects. Here we present preliminary data on the possible use of a proteomics derived desmoglein peptide which appears promising in halting disease progression without adverse effects., Methods: The low-similarity Dsg349-60REWVKFAKPCRE peptide was topically applied for 1 wk onto a lesion in a patient with a late-stage Pemphigus vulgaris (PV) complicated by diabetes and cataract disease. The peptide was applied as an adjuvant in combination with the standard corticosteroid-based immunosuppressive treatment., Results: After 1 wk, the treated PV eroded lesion appeared dimensionally reduced and with an increased rate of re-epithelization when compared to adjacent non-treated lesions. Short-term benefits were: decrease of anti-Dsg antibody titer and reduction of the corticosteroid dosage. Long-term benefits: after two years following the unique 1-wk topical treatment, the decrease of anti-Dsg antibody titer persists. The patient is still at the low cortisone dosage. Adverse effects: no adverse effect could be monitored., Conclusion: With the limits inherent to any preliminary study, this case report indicates that topical treatment with Dsg349-60REWVKFAKPCRE peptide may represent a feasible first step in the search for a simple, effective and safe treatment of PV.

Background: A number of autoimmune diseases have been clinically and pathologically characterized. In contrast, target antigens have been identified only in a few cases and, in these few cases, the knowledge of the exact epitopic antigenic sequence is still lacking. Thus the major objective of current work in the autoimmunity field is the identification of the epitopic sequences that are related to autoimmune reactions. Our labs propose that autoantigen peptide epitopes able to evoke humoral (auto)immune response are defined by the sequence similarity to the host proteome. The underlying scientific rationale is that antigen peptides acquire immunoreactivity in the context of their proteomic similarity level. Sequences uniquely owned by a protein will have high potential to evoke an immune reaction, whereas motifs with high proteomic redundancy should be immunogenically silenced by the tolerance phenomenon. The relationship between sequence redundancy and peptide immunoreactivity has been successfully validated in a number of experimental models. Here the hypothesis has been applied to pemphigus diseases and the corresponding desmoglein autoantigens., Methods: Desmoglein 3 sequence similarity analysis to the human proteome followed by dot-blot/NMR immunoassays were carried out to identify and validate possible epitopic sequences., Results: Computational analysis led to identifying a linear immunodominant desmoglein-3 epitope highly reactive with the sera from Pemphigus vulgaris as well as Pemphigus foliaceous. The epitopic peptide corresponded to the amino acid REWVKFAKPCRE sequence, was located in the extreme N-terminal region (residues 49 to 60), and had low redundancy to the human proteome. Sequence alignment showed that human desmoglein 1 and 3 share the REW-KFAK-RE sequence as a common motif with 75% residue identity., Conclusion: This study 1) validates sequence redundancy to autoproteome as a main factor in shaping desmoglein peptide immunogenicity; 2) offers a molecular mechanicistic basis in analyzing the commonality of autoimmune responses exhibited by the two forms of pemphigus; 3) indicates possible peptide-immunotherapeutical approaches for pemphigus diseases.

Background: Cruciferous vegetables have been found to have anti-prostate cancer effects. The active compounds mediating these effects include indoles such as indole-3-carbinol (I3C) and isothiocyanates. I3C is unstable having tissue tropic effects and clinical utility has been partly addressed by the synthesis of a more stable dimer diindolylmethane (DIM)., Methods: Anti-proliferative activity was measured by XTT assay and cytosolic proteins quantitated by Western blot analysis., Results: DIM (IC(50) 50 microM) is a better anti-proliferative agent than I3C (IC(50) 150 microM) in androgen dependent LNCaP cells, inhibits DNA synthesis, and growth of R1881 stimulated LNCaP cells. Androgen receptor (AR), cyclin D1, and cdk4, induced by R1881, are downregulated by DIM. DIM downregulates phosphorylated Akt and phosphatidyl inositol 3-kinase and downstream inhibition of cyclin D1 and cdk4., Conclusion: These studies provide evidence that DIM is a second-generation chemopreventive agent with a viable cellular target and has clinical potential as an anti-prostate cancer chemopreventive., ((c) 2005 Wiley-Liss, Inc.)

Epidemiological evidences suggest that the progression and promotion of prostate cancer (CaP) can be modulated by diet. Since all men die with prostate cancer rather than of the disease, it is of particular interest to prevent or delay the progression of the disease by chemopreventive strategies. We have been studying the anticancer properties of compounds present in cruciferous vegetables such as indole-3-carbinol (I3C). Diindolylmethane (DIM) is a dimer of I3C that is formed under acidic conditions and unlike I3C is more stable with higher anti-cancer effects. In the present report, we demonstrate that DIM is a potent anti-proliferative agent compared to I3C in the hormone independent DU 145 CaP cells. The anti-prostate cancer effect is mediated by the inhibition of the Akt signal transduction pathway as DIM, in sharp contrast to I3C, induces the downregulation of Akt, p-Akt, and PI3 kinase. DIM also induced a G1 arrest in DU 145 cells by flow cytometry and downstream concurrent inhibition of cell cycle parameters such as cyclin D1, cdk4, and cdk6. Our data suggest a need for further development of DIM, as a chemopreventive agent for CaP, which justifies epidemiological evidences and molecular targets that are determinants for CaP dissemination/progression. The ingestion of DIM may benefit CaP patients and reduce disease recurrence by eliminating micro-metastases that may be present in patients who undergo radical prostatectomy.

Immune responses contribute to the pathogenesis of vitiligo and target melanoma sometimes associated with vitiligo-like depigmentation in some melanoma patients. We analyzed the sera from patients with vitiligo and cutaneous melanoma for reactivity toward tyrosinase peptide sequences 1) endowed with low level of similarity to human proteome, and 2) potentially able to bind HLA-DR1 Ags. We report that the tyrosinase autoantigen was immunorecognized with the same molecular pattern by sera from vitiligo and melanoma patients. Five autoantigen peptides composed the immunodominant anti-tyrosinase response: aa95-104FMGFNCGNCK; aa175-182 LFVWMHYY; aa176-190FVWMHYYVSMDALLG; aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC. All of the five antigenic peptides were characterized by being (or containing) a sequence with low similarity level to the self proteome. Sera from healthy subjects were responsive to aa95-104FMGFNCGNCK, aa222-236IQKLTGDENFTIPYW, and aa233-247 IPYWDWRDAEKCDIC, but did not react with the aa175-182LFVWMHYY and aa176-190FVWMHYYVSMDALLG peptide sequences containing the copper-binding His180 and the oculocutaneous albinism I-A variant position F176. Our results indicate a clear-cut link between peptide immunogenicity and low similarity level of the corresponding amino acid sequence, and are an example of a comparative analysis that might allow to comprehensively distinguish the epitopic peptide sequences within a disease from those associated to natural autoantibodies. In particular, these data, for the first time, delineate the linear B epitope pattern on tyrosinase autoantigen and provide definitive evidence of humoral immune responses against tyrosinase.

Heat shock protein gp96 induces a tumor-specific protective immunity in a variety of experimental tumor models. Because the primary sequences of the glycoprotein, gp96 are identical between tumor and normal tissues, the peptides associated with gp96 and/or the posttranslational modifications of gp96, determine its immunogenicity. Gp96-associated peptides constitute the antigenic repertoire of the source tissue; thus, purified gp96-peptide complexes have clinical significance as autologous cancer vaccines. However, the role of altered glycosylation and its contribution in the biological as well as immunologic activity of gp96 still remains uncharacterized. We examined the cancer-specific glycosylation patterns of gp96. To this end, monosaccharide compositions of gp96 were compared between normal rat prostate and two cancerous rat prostate tissues, nonmetastatic/androgen-dependent Dunning G and metastatic/androgen-independent MAT-LyLu, as well as two human nonmetastatic prostate cancer cell lines, androgen-dependent LnCaP and androgen-independent DU145. Marked differences were observed between the gp96 monosaccharide compositions of the normal and cancerous tissues. Furthermore, gp96 molecules from more aggressive cellular transformations were found to carry decreasing quantities of several monosaccharides as well as sum total content of neutral and amino sugars. We believe that the unique glycosylation patterns contribute to cellular phenotype and that the posttranslational modifications of gp96 may affect its functional attributes.

Using non-self discrimination as a driving force in generating peptide immunogenicity, we have developed a computer-assisted proteomic analysis in order to identify the protein antigenic regions that have evoked humoral response. The purpose of this study was to further validate the computational analysis for melanoma-associated antigens and, at the same time, to assess the efficacy of the methodology in defining antigenic regions of autoantigens associated to autoimmune diseases. To achieve this two-fold objective, we have examined the enzyme tyrosinase, a protein that represents an important autoantigen in patients with vitiligo or melanoma. Here, we report that the antigenic linear determinant of the monoclonal antibody (Mab) T311 raised against the melanoma/vitiligo tyrosinase autoantigen is located in the low similarity 15-mer amino acid sequence tyrosinase 233-247 IPYWDWRDAEKCDIC, within the fragment 237-247. These data confirm non-similarity to the host proteome as a factor that participates in shaping peptide immune reactivity and may be a first step towards designing tyrosinase antigenic peptides to be used for (i) direct neutralization of harmful melanocytes-attacking autoantibodies in vitiligo, or (ii) production of antibodies against tyrosinase-positive melanomas. Moreover tyrosinase peptide antigens might be used as key tools in studying the boundaries between self-tolerance and autoimmunity phenomena.

Background: Prostate cancer is the most frequently diagnosed neoplasm and the second leading cause of cancer-related mortality in men. Although the incidence of prostate cancer increases with age, the link between aging and prostate cancer is poorly understood., Methods: Affymetrix oligonucleotide microarrays were used to analyze the mRNA expression levels in the dorsolateral prostates from 6- and 18-month-old ACI rats. Real-time RT-PCR and immunohistochemistry was performed to validate microarray data in a select set of genes., Results: Microarray analysis revealed changes in gene expression associated with inflammation, oxidative stress, tissue remodeling, and energy metabolism. Most of these changes have been related to increased proliferative status of the prostate, anti-apoptosis, activated stroma, and alteration of the energy metabolism., Conclusions: Age-associated alterations in the gene expression profile may put the aging prostate in risk for the initiation, promotion, and progression of neoplastic transformation in both our animal model and humans., ((c) 2004 Wiley-Liss, Inc.)

The goal of this study was to determine the effects of various compounds on the 17-beta-estradiol-induced dimerization of the human estrogen receptor alpha (hERalpha), a nuclear transcription factor. For this purpose, we used a modified yeast two-hybrid (YTH) bioassay designed to study protein-protein interactions, based on the electrochemical monitoring of hERalpha dimerization and detected as beta-D-galactosidase reporter gene activity in a synthetic substrate p-aminophenyl-beta-D-galactopyranoside (pAPG). Compared with 17-beta-estradiol activity, genistein, bisphenol-A (BPA), and naringenin induced dimerization to a lower extent by four, five and six magnitudes of orders of magnitude, respectively. In the presence of physiological concentrations of 17-beta-estradiol, both tamoxifen and the analgesic drug acetaminophen inhibited hER dimerization in an antiestrogenic manner.

Over the last century cancer research has produced data leading to a composite picture where gene mutations and epigenetic phenomena strictly relate and overlap. This complexity has repercussions on the anti-cancer therapeutical strategies. The therapeutic pathway paved by the kochian one-cause/one disease principle fails in front of a multigenic multiphenomena disease like cancer. We still do not know what target(s) to hit/modify in order to prevent/stop the carcinogenic progression. On the light of cancer statistics 2005, we discuss the need of exactly defining the cancer targets in order to exploit the high potential of gene therapy.

Our labs are pursueing the goal of exactly defining the qualities of peptide antigenicity, immunogenicity and pathogenicity in order to develop safe specific immunotherapies by utilizing specific portions of disease-associated-proteins (DAPs). Using as a model the Pemphigus vulgaris antigen (PVA) desmoglein 3 (Dsg3), we have studied the murine humoral response against the Dsg3 amino acid 49-60 peptide sequence, previously characterized as sequence having low similarity to the mouse proteome. The results show that the low-similarity Dsg3(49-60)REWVKFAKPCRE peptide does not elicit pathogenic antibodies.

Indole-3-carbinol (I3C), a compound present as glucobracissin in cruciferous vegetables has anticancer activities which is in line with some of the epidemiological evidence that suggests a beneficial effect of consumption of cruciferous vegetables on cancer incidence and progression. The precise target of indole-3-carbinol has not been determined. We examined the effect of I3C on prostate cancer in a well-defined R3327 model using Copenhagen rats and the transplantable cell line, MAT-LyLu. This cell line derived from a tumor in Copenhagen rats is androgen independent and metastasizes to the lung and lymph nodes. Tumors were induced in Copenhagen rats by injecting MAT-LyLu subcutaneously and the animals treated with I3C that was administered either intraperitoneally or intravenously, in order to achieve maximal systemic exposure. This was a departure from the traditional chemopreventive route of indole-3-carbinol where the compound was incorporated in the diet. Our results indicate that I3C inhibited the incidence, growth and metastases of MAT-LyLu cells and both i.p. and i.v. injections of I3C were equally effective. Statistical analysis (Kaplan-Meier curves) clearly indicates a tumor-free and overall survival benefit as a result of treatment with I3C. These studies show for the first time that I3C in an injectible form has anti-prostate cancer activity.

BACKGROUND: Walking along disease-associated protein sequences in the search for specific segments able to induce cellular immune response may direct clinical research towards effective peptide-based vaccines. To this aim, we are studying the targets of the immune response in autoimmune diseases by applying the principle of non-self-discrimination as a driving concept in the identification of the autoimmunogenic peptide sequences. METHODS: Computer-assisted proteomic analysis of the autoantigen protein sequence and dot-blot/NMR immunoassays are applied to the prediction and subsequent validation of the epitopic sequences. RESULTS: Using the experimental model Pemphigus vulgaris/desmoglein 3, we have identified the antigenic linear determinant recognized by MAb 5H10, a monoclonal antibody raised against the extracellular domain of human desmoglein-3. The computer-assisted search for the Dsg3 epitope was conducted by analyzing the similarity level to the mouse proteome of the human desmoglein protein sequence. Dot-blot immunoassay analyses mapped the epitope within the sequence Dsg349-60 REWVKFAKPCRE, which shows low similarity to the mouse proteome. NMR spectroscopy analyses confirmed the specificity of MAb 5H10 for the predicted epitope. CONCLUSIONS: This report promotes the concept that low level of sequence similarity to the host's proteome may modulate peptide epitopicity.

We have mapped the linear antigenic determinant of a commercial MAb raised in the mouse against the melanoma-associated-antigen Melan-A/MART-1. The B cell epitope on the Melan-A/MART-1 oncoprotein is located in the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP, within residues 102-106. The definition of the antigenic sequence on Melan-A/MART-1 oncoprotein was reached following analyses of MHC II binding potential and similarity level to the mouse proteome, that put into evidence the 15-mer amino acid sequence 101-115 PPAYEKLSAEQSPPP as the top scoring peptide in binding H2-A(d) molecules and the epitopic sequence residues 102-106 (i.e., the peptide sequence PAYEK) as having low-similarity level to the mouse proteome. Dot-blot epitope mapping immunoassay identified proline residue 102 as critical, based on its effect on antibody recognition. The present study adds to previous companion reports in validating the hypothesis that low-similarity to the host's proteome and binding potential to MHC II molecules are essential concurring factors in the modulation of the pool of epitopic sequences.

An efficient strategy is presented for the identification of antigenic sequences in the context of given MHC molecules of interest. The proteomic analysis of the antigenic peptide repertoire is described and demonstrated by using high-molecular weight melanoma-associated antigen. The identification of the epitopic sequence of a monoclonal antibody raised against the 250 kDa tumor associated antigen was reached by using only seven short synthetic peptide fragments, instead of the 155 non-overlapping 15-mer peptides theoretically necessary as minimum screening library. The present result has been obtained by applying as driving criteria the analysis of the peptide affinity to MHC class II molecules and the non-self discrimination concept.

We analyzed the sera of patients with melanoma to define the human humoral autoantibody profile towards HMW-MAA. Computational proteome scanning using the non-self-discrimination principle as a guide led to the individuation of the low-similarity HMW-MAA781-789RATVWMLRL peptide fragment as an immunodominant B-cell epitope. Linear B-cell determinant individuation was experimentally validated by dot blot immunoassay and NMR spectroscopy analysis. Regulation of physiologic self-reactivity by the non-self-discrimination principle is discussed., (Copyright 2004 Wiley-Liss, Inc.)