Does the Ingestion of Isoxaflutole Herbicide Affect the Midgut and Salivary Glands of Pentatomidae Predators?

Podisus nigrispinus is a generalist predator naturally occurring in agricultural and forestry systems that effectively contributes to the population balance of phytophagous insects, especially defoliating caterpillars. Histological changes were evaluated in the salivary glands and midgut of P. nigrispinus caused by ingestion of systemic herbicide isoxaflutole. These predator females were fed with leaves of eucalyptus plants, Tenebrio molitor pupae or water, contaminated or not by herbicide. Salivary glands and midguts were dissected, processed and analyzed under a light microscope. Activity level and cell morphology of the salivary glands and midgut showed differences among insects fed on plants, contaminated water or pupae. The epithelia of the salivary gland and midgut of individuals which had no contact with the herbicide showed homogeneous cytoplasm, nucleus with predominance of decondensed chromatin and evident nucleoli, intense cell activity features. As for the insects in contact with contaminated food, they presented undeveloped nucleus and condensed chromatin. The luminal contents of the salivary glands in the contaminated insects had become more acidophilus than in insects without poisoning, as well as having heterogeneous and granular secretion, being more evident in the bioassay in which the insects fed on contaminated water. There was a marked morphological change in the midgut cells in contaminated insects. High degree of apoptosis, disorganization and secretory vacuoles in the epithelial cytoplasm were observed. The apical portion of the midgut cells proved undeveloped, irregular and partially destroyed. It is concluded that isoxaflutole causes morphological changes in the digestive system of the predator P. nigrispinus.

Growth and reproduction of the understory palm Geonoma schottiana Mart. in the gallery forest in Central Brazil

To understand the growth and reproduction of the palm Geonoma schottiana in the gallery forest of Central Brazil, two hypotheses were raised: (i) production of leaves and reproductive structures are concentrated in the period of the year with high light availability; (ii) leaf production and reproductive activity are related to plant length. However, it is expected that senility effects will cause fast reduction in growth and reproduction activities in higher plants. Growth and reproduction were concentrated in the dry season, when insolation is higher than in the wet season, suggesting that leaf and inflorescence production on G. schottiana in the gallery forest understory is more limited by light than by soil humidity. As the individual grows, leaf number and blade area increase, but reproduction activity is independent of plant length. Resources stored in the stem are important to growth, but exogenous factors, as canopy openness, should be more important to reproduction. Plant senescence seems to have a lesser effect on the production of vegetative and reproductive structures in G. schottiana than has been detected in congeneric species with greater clonal activity.

Relationship of cytokines and cytokine signaling to immunodeficiency disorders in the mouse

The contributions of cytokines to the development and progression of disease in a mouse model of retrovirus-induced immunodeficiency (MAIDS) are controversial. Some studies have indicated an etiologic role for type 2 cytokines, while others have emphasized the importance of type 1 cytokines. We have used mice deficient in expression of IL-4, IL-10, IL-4 and IL-10, IFN-g, or ICSBP - a transcriptional protein involved in IFN signaling - to examine their contributions to this disorder. Our results demonstrate that expression of type 2 cytokines is an epiphenomenon of infection and that IFN-g is a driving force in disease progression. In addition, exogenously administered IL-12 prevents many manifestations of disease while blocking retrovirus expression. Interruption of the IFN signaling pathways in ICSBP-/- mice blocks induction of MAIDS. Predictably, ICSBP-deficient mice exhibit impaired responses to challenge with several other viruses. This immunodeficiency is associated with impaired production of IFN-g and IL-12. Unexpectedly, however, the ICSBP-/- mice also develop a syndrome with many similarities to chronic myelogenous leukemia in humans. The chronic phase of this disease is followed by a fatal blast crisis characterized by clonal expansions of undifferentiated cells. ICSBP is thus an important determinant of hematopoietic growth and differentiation as well as a prominent signaling molecule for IFNs

MHC-restricted antigen presentation and recognition: constraints on gene, recombinant and peptide vaccines in humans

The target of any immunization is to activate and expand lymphocyte clones with the desired recognition specificity and the necessary effector functions. In gene, recombinant and peptide vaccines, the immunogen is a single protein or a small assembly of epitopes from antigenic proteins. Since most immune responses against protein and peptide antigens are T-cell dependent, the molecular target of such vaccines is to generate at least 50-100 complexes between MHC molecule and the antigenic peptide per antigen-presenting cell, sensitizing a T cell population of appropriate clonal size and effector characteristics. Thus, the immunobiology of antigen recognition by T cells must be taken into account when designing new generation peptide- or gene-based vaccines. Since T cell recognition is MHC-restricted, and given the wide polymorphism of the different MHC molecules, distinct epitopes may be recognized by different individuals in the population. Therefore, the issue of whether immunization will be effective in inducing a protective immune response, covering the entire target population, becomes an important question. Many pathogens have evolved molecular mechanisms to escape recognition by the immune system by variation of antigenic protein sequences. In this short review, we will discuss the several concepts related to selection of amino acid sequences to be included in DNA and peptide vaccines.

Long-term outcome of 25 children and adolescents with severe aplastic anemia treated with antithymocyte globulin

Severe aplastic anemia (SAA) is probably an immune-mediated disorder, and immunosuppressive therapy is recommended for patients with no available donor for bone marrow transplant. Between October 1984 and November 1987, 25 consecutive children and adolescents with SAA with no HLA-compatible marrow donor received equine antithymocyte globulin (ATG) (15 mg kg-1 day-1) for 10 days. The patients were evaluated 6 weeks, 6 months, and 12 months after starting ATG treatment. Thereafter, patients were evaluated yearly until July 1998. Median age was 10 years (range, 1.5-20 years), granulocyte counts on referral ranged from 0.032 to 1.4 x 10(9)/l (median 0.256 x 10(9)/l), and 12 patients had granulocyte counts <0.2 x 10(9)/l. At a median follow-up of 9.6 years (range, 8.6-11.8 years), 10 patients (40%) remained alive with good marrow function. No morphologic evidence of hematological clonal disorders has been observed, although two patients probably have acquired clonal chromosomal abnormalities (trisomy 8 and del(6)q21, respectively). Responses to ATG were observed between 6 weeks and 6 months from the start of treatment in 60% of evaluable patients. The response rate was not different in patients whose granulocyte count at diagnosis was <0.2 x 10(9)/l, or in those who were <10 years of age. This study supports the view that, when compared with supportive measures, ATG is an effective treatment for children or adolescents with SAA. Although these results are inferior to those reported for marrow transplantation or more intensive immunosuppressive regimens, these patients who responded to ATG are long-term survivors with stable peripheral blood counts and a low rate of relapse.

No evidence for mutations in exons 1, 8 and 18 of the patched gene in sporadic skin lesions of Brazilian patients

There is strong evidence that the patched (PTCH) gene is a gene for susceptibility to the nevoid basal cell carcinoma syndrome. PTCH has also been shown to mutate in both familial and sporadic basal cell carcinomas. However, mutations of the gene seem to be rare in squamous cell carcinomas. In order to characterize the role of the gene in the broader spectrum of sporadic skin malignant and pre-malignant lesions, we performed a polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis of genomic DNA extracted from 105 adult patients (46 females and 59 males). There were 66 patients with basal cell carcinomas, 30 with squamous cell carcinomas, 2 with malignant melanomas and 7 patients with precancerous lesions. Two tissue samples were collected from each patient, one from the central portion of the tumor and another from normal skin. Using primers that encompass the entire exon 1, exon 8 and exon 18, where most of the mutations have been detected, we were unable to demonstrate any band shift. Three samples suspected to present aberrant migrating bands were excised from the gel and sequenced directly. In addition, we sequenced 12 other cases, including tumors and corresponding normal samples. A wild-type sequence was found in all 15 cases. Although our results do not exclude the presence of clonal alterations of the PTCH gene in skin cancers or mutations in other exons that were not screened, the present data do not support the presence of frequent mutations reported for non-melanoma skin cancer of other populations.

Taxonomy and Species Richness of Amazonian Pimplinae and Rhyssinae (hymenoptera: ichneumonidae)

The Amazonian region, the biggest rain forest of our planet, is known for its extraordinary biodiversity. Most of this diversity is still unexplored and new species of different taxa are regularly found there. In this region, as in most areas of the world, insects are some of the most abundant organisms. Therefore, studying this group is important to promote the conservation of these highly biodiverse ecosystems of the planet. Among insects, parasitoid wasps are especially interesting because they have potential for use as biodiversity indicators and biological control agents in agriculture and forestry. The parasitoid wasp family Ichneumonidae is one of the most species rich groups among the kingdom Animalia. This group is still poorly known in many areas of the world; the Amazonian region is a clear example of this situation. Ichneumonids have been thought to be species poor in Amazonia and other tropical areas. However, recent studies are suggesting that parasitoid wasps may be quite abundant in Amazonia and possibly in most tropical areas of the world. The aim of my doctoral thesis is to study the species richness and taxonomy of two of the best known ichneumonid subfamilies in the Neotropical region, Pimplinae and Rhyssinae. To do this I conducted two extensive sampling programs in the Peruvian Amazonia. I examined also a large number of Neotropical ichneumonids deposited to different natural history museums. According to the results of my thesis, the species richness of these parasitoids in the Amazonian region is considerably higher than previously reported. In my research, I firstly further develop the taxonomy of these parasitoids by describing many new species and reporting several new faunistic records (I, II, III). In this first part I focus on two genera (Xanthopimpla and Epirhyssa) which were thought to be rather species poor. My thesis demonstrates that these groups are actually rather species rich in the Amazonian region. Secondly, I concentrate on the species richness of these parasitoids in a global comparison showing that the Neotropical region and especially the Peruvian Amazonia is one of the most species rich areas of Pimpliformes ichneumonids (V). Furthermore, I demonstrate that with the data available to date no clear latitudinal gradient in species richness is visible. Thirdly, increasing the macroecological knowledge of these parasitoids I show that some previously unreported ichneumonid subfamilies are present in the Amazonian region (IV). These new insights and the results of the global comparison of ichneumonid inventories suggest that the previous belief of low diversity in the tropics is most likely related to a lack of sampling effort in the region. Overall, my research increases the knowledge of Neotropical ichneumonids highlighting the importance of Peruvian Amazonia as one of the diversity hotspots of parasitoid wasps.

Typing of Pseudomonas aeruginosa from hospitalized patients: a comparison of susceptibility and biochemical profiles with genotype

Typing techniques are essential for understanding hospital epidemiology, permitting the elucidation of the source of infection and routes of bacterial transmission. Although DNA-based techniques are the "gold standard" for the epidemiological study of Pseudomonas aeruginosa, antibiotic profiles and biochemical results are used because they are easy to perform and to interpret and relatively inexpensive. Antibiotypes (susceptibility profiles) and biotypes (biochemical profiles) were compared to genotypes established by DNA restriction enzyme analysis in 81 clinical isolates of P. aeruginosa from three hospitals in Porto Alegre, Brazil. The epidemiological relationship among patients was also evaluated. Susceptibility and restriction profiles were discrepant in more than 50% of the cases, and many antibiotypes were observed among isolates from the same genotype. Furthermore, susceptibility profiles did not allow the distinction of isolates from unrelated genotypes. Since a large number of isolates (63%) yielded the same biochemical results, only 10 biotypes were detected, showing that this typing method has a low discriminatory power. On the other hand, DNA restriction enzyme typing allowed us to establish 71 distinct types. Epidemiological data about the relation among P. aeruginosa isolates were not conclusive. The results of the present study indicate that the only method that can establish a clonal relation is DNA restriction enzyme typing, whereas the other methods may cause misleading interpretations and are inadequate to guide proper infection control measures.

CaV 3.1 and CaV 3.3 account for T-type Ca2+ current in GH3 cells

T-type Ca2+ channels are important for cell signaling by a variety of cells. We report here the electrophysiological and molecular characteristics of the whole-cell Ca2+ current in GH3 clonal pituitary cells. The current inactivation at 0 mV was described by a single exponential function with a time constant of 18.32 ± 1.87 ms (N = 16). The I-V relationship measured with Ca2+ as a charge carrier was shifted to the left when we applied a conditioning pre-pulse of up to -120 mV, indicating that a low voltage-activated current may be present in GH3 cells. Transient currents were first activated at -50 mV and peaked around -20 mV. The half-maximal voltage activation and the slope factors for the two conditions are -35.02 ± 2.4 and 6.7 ± 0.3 mV (pre-pulse of -120 mV, N = 15), and -27.0 ± 0.97 and 7.5 ± 0.7 mV (pre-pulse of -40 mV, N = 9). The 8-mV shift in the activation mid-point was statistically significant (P < 0.05). The tail currents decayed bi-exponentially suggesting two different T-type Ca2+ channel populations. RT-PCR revealed the presence of a1G (CaV3.1) and a1I (CaV3.3) T-type Ca2+ channel mRNA transcripts.

Clonal dissemination of VanA-type glycopeptide-resistant Enterococcus faecalis between hospitals of two cities located 100 km apart

Nosocomial dissemination of glycopeptide-resistant enterococci represents a major problem in hospitals worldwide. In Brazil, the dissemination among hospitals in the city of São Paulo of polyclonal DNA profiles was previously described for vancomycin-resistant Enterococcus faecium. We describe here the dissemination of VanA phenotype E. faecalis between two hospitals located in different cities in the State of São Paulo. The index outbreak occurred in a tertiary care university hospital (HCUSP) in the city of São Paulo and three years later a cluster caused by the same strain was recognized in two patients hospitalized in a private tertiary care hospital (CMC) located 100 km away in the interior of the state. From May to July 1999, 10 strains of vancomycin-resistant E. faecalis were isolated from 10 patients hospitalized in the HCUSP. The DNA genotyping using pulsed-field gel electrophoresis (PFGE) showed that all isolates were originated from the same clone, suggesting nosocomial dissemination. From May to July 2002, three strains of vancomycin-resistant E. faecalis were isolated from two patients hospitalized in CMC and both patients were colonized by the vancomycin-resistant Enterococcus in skin lesions. All isolates from CMC and HCUSP were highly resistant to vancomycin and teicoplanin. The three strains from CMC had minimum inhibitory concentration >256 µg/ml for vancomycin, and 64 (CMC 1 and CMC 2) and 96 µg/ml (CMC 3) for teicoplanin, characterizing a profile of VanA resistance to glycopeptides. All strains had the presence of the transposon Tn1546 detected by PCR and were closely related when typed by PFGE. The dissemination of the E. faecalis VanA phenotype among hospitals located in different cities is of great concern because E. faecalis commonly colonizes the gastrointestinal tract of patients and healthy persons for periods varying from weeks to years, which, together with the persistence of vancomycin-resistant Enterococcus in hospital rooms after standard cleaning procedures, increases the risk of the dissemination and reservoir of the bacteria.

From genotype to phenotype : diversity and population structure of Flavobacterium psychrophilum

Flavobacterium psychrophilum is the etiological agent of bacterial cold-water disease (BCWD) causing high fish mortalities and significant economic losses to the freshwater salmonid aquaculture industry around the world. Today BCWD outbreaks are mainly treated with environmentally hazardous antimicrobial agents and alternative preventative measures are urgently needed in order to ensure the well-being of animals and the sustainability of aquaculture. The diversity of pathogenic bacteria challenges the development of universal control strategies and in many cases the pathogen population structure, i.e. the total genetic diversity of the species must be taken into account. This work integrates the tools of modern molecular biology and conventional phenotypic microbiology to gain knowledge about the diversity and population structure of F. psychrophilum. The present work includes genetic characterization of a large collection of isolates collected from diverse origins and years, from aquaculture in a whole region including different countries, and provides the first international validation of a universal multilocus sequence typing (MLST) approach for unambiguous genetic typing of F. psychrophilum. Population structure analyses showed that the global F. psychrophilum population is subdivided into pathogenic species-specific clones, of which one particular genetic lineage, clonal complex CC-ST2, has been responsible for the majority of BCWD outbreaks in rainbow trout (Oncorhynchus mykiss) in European aquaculture facilities over several decades. Genotypic and phenotypic population heterogeneity affecting antimicrobial resistance in F. psychrophilum within BCWD outbreaks was discovered. Specific genotypes were associated with severe infections in farmed rainbow trout and Atlantic salmon (Salmo salar), and in addition to high adherence, antimicrobial resistance was strongly associated with outbreak strains. The study brought additional support for the hypothesis of an epidemic F. psychrophilum population structure, where recombination is an important force for the generation and maintenance of genetic diversity, and a significant contribution towards mapping the genetic diversity of this important fish pathogen. Evidence indicating dissemination of virulent strains with commercial movement of fish and fish products was strengthened.

Wooden packaging waste reuse and refining

The aim of the thesis was both to study wooden packaging waste reuse and refining generated in the forestry machine factory environment, and to find alternative wooden packaging waste utilization options in order to create a new operating model which would decrease the overall amount of waste produced. As environmental and waste legislation has become more rigid and companies' own environmental management systems’ requirements and control have increased, companies have had to consider their environmental aspects more carefully. Companies have to take into account alternative ways of reducing waste through an increase in reuse and recycling. A part of this waste is from different forms of packaging. In the metal industry the most heavily used packaging material is wooden packaging, as such material is heavy and the packaging has to be able to bear heavy stress. In the theoretical part of the thesis, the requirements of packaging and packaging waste legislation, as well as environmental management systems governing companies’ processing of their packaging waste, are studied. The theoretical part includes a process study of systems, which direct packaging waste and wooden packaging waste refining. In addition, methods related to the continuous improvement of these processes are introduced. This thesis concentrates on designing and creating a new operating model in relation to wooden packaging waste processing. The main target was to find an efficient model in order to decrease the total amount of wooden packaging waste and to increase refining. The empirical part introduces methods for approaches to wooden packaging waste re-utilization, as well as a description of a new operating model and its impact.

Epidemiological characterization of resistance and PCR typing of Shigella flexneri and Shigella sonnei strains isolated from bacillary dysentery cases in Southeast Brazil

Shigella spp are Gram-negative, anaerobic facultative, non-motile, and non-sporulated bacilli of the Enterobacteriaceae family responsible for "Shigellosis" or bacillary dysentery, an important cause of worldwide morbidity and mortality. However, despite this, there are very few epidemiological studies about this bacterium in Brazil. We studied the antibiotic resistance profiles and the clonal structure of 60 Shigella strains (30 S. flexneri and 30 S. sonnei) isolated from shigellosis cases in different cities within the metropolitan area of Campinas, State of São Paulo, Brazil. We used the following well-characterized molecular techniques: enterobacterial repetitive intergenic consensus, repetitive extragenic palindromic, and double-repetitive element-polymerase chain reaction to characterize the bacteria. Also, the antibiotic resistance of the strains was determined by the diffusion disk method. Many strains of S. flexneri and S. sonnei were found to be multi-resistant. S. flexneri strains were resistant to ampicillin in 83.3% of cases, chloramphenicol in 70.0%, streptomycin in 86.7%, sulfamethoxazole in 80.0%, and tetracycline in 80.0%, while a smaller number of strains were resistant to cephalothin (3.3%) and sulfazotrim (10.0%). S. sonnei strains were mainly resistant to sulfamethoxazole (100.0%) and tetracycline (96.7%) and, to a lesser extent, to ampicillin (6.7%) and streptomycin (26.7%). Polymerase chain reaction-based typing supported the existence of specific clones responsible for the shigellosis cases in the different cities and there was evidence of transmission between cities. This clonal structure would probably be the result of selection for virulence and resistance phenotypes. These data indicate that the human sanitary conditions of the cities investigated should be improved.

Potential pathogenic role of aggregative- adhering Corynebacterium diphtheriae of different clonal groups in endocarditis

Invasive diseases caused by Corynebacterium diphtheriae have been described increasingly. Several reports indicate the destructive feature of endocarditis attributable to nontoxigenic strains. However, few reports have dealt with the pathogenicity of invasive strains. The present investigation demonstrates a phenotypic trait that may be used to identify potentially invasive strains. The study also draws attention to clinical and microbiological aspects observed in 5 cases of endocarditis due to C. diphtheriae that occurred outside Europe. Four cases occurred in female school-age children (7-14 years) treated at different hospitals in Rio de Janeiro, Brazil. All patients developed other complications including septicemia, renal failure and/or arthritis. Surgical treatment was performed on 2 patients for valve replacement. Lethality was observed in 40% of the cases. Microorganisms isolated from 5 blood samples and identified as C. diphtheriae subsp mitis (N = 4) and C. diphtheriae subsp gravis (N = 1) displayed an aggregative adherence pattern to HEp-2 cells and identical one-dimensional SDS-PAGE protein profiles. Aggregative-adhering invasive strains of C. diphtheriae showed 5 distinct RAPD profiles. Despite the clonal diversity, all 5 C. diphtheriae invasive isolates seemed to display special bacterial adhesive properties that may favor blood-barrier disruption and systemic dissemination of bacteria. In conclusion, blood isolates from patients with endocarditis exhibited a unique adhering pattern, suggesting a pathogenic role of aggregative-adhering C. diphtheriae of different clones in endocarditis. Accordingly, the aggregative-adherence pattern may be used as an indication of some invasive potential of C. diphtheriae strains.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R), with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.