Update of the PANCCO clinical practice guidelines for the treatment of ulcerative colitis in the adult population.

Ulcerative colitis (US) is a chronic disease of unknown etiology. It is incurable and its clinical course is intermittent, characterized by periods of remission and relapse. The prevalence and incidence of the disease has been increasing worldwide. The update presented herein includes the participation of healthcare professionals, decision-makers, and a representative of the patients, all of whom declared their conflicts of interest. Answerable clinical questions were formulated, and the outcomes were graded. The information search was conducted on the Medline/PubMed, Embase, Epistemonikos, and LILACS databases, and covered grey literature sources, as well. The search was updated on November 30, 2020, with no restrictions regarding date or language. The Grading of Recommendations Assessment, Development and Evaluation (GRADE) classification system was implemented to establish the strength of the recommendation and quality of evidence. A formal consensus was developed, based on the RAND/UCLA methodology and the document was peer reviewed. The short version of the Clinical Practice Guidelines for the Treatment of Ulcerative Colitis in the Adult Population is presented herein, together with the supporting evidence and respective recommendations. In mild-to-moderate UC, budesonide MMX is an option when treatment with 5-ASA fails, and before using systemic steroids. In moderate-to-severe UC, infliximab, adalimumab, vedolizumab, ustekinumab, and tofacitinib can be used as first-line therapy. If there is anti-TNF therapy failure, ustekinumab and tofacitinib provide the best results. In patients with antibiotic-refractory pouchitis, anti-TNFs are the treatment of choice.

Factores predictivos de infección de herida quirúrgica en cirugía arterial de extremidades inferiores / Predictive factors of surgical wound infection in lower limb arterial surgery

INTRODUCCIÓN: La infección de herida quirúrgica (IHQ) es una complicación grave de la cirugía reconstructiva arterial periférica, que genera un gran coste hospitalario y se asocia, si hay afectación del injerto, a altas tasas de amputación y mortalidad. OBJETIVOS: Análisis de incidencia y factores predictivos de IHQ, identificación de agente causal y supervivencia libre de amputación en pacientes con IHQ intervenidos de reparación arterial de extremidades inferiores. MATERIAL Y MÉTODO: Estudio observacional prospectivo sobre infecciones de cirugía arterial directa revascularizadora por arteriopatía obliterante de extremidades inferiores realizadas en nuestro centro desde enero de 2011 hasta diciembre de 2014. Se incluyeron como variables a estudio: datos demográficos, factores de riesgo cardiovascular, inmunosupresión, estancia hospitalaria, clínica, reintervenciones, ASA, tiempo quirúrgico, grado de urgencia, uso de drenajes, tipo de injerto, transfusiones sanguíneas y preparación preoperatoria. Las variables resultado fueron la incidencia, factores predictivos, agente causal y supervivencia libre de amputación de IHQ. RESULTADOS: Se reclutó a 652 pacientes, con IHQ en 94 procedimientos (mediana de seguimiento 618 días): 63 (9,7%) infecciones superficiales, 18 (2,8%) profundas y 13 (2%) protésicas. Los gérmenes aislados más frecuentes fueron enterobacterias (27,9%) y Staphylococcus aureus (20,5%). Se identificaron como factores predictivos de infección: obesidad (OR: 2,34; IC 95%: 1,26-4,34), hemodiálisis (OD: 3,21; IC 95%: 1,26-8,20), tiempo quirúrgico >180 min (OR: 2,05; IC 95%: 1,13-3,71) y edad mayor de 65 años (OD: 2,35; IC 95%: 1,12-4,92). El análisis de supervivencia señala una menor supervivencia libre de amputación en los pacientes con IHQ, pese a no ser significativo (p = 0,09). CONCLUSIÓN: La supervivencia libre de amputación fue menor en los pacientes con infección de injerto que en aquellos con infección superficial. Encontramos como factores predictivos de IHQ la obesidad, hemodiálisis, edad mayor de 65 años e intervención >180 min. La identificación correcta de estos permitiría establecer estrategias para prevenir la IHQ en cirugía arterial y evitar sus consecuencias

INTRODUCTION: Surgical site infection (SSI) is a serious complication of peripheral arterial surgery. It generates increased hospital costs, and associated with high amputation and mortality rates if the graft is affected. AIMS: The aim of this study is to analyse the incidence rate and risk factors of SSI, to identify causal bacterial agents, and amputation free survival rates in patients with SSI undergoing lower limb arterial surgery. MATERIAL AND METHODS: A prospective observational study of SSI in peripheral open arterial surgery was performed in our centre from January 2011 to December 2014. The variables studied were: Demographic data, cardiovascular risk factors, immunosuppression, hospital stay, clinical symptoms, re-operations, ASA, surgical time, level of urgency, use of drains, type of graft, blood transfusion, and preoperative skin preparation. Outcome variables were incidence rate and risk factors of SSI, causal agent identification, and amputation free survival rate. RESULTS: A total of 652 patients were registered, identifying 94 SSI (median follow-up 618 days): 63 (9.7%) superficial infection, 18 (2.8%) deep infection, and 13 (2%) graft infection. The most common bacterial agent was enterobacter species (27.9%), followed by Staphylococcus aureus (20.5%). Obesity (OR: 2.34, 95% CI: 1.26-4.34), haemodialysis (OR: 3.21, 95% CI: 1.26-8.20), surgical time > 180 minutes (OR: 2.05, 95% CI: 1.13-3.71) and older than 65 years (OR: 2.35, 95% CI: 1.12-4.92) were identified as predictors of SSI. Survival analysis suggests a lower amputation-free survival rate in patients with SSI, although it is not statistically significant (P=.09). CONCLUSION: Amputation free survival rates are lower in patients with graft infection than those with superficial infection. Obesity, haemodialysis, over 65 years old, and surgical time > 180 min, were found to be predictors of SSI. The correct identification of these factors could help in establishing strategies to prevent SSI and avoid its consequences

Degeneración aneurismática de la arteria poplítea por síndrome de atrapamiento poplíteo / Aneurysmal degeneration of the popliteal artery due to popliteal entrapment syndrome

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Metal-specific CD4+ T-cell responses induced by beryllium exposure in HLA-DP2 transgenic mice.

Chronic beryllium disease (CBD) is a granulomatous lung disorder that is associated with the accumulation of beryllium (Be)-specific CD4(+) T cells into the lung. Genetic susceptibility is linked to HLA-DPB1 alleles that possess a glutamic acid at position 69 (ßGlu69), and HLA-DPB1*02:01 is the most prevalent ßGlu69-containing allele. Using HLA-DP2 transgenic (Tg) mice, we developed a model of CBD that replicates the major features of the human disease. Here we characterized the T-cell receptor (TCR) repertoire of Be-responsive CD4(+) T cells derived from the lungs of Be oxide-exposed HLA-DP2 Tg mice. The majority of Be-specific T-cell hybridomas expressed TCR Vß6, and a subset of these hybridomas expressed identical or nearly identical ß-chains that were paired with different α-chains. We delineated mimotopes that bind to HLA-DP2 and form a complex recognized by Be-specific CD4(+) T cells in the absence of Be. These Be-independent peptides possess an arginine at p5 and a tryptophan at p7 that surround the Be-binding site within the HLA-DP2 acidic pocket and likely induce charge and conformational changes that mimic those induced by the Be(2+) cation. Collectively, these data highlight the interplay between peptides and Be in the generation of an adaptive immune response in metal-induced hypersensitivity.

A small molecule inhibitor of XIAP induces apoptosis and synergises with vinorelbine and cisplatin in NSCLC.

BACKGROUND: Evasion of apoptosis contributes to the pathogenesis of solid tumours including non-small cell lung cancer (NSCLC). Malignant cells resist apoptosis through over-expression of inhibitor of apoptosis proteins (IAPs), such as X-linked IAP (XIAP). METHODS: A phenylurea-based small molecule inhibitor of XIAP, XIAP antagonist compound (XAC) 1396-11, was investigated preclincally to determine its ability to sensitise to clinically relevant cytotoxics, potentially allowing dose reduction while maintaining therapeutic efficacy. RESULTS: XIAP protein expression was detected in six NSCLC cell lines examined. The cytotoxicity of XAC 1396-11 against cultured NSCLC cell lines in vitro was concentration- and time-dependent in both short-term and clonogenic assays. XAC 1396-11-induced apoptosis was confirmed by PARP cleavage and characteristic nuclear morphology. XAC 1396-11 synergised with vinorelbine+/-cisplatin in H460 and A549 NSCLC cells. The mechanism of synergy was enhanced apoptosis, shown by increased cleavage of caspase-3 and PARP and by the reversal of synergy by a pan-caspase inhibitor. Synergy between XAC 1396-11 and vinorelbine was augmented by optimising drug scheduling with superior effects when XAC 1396-11 was administered before vinorelbine. CONCLUSION: These preclinical data suggest that XIAP inhibition in combination with vinorelbine holds potential as a therapeutic strategy in NSCLC.

[Profile of the patient who wants to quick smoking seen in the Primary Care and Specialized consultations in Spain]. / Perfil del paciente que quiere dejar de fumar atendido en consultas de Atención Primaria y especializada de España.

OBJECTIVE: To determine the profile of the smoker who wants to stop smoking. Subjects and methods. A cross-sectional, multicenter study was conducted in 1634 smokers attended by General Practitioners and Specialized Physicians in Spain who had expressed their desire to stop smoking when explicitly asked. Sociodemographic data, background of smoking and related consequences were collected. A descriptive statistical analysis was performed. RESULTS: 67.1% of patients were seen by General Practitioners. Mean age +/- standard deviation was 45.6 +/- 12.0 years old and mean age of onset of usual consumption was 20.6 +/- 7.0. Fifty-six percent of the population studied were men. Most (35.2%) had studied beyond high school and 76.4% were active workers. Seventy-one percent had previously tried to stop smoking (2.7+/- 3.0 mean attempts). Out of 39.2% smokers who had participated in some type of smoking cessation treatment the year before, 70.7% were on substitutive therapy with nicotine. At the time of the visit, 80% admitted they could not stop smoking by themselves. CONCLUSIONS: The profile of the smoker seen in Primary Care and Specialized Consultation in Spain who wants to quit smoking corresponds to a male subject in his 40's, with studies beyond high school, actively working who has made more that two previous attempts to stop smoking, mostly with substitutive therapy with nicotine and who currently believes he is not being able to achieve it by himself.

Local and semilocal density functional computations for crystals of 1-alkyl-3-methyl-imidazolium salts.

The accuracy and reliability of popular density functional approximations for the compounds giving origin to room temperature ionic liquids have been assessed by computing the T=0 K crystal structure of several 1-alkyl-3-methyl-imidazolium salts. Two prototypical exchange-correlation approximations have been considered, i.e., the local density approximation (LDA) and one gradient corrected scheme [PBE-GGA, Phys. Rev. Lett. 77, 3865 (1996)]. Comparison with low-temperature x-ray diffraction data shows that the equilibrium volume predicted by either approximations is affected by large errors, nearly equal in magnitude (approximately 10%), and of opposite sign. In both cases the error can be traced to a poor description of the intermolecular interactions, while the intramolecular structure is fairly well reproduced by LDA and PBE-GGA. The PBE-GGA optimization of atomic positions within the experimental unit cell provides results in good agreement with the x-ray structure. The correct system volume can also be restored by supplementing PBE-GGA with empirical dispersion terms reproducing the r-6 attractive tail of the van der Waals interactions.

Neutral and charged 1-butyl-3-methylimidazolium triflate clusters: equilibrium concentration in the vapor phase and thermal properties of nanometric droplets.

Ground state energy, structure, and harmonic vibrational modes of 1-butyl-3-methylimidazolium triflate ([bmim][Tf]) clusters have been computed using an all-atom empirical potential model. Neutral and charged species have been considered up to a size (30 [bmim][Tf] pairs) well into the nanometric range. Free energy computations and thermodynamic modeling have been used to predict the equilibrium composition of the vapor phase as a function of temperature and density. The results point to a nonnegligible concentration of very small charged species at pressures (P approximately 0.01 Pa) and temperatures (T >or= 600 K) at the boundary of the stability range of [bmim][Tf]. Thermal properties of nanometric neutral droplets have been investigated in the 0

Implicancias del diagnóstico antenatal de dilatación ureteral / Prenatal diagnosis of ureteral dilation

Muchos de los hallazgos del diagnóstico antenatal de patología urológica son inespecíficos y sólo nos permiten diferenciar pacientes con un mayor riesgo de infección urinaria o de perder función renal. El hecho de identificar un uréter en la ecografía prenatal es un hallazgo inespecífico, que aún no tenemos claro qué significa y qué pronóstico tiene para nuestros pacientes. A partir de noviembre de 1999, tenemos un protocolo prospectivo para el seguimiento y manejo de pacientes con diagnóstico antenatal de patología urológica. Se revisó en forma retrospectiva los pacientes en que hubo dilatación ureteral en la ecografía prenatal.180 pacientes han sido referidos y seguidos según protocolo. En 21 pacientes (11.7 por ciento) se ha hecho diagnóstico de dilatación ureteral en la ecografía prenatal. Once hombres y 10 mujeres. Los diagnósticos postnatales de estos pacientes fueron: 4 pacientes con doble sistema pieloureteral; 2 pacientes con megauréteres; 1paciente con una cloaca; 5 pacientes con dilataciones transitorias; 2 pacientes con ureterocele en sistemaúnico; 1 paciente con reflujo vesicoureteral en un sistema único y con un riñón multicístico en el contralateral; 1 paciente con valvas de uretra posterior y 5 pacientes con obstrucción pieloureteral. Un total de 13 pacientes de los 21 requirieron cirugía (62por ciento) y 8 se manejaron médicamente (38.09 por ciento).El hecho de identificar el uréter dilatado en la ecografía prenatal nos permite identificar una población prenatal de riesgo, ya que un 57,1 por ciento de nuestros pacientes requirieron de cirugía. Llama la atención que 5 pacientes en que se vio el uréter dilatado en la ecografía prenatal, terminaron operándose de obstrucción pieloureteral. Esto apoya la hipótesis de que estas obstrucciones sean secundarias a dilataciones ureterales prenatales. El consejo prenatal tiene que ser dado con cautela y por gente con experiencia, ya que a pesar de existir un riesgo elevado de cirugía...

Prenatal diagnosis is usually non specific and only allows for early detection of patients with a higher risk of developing urinary tract infections or loss of renal function. The identification of the ureter in a prenatal ultrasound is a nonspecific finding. The relevance of this is unknown. A prospective protocol for the management and follow up of patients with antenatal diagnosis of urologicmal formations was started in 1999. We retrospectively review our patients who presented a ureteral dilatation in the antenatal ultrasound.180 patients were referred and follow up was carried out according to protocol. 21 patients (11.7 percent) had ureteral dilatation on the prenatal ultrasound, 11 male and 10 female. The following post natal diagnoses were registered: 4 patients had a duplex kidney, 2 patients had megaureter, 1 patient had a cloaca, 5 patients had transitory dilatations, 2 patients had ureterocele in an single system, 1 patient had VUR in a single system and a contralateral muticystic kidney, 1 patient had posterior urethral valves, and 5 patients had ureteropelvic obstruction.13 of 21 patients required surgery (62 percent), the other 8 required only prophylaxis and follow up (38.09 percent). Being able to identify a dilated ureter in an prenatal ultrasound allowed us to define a high risk group, in which 62 percent required surgery. Of the patients in this group, it is noteworthy that 5 patients in whom a dilated ureter was visualized were operated on for ureteropelvic obstruction. This fact supports the idea that obstructions are secondary to antenatal ureteral dilations. Antenatal advice should be given carefully and by experienced personnel because, although there was a high percentage of surgery in this group, there was also a group of patients that only required antibiotics prophylaxis and image monitoring.

[Hypertensive crises: prevalence and clinical aspects]. / Crisis hipertensiva: prevalencia y aspectos clínicos.

OBJECTIVE: To analyze the prevalence and characteristics of patients with hypertensive crises and to know the clinical differences between patients with hypertensive urgencies and patients with hypertensive emergencies. PATIENTS AND METHODS: Three-months prospective study in which all patients attended at the Emergency Department with an hypertensive crisis (arterial blood pressure of at least 210/120 mmHg) were included. From each patient, a clinical history, physical examination, eye fundus examination, blood analysis, electrocardiogram, and a chest X-ray were obtained. RESULTS: A total of 118 patients were included in the study, representing 0.65% of all attended emergencies. Twenty-two percent of them had an emergency hypertensive crisis. Coronary heart disease was the most common cause for this emergency crisis. Hypertension was unknown to 12.7% of patients and 12.6% of patients aware of their condition were not taking any medication. Twenty-four percent of patients were diabetic. Patients with hypertensive emergencies had more involvement of target organs. Twenty-four percent of crises resolved with no therapy, and captopril was the most commonly used drug. CONCLUSIONS: Hypertensive crises accounted for 0.65% of attended emergencies at our institution. Coronary heart disease was the most common condition for hypertensive emergencies. Patients with hypertensive emergencies had a more severe involvement of target organs. Twenty four percent of crisis resolved with rest alone.

Crisis hipertensiva: prevalencia y aspectos clínicos / Hypertensive crises: prevalence and clinical aspects

Fundamento. Analizar la prevalencia y las características de los pacientes que sufren una crisis hipertensiva y conocer qué diferencias clínicas existen entre los pacientes con urgencia y con emergencia hipertensiva. Pacientes y métodos. Estudio prospectivo durante 3 meses en el que se incluyen todos los pacientes que acuden al servicio de Urgencias con una crisis hipertensiva (presión arterial 210/120 mmHg).Se realiza anamnesis, exploración física, funduscopia, analítica, electrocardiograma y radiografía de tórax. Resultados. Se incluyen 118 pacientes, que representan el 0,65 por ciento de los atendidos en el área de Medicina de Urgencias. El 22 por ciento presentaron una emergencia hipertensiva, siendo la cardiopatía isquémica la emergencia más frecuente. El 12,7 por ciento de los pacientes desconocía ser hipertenso y el 12,6 por ciento de los conocidos no recibían tratamiento farmacológico. El 23,7 por ciento de los pacientes era diabético. Los enfermos con emergencia hipertensiva presentaron una mayor afectación de los órganos diana. Un 23,9 por ciento de las urgencias hipertensivas no precisaron tratamiento farmacológico para su resolución. El fármaco más empleado fue el captopril por vía oral. Conclusiones. En nuestro estudio el 0,65 por ciento de las urgencias atendidas en el área de medicina fueron crisis hipertensivas. La emergencia hipertensiva más frecuente fue la cardiopatía isquémica. La afectación visceral fue más frecuente en los pacientes con emergencia hipertensiva. El 23,9 por ciento de las urgencias hipertensivas se resolvió únicamente con reposo. (AU)

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

Combinatorial peptide libraries as an alternative approach to the identification of ligands for tumor-reactive cytolytic T lymphocytes.

The recent identification of molecularly defined human tumor antigens recognized by autologous CTLs has opened new opportunities for the development of antigen-specific cancer vaccines. Despite extensive work, however, the number of CTL-defined tumor antigens that are suitable targets for generic vaccination of cancer patients is still limited, mostly because of the painstaking and lengthy nature of the procedures currently used for their identification. A novel approach is based on the combined use of combinatorial peptide libraries in positional scanning format (positional scanning synthetic combinatorial peptide libraries, PS-SCLs) and tumor-reactive CTL clones. To validate this approach, we herein analyzed in detail the recognition of PS-SCLs by Melan-A-specific CTL clones. Our results indicate that, at least for some clones, most of the amino acids composing the native antigenic peptide can be identified through the use of PS-SCLs. Interestingly, this analysis also allowed the identification of peptide analogues with increased antigenic activity as well as agonist peptides containing multiple amino-acid substitutions. In addition, biometrical analysis of the data generated by PS-SCL screening allowed the identification of the native ligand in a public database. Overall, these data demonstrate the successful use of PS-SCLs for the identification and optimization of tumor-associated CTL epitopes.

Molecular mimicry and antigen-specific T cell responses in multiple sclerosis and chronic CNS Lyme disease.

The concept of molecular mimicry provides and elegant framework as to how cross-reactivity between antigens from a foreign agent with self proteins may trigger autoimmune diseases. While it was previously thought that sequence and structural homology between foreign and self proteins or the sharing of T cell receptor (TCR) and MHC-binding motifs are required for molecular mimicry to occur, we have shown that even completely unrelated peptide sequences may lead to cross-recognition by T cells. The use of synthetic combinatorial peptide libraries in the positional scanning format (PS-SCL) together with novel biometric prediction approaches has allowed us to describe the recognition profiles of individual autoreactive T cell clones (TCC) with unprecedented accuracy. Through studies of myelin-specific TCC as well as clones from the nervous system of patients suffering from chronic central nervous (CNS) Lyme disease it has become clear that at least some T cells are more degenerate than previously anticipated. These data will not only help us to redefine what constitutes specific T cell recognition, but also allow us to study in more detail the biological role of molecular mimicry. A recent clinical trial with an altered peptide ligand (APL) of one of the candidate myelin basic protein (MBP) epitopes in MS (amino acids 83-99) has shown that such a modified MBP peptide may not only have therapeutic efficacy, but also bears the potential to exacerbate disease. Thus, we provide firm evidence that the basic principles of cross-recognition and their pathogenetic significance are relevant in MS.

Enhanced immune activity of cytotoxic T-lymphocyte epitope analogs derived from positional scanning synthetic combinatorial libraries.

The pp65(495-503) cytotoxic T-lymphocyte (CTL) epitope from cytomegalovirus (CMV) is universally recognized among CMV+ individuals who express an allele of the human leukocyte antigen A (HLA-A*0201). The relative binding affinity of the epitope to HLA-A*0201 is moderate, and its increased activity might prove beneficial in its use as a CTL epitope vaccine. A new approach to enhance the activity of T-cell epitopes is the use of positional scanning synthetic combinatorial libraries (PS-SCLs). Using a nonamer PS-SCL, the pp65(495-503) epitope was modified after screening a CMV-specific T-cell clone (TCC) (3-3F4) from which the native peptide sequence was derived. Two peptides with amino acid substitutions at P1, P3, P7, and P8 are between 10(3) and 10(4) more active than the native epitope. Although the native CTL epitope terminates as a free acid, both tetrasubstituted peptides only function as CTL epitopes when the carboxyl terminus is amidated. Selective substitution of the native sequence based on PS-SCL screening results identified 3 amidated monosubstituted and disubstituted peptides that are better recognized than the native epitope by TCCs from a cohort expressing HLA-A*0201. In vitro stimulation of peripheral blood mononuclear cells with each of the peptide epitope analogs stimulated memory CTLs, which recognized CMV-infected targets among a high percentage of CMV+ individuals. Binding studies of peptide analogs with HLA-Ig (immunoglobulin) dimers and 2 different TCCs correlated with in vitro lysis results. These data suggest that increasing the activity of CTL epitopes while maintaining broad recognition is possible, which holds promise for vaccine development in infectious disease and cancer.

Identification of MHC class II-restricted peptide ligands, including a glutamic acid decarboxylase 65 sequence, that stimulate diabetogenic T cells from transgenic BDC2.5 nonobese diabetic mice.

Nonobese diabetic (NOD) mice spontaneously develop insulitis and destruction of pancreatic islet beta cells similar to type 1 diabetes mellitis in humans. Insulitis also occurs in the BDC2.5 TCR transgenic line of NOD mice that express the rearranged TCR alpha- and beta-chain genes of a diabetogenic NOD CD4 T cell clone. When activated with syngeneic islet cells in culture, BDC2.5 T cells adoptively transfer disease to NOD recipients, but the identity of the islet cell Ag responsible for pathogenicity is not known. To characterize the autoantigen(s) involved, BDC2.5 T cells were used to screen a combinatorial peptide library arranged in a positional scanning format. We identified more than 100 decapeptides that stimulate these T cells at nanomolar concentrations; they are then capable of transferring disease to NOD-scid mice. Surprisingly, some of the peptides include sequences similar (8 of 10 residues) to those found within the 528-539 fragment of glutamic acid decarboxylase 65. Although this 12-mer glutamic acid decarboxylase 65 fragment is only slightly stimulatory for BDC2.5 T cells (EC(50) > 100 microM), a larger 16-mer fragment, 526-541, shows activity in the low micromolar range (EC(50) = 2.3 microM). Finally, T cells from prediabetic NOD mice respond spontaneously to these peptide analogs in culture; this finding validates them as being related to a critical autoantigen involved in the etiology of spontaneous diabetes and indicates that their further characterization is important for a better understanding of underlying disease mechanisms.

Drug discovery and vaccine development using mixture-based synthetic combinatorial libraries.

The approaches and concepts that encompass combinatorial chemistry represent a paradigm shift in drug discovery and basic research. Viewed initially as a curiosity by the pharmaceutical industry, combinatorial chemistry approaches are now recognized as essential drug discovery tools that decrease the time taken for discovery and increase the throughput of chemical screening by as much as 1000-fold. Although the use of mixture-based synthetic combinatorial libraries was one of the first approaches presented, its inherent strengths are only recently being recognized. Numerous mixture-based libraries of peptides, peptidomimetics and heterocycles have been synthesized and deconvoluted using the positional scanning approach. Mixture-based library approaches for drug discovery and vaccine development will be reviewed herein.

Dissection of bacteriophage lambda site-specific recombination using synthetic peptide combinatorial libraries.

A wide variety of tools have been used to dissect biochemical pathways, inhibitors being chief among them. Combinatorial approaches have made the search for inhibitors much more efficient. We have applied such an approach to identify hexapeptides which inhibit different steps in a site-specific recombination reaction mediated by the bacteriophage lambda integrase protein. Integrase's mechanism is still incompletely understood, in large part because several pathway intermediates remain hard to isolate. Integrase-catalyzed recombination is very efficient, but if blocked, it is highly reversible to substrates; this combination makes some intermediates exceedingly transient. We have used synthetic peptide combinatorial libraries to screen for hexapeptides that affect the recombination pathway at different stages, and have identified two families of peptides: one probably blocks DNA cleavage, the other may stabilize the Holliday junction intermediates. These peptides do not resemble parts of integrase or any of the other helper functions in the pathway. The deconvolution of hexapeptide libraries based both on inhibition of an enzymatic reaction as well as on accumulation of reaction intermediates is a novel approach to finding useful tools for dissecting a biochemical pathway.