Resistance to experimental autoimmune encephalomyelitis development in Lewis rats from a conventional animal facility

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the brain and spinal cord that is mediated by CD4+ T lymphocytes specific to myelin components. In this study we compared development of EAE in Lewis rats from two colonies, one kept in pathogen-free conditions (CEMIB colony) and the other (Botucatu colony) kept in a conventional animal facility. Female Lewis rats were immunized with 100 µl of an emulsion containing 50 µg of myelin, associated with incomplete Freund's adjuvant plus Mycobacterium butyricum. Animals were daily evaluated for clinical score and weight. CEMIB colony presented high EAE incidence with clinical scores that varied from three to four along with significant weight losses. A variable disease incidence was observed in the Botucatu colony with clinical scores not higher than one and no weight loss. Immunological and histopathological characteristics were also compared after 20 days of immunization. Significant amounts of IFN-gamma, TNF-alpha and IL-10 were induced by myelin in cultures from CEMIB animals but not from the Botucatu colony. Significantly higher levels of anti-myelin IgG1 were detected in the CEMIB colony. Clear histopathological differences were also found. Cervical spinal cord sections from CEMIB animals showed typical perivascular inflammatory foci whereas samples from the Botucatu colony showed a scanty inflammatory infiltration. Helminths were found in animals from Botucatu colony but not, as expected, in the CEMIB pathogen-free animals. As the animals maintained in a conventional animal facility developed a very discrete clinical, and histopathological EAE in comparison to the rats kept in pathogen-free conditions, we believe that environmental factors such as intestinal parasites could underlie this resistance to EAE development, supporting the applicability of the hygiene hypothesis to EAE.

Addition of inspiratory resistance increases the amplitude of the slow component of O2 uptake kinetics.

The contribution of respiratory muscle work to the development of the O(2) consumption (Vo(2)) slow component is a point of controversy because it has been shown that the increased ventilation in hypoxia is not associated with a concomitant increase in Vo(2) slow component. The first purpose of this study was thus to test the hypothesis of a direct relationship between respiratory muscle work and Vo(2) slow component by manipulating inspiratory resistance. Because the conditions for a Vo(2) slow component specific to respiratory muscle can be reached during intense exercise, the second purpose was to determine whether respiratory muscles behave like limb muscles during heavy exercise. Ten trained subjects performed two 8-min constant-load heavy cycling exercises with and without a threshold valve in random order. Vo(2) was measured breath by breath by using a fast gas exchange analyzer, and the Vo(2) response was modeled after removal of the cardiodynamic phase by using two monoexponential functions. As anticipated, when total work was slightly increased with loaded inspiratory resistance, slight increases in base Vo(2), the primary phase amplitude, and peak Vo(2) were noted (14.2%, P < 0.01; 3.5%, P > 0.05; and 8.3%, P < 0.01, respectively). The bootstrap method revealed small coefficients of variation for the model parameter, including the slow-component amplitude and delay (15 and 19%, respectively), indicating an accurate determination for this critical parameter. The amplitude of the Vo(2) slow component displayed a 27% increase from 8.1 +/- 3.6 to 10.3 +/- 3.4 ml. min(-1). kg(-1) (P < 0.01) with the addition of inspiratory resistance. Taken together, this increase and the lack of any differences in minute volume and ventilatory parameters between the two experimental conditions suggest the occurrence of a Vo(2) slow component specific to the respiratory muscles in loaded condition.

Analysis of hepatitis C virus resistance to silibinin in vitro and in vivo points to a novel mechanism involving nonstructural protein 4B.

Intravenous silibinin (SIL) is an approved therapeutic that has recently been applied to patients with chronic hepatitis C, successfully clearing hepatitis C virus (HCV) infection in some patients even in monotherapy. Previous studies suggested multiple antiviral mechanisms of SIL; however, the dominant mode of action has not been determined. We first analyzed the impact of SIL on replication of subgenomic replicons from different HCV genotypes in vitro and found a strong inhibition of RNA replication for genotype 1a and genotype 1b. In contrast, RNA replication and infection of genotype 2a were minimally affected by SIL. To identify the viral target of SIL we analyzed resistance to SIL in vitro and in vivo. Selection for drug resistance in cell culture identified a mutation in HCV nonstructural protein (NS) 4B conferring partial resistance to SIL. This was corroborated by sequence analyses of HCV from a liver transplant recipient experiencing viral breakthrough under SIL monotherapy. Again, we identified distinct mutations affecting highly conserved amino acid residues within NS4B, which mediated phenotypic SIL resistance also in vitro. Analyses of chimeric viral genomes suggest that SIL might target an interaction between NS4B and NS3/4A. Ultrastructural studies revealed changes in the morphology of viral membrane alterations upon SIL treatment of a susceptible genotype 1b isolate, but not of a resistant NS4B mutant or genotype 2a, indicating that SIL might interfere with the formation of HCV replication sites. CONCLUSION: Mutations conferring partial resistance to SIL treatment in vivo and in cell culture argue for a mechanism involving NS4B. This novel mode of action renders SIL an attractive candidate for combination therapies with other directly acting antiviral drugs, particularly in difficult-to-treat patient cohorts.

Susceptibility of Triatoma brasiliensis from state of Ceará, Northeastern Brazil, to the pyrethroid deltamethrin

After controlling Triatoma infestans in Brazil, other species of triatomine that were considered minor in the transmission of Chagas disease became important. The persistence of Triatoma brasiliensis in Northeastern Brazil, associated with reinfection of domestic environments recently sprayed with pyrethroids, may be a signal of susceptibility alteration of this species to this insecticide. Specimens of T. brasiliensis from the municipality of Tauá, state of Ceará, were captured before and one year after spraying. They were submitted to bioassays using deltamethrin. The LD50 ranged from 0.19-0.33 ng of deltamethrin/nymph. The resistance ratio among samples from Tauá varied from 1.16-1.79 in the samples captured before the spraying and 1.00-1.74 in the samples captured one year after spraying, demonstrating that the two populations were equally susceptible to deltamethrin. The small difference in susceptibility between the two captures suggests that T. brasiliensis obtained in the second capture are from new invasions of the domestic environment and that the insecticide did not select resistant individuals. Therefore, it is suggested that T. brasiliensis control be carried out supplementing the regular use of pyrethroids with complementary measures, such as improvement of the dwellings and health education.

Alternative ways to estimate change points in multinomial sequences. An application to an authorship attribution problem

The statistical analysis of literary style is the part of stylometry that compares measurable characteristicsin a text that are rarely controlled by the author, with those in other texts. When thegoal is to settle authorship questions, these characteristics should relate to the author’s style andnot to the genre, epoch or editor, and they should be such that their variation between authors islarger than the variation within comparable texts from the same author.For an overview of the literature on stylometry and some of the techniques involved, see for exampleMosteller and Wallace (1964, 82), Herdan (1964), Morton (1978), Holmes (1985), Oakes (1998) orLebart, Salem and Berry (1998).Tirant lo Blanc, a chivalry book, is the main work in catalan literature and it was hailed to be“the best book of its kind in the world” by Cervantes in Don Quixote. Considered by writterslike Vargas Llosa or Damaso Alonso to be the first modern novel in Europe, it has been translatedseveral times into Spanish, Italian and French, with modern English translations by Rosenthal(1996) and La Fontaine (1993). The main body of this book was written between 1460 and 1465,but it was not printed until 1490.There is an intense and long lasting debate around its authorship sprouting from its first edition,where its introduction states that the whole book is the work of Martorell (1413?-1468), while atthe end it is stated that the last one fourth of the book is by Galba (?-1490), after the death ofMartorell. Some of the authors that support the theory of single authorship are Riquer (1990),Chiner (1993) and Badia (1993), while some of those supporting the double authorship are Riquer(1947), Coromines (1956) and Ferrando (1995). For an overview of this debate, see Riquer (1990).Neither of the two candidate authors left any text comparable to the one under study, and thereforediscriminant analysis can not be used to help classify chapters by author. By using sample textsencompassing about ten percent of the book, and looking at word length and at the use of 44conjunctions, prepositions and articles, Ginebra and Cabos (1998) detect heterogeneities that mightindicate the existence of two authors. By analyzing the diversity of the vocabulary, Riba andGinebra (2000) estimates that stylistic boundary to be near chapter 383.Following the lead of the extensive literature, this paper looks into word length, the use of the mostfrequent words and into the use of vowels in each chapter of the book. Given that the featuresselected are categorical, that leads to three contingency tables of ordered rows and therefore tothree sequences of multinomial observations.Section 2 explores these sequences graphically, observing a clear shift in their distribution. Section 3describes the problem of the estimation of a suden change-point in those sequences, in the followingsections we propose various ways to estimate change-points in multinomial sequences; the methodin section 4 involves fitting models for polytomous data, the one in Section 5 fits gamma modelsonto the sequence of Chi-square distances between each row profiles and the average profile, theone in Section 6 fits models onto the sequence of values taken by the first component of thecorrespondence analysis as well as onto sequences of other summary measures like the averageword length. In Section 7 we fit models onto the marginal binomial sequences to identify thefeatures that distinguish the chapters before and after that boundary. Most methods rely heavilyon the use of generalized linear models

Combined low initial DNA damage and high radiation-induced apoptosis confers clinical resistance to long-term toxicity in breast cancer patients treated with high-dose radiotherapy

BACKGROUND. Either higher levels of initial DNA damage or lower levels of radiation-induced apoptosis in peripheral blood lymphocytes have been associated to increased risk for develop late radiation-induced toxicity. It has been recently published that these two predictive tests are inversely related. The aim of the present study was to investigate the combined role of both tests in relation to clinical radiation-induced toxicity in a set of breast cancer patients treated with high dose hyperfractionated radical radiotherapy. METHODS. Peripheral blood lymphocytes were taken from 26 consecutive patients with locally advanced breast carcinoma treated with high-dose hyperfractioned radical radiotherapy. Acute and late cutaneous and subcutaneous toxicity was evaluated using the Radiation Therapy Oncology Group morbidity scoring schema. The mean follow-up of survivors (n = 13) was 197.23 months. Radiosensitivity of lymphocytes was quantified as the initial number of DNA double-strand breaks induced per Gy and per DNA unit (200 Mbp). Radiation-induced apoptosis (RIA) at 1, 2 and 8 Gy was measured by flow cytometry using annexin V/propidium iodide. RESULTS. Mean DSB/Gy/DNA unit obtained was 1.70 ± 0.83 (range 0.63-4.08; median, 1.46). Radiation-induced apoptosis increased with radiation dose (median 12.36, 17.79 and 24.83 for 1, 2, and 8 Gy respectively). We observed that those "expected resistant patients" (DSB values lower than 1.78 DSB/Gy per 200 Mbp and RIA values over 9.58, 14.40 or 24.83 for 1, 2 and 8 Gy respectively) were at low risk of suffer severe subcutaneous late toxicity (HR 0.223, 95%CI 0.073-0.678, P = 0.008; HR 0.206, 95%CI 0.063-0.677, P = 0.009; HR 0.239, 95%CI 0.062-0.929, P = 0.039, for RIA at 1, 2 and 8 Gy respectively) in multivariate analysis. CONCLUSIONS. A radiation-resistant profile is proposed, where those patients who presented lower levels of initial DNA damage and higher levels of radiation induced apoptosis were at low risk of suffer severe subcutaneous late toxicity after clinical treatment at high radiation doses in our series. However, due to the small sample size, other prospective studies with higher number of patients are needed to validate these results.

Schistosoma mansoni: a method for inducing resistance to praziquantel using infected Biomphalaria glabrata snails

To elucidate the mechanisms of antischistosoma resistance, drug-resistant Schistosoma mansoni laboratory isolates are essential. We developed a new method for inducing resistance to praziquantel (PZQ) using successive drug treatments of Biomphalaria glabrata snails infected with S. mansoni. Infected B. glabrata were treated three times with 100 mg/kg PZQ for five consecutive days with a one-week interval between them. After the treatment, the cercariae (LE-PZQ) produced from these snails and the LE strains (susceptible) were used to infect mice. Forty-five days after infection, mice were treated with 200, 400 or 800 mg/kg PZQ. Thirty days post-treatment, we observed that the mean number of worms recovered by perfusion was significantly higher in the group of mice infected with the LE-PZQ isolate treated with 200 and 400 mg/kg in comparison to the LE strain with the same treatment. Moreover, there was a significant difference between the ED50 (effective dose required to kill 50% of the worms) of the LE-PZQ isolate (362 mg/kg) and the LE strain (68 mg/kg). In the in vitro assays, the worms of the LE-PZQ isolate were also less susceptible to PZQ. Thus, the use of infected snails as an experimental model for development of resistance to S. mansoni is effective, fast, simple and cheap.

Progression from high insulin resistance to type 2 diabetes does not entail additional visceral adipose tissue inflammation.

Obesity is associated with a low-grade chronic inflammation state. As a consequence, adipose tissue expresses pro-inflammatory cytokines that propagate inflammatory responses systemically elsewhere, promoting whole-body insulin resistance and consequential islet β-cell exhaustation. Thus, insulin resistance is considered the early stage of type 2 diabetes. However, there is evidence of obese individuals that never develop diabetes indicating that the mechanisms governing the association between the increase of inflammatory factors and type 2 diabetes are much more complex and deserve further investigation. We studied for the first time the differences in insulin signalling and inflammatory pathways in blood and visceral adipose tissue (VAT) of 20 lean healthy donors and 40 equal morbidly obese (MO) patients classified in high insulin resistance (high IR) degree and diabetes state. We studied the changes in proinflammatory markers and lipid content from serum; macrophage infiltration, mRNA expression of inflammatory cytokines and transcription factors, activation of kinases involved in inflammation and expression of insulin signalling molecules in VAT. VAT comparison of these experimental groups revealed that type 2 diabetic-MO subjects exhibit the same pro-inflammatory profile than the high IR-MO patients, characterized by elevated levels of IL-1β, IL-6, TNFα, JNK1/2, ERK1/2, STAT3 and NFκB. Our work rules out the assumption that the inflammation should be increased in obese people with type 2 diabetes compared to high IR obese. These findings indicate that some mechanisms, other than systemic and VAT inflammation must be involved in the development of type 2 diabetes in obesity.

Resistance to thyroid hormone and down syndrome: coincidental association or genetic linkage?

We report the first case of RTH and DS. Although this congruence could be coincidental, we cannot exclude a possible linkage between both syndromes.

Association of hepatitis C virus NS5B variants with resistance to new antiviral drugs among untreated patients

Mutations located in the 109-amino acid fragment of NS5B are typically associated with resistance to interferon (IFN) and ribavirin (RIB) and to new antiviral drugs. The prevalence of these mutations was examined in 69 drug-naïve individuals with hepatitis C virus (HCV) infections in Rio de Janeiro, Brazil. Mutations related to non-response to IFN/RIB were observed in all subtypes studied (1a, 1b, 2b, 3a and 4). The most common mutation was Q309R, present in all subtypes, except subtype 2b with frequency above 20%. D244N was detected only in subtype 3a and A333E was detected only in subtype 2b. We did not detect the S282T, S326G or T329I mutations in any of the samples analysed. Of note, the C316N mutation, previously related to a new non-nucleoside compound (HCV796 and AG-021541), was observed in only eight of 33 (24%) samples from subtype 1b. Site 316 was under positive selection in this HCV variant. Our data highlight the presence of previously described resistance mutations in HCV genotypes from drug-naïve patients.

Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance

To study the potential for the emergence of resistance in Aedes aegypti populations, a wild colony was subjected to selective pressure with Cry11Aa, one of four endotoxins that compose the Bacillus thuringiensis serovar israelensis toxin. This bacterium is the base component of the most important biopesticide used in the control of mosquitoes worldwide. After 54 generations of selection, significant resistance levels were observed. At the beginning of the selection experiment, the half lethal concentration was 26.3 ng/mL and had risen to 345.6 ng/mL by generation 54. The highest rate of resistance, 13.1, was detected in the 54th generation. Because digestive proteases play a key role in the processing and activation of B. thuringiensis toxin, we analysed the involvement of insect gut proteases in resistance to the Cry11Aa B. thuringiensis serovar israelensis toxin. The protease activity from larval gut extracts from the Cry11Aa resistant population was lower than that of the B. thuringiensisserovar israelensis susceptible colony. We suggest that differences in protoxin proteolysis could contribute to the resistance of this Ae. aegypti colony.

The classification of esterases: an important gene family involved in insecticide resistance - A review

The use of chemical insecticides continues to play a major role in the control of disease vector populations, which is leading to the global dissemination of insecticide resistance. A greater capacity to detoxify insecticides, due to an increase in the expression or activity of three major enzyme families, also known as metabolic resistance, is one major resistance mechanisms. The esterase family of enzymes hydrolyse ester bonds, which are present in a wide range of insecticides; therefore, these enzymes may be involved in resistance to the main chemicals employed in control programs. Historically, insecticide resistance has driven research on insect esterases and schemes for their classification. Currently, several different nomenclatures are used to describe the esterases of distinct species and a universal standard classification does not exist. The esterase gene family appears to be rapidly evolving and each insect species has a unique complement of detoxification genes with only a few orthologues across species. The examples listed in this review cover different aspects of their biochemical nature. However, they do not appear to contribute to reliably distinguish among the different resistance mechanisms. Presently, the phylogenetic criterion appears to be the best one for esterase classification. Joint genomic, biochemical and microarray studies will help unravel the classification of this complex gene family.

Evolution of insecticide resistance in non-target black flies (Diptera: Simuliidae) from Argentina

Black flies, a non-target species of the insecticides used in fruit production, represent a severe medical and veterinary problem. Large increases in the level of resistance to the pyrethroids fenvalerate (more than 355-fold) and deltamethrin (162-fold) and a small increase in resistance to the organophosphate azinphos methyl (2-fold) were observed between 1996-2008 in black fly larvae under insecticide pressure. Eventually, no change or a slight variation in insecticide resistance was followed by a subsequent increase in resistance. The evolution of pesticide resistance in a field population is a complex and stepwise process that is influenced by several factors, the most significant of which is the insecticide selection pressure, such as the dose and frequency of application. The variation in insecticide susceptibility within a black fly population in the productive area may be related to changes in fruit-pest control. The frequency of individuals with esterase activities higher than the maximum value determined in the susceptible population increased consistently over the sampling period. However, the insecticide resistance was not attributed to glutathione S-transferase activity. In conclusion, esterase activity in black flies from the productive area is one mechanism underlying the high levels of resistance to pyrethroids, which have been recently used infrequently. These enzymes may be reselected by currently used pesticides and enhance the resistance to these insecticides.

Susceptibility and resistance to deltamethrin of wild and domestic populations of Triatoma infestans (Reduviidae: Triatominae) in Bolivia: new discoveries

Bolivia is a high-endemic country for Chagas disease, for which the principal vector is Triatoma infestans (Triatominae). This is a mainly domestic species that is also found in the wild environment. Recently, an increasing number of studies have shown the importance of Triatominae resistance to insecticides, especially in Bolivia. Data regarding the susceptibility/resistance of wild and domestic populations of T. infestans to deltamethrin are presented. For the first time, domestic populations of the department of Santa Cruz were tested, showing low resistance. Although most of the wild populations were found to be susceptible to deltamethrin, three populations from three departments showed a mortality rate of less than 100%. This result is emphasised here.

In vivo expansion of T reg cells with IL-2-mAb complexes: induction of resistance to EAE and long-term acceptance of islet allografts without immunosuppression.

Via a transcription factor, Foxp3, immunoregulatory CD4(+)CD25(+) T cells (T reg cells) play an important role in suppressing the function of other T cells. Adoptively transferring high numbers of T reg cells can reduce the intensity of the immune response, thereby providing an attractive prospect for inducing tolerance. Extending our previous findings, we describe an in vivo approach for inducing rapid expansion of T reg cells by injecting mice with interleukin (IL)-2 mixed with a particular IL-2 monoclonal antibody (mAb). Injection of these IL-2-IL-2 mAb complexes for a short period of 3 d induces a marked (>10-fold) increase in T reg cell numbers in many organs, including the liver and gut as well as the spleen and lymph nodes, and a modest increase in the thymus. The expanded T reg cells survive for 1-2 wk and are highly activated and display superior suppressive function. Pretreating with the IL-2-IL-2 mAb complexes renders the mice resistant to induction of experimental autoimmune encephalomyelitis; combined with rapamycin, the complexes can also be used to treat ongoing disease. In addition, pretreating mice with the complexes induces tolerance to fully major histocompatibility complex-incompatible pancreatic islets in the absence of immunosuppression. Tolerance is robust and the majority of grafts are accepted indefinitely. The approach described for T reg cell expansion has clinical potential for treating autoimmune disease and promoting organ transplantation.