Localizing coordinates of cerebral ischemic tissue without the need of staining in a rat model of focal cerebral infarct.

Biochemical and metabolic analysis of ischemic cerebral tissue is central in stroke investigation and is usually performed in animal stroke models, such as the permanent occlusion of the middle cerebral artery (MCAO) in the rat that we have used. To be sure that the sample is from infarct tissue, it is differentiated from the surrounding normal tissue by staining, usually with 2,3,5-triphenyltetrazolium chloride (TTC), but staining can hamper biochemical colorimetric analysis. We performed this study to avoid this obstacle. A cerebral infarct was provoked in a sample of 10 rats and the brain was cut in coronal sections that were stained with TTC so that the unstained, infarct areas could be delineated in a template of each section in which areas with infarct in all animals were delineated. We calculated infarct coordinates and depth so that the infarct tissue can be sampled without staining. For more precision, the ischemic cortex can be delimited staining its surface before sectioning and cortical tissue into which TTC diffuses can be afterwards discarded, as we had previously measured the TTC diffusion depth in rat brains.

Reliability of telemedicine in the diagnosis and treatment of sleep apnea syndrome.

BACKGROUND: Advances in information technology and telecommunications have provided the option of making it easier to diagnose and treat obstructive sleep apnea syndrome (OSAS) using telemedicine techniques. This study assessed the feasibility and reliability of respiratory polygraphy and prescription of treatment by pressure adjustment with auto-continuous positive airway pressure (CPAP) systems, both being transmitted telematically to the Sleep Unit, with teleconsultation as a support method. SUBJECTS AND METHODS: Forty patients were studied from a population 80 km from the Sleep Unit using respiratory polygraphy transmitted in real time. They were divided into two groups: one was seen by conventional consultation, and the other was seen using teleconsultation. We also estimated satisfaction with this system and its costs. RESULTS: The mean patient age was 53 ± 10.3 years, with a body mass index of 31 ± 6.2 kg/m(2) and an Epworth score of 12 ± 5.3. In total, 35 patients were diagnosed with OSAS, with an Apnea-Hypopnea Index of ≥10, and CPAP treatment was started in 16 of them. The agreement in the Apnea-Hypopnea Index, total apneas and hypopneas, mean oxygen saturation, and time with an oxygen saturation <90% was greater than 90% between the studies transmitted in real time and those stored in the polygraph. The level of compliance with CPAP treatment was 85% for the patients who were seen in a conventional clinic and 75% in those seen by teleconsultation. CONCLUSIONS: The use of telematic techniques is useful to establish a diagnostic and therapeutic strategy for OSAS with the creation of a Wide Core Sleep Laboratory as a process controller.

Changes in the levels of thioredoxin and indoleamine-2,3-dioxygenase activity in plasma of patients with colorectal cancer treated with chemotherapy.

Increased oxidative stress and indoleamine-2,3-dioxygenase (IDO) activity have been reported in cancer, but their relationship with chemotherapy remains unknown. The aim of the present study was to examine wether the chemotherapy treatments used in colorectal cancer had an additional effect on oxidative stress and on IDO activity. Plasma samples were collected from 27 colorectal cancer patients on cytostatic treatment, 27 with cytostatic drugs plus monoclonal antibodies (cytostatic-Mabs) and 15 non-treated patients. All patients with colorectal cancer had high plasma malondialdehyde (MDA), thioredoxin (Trx) levels, and elevated IDO activity in plasma (IDOp) and in dendritic cells (IDOc). This study shows that treatment with cytostatics have an effect on oxidative stress by increasing MDA levels and by decreasing Trx levels and IDO activity. However, treatment with cytostatic-Mabs showed no effect on MDA levels but decreased Trx levels, and the IDO activity showed values similar to the healthy group. Significant correlations between plasma IDO activity and the levels of Trx (r = 0.2062, p < 0.05) and MDA (r = 0.2873, p < 0.005) were observed. Furthermore, our study suggests that IDO activity measured as kynurenine levels could be used as a marker of the response to the chemotherapy treatments, although further studies are necessary.

KRAS, BRAF, EGFR and HER2 gene status in a Spanish population of colorectal cancer.

To evaluate the KRAS, BRAF, EGFR, and HER2 gene status in colorectal cancer by novel techniques and evaluate whether anti-HER2 therapies could be offered in the treatment of these patients. There are conflicting data on the prevalence of BRAF mutations and EGFR and HER2 gene amplification in colorectal KRAS wild type patients. In our study we tried to evaluate these expressions and their relationship to future treatment assays. Clinical-pathological data and paraffin-embedded specimens were collected from 186 patients who underwent colorectal resections at General Yagüe Hospital in Burgos, Spain. KRAS and BRAF status was analyzed by real-time PCR in all patients. EGFR and HER2/NEU gene amplification was detected using fluorescent in situ hybridisation technique (FISH) in 38 KRAS and BRAF wild type patients. KRAS mutations were present in 48% of the colorectal cancer patients. BRAF mutations were present in 6.25% of the KRAS wild type patients. EGFR and HER2 gene amplification was observed in 5.3% and 26.3%, respectively, of KRAS and BRAF wild type colorectal cancer patients. HER2, but not EGFR gene amplification, was frequently observed in KRAS and BRAF wild type colorectal cancer patients. These data indicate that HER2 amplification could be one of the genes to be considered in the therapeutic management of colorectal cancer.

PIK3CA mutations in KRAS and BRAF wild type colorectal cancer patients. A study of Spanish population.

The objective of the work was to study PIK3CA mutations in wild type KRAS and BRAF colorectal cancer. Clinicopathological data and paraffin-embedded specimens were collected on 73 patients who underwent colorectal resections at General Yagüe Hospital in Burgos. KRAS, BRAF and PIK3CA status were analyzed by real-time PCR in all patients. PIK3CA mutations were present in 8.22% of wild type KRAS and BRAF colorectal cancers. The most frequent mutation is E545K/D in exon 9 which represents 83.3% of all mutations. By contrast, we did not found any tumour harbouring H1047R mutation in exon 20. Among the patients who undergo a curative resection of colorectal cancer, PIK3CA mutation is present in an important percentage of KRAS and BRAF wild type tumours. PIK3CA mutation may be considered as it could be a hypothetic reason to be not responder to anti-EGFR antibodies.

Discovery of a benzothiophene-flavonol halting miltefosine and antimonial drug resistance in Leishmania parasites through the application of medicinal chemistry, screening and genomics.

Leishmaniasis, a major health problem worldwide, has a limited arsenal of drugs for its control. The appearance of resistance to first- and second-line anti-leishmanial drugs confirms the need to develop new and less toxic drugs that overcome spontaneous resistance. In the present study, we report the design and synthesis of a novel library of 38 flavonol-like compounds and their evaluation in a panel of assays encompassing parasite killing, pharmacokinetics, genomics and ADME-Toxicity resulting in the progression of a compound in the drug discovery value chain. Compound 19, 2-(benzo[b]thiophen-3-yl)-3-hydroxy-6-methoxy-4H-chromen-4-one, exhibited a broad-spectrum activity against Leishmania spp. (EC50 1.9 µM for Leishmania infantum, 3.4 µM for L. donovani, 6.7 µM for L. major), Trypanosoma cruzi (EC50 7.5 µM) and T. brucei (EC50 0.8 µM). Focusing on anti-Leishmania activity, compound 19 challenge in vitro did not select for resistance markers in L. donovani, while a Cos-Seq screening for dominant resistance genes identified a gene locus on chromosome 36 that became ineffective at concentrations beyond EC50. Thus, compound 19 is a promising scaffold to tackle drug resistance in Leishmania infection. In vivo pharmacokinetic studies indicated that compound 19 has a long half-life (intravenous (IV): 63.2 h; per os (PO): 46.9 h) with an acceptable ADME-Toxicity profile. When tested in Leishmania infected hamsters, no toxicity and limited efficacy were observed. Low solubility and degradation were investigated spectroscopically as possible causes for the sub-optimal pharmacokinetic properties. Compound 19 resulted a specific compound based on the screening against a protein set, following the intrinsic fluorescence changes.

Accelerating Drug Discovery Efforts for Trypanosomatidic Infections Using an Integrated Transnational Academic Drug Discovery Platform.

According to the World Health Organization, more than 1 billion people are at risk of or are affected by neglected tropical diseases. Examples of such diseases include trypanosomiasis, which causes sleeping sickness; leishmaniasis; and Chagas disease, all of which are prevalent in Africa, South America, and India. Our aim within the New Medicines for Trypanosomatidic Infections project was to use (1) synthetic and natural product libraries, (2) screening, and (3) a preclinical absorption, distribution, metabolism, and excretion-toxicity (ADME-Tox) profiling platform to identify compounds that can enter the trypanosomatidic drug discovery value chain. The synthetic compound libraries originated from multiple scaffolds with known antiparasitic activity and natural products from the Hypha Discovery MycoDiverse natural products library. Our focus was first to employ target-based screening to identify inhibitors of the protozoan Trypanosoma brucei pteridine reductase 1 ( TbPTR1) and second to use a Trypanosoma brucei phenotypic assay that made use of the T. brucei brucei parasite to identify compounds that inhibited cell growth and caused death. Some of the compounds underwent structure-activity relationship expansion and, when appropriate, were evaluated in a preclinical ADME-Tox assay panel. This preclinical platform has led to the identification of lead-like compounds as well as validated hits in the trypanosomatidic drug discovery value chain.

Methoxylated 2'-hydroxychalcones as antiparasitic hit compounds.

Chalcones display a broad spectrum of pharmacological activities. Herein, a series of 2'-hydroxy methoxylated chalcones was synthesized and evaluated towards Trypanosoma brucei, Trypanosoma cruzi and Leishmania infantum. Among the synthesized library, compounds 1, 3, 4, 7 and 8 were the most potent and selective anti-T. brucei compounds (EC50 = 1.3-4.2 µM, selectivity index >10-fold). Compound 4 showed the best early-tox and antiparasitic profile. The pharmacokinetic studies of compound 4 in BALB/c mice using hydroxypropil-ß-cyclodextrins formulation showed a 7.5 times increase in oral bioavailability.

Immunomodulatory nanoparticles ameliorate disease in the Leishmania (Viannia) panamensis mouse model.

Leishmania (Viannia) panamensis (L. (V.) panamensis) is a species of protozoan parasites that causes New World leishmaniasis, which is characterized by a hyper-inflammatory response. Current treatment strategies, mainly chemotherapeutic, are suboptimal due to adverse effects, long treatment regimens, and increasing drug resistance. Recently, immunotherapeutic approaches have shown promise in preclinical studies of leishmaniasis. As NPs may enable broad cellular immunomodulation through internalization in phagocytic and antigen-presenting cells, we tested the therapeutic efficacy of biodegradable NPs encapsulating a pathogen-associated molecular pattern (PAMP), CpG-rich oligonucleotide (CpG; NP-CpG), in mice infected with L. (V.) panamensis. NP-CpG treatment reduced lesion size and parasite burden, while neither free CpG nor empty NP showed therapeutic effects. NP-encapsulation led to CpG persistence at the site of infection along with an unexpected preferential cellular uptake by myeloid derived suppressor cells (MDSCs; CD11b(+)Ly6G(+)Ly6C(-)) as well as CD19(+) dendritic cells. This corresponded with the suppression of the ongoing immune response measured by the reduction of pathogenic cytokines IL-10 and IL-13, as well as IL-17 and IFNγ, in comparison to other treatment groups. As chronic inflammation is generally associated with the accumulation of MDSCs, this study may enable the rational design of cost-effective, safe, and scalable delivery systems for the treatment of inflammation-mediated diseases.

Risk factors for methicillin-resistant Staphylococcus aureus colonisation or infection in intensive care units and their reliability for predicting MRSA on ICU admission.

Predicting methicillin-resistant Staphylococcus aureus (MRSA) in intensive care units (ICUs) avoids inappropriate antimicrobial empirical treatment and enhances infection control. We describe risk factors for colonisation/infection related to MRSA (MRSA-C/I) in critically ill patients once in the ICU and on ICU admission, and search for an easy-to-use predictive model for MRSA colonisation/infection on ICU admission. This multicentre cohort study included 69,894 patients admitted consecutively (stay>24h) in April-June in the five-year period 2006-2010 from 147 Spanish ICUs participating in the National Surveillance Study of Nosocomial Infections in ICUs (ENVIN-HELICS). Data from all patients included were used to identify risk factors for MRSA-C/I during ICU stays, from admission to discharge, using uni- and multivariable analysis (Poisson regression) to check that the sample to be used to develop the predictive models was representative of standard critical care population. To identify risk factors for MRSA-C/I on ICU admission and to develop prediction models, multivariable logistic regression analysis were then performed only on those admitted in 2010 (n=16950, 2/3 for analysis and 1/3 for subsequent validation). We found that, in the period 2006-2010, 1046 patients were MRSA-C/I. Independent risk factors for MRSA-C/I in ICU were: age>65, trauma or medical patient, high APACHE-II score, admitted from a long-term care facility, urinary catheter, previous antibiotic treatment and skin-soft tissue or post-surgical superficial skin infections. Colonisation with several different MDRs significantly increased the risk of MRSA-C/I. Risk factors on ICU admission were: male gender, trauma critical patient, urgent surgery, admitted from other ICUs, hospital ward or long-term facility, immunosuppression and skin-soft tissue infection. Although the best model to identify carriers of MRSA had a good discrimination (AUC-ROC, 0.77; 95% CI, 0.72-0.82), sensitivity was 67% and specificity 76.5%. Including more complex variables did not improve prediction capability. Our conclusion is that clinical-demographic risk factors for colonisation/infection related to MRSA should not be used to accurately identify patients who would benefit from empirical anti-MRSA treatment or from specific preventive measures. Independent risk factors for MRSA colonisation/infection during ICU stay and on ICU admission are described. The latter should be considered in future studies for MRSA prediction.

Utility of a thematic network in primary health care: a controlled interventional study in a rural area.

BACKGROUND: UniNet is an Internet-based thematic network for a virtual community of users (VCU). It supports a virtual multidisciplinary community for physicians, focused on the improvement of clinical practice. This is a study of the effects of a thematic network such as UniNet on primary care medicine in a rural area, specifically as a platform of communication between specialists at the hospital and doctors in the rural area. METHODS: In order to study the effects of a thematic network such as UniNet on primary care medicine in a rural area, we designed an interventional study that included a control group. The measurements included the number of patient displacements due to disease, number of patient hospital stays and the number of prescriptions of drugs of low therapeutic utility and generic drug prescriptions by doctors. These data were analysed and compared with those of the control center. RESULTS: Our study showed positive changes in medical practice, reflected in the improvement of the evaluated parameters in the rural health area where the interventional study was carried out, compared with the control area. We discuss the strengths and weaknesses of UniNet as a potential medium to improve the quality of medical care in rural areas. CONCLUSION: The rural doctors had an effective, useful, user-friendly and cheap source of medical information that may have contributed to the improvement observed in the medical quality indices.

Vaccination of lambs with the recombinant protein rHc23 elicits significant protection against Haemonchus contortus challenge.

Gene encoding a somatic protein of Haemonchus contortus (Hc23) known to confer significant protection against experimental haemonchosis has been cloned and expressed in a prokaryotic system. A cDNA library of H. contortus using the vector λ ZAP II was obtained. Full-length gene was amplified, cloned and expressed in Escherichia coli BL21. The recombinant protein was purified in Ni-NTA column. Recombinant protein (rHc23) had 203 aminoacids and a molecular mass of 24.15 kDa. Recombinant protein (100 µg/dose) with aluminum hydroxide as adjuvant was administered to 5-6 months age female Assaf lambs on days -42, -28 and -14. On day 0 animals were infected with 15,000 L3 of H. contortus. Vaccination with rHc23 elicited a significant protection against challenge, with >80% reductions in both fecal egg counts and average abomasal parasite burdens at the end of the experiment (45 days post challenge) besides lack of variations in packed cell volume. Results support the feasibility of vaccination against lamb haemonchosis with a recombinant product from an exposed antigen.

Vaccination of lambs against Haemonchus contortus infection with a somatic protein (Hc23) from adult helminths.

A somatic protein from adult Haemonchus contortus (Hc23), the most abundant component in a low molecular weight fraction with known immunizing effect against experimental haemonchosis, has been purified by immunochromatography. The immunoprophylactic value of Hc23 was tested in groups of 5-6 months old Assaf lambs using Al(OH)(3) or Escherichia coli lipopolysaccharide+inactivated Propionibacterium acnes as adjuvant and the results compared with uninfected control, uninfected and challenged or infected and challenged lambs. Immunization with Hc23 in either adjuvant elicited significant reductions in fecal egg counts after challenge with 15,000 L3s (70.67%-85.64%, respectively) and reduced (67.1% and 86%) abomasal worm counts (45 days post-challenge). Immunized lambs displayed higher peripheral eosinophil counts, were less anaemic and had weight gains than challenged controls. The results suggest that the Hc23 antigen can induce a partially protective response against haemonchosis in lambs.

LPS and inactivated Propionibacterium acnes elicit a partially protective response in primary infections of Heligmosomoides polygyrus.

Intestinal helminth infections are common and of paramount economic importance in domestic animals. Available chemotherapy is limited and anthelmintic resistance is widespread in some hosts. This scenario favors the exploration of alternative methods of control, among them immune modulators. The effect of Escherichia coli LPS+Propionibacterium acnes on a primary infection of Heligmosomoides polygyrus (Trichostongyloidea) in mice has been tested. Nematode infection induced a rise of specific IgG1, both serum and intestinal, and a significant reduction in the unspecific (ConA) lymphoproliferative response. Treatment with the immune modulator (days -2, 0, 7 and 14 post infection) elicited an apparent delay of larval intramucosal development. Moreover cumulative nematode egg shedding in treated mice was significantly lower (p=0.0041). Preliminary results point toward the interest of immune modulators to control intestinal helminths.

Improvement of 96-well microplate assay for estimation of cell growth and inhibition of Leishmania with Alamar Blue.

The value of resazurin-based Alamar Blue redox indicator to determine multiplication of Leishmania promastigotes in 96-well microtiter plates was examined. In addition, assay was validated with amphotericin B (AmB) and allicin. The method was tested on L.donovani and L.infantum promastigotes under different culture conditions (variable air-phase, presence of phenol red, initial cell density, incubation time, use of Hepes buffer). Results showed that the gas-phase of promastigote cultures was critical. The method yielded consistent results with initial plating cell densities of 2.5 × 105 promastigotes/well, up to 72 h incubation and 5% CO2 atmosphere or reduced air availability (sealed plastic bags, film-sealed microplates). Detection of low numbers of promastigotes and earlier results could be obtained using fluorimetry instead of spectrophotometry. The addition of 20 mM Hepes improved the results. Fluorescence intensity correlated to promastigotes number in both Leishmania spp. Inhibitory concentration (IC50) values for AmB and allicin using cell counting and fluorimetry were comparable. Under these conditions this one-step, low-cost redox indicator can be used in drug sensitivity assays and studies of differential proliferation rates of Leishmania isolates or strains in a 96-well format.

Toxicity of betulin derivatives and in vitro effect on promastigotes and amastigotes of Leishmania infantum and L. donovani.

The toxicity and antileishmanial activity of 20 betulin derivatives were studied. The toxicity of betulin and synthesized compounds was determined using a bacterial test (Microtox) and two mammalian cell lines (CHO-K1 and J774). The antileishmanial activity of compounds (50 µM) was examined in both the promastigote and intracellular amastigote stages of Leishmania infantum and L. donovani. No correlation was found among the toxicity tests. All the compounds showed significant antipromastigote activity. The antiproliferative capacity of derivatives was dependent on the parasite stage studied, and no substantial differences were found between Leishmania species. Betulin, 3,28-di-O-acetylbetulin and L-aspartyl amide of betulonic acid showed moderate activity against amastigotes. The highest inhibition of intracellular amastigote multiplication was achieved with a low micromolar concentration (IC(50) ca 9 µM) of heterocyclic betulin derivative 3,28-di-O-acetyllup-13(18)-ene with N-ethyltriazolo moiety 16, without significant toxicity for mammalian cells. These results point to the interest of this lead compound for further in vitro and in vivo tests.

Usefulness of an Internet-based thematic learning network: comparison of effectiveness with traditional teaching.

UniNet is an Internet-based thematic network for a virtual community of users (VCU). It supports one multidisciplinary community of doctoral students, who receive most of the courses on the network. The evident advantages of distance learning by Internet, in terms of costs, comfort, etc., require a previous evaluation of the system, focusing on the learning outcomes of the student. The aim was to evaluate the real learning of the students of doctorate courses, by comparing the effectiveness of distance learning in UniNet with traditional classroom-based teaching. Five doctorate courses were taught simultaneously to two independent groups of students in two ways: one, through the UniNet Network, and the other in a traditional classroom. The academic knowledge of students was evaluated at the beginning and end of each course. The difference in score was considered as a knowledge increase. The comparison was made using Student's t-test for independent groups. There were no significant statistical differences in the outcomes of the two groups of students. This suggests that both teaching systems were equivalent in increasing the knowledge of the students. Both educational methods, the traditional system and the online system in a thematic network, are effective and similar for increasing knowledge.