Plasma cell-free DNA as a monitoring tool for high-risk pregnancies associated with antiphospholipid syndrome.

INTRODUCTION: Despite thromboprophylaxis, women with antiphospholipid syndrome (APS) face high-risk pregnancies due to proinflammatory and prothrombotic states. This highlights the need for new monitoring and prognostic tools. Recent insights into the pathophysiological role of neutrophil activation and extracellular trap (NET) formation in this syndrome led to the exploration of plasma cell-free DNA (cfDNA), a derivative of NETosis, as a promising biomarker. MATERIALS AND METHODS: cfDNA was isolated and quantified from plasma samples of healthy pregnant women (control group, HC) and women with APS (APS group). We assessed the physiological variability of cfDNA across the three trimesters in HC. Levels of cfDNA were compared between APS and HC by gestational trimester. ROC curve analysis was performed to evaluate the efficacy of cfDNA levels for classifying APS patients. Furthermore, cfDNA levels in pregnant women with APS with obstetric complications were compared to those from uncomplicated pregnancies. RESULTS: Among HC, cfDNA significantly increased in the third trimester compared to the first and second. Elevated cfDNA levels in APS compared to HC were observed in the first and second trimesters. First-trimester cfDNA levels demonstrated the highest classification ability to discriminate between APS and HC patients (AUC: 0.906). Among APS, those with complicated pregnancies (fetal growth restriction, preeclampsia, placenta accreta) exhibited significantly elevated cfDNA levels in the second trimester. CONCLUSIONS: Elevated levels of cfDNA in pregnant women with APS, particularly among those with obstetric complications, supports further investigation into the potential of cfDNA as a valuable tool in the obstetric management of women with APS.

Plant growth promoting activities of endophytic bacteria from Melia azedarach (Meliaceae) and their influence on plant growth under gnotobiotic conditions.

Bacteria that live asymptomatically within plant tissues are known as endophytes. Because of the close relation with the plant host, they have been a matter of interest for application as plant growth promoters. Melia azedarach is a widely distributed medicinal tree with proven insecticidal, antimicrobial, and antiviral activity. The aim of this study was to isolate and characterize endophytic bacteria from M. azedarach and analyze their plant growth promoting activities for the potential application as biological products. Bacteria were isolated from roots and leaves of trees growing in two locations of Northeastern Argentina. The isolates were characterized by repetitive extragenic palindromic sequence PCR and 16S rDNA sequence analysis. The plant growth-promoting activities were assayed in vitro, improvement of plant growth of selected isolates was tested on M. azedarach plantlets, and the effect of selected ACC deaminase producing isolates was tested on tomato seedlings under salt-stress conditions. The highest endophytic bacterial abundance and diversity were obtained from the roots. All isolates had at least one of the assayed plant growth-promoting activities and 80 % of them had antagonistic activity. The most efficient bacteria were Pseudomonas monteilii, Pseudomonas farsensis, Burkholderia sp. and Cupriavidus sp. for phosphate solubilization (2064 µg P ml-1), IAA production (94.7 µg ml-1), siderophore production index (5.5) and ACC deaminase activity (1294 nmol α-ketobutyrate mg-1 h-1). M. azedarach inoculation assays revealed the bacterial growth promotion potential, with Pseudomonas monteilii, Pseudomonas farsensis and Cupriavidus sp. standing out for their effect on leaf area, leaf dry weight, specific leaf area, and total Chl, Mg and N content, with increases of up to 149 %, 58 %, 65 %, 178 %, 76 % and 97.7 %, respectively, compared to NI plants. Efficient ACC deaminase-producing isolates increased stress tolerance of tomato plants under saline condition. Overall, these findings indicate the potential of the endophytic isolates as biostimulant and biocontrol agents.

A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Antisepsys and profilatic antimicrobial therapy in prevention of surgical site infection of horses / [Antissepsia e terapia antimicrobiana profilática no controle infeccioso do foco cirúrgico em equinos

The efficacy of an antisepsis protocol comprising chlorhexidine gluconate and ethyl alcohol in combination with prophylactic antimicrobial therapy in controlling surgical site infection in horses was studied. To that purpose, seven mixed breed horses received potassium penicillin and gentamicin at least 30 minutes prior to surgery. The surgical site was scrubbed with chlorhexidine gluconate and rinsed with ethyl alcohol. Samples were collected at four time points: (A) - before and (B) - immediately following shaving of the hair coat, (C) - at the end of antisepsis procedures, and (D) - at the end of the surgical procedure. Duration of surgery was recorded. Samples were cultured in three different culture mediums: Mitis Salivarus (Streptococcus sp.), Staphylococcus 110 (Staphylococcus sp.), and Mac Conkey (Enterobacteria). A high level of bacterial growth was observed in all culture mediums at (A) and (B), with no bacterial growth in (C). Staphylococcus sp. growth was observed in (D) in a single patient whose surgical procedure lasted for 120 minutes. Shaving of the hair coat reduced microbial flora on the surface of the skin. Antisepsis in combination with prophylactic antimicrobial therapy was effective in controlling surgical site infection in elective procedures with an average duration of 90 minutes.(AU)

Objetivou-se averiguar a eficácia do protocolo de antissepsia com clorexidina degermante e álcool etílico hidratado 70%, em associação com terapia antimicrobiana profilática, no controle microbiano do foco cirúrgico de equinos submetidos a procedimentos cirúrgicos. Foram utilizados 07 cavalos adultos de raças variadas, onde ambos receberam o mesmo tratamento (terapia antimicrobiana profilática e antissepsia com clorexidina degermante 2% e álcool etílico hidratado 70%), coletando-se amostras em quatro tempos distintos [(A - antes da tricotomia), (B - imediatamente após tricotomia), (C - ao término da antissepsia), (D - ao término do procedimento cirúrgico)]. O tempo de cada procedimento cirúrgico foi contabilizado. Foram utilizados três meios de cultura diferentes, cada um com especificidade para um tipo de crescimento bacteriano. Constatou-se alta incidência de crescimento bacteriano nos três meios utilizados nos tempos de coleta A e B. Para o tempo C, não foi observado crescimento bacteriano. No tempo D averiguou-se crescimento bacteriano do tipo Staphylococcus sp. em um único paciente, cujo tempo cirúrgico foi de 120 minutos de duração. Desta forma, a tricotomia reduziu a carga microbiana na superfície da pele. A antissepsia associada à terapia antimicrobiana profilática mostrou-se eficaz no controle microbiano do foco cirúrgico em procedimentos eletivos, com duração média de 90 minutos.(AU)

Next generation sequencing characterizes the extent of HLA diversity in an Argentinian registry population.

Next generation DNA sequencing is used to determine the HLA-A, -B, -C, -DRB1, and -DQB1 assignments of 1472 unrelated volunteers for the unrelated donor registry in Argentina. The analysis characterized all HLA exons and introns for class I alleles; at least exons 2, 3 for HLA-DRB1; and exons 2 to 6 for HLA-DQB1. Of the distinct alleles present, there are 330 class I and 98 class II. The majority (~98%) of the cumulative allele frequency at each locus is contributed by alleles that appear at a frequency of at least 1 in 1000. Fourteen (18.2%) of the 77 novel class I and II alleles carry nonsynonymous variation within their exons; 52 (75.4%) class I novel alleles carry only single, apparently random, nucleotide variation within their introns/untranslated regions. Alleles encoding protein variation not usually detected by typing focused only on the exons encoding the antigen recognition domain are 1.0% of the class I assignments and 7.3% of the class II assignments (predominantly DQB1*02:02:01, DQB1*03:19:01, and DRB1*14:54:01). Updates to the common and well documented list of alleles include 10 alleles previously thought to be uncommon but that are found at least 30 times. Five locus haplotypes estimated using the expectation-maximization algorithm as present 3 or more times total 187. While the known HLA diversity continues to increase, the conservation of known allele sequences is remarkable. Overall, the HLA diversity observed in the Argentinian population reflects its European and Native American ancestry.

Multidrug-resistant pathogenic Escherichia coli isolated from wild birds in a veterinary hospital.

Wild birds are carriers of Escherichia coli. However, little is known about their role as reservoirs for extra-intestinal pathogenic E. coli (ExPEC). In this work we investigated E. coli strains carrying virulence genes related to human and animal ExPEC isolated from free-living wild birds treated in a veterinary hospital. Multidrug resistance was found in 47.4% of the strains, but none of them were extended-spectrum beta-lactamase producers. Not only the virulence genes, but also the serogroups (e.g. O1 and O2) detected in the isolates of E. coli have already been implicated in human and bird diseases. The sequence types detected were also found in wild, companion and food animals, environmental and human clinical isolates in different countries. Furthermore, from the 19 isolates, 17 (89.5%) showed a degree of pathogenicity on an in vivo infection model. The isolates showed high heterogeneity by pulsed-field gel electrophoresis indicating that E. coli from these birds are clonally diverse. Overall, the results showed that wild birds can be reservoirs and/or vectors of highly pathogenic and multidrug-resistant E. coli that have the potential to cause disease in humans and poultry.

Morfologia e quantificação da microbiota intestinal do curimbatá (Prochilodus lineatus) e do cascudo cinza (Pterygoplichthys anisitsi) cultivados em cativeiro / Morphology and quantification of intestinal microbiota of curimbatá (Prochilodus lineatus) and the gray armored catfish (Pterygoplichthys anisitsi) reared in captivity

Todos os animais vivem em íntima associação com micro-organismos que desempenham importantes funções em seu desenvolvimento normal. Nos vertebrados, a mais populosa e complexa comunidade de micro-organismos reside no trato intestinal. O intuito do estudo foi quantificar, classificar e verificar morfologicamente a população microbiana intestinal de duas importantes espécies de peixes de água doce, o curimbatá (Prochilodus lineatus) e o cascudo cinza (Pterygoplichthys anisitsi). As amostras foram coletadas por meio de raspagens da mucosa intestinal, diluídas seriadamente até 10-4, semeadas em placas contendo ágar soja tripticaseína (TSA) e ágar chocolate (AC) para contagem de bactérias totais e identificação morfológica por Gram, em aerobiose e em anaerobiose facultativa, respectivamente. As contagens de bactérias totais mostraram resultados que variaram entre 10³ e 10(4)ufc.mL-1. Os tipos morfológicos encontrados foram cocos, leveduras e bastonetes Gram negativos e positivos. Estudos adicionais sobre os padrões de colonização microbiana e a morfologia dos micro-organismos aderidos à mucosa intestinal foram possíveis com o uso da microscopia eletrônica de varredura (MEV), sendo encontradas formas variadas de micro-organismos, tais como leveduras, formas cocoides e bacilares flageladas e não flageladas. A microbiota intestinal do curimbatá e a do cascudo cinza provaram ser bastante diversas e populosas, com o predomínio de micro-organismos Gram negativos.

All animals exist in intimate associations with microorganisms that play important roles in the hosts' normal development. In vertebrates, the most populous and complex community of microbes resides in the digestive tract.The aim of this research was to morphologically quantify, classify and verify the composition of intestinal microbiota of two species of freshwater fish, the Prochilodus lineatus and the Pterygoplichthys anisitsi. The samples were collected by scrapings of intestinal mucosa, serially diluted to 10-4, plated on tryptic soy agar (TSA) and chocolate agar (CA) for total bacterial counting and morphological identification by Gram, in aerobiosis and facultative anaerobiosis conditions, respectively. In the total bacterial counting results ranged between 10³ to 10(4) cfu.mL-1. The morphological types found were cocci, yeasts and Gram negative and positive rods. Additional studies about patterns of microbial colonization and the morphology of the adhered microorganisms to the intestinal mucosa were possible using the scanning electron microscopy (SEM) and several forms of microorganisms, such as yeasts, cocci and bacillary shapes flagellated and non-flagellated were found. The intestinal microbiota of P. lineatus and P. anisitsi was diverse and populous, with a predominance of Gram negative microorganisms.

Efficacy of geraniol but not of β-ionone or their combination for the chemoprevention of rat colon carcinogenesis

β-ionone (βI), a cyclic isoprenoid, and geraniol (GO), an acyclic monoterpene, represent a promising class of dietary chemopreventive agents against cancer, whose combination could result in synergistic anticarcinogenic effects. The chemopreventive activities of βI and GO were evaluated individually or in combination during colon carcinogenesis induced by dimethylhydrazine in 48 3-week-old male Wistar rats (12 per group) weighing 40-50 g. Animals were treated for 9 consecutive weeks with βI (16 mg/100 g body weight), GO (25 mg/100 g body weight), βI combined with GO or corn oil (control). Number of total aberrant crypt foci (ACF) and of ACF ≥4 crypts in the distal colon was significantly lower in the GO group (66 ± 13 and 9 ± 2, respectively) compared to control (102 ± 9 and 17 ± 3) and without differences in the βI (91 ± 11 and 14 ± 3) and βI+GO groups (96 ± 5 and 19 ± 2). Apoptosis level, identified by classical apoptosis morphological criteria, in the distal colon was significantly higher in the GO group (1.64 ± 0.06 apoptotic cells/mm²) compared to control (0.91 ± 0.07 apoptotic cells/mm²). The GO group presented a 0.7-fold reduction in Bcl-2 protein expression (Western blot) compared to control. Colonic mucosa concentrations of βI and GO (gas chromatography/mass spectrometry) were higher in the βI and GO groups, respectively, compared to the control and βI+GO groups. Therefore, GO, but not βI, represents a potential chemopreventive agent in colon carcrvpdate=20110329inogenesis. Surprisingly, the combination of isoprenoids does not represent an efficient chemopreventive strategy.

Efficacy of geraniol but not of ß-ionone or their combination for the chemoprevention of rat colon carcinogenesis.

ß-ionone (ßI), a cyclic isoprenoid, and geraniol (GO), an acyclic monoterpene, represent a promising class of dietary chemopreventive agents against cancer, whose combination could result in synergistic anticarcinogenic effects. The chemopreventive activities of ßI and GO were evaluated individually or in combination during colon carcinogenesis induced by dimethylhydrazine in 48 3-week-old male Wistar rats (12 per group) weighing 40-50 g. Animals were treated for 9 consecutive weeks with ßI (16 mg/100 g body weight), GO (25 mg/100 g body weight), ßI combined with GO or corn oil (control). Number of total aberrant crypt foci (ACF) and of ACF ≥4 crypts in the distal colon was significantly lower in the GO group (66 ± 13 and 9 ± 2, respectively) compared to control (102 ± 9 and 17 ± 3) and without differences in the ßI (91 ± 11 and 14 ± 3) and ßI+GO groups (96 ± 5 and 19 ± 2). Apoptosis level, identified by classical apoptosis morphological criteria, in the distal colon was significantly higher in the GO group (1.64 ± 0.06 apoptotic cells/mm²) compared to control (0.91 ± 0.07 apoptotic cells/mm²). The GO group presented a 0.7-fold reduction in Bcl-2 protein expression (Western blot) compared to control. Colonic mucosa concentrations of ßI and GO (gas chromatography/mass spectrometry) were higher in the ßI and GO groups, respectively, compared to the control and ßI+GO groups. Therefore, GO, but not ßI, represents a potential chemopreventive agent in colon carcinogenesis. Surprisingly, the combination of isoprenoids does not represent an efficient chemopreventive strategy.

Ocorrência de Aeromonas spp. em abatedouro bovino e sensibilidade a antimicrobianos / Occurrence of Aeromonas spp. in bovine abattoir and antimicrobial sensitivity

O gênero Aeromonas compreende espécies consideradas importantes patógenos para os seres humanos, sendo as gastroenterites as infecções mais comumente atribuídas a estas bactérias. Tendo em vista sua importância como patógeno de origem alimentar, a ocorrência deAeromonas spp. foi estudada em carcaças bovinas e ambiente do abatedouro em uma indústria do Estado de São Paulo. Foram colhidas 285 amostras de 19 locais. Foi realizada a contagem direta por semeadura em meio seletivo, caracterização bioquímica das espécies após enriquecimento seletivo e teste de sensibilidade a antimicrobianos. A contagem direta permitiu a quantificação dessas bactérias em apenas 12 amostras, variando de 0,5 a 9,2 x 100 UFC/cm2, sendo cinco delas de ambiente, com populações que variaram de 1,0 x 100 a 3,0 x 100 UFC/placa. Entretanto, após o enriquecimento seletivo, Aeromonas spp. foram isoladas de 38 amostras que, somadas às amostras de ambiente não submetidas ao enriquecimento, forneceram 62 isolados para análise. A caracterização bioquímica das espécies permitiu verificar a ocorrência de 59 isolados de A. caviae, um de A. sobria, um de A. trota e um de A. schubertii. O teste com antimicrobianos revelou resistência de todos os isolados pela ampicilina e cefalotina, enquanto, para os demais antimicrobianos, esta foi variável. A resistência da totalidade dos isolados a determinados antimicrobianos indica que estes devem ser utilizados criteriosamente com a finalidade de evitar o surgimento precoce de cepas deAeromonas spp. multirresistentes. Ainda, a maior prevalência deA. caviae deve ser considerada relevante, pois trata-se de uma das espécies causadoras de gastroenterite em humanos.

The genus Aeromonas comprises important human pathogens, gastroenteritis being the most common type of infection attributed to these bacteria. Considering their importance as foodborne pathogens, the occurrence of Aeromonas spp. was studied on bovine carcasses and the environment of a slaughterhouse in São Paulo State, Brazil. A total of 285 samples were collected from 19 points. Direct counting was carried out by seeding on selective medium, with biochemical characterization of the species after selective enrichment and antimicrobial sensitivity testing. The direct count permitted bacteria quantification only in 12 samples ranging from 0.5 to 9.2 x 100 CFU/ cm2, of which 5 were from environment, ranging from 1.0 x 100 to 3.0 x 100 CFU/plate. However, after selective enrichment,Aeromonas spp. was isolated from 38 samples, which when added to the environmental samples not subjected to the enrichment provided 62 isolates for analysis. Biochemical characterization allowed for the verification of the occurrence of 59 isolates of A. caviae, 1 ofA. sobria, 1 ofA. trota and 1 ofA. schubertii. Antimicrobial testing revealed that all isolates were resistant to ampicillin and cephalotin, while the results for the other antimicrobials were variable. Resistance of all isolates to some antimicrobials indicates that these must be used carefully to avoid early development of multiresistantAeromonas spp. Also, the greatest prevalence of A. caviae must be considered relevant because it is one of the causative species of human gastroenteritis.

[Hyperhomocysteinaemia and type 2 diabetes]. / Hyperhomocystéinémie et diabète de type 2.

OBJECTIVES: Hyperhomocysteinaemia is associated with cardiovascular events in nondiabetic individuals. The present study was aimed to explore the implication of hyperhomocysteinemia in development of cardiovascular events in patients with type 2 diabetes. DESIGN: A total of 185 patients with type 2 diabetes (115 women and 70 men, 30 to 93 years of age) have been included consecutively in the ambulatory unit at the Saint-Philibert Hospital. For each patient the concentration of homocysteine, cholesterol and triglyceride levels and HbA1c have been measured. In the studied population, 121 patients presented cardiovascular events (myocardial infarctus, peripheric arteriopathy, cerebrovascular accident). RESULTS: The patients with cardiovascular events were older, the concentration of homocysteine and creatinine were higher. The plasma homocysteine levels adjusted for age and creatinine levels were higher in patients with cardiovascular events than in patients without cardiovascular events (15.4 +/- 3.52 micromol/L and 13.13 +/- 2.26 micromol/L respectively; p = 2. 10(-5)). CONCLUSIONS: Hyperhomocysteinemia is an independant risk factor for cardiovascular events in type 2 diabetes, independent of age and renal function.

Use of superficial femoral vein in the treatment of infected aortoiliofemoral prosthetic grafts.

PURPOSE: To assess the use of the superficial femoral vein for the reconstruction of the aortoiliofemoral sector in the treatment of prosthetic infections. METHODS: From December 1995 to November 1999, 12 patients with infection involving a synthetic vascular prosthesis were submitted to thirteen surgical procedures involving partial or total resection of a synthetic vascular prosthesis and restoration of arterial flow with an in situ femoral vein. RESULTS: The overall operative mortality rate was 15.3% (2 out of 3 patients who underwent total graft replacement and 0 out of 9 patients who underwent unilateral graft replacement). Major amputations related to the surgical procedures were performed in two cases (an 87.5% rate of limb salvage). The series was followed up on average for 22 months (range: 6-65 months). No patient presented clinically significant edema or signs of chronic venous insufficiency in the lower limbs used as donors of autogenous venous grafts. All surviving patients presented complete resolution of the infectious signs and symptoms and none of them presented late thrombosis of the venous graft. CONCLUSION: The use of the superficial femoral vein is a good surgical alternative for the treatment of prosthetic infections with minimal venous morbidity of the lower limbs used as venous graft donors. The autogenous venous grafts present good long-term patency and excellent adaptation to the aortoiliofemoral position.

Symptomatic mucocele of a surgically excluded esophagus.

Isolated esophageal segments following esophageal bypass surgery may develop into mucus-filled dilatations (mucoceles) of the esophagus. They usually remain small and asymptomatic. This report describes a patient who developed a symptomatic esophageal mucocele 1 year after surgical exclusion of the thoracic esophagus for Boerhaave's syndrome.

Traqueopatía osteocondroplástica Presentación de un caso / Osteocondroplastic tracheobronchopathy

La traqueobroncopatía osteocondroplástica o traqueopatía osteoplástica es una enfermedad benigna de origen desconocido caracterizada por el depósito de tejido osteocartilaginoso en la submucosa del tracto respiratorio inferior provocando una estrechez de la vía aérea. Presentamos aspectos relevantes de esta patología y un caso clínico de un paciente, que al ser sometido a un procedimiento quirúrgico por parte de otorrinolaringología, se detecta una alteración de la vía aérea durante la inducción anestésica

A novel frequency distribution selection method for efficient plate layout of a diverse combinatorial library.

A deterministic method (frequency distribution method) for selecting compounds from a partitioned virtual combinatorial library for efficient synthesis is presented here. The method is based on reagent frequency analysis and can be applied to any library of molecules distributed in any given partitioned chemical space (cluster, cell-based, etc.). Compound selection by reagent frequency distribution can produce a unique, diverse set of molecules that adequately represents the library while requiring the least amount of compounds to be synthesized and minimizing the number of different reagents that must be used. This method also provides a practical solution to the configuration of plate layout. Because the method essentially identifies "expensive" regions in the chemical space to synthesize for a desired diversity or similarity coverage, decisions concerning the necessity to synthesize these compounds can be addressed. Minimum compound generation and efficient plate layout results in savings both in time of synthesis and cost of materials. This method always results in a discrete solution, which can be used for any given library size as well as any combination of reagents and is also readily adaptable to robotic automation.

Nipple-sparing mastectomy in breast cancer: a viable option?

BACKGROUND: In women with breast cancer for whom breast-conserving therapy (BCT) is not the best option, a nipple and areola complex-(NAC) sparing mastectomy with immediate reconstruction has been proposed as a good and safe alternative to conventional, more radical mastectomy. Surgeons hesitate to perform this operation for fear of recurrence of tumour in the NAC due to undetected nipple involvement (NI) of the tumour. In order to determine whether a NAC-sparing mastectomy is a viable option, the frequency and predictive factors of NI by the tumour were studied in the literature. METHODS: A literature survey was performed by searching the Medline database. Other references were derived from the material perused. RESULTS AND CONCLUSIONS: NI is found in up to 58% of mastectomy specimens and correlates with tumour size, tumour-areola or tumour-nipple distance, positive lymph nodes and clinical suspicion. Best candidates for NAC-sparing mastectomy are patients with a small tumour (T1) at a large distance (>4-5 cm) from the nipple. However, in these patients BCT has excellent results with low complications and recurrence rates. Considering the incidence of NI in larger tumours (T2 average 33%, T3 average >50%) a NAC-sparing mastectomy may carry an unacceptable high risk for local relapse and should therefore not be advocated.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Binding site elucidation of hydantoin-based antagonists of LFA-1 using multidisciplinary technologies: evidence for the allosteric inhibition of a protein--protein interaction.

The binding site on the lymphocyte function-associated antigen-1 (LFA-1) of a class of hydantoin-based antagonists of leukocyte cell adhesion has been identified. This site resides in the inserted-domain (I-domain) of the CD11a chain at a location that is distal to residues known to be required for interactions with the intercellular adhesion molecules. This finding supports the hypothesis that the molecules are antagonizing cell adhesion via an allosteric modification of LFA-1. The binding site was identified using an integrated immunochemical, chemical, and molecular modeling approach. Antibodies that map to epitopes on the I-domain were blocked from binding to the purified protein by the hydantoins, indicating that the hydantoin-binding site resides on the I-domain. Photoaffinity labeling of the I-domain followed by LC/MS and LC/MS/MS analysis of the enzymatic digest identified proline 281 as the primary amino acid residue covalently attached to the photoprobe. Distance constraints derived from this study coupled with known SAR considerations allowed for the construction of a molecular model of the I-domain/inhibitor complex. The atomic details of the protein/antagonist interaction were accurately predicted by this model, as subsequently confirmed by the X-ray crystal structure of the complex.

Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Phosphotyrosine-containing dipeptides as high-affinity ligands for the p56lck SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.