Chemically synthesized peptide libraries as a new source of BBB shuttles. Use of mass spectrometry for peptide identification.

The blood-brain barrier (BBB) is a biological barrier that protects the brain from neurotoxic agents and regulates the influx and efflux of molecules required for its correct function. This stringent regulation hampers the passage of brain parenchyma-targeting drugs across the BBB. BBB shuttles have been proposed as a way to overcome this hurdle because these peptides can not only cross the BBB but also carry molecules which would otherwise be unable to cross the barrier unaided. Here we developed a new high-throughput screening methodology to identify new peptide BBB shuttles in a broadly unexplored chemical space. By introducing d-amino acids, this approach screens only protease-resistant peptides. This methodology combines combinatorial chemistry for peptide library synthesis, in vitro models mimicking the BBB for library evaluation and state-of-the-art mass spectrometry techniques to identify those peptides able to cross the in vitro assays. BBB shuttle synthesis was performed by the mix-and-split technique to generate a library based on the following: Ac-d-Arg-XXXXX-NH2 , where X were: d-Ala (a), d-Arg (r), d-Ile (i), d-Glu (e), d-Ser (s), d-Trp (w) or d-Pro (p). The assays used comprised the in vitro cell-based BBB assay (mimicking both active and passive transport) and the PAMPA (mimicking only passive diffusion). The identification of candidates was determined using a two-step mass spectrometry approach combining LTQ-Orbitrap and Q-trap mass spectrometers. Identified sequences were postulated to cross the BBB models. We hypothesized that some sequences cross the BBB through passive diffusion mechanisms and others through other mechanisms, including paracellular flux and active transport. These results provide a new set of BBB shuttle peptide families. Furthermore, the methodology described is proposed as a consistent approach to search for protease-resistant therapeutic peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

Mortality and recurrence patterns of breast cancer patients diagnosed under a screening programme versus comparable non-screened breast cancer patients from the same population: analytical survey from 2002 to 2012.

Breast cancer screening programmes seem to bring about significant benefits, including decreased mortality, although they may also have some drawbacks such as false-negative and false-positive results. This study aims to compare the clinical outcome of a group of patients undergoing a breast cancer screening programme with that of a synchronous non-screened group of patients matched for age and follow-up period. We studied basic characteristics of epidemiology, immunohistochemistry, loco-regional relapse, distant metastases, disease-free interval and overall and specific mortality. We compared 510 patients in the screened group with 394 non-screened patients, along the period of 2002-2012. Screening was applied on a target population of 49,847 and was based on double-projection, double-read mammograms. Two years were allowed per round. Overall participation for the five rounds considered was 75.2%, with 86.5% coverage, and a total cumulative population of 123,445. The non-participant women amounted 40,794. Tumour detection rate for the screened women was 3.8 per thousand (475/123,445), while the corresponding rate for non-participants was 9.4 per thousand (382/40,797). Incidence of luminal A subtype was 15% higher in screened than that in non-screened patients (95% confidence interval (CI) 8-22%). Conversely, the triple-negative subtype was 6% higher in the non-screened group (95% CI 2-10%). Incidence of breast conservative treatments and sentinel node biopsies was significantly higher in the screened group. Overall mortality was 2.6 times higher in non-screened than that in screened group (95% CI 1.2-5.6) After 10 years of experience with our own screening programme, we believe that included patients receive a benefit versus comparable non-screened breast cancer patients, with acceptable benefit-risk relation.

Migraine and benign paroxysmal positional vertigo: a single-institution review.

BACKGROUND: Benign paroxysmal positional vertigo and migraine-associated dizziness are common. The prevalence of benign paroxysmal positional vertigo seems to be higher in patients with migraine-associated dizziness than in those without migraine. METHODS: A database of 508 patients seen at the primary author's balance clinic was analysed to determine the prevalence of migraine, as defined by International Headache Society criteria, in patients with benign paroxysmal positional vertigo. RESULTS: The percentage of patients with dizziness or vertigo who met criteria for migraine was 33.7 per cent, with a prevalence of benign paroxysmal positional vertigo of 42.3 per cent. When excluding patients with migrainous vertigo, patients with migraine frequently had benign paroxysmal positional vertigo (66.7 per cent vs 55.8 per cent), although this finding was not statistically significant. CONCLUSION: The results for the entire sample suggest that, after excluding patients with migrainous vertigo, patients with migraine seem more likely to have benign paroxysmal positional vertigo; however, this association was not significant, probably because of the small sample size.

Using peptides to increase transport across the intestinal barrier.

The oral route is the preferred for the administration of drugs; however, it has some serious limitations. One of the main disadvantages is poor permeability across the intestinal barrier. Various approaches are currently being adopted to overcome this issue. In this review, we describe the alternatives that use peptides to enhance intestinal absorption. First, we define the various sources of peptide enhancers followed by the analysis of the absorption mechanism used. We then comment on the possible toxic effects derived from their use as permeation enhancers, as well as potential formulation strategies. Finally, the advantages and drawbacks of peptides as intestinal enhancers are examined.

A pyridinium-substituted analog of the TRH-like tripeptide pGlu-Glu-Pro-NH2 and its prodrugs as central nervous system agents.

A metabolically stable and centrally acting analog of pGlu-Glu-Pro-NH2 ([Glu2]TRH, a tripeptide structurally related to TRH (thyrotropin-releasing hormone)) was designed by replacing the amino-terminal pyroglutamyl residue with a pyridinium moiety. The analeptic action of the analog was used to optimize the efficacy of this novel CNS agent when administered intravenously in its CNS-permeable prodrug forms obtained via the reduction of the pyridinium moiety to the nonionic dihydropyridine and esterifying the central Glu with various alcohols. The maximum effect in antagonizing pentobarbital-induced narcosis in mice was achieved with the hexyl ester that was used subsequently for a comparative evaluation with a prodrug of the parent neuropeptide in the Porsolt swim test as a paradigm for antidepressant effect. The novel analog maintained its antidepressant potency but showed reduced analeptic action compared to [Glu2]TRH; thus, an increase in the selectivity of CNS-action was obtained by the incorporation of the pyridinium moiety.

Synthesis and photochemical reactions of nitroestrogens: possible photoaffinity labels of zero length.

The syntheses of 3,4-dimethoxy-1,3,5(10)-estratrien-17-one and 4-bromo-3-methoxy-2-nitro-1,3,5(10)-estratrein-17-one are described and their photoreactions with amines and hydroxide ion studied. The possible usefulness of these new steroids as photoaffinity labels of zero length is discussed.

Hearing results in retrolabyrinthine vestibular neurectomy.

Patients having retrolabyrinthine vestibular neurectomy (RLVN) may have complications that compromise hearing. While most reviews have emphasized sensorineural loss, less attention has been given to conductive hearing loss, which may complicate RLVN. Hearing results of 25 consecutive cases of RLVN performed for Meniere's disease with incapacitating vertigo were tabulated according to 1985 American Academy of Otolaryngology (AAO) guidelines. Nine patients (36%) had improved hearing postoperatively, 5 (20%) had no change in hearing, and 11 (44%) had worse hearing postoperatively. The most commonly observed audiometric change was low-frequency conductive hearing loss, presumably secondary to partial ossicular fixation by bone dust or fat fibrosis in the attic and antrum. Five patients (20%) had low-frequency conductive hearing losses that increased by 10 dB or greater over preoperative levels. An additional 7 patients had lesser losses at low frequencies. One patient had a flat conductive hearing loss. Six (24%) of the patients had a decrease in bone levels of greater than 10 dB. Overall hearing results in this study are comparable to those of other series in the literature. Causes and prevention of conductive hearing loss in RLVN are discussed, and a format for presentation of hearing data that will highlight conductive hearing loss after surgery for Meniere's disease is presented.

Head and neck sequelae of cardiac transplantation.

Cardiac transplantation has become the treatment of choice for end-stage cardiomyopathies. In 1987, nearly 2000 cardiac transplants were performed in the United States. Otolaryngologists will be asked with increasing frequency to evaluate and treat these patients. The otolaryngology service at Loyola University Medical Center has been involved in the follow-up and treatment of head and neck complications in 100 transplant patients. Sixty percent of these patients manifest head and neck sequelae. The results of this review are presented. The otolaryngologist should be aware of the special features of this patient population that require modification of the treatment approach, such as 1. the need to avoid the drugs erythromycin, trimethoprim/sulfamethoxazole, and ketoconazole, 2. the need to preserve the right internal jugular vein, and 3. the high risk of silent myocardial infarction. A discussion of these treatment modifications is provided. All patients should be treated in close communication with the medical transplant treatment team.

Recurrent laryngeal nerve paralysis associated with thoracic aortic aneurysm.

The association of vocal cord dysfunction with thoracic aortic aneurysm (TAA) has been noted in the cardiovascular and otolaryngologic literature. A retrospective review of 168 cases of TAA was performed in order to: (1) define the natural history of associated recurrent laryngeal nerve paralysis (RLNP) and (2) propose mechanisms for the development of RLNP in operated and nonoperated aneurysms. Of 168 aneurysms, 5% manifested hoarseness secondary to RLNP. All had type I aneurysms. Only one patient regained vocal cord function after surgical treatment of the aneurysm. RLNP developed as a sequela of TAA repair in 12% of the patients managed surgically. RLNP associated with TAA type III repair had a higher incidence of recovery than paralysis that occurred after TAA type I repair (40% vs. 0% recovery). Sixty-six percent of all patients with permanently paralyzed larynges in this series attained glottic competence sufficient to avoid Teflon injection, and 27% of all RLNP associated with TAA in this series required Teflon injection for aspiration, severe dysphonia, or both. Seventeen percent of the patients with vocal cord paralysis associated with TAA recovered within 12 months. Aneurysm classification and pertinent anatomic relationships are discussed with reference to various mechanisms of recurrent laryngeal nerve paralysis.

Complications in acoustic neuroma surgery.

There has been a continuous lowering of mortality and morbidity rates in the 20th century, thanks to increasing experience and newer technology. Despite this, the readership should be aware of the hazards involved in this challenging field. Complications occur, and their incidence, mechanism, management, and prevention have been outlined here so physicians performing these procedures will be better prepared to manage them. We have found that the occurrence of complications can be greatly reduced through a team approach using the combined talents of a neurotologist, neurosurgeon, and frequently an internist. It is apparent from this article that the morbidity of removing larger tumors is significantly greater than the morbidity of removing small tumors. The recent introduction of MRI to the diagnostic armamentarium of the neurotologist may permit earlier detection and a further reduction of morbidity and mortality rates. Only continued study of prevention of complications will assure the improved quality of our results for patients undergoing microsurgical removal of acoustic neuromas.

[Vaccination against the hepatitis B virus in alcoholics]. / Vacuna contra el virus de la hepatitis B en alcohólicos.

BACKGROUND: The risk of hepatitis B virus infections is high among alcohol abusers, and vaccination has been evaluated in previous studies. However, the response has been low with the usual doses and schedules. METHODS: In the present controlled trail a group of 14 alcohol abusers was vaccinated following a rapid schedule with a dose twice the usual one. Their response was compared to that of a second group of 16 alcohol abusers vaccinated in the usual way. Both groups were compared to a third one consisting of 30 healthy individuals. RESULTS: At the end of the program 83% of the healthy controls responded, with a mean anti-HBs titer of 1130.3 mU/ml. The anti-HBs titer achieved in both groups of alcohol abusers was lower that in healthy individuals (p less than 0.05). 56% of the alcohol abusers vaccinated with the usual program responded (with a mean titer of 80.1 mU/ml), and so did 66% of those vaccinated with high doses (mean titer 63.3 mU/ml). CONCLUSIONS: These results indicate that the poor response of alcohol abusers to HBV vaccine does not improve with increased doses and a rapid schedule.

[Spontaneous bacterial peritonitis: a comparative study of 2 methods of ascitic fluid culture]. / Peritonitis bacteriana espontánea: estudio comparativo de dos métodos de cultivo del líquido ascítico.

The sensitivity in the diagnosis of spontaneous bacterial peritonitis of ascitic fluid inoculation in blood culture bottles has been compared with the conventional method. We have analyzed 74 positive ascitic fluid cultures from 64 patients, the samples being processed by both techniques simultaneously. While all the ascitic fluid cultures performed by inoculation in blood culture bottles were positive, in only 42 from the 74 conventional cultures (56, 75%) bacterial growth was detected (p less than 0.001). Nineteen bacterascites were diagnosed by inoculation in blood culture bottles and 10 by the conventional method (p = NS). Gram stain was only positive in 3 spontaneous bacterial peritonitis (6%). We conclude that ascitic fluid inoculation in blood culture bottles improves significantly the sensitivity of the microbiological diagnosis of spontaneous bacterial peritonitis, without increasing the diagnosis of bacterascites.

[An analysis of hospital bacterial infections in cirrhotic patients undergoing selective intestinal decontamination]. / Análisis de las infecciones bacterianas intrahospitalarias en pacientes cirróticos sometidos a descontaminación intestinal selectiva.

Cirrhotic patients with ascites and low levels of ascitic fluid C3 and total protein and cirrhotic patients with gastrointestinal hemorrhage are at high risk of infection. Selective intestinal decontamination with oral norfloxacin is useful to decrease the incidence of infections in cirrhotic patients at high risk. This study analyzes hospital acquired bacterial infections in cirrhotic patients with ascites and low levels of total protein in ascitic fluid (n = 53) and cirrhotic patients with gastrointestinal hemorrhage (n = 26), both submitted to selective intestinal decontamination with norfloxacin during the hospitalization. Seven patients developed eight infections (8.8%): three patients with ascites and low levels of total protein in ascitic fluid and four patients with gastrointestinal hemorrhage (5.6% vs 15.3%, pNS). Gram negative bacilli were not isolated in any case, but Gram positive cocci were isolated in seven cases. These results suggest that Gram positive cocci must be empirically covered when infection is suspected in cirrhotic patients submitted to selective intestinal decontamination. The analysis of antibiograms in these infections showed a high sensitivity of Gram positive cocci to amoxycillin and clavulanic acid, which could be used as empirical treatment when infection is suspected in these patients.

[Duodenal duplication]. / Duplicación duodenal.

Cystic duplication of the duodenum is a rare anomaly of the gastrointestinal tract. This is a report of a newborn with a cystic duplication of duodenum diagnosed prenatally. It's relevant the few clinical symptoms of a such big mass. The surgical procedure was excision of the cyst, with a good post operative curse.

Sorption of tetracyclines onto natural soils: data analysis and prediction.

Residues of tetracyclines reach soils as a result of animal waste application. Sorption is a key process in transport, fate, and effects of contaminants in the environment. In this work, we have attempted to predict the sorption of four widely used tetracyclines (oxytetracycline, tetracycline, chlortetracycline, and doxycycline) from soil physicochemical properties. Batch sorption experiments were performed on 15 natural soils with a broad range of physicochemical properties, and the data were fitted to several isotherm models. Multivariate analysis methods were conducted to identify the main factors affecting the sorption distribution coefficients (K (d)) of the tetracyclines at two aqueous concentration levels (100 and 400 µg L(-1)). All four tetracycline sorption isotherms in alkaline and acidic soils were well described by the Freundlich and Langmuir equation, respectively. At intermediate soil pH (from 5.3 to 7), oxytetracycline and tetracycline exhibited Freundlich behavior, whereas chlortetracycline and doxycycline followed a Langmuir model. Two partial least squares (PLS) models were developed. The first one uses five soil descriptors as input variables; the second uses, pH, cation exchange capacity (CEC), and log K (d,OTC). Both models satisfactorily predicted distribution coefficients within a factor of 1.5. Sorption of tetracyclines in soil is governed by several factors, in the following order of importance: solution speciation, CEC (dominant at acidic-neutral soil pH), transition metal content, and texture. The PLS models indicated that tetracycline sorption can be predicted using a minimal set of soil descriptors including oxytetracycline sorption data.

Solid-phase synthesis and characterization of N-methyl-rich peptides.

A library of peptides required for a project investigating the factors relevant for blood-brain barrier transport was synthesized on solid phase. As a result of the high N-methylamino acid content in the peptides, their syntheses were challenging and form the basis of the work presented here. The coupling of protected N-methylamino acids with N-methylamino acids generally occurs in low yield. (7-azabenzotriazol-1-yloxy)-tris(pyrrolidino)phosphonium hexafluorophosphate (PyAOP) or PyBOP/1-hydroxy-7-azabenzotriazole (HOAt), are the most promising coupling reagents for these couplings. When a peptide contains an acetylated N-methylamino acid at the N-terminal position, loss of Ac-N-methylamino acid occurs during trifluoroacetic acid (TFA) cleavage of the peptide from the resin. Other side reactions resulting from acidic cleavage are described here, including fragmentation between consecutive N-methylamino acids and formation of diketopiperazines (DKPs). The time of cleavage is shown to greatly influence synthetic results. Finally, high-performance liquid chromatography (HPLC) profiles of N-methyl-rich peptides show multiple peaks because of slow conversion between conformers.

Chronic liver disease: comparison of ultrasound patterns with laparoscopy and biopsy.

We studied the correlation of ultrasound patterns with laparoscopy and biopsy results in 140 patients with chronic liver disease (CLD). Of the 23 patients with a normal ultrasound pattern (N), biopsies revealed CLD in 18; in the 22 patients with unspecified hepatomegaly (H), biopsies disclosed CLD in 20; and in the 64 patients with a homogeneous bright pattern (HB), biopsies showed CLD in 62. All 22 cases of heterogeneous bright pattern (HTB) and all 9 patients with nodular pattern (ND) had CLD. In conclusion, it appears that the HTB and nodular ultrasound patterns confirm the presence of CLD, the HB pattern is suggestive of CLD, but diagnosis of CLD cannot be made from N and H patterns.

Selective intestinal decontamination prevents spontaneous bacterial peritonitis.

In a prospective randomized study, selective intestinal decontamination with norfloxacin was performed during hospitalization in 32 cirrhotic patients with low ascitic fluid total protein levels. The incidence of infections was compared with that in a control group of 31 nontreated cirrhotic patients of similar characteristics. We found a significantly lower incidence of infections [1/32 (3.1%) vs. 13/31 (41.9%); P less than 0.005] and spontaneous bacterial peritonitis [0/32 (0%) vs. 7/31 (22.5%); P less than 0.05] in patients receiving norfloxacin. The lower incidence of extraperitoneal infections [1/32 (3.1%) vs. 7/31 (22.5%); P = 0.052] in the treated group did not reach statistical significance. The incidence of infections [1/28 (3.6%) vs. 9/22 (40.9%); P less than 0.01] and spontaneous bacterial peritonitis [0/28 (0%) vs. 5/22 (22.7%); P less than 0.05] in cirrhotic patients admitted because of ascites was also significantly lower in the treated group. The decrease in the rate of mortality observed in the group undergoing selective intestinal decontamination did not reach statistical significance. These data show that selective intestinal decontamination is useful to prevent spontaneous bacterial peritonitis and extraperitoneal infections in hospitalized cirrhotic patients with low ascitic fluid total protein levels.

Quantification of foetomaternal haemorrhage. An analysis of two cytometric techniques and a semiquantitative gel agglutination test.

Traditional tests to screen for foetomaternal haemorrhage are time-consuming and difficult to perform. The Kleihauer test is widely used but difficult to standardize. We evaluated three techniques for quantifying foetomaternal haemorrhage: a semiquantitative gel agglutination test and two flow cytometric techniques. The gel agglutination test is based on the consumption of anti-D reagent by D+ cells, analysing the reaction of the supernatant against indicator cells in a Coombs-gel card. In the two colour direct immunofluorescent technique, the sample is incubated with Per-CP labelled anti CD45 antibody, fixed with glutaraldehyde and permeabilized by exposure to Triton X-100. An aliquot is stained with an antibody to foetal haemoglobin, conjugated with fluorescein isothiocyanate or phycoerythrin. The indirect immunofluorescent technique is based on the labelling of Rh (D) antigen with an anti D reagent, followed by the addition of an anti IgG antibody conjugated with phycoerythrin. Foetomaternal haemorrhage was not detected in 75 of the 85 samples analysed by the direct immunofluorescent technique. In the remaining 10 samples, the volume was very low. Thirty-five samples with Rh (D) antigen incompatibility were analysed in parallel by the indirect immunofluorescent technique and in 15 of the 35 samples, the gel agglutination technique was also carried out. The three techniques gave similar results. The gel agglutination test can be used to screen for foetomaternal haemorrhage, while greater volumes should be quantified by flow cytometric techniques.