Bleaching of melanin in the epidermis of South American fur seal and its application on enzyme immunohistochemistry

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immuno-histochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

TOLL-LIKE RECEPTORS (TLR) 2 AND 4 EXPRESSION OF KERATINOCYTES FROM PATIENTS WITH LOCALIZED AND DISSEMINATED DERMATOPHYTOSIS

There are few studies on the role of innate immune response in dermatophytosis. An investigation was conducted to define the involvement of Toll-Like Receptors (TLRs) 2 and 4 in localized (LD) and disseminated (DD) dermatophytosis due to T. rubrum. Fifteen newly diagnosed patients, eight patients with LD and seven with DD, defined by involvement of at least three body segments were used in this study. Controls comprised twenty skin samples from healthy individuals undergoing plastic surgery. TLR2 and TLR4 were quantified in skin lesions by immunohistochemistry. A reduced expression of TLR4 in the lower and upper epidermis of both LD and DD patients was found compared to controls; TLR2 expression was preserved in the upper and lower epidermis of all three groups. As TLR4 signaling induces the production of inflammatory cytokines and neutrophils recruitment, its reduced expression likely contributed to the lack of resolution of the infection and the consequent chronic nature of the dermatophytosis. As TLR2 expression acts to limit the inflammatory process and preserves the epidermal structure, its preserved expression may also contribute to the persistent infection and limited inflammation that are characteristic of dermatophytic infections.

Atividade antibacteriana e antifúngica de espécies de Gnetum.

Este trabalho trata da determinação da atividade antibiótica dos extratos de Gnetum paniculatum e G. schwackeanum e dos constituintes químicos isolados deste último como resperatrol, gnetina C e E, os quais foram testados contra várias bactérias e fungos. O extrato de Gnetum schwackeanum e dos constituintes químicos isolados deste último como resveratrol, gnetina C e E, os quais foram testados contra várias bactérias e fungos. O extrato de Gnetum schwackeanum e todas as substâncias dele isoladas foram ativos a Staphylococcus aureus. S. epidermis e Mycobacterium smegmatis. Resveratrol e gnetina C são ativos contra a Candida albicans, mas somente gnetina C possui atividade em relação a Candida parapsilosis e Saccharomyces cervisiae. O derivado sintético de gnetina E não mostrou nenhuma atividade. O extrato de G. paniculatum, é completamente inativo à bactéria e fungos o que sugere que a atividade de G. paniculatum, é completamente inativo à bactéria e fungos o que sugere que a atividade de G. schwackeanum deve-se à presença dos hidroxiestilbenos e seus derivados, uma vez que G. paniculatum não contêm esses tipos de substâncias.

Atividade antibacteriana e antifúngica de espécies de Gnetum

Este trabalho trata da determinação da atividade antibíotica dos extratos de Gnetum paniculatum e G. schwackeanum e dos constituintes químicos isolados deste último como resveratrol, gnetina C e E, os quais foram testados contra vários bactérias e fungos. O extrato de Gnetum schwackeanum e todas as substâncias dele isoladas foram ativos a Staphylococcus aureus, S. epidermis e Mycobacterium smegmatis. Resveratrol e gnetina C são ativos contra Candida albicans, mas somente gnetina C possui atividade em relação a Candida parapsilosis e Saccharomyces cerevisiae. O derivado sintético de gnetina E não mostrou nenhuma atividade. O extrato de G. paniculatum, é completamente inativo à bactéria e fungos o que sugere que a atividade de G. schwackeanum deve-se à presença dos hiroxiestilbenos e seus derivados, uma vez que G. paniculatum não contém esses tipos de substâncias.

Leaf anatomical features of three Theobroma species (Malvaceae s.l.) native to the Brazilian Amazon

Theobroma species have economic importance due to their use in the cosmetic and food industries, mainly in the production of chocolate. However, the anatomy of their vegetative structures remains poorly studied. The goal of this study was to describe the anatomical features of Theobroma grandiflorum, T. speciosum and T. subincanum to contribute to the biological knowledge of these species, as well as provide support to the biotechnological studies of native fruit plants of the Amazon. Leaves at different developmental stages were collected and analyzed under light microscopy and scanning electron microscopy. Sessile and stalked stellate trichomes and digitiform glandular trichomes were observed in the expanded leaves of T. grandiflorum and T. subincanum. These species were also similar in the morphology of the midrib, the organization of the mesophyll and the presence of starch grains in the midrib pith cells. Claviform glandular trichomes and mucilage cells in the epidermis occurred only in the expanded leaves of T. speciosum. The presence of mucilage secretory trichomes in shoot apices (colleters) of all species is a new finding for the genus Theobroma.

Leaf anatomy of Qualea parviflora (Vochysiaceae) in three phytophysiognomies of the Mato Grosso State, Brazil

ABSTRACT Leaves have a variety of morphological and anatomical characters mainly influenced by climatic, edaphic and biotic factors. The aim of this study was to describe the anatomical leaf traits of Qualea parviflora from three phytophysiognomies. The studied phytophysiognomies were Amazon Savannah on rocky outcrops (ASR), Transition Rupestrian Cerrado (TRC), and Cerradão (CDA). Freehand sections of the leaf blade were made and stained with 0.5% astra blue and with basic fuchsin. From the adaxial and abaxial leaf surface, freehand paradermal sections were made for epidermis analysis. The Jeffrey´s method, with modifications, was used in the epidermis dissociation process. The samples from the TRC phytophysiognomy had relatively smaller ordinary epidermal cells, higher abundance of trichomes, and mesophyll with few intercellular spaces, in comparison to the other phytophysiognomies. The leaves from the ASR phytophysiognomy had higher stomatal index (SI = 21.02), and five to six layers of sclerenchyma surrounding the midrib vascular bundle. The secondary vascular bundles had thicker cell walls and the bundle sheath extended up to the epidermal tissue of both leaf sides. Leaves from the CDA phytophysiognomy had mesomorphic environmental traits, such as a thinner cuticle. It is concluded that trees from ASR and TRC phytophysiognomies have xeromorphic traits following the environmental conditions where they occur.

Dissecando o GEN

In thee present paper the classical concept of the corpuscular gene is dissected out in order to show the inconsistency of some genetical and cytological explanations based on it. The author begins by asking how do the genes perform their specific functions. Genetists say that colour in plants is sometimes due to the presence in the cytoplam of epidermal cells of an organic complex belonging to the anthocyanins and that this complex is produced by genes. The author then asks how can a gene produce an anthocyanin ? In accordance to Haldane's view the first product of a gene may be a free copy of the gene itself which is abandoned to the nucleus and then to the cytoplasm where it enters into reaction with other gene products. If, thus, the different substances which react in the cell for preparing the characters of the organism are copies of the genes then the chromosome must be very extravagant a thing : chain of the most diverse and heterogeneous substances (the genes) like agglutinins, precipitins, antibodies, hormones, erzyms, coenzyms, proteins, hydrocarbons, acids, bases, salts, water soluble and insoluble substances ! It would be very extrange that so a lot of chemical genes should not react with each other. remaining on the contrary, indefinitely the same in spite of the possibility of approaching and touching due to the stato of extreme distension of the chromosomes mouving within the fluid medium of the resting nucleus. If a given medium becomes acid in virtue of the presence of a free copy of an acid gene, then gene and character must be essentially the same thing and the difference between genotype and phenotype disappears, epigenesis gives up its place to preformation, and genetics goes back to its most remote beginnings. The author discusses the complete lack of arguments in support of the view that genes are corpuscular entities. To show the emharracing situation of the genetist who defends the idea of corpuscular genes, Dobzhansky's (1944) assertions that "Discrete entities like genes may be integrated into systems, the chromosomes, functioning as such. The existence of organs and tissues does not preclude their cellular organization" are discussed. In the opinion of the present writer, affirmations as such abrogate one of the most important characteristics of the genes, that is, their functional independence. Indeed, if the genes are independent, each one being capable of passing through mutational alterations or separating from its neighbours without changing them as Dobzhansky says, then the chromosome, genetically speaking, does not constitute a system. If on the other hand, theh chromosome be really a system it will suffer, as such, the influence of the alteration or suppression of the elements integrating it, and in this case the genes cannot be independent. We have therefore to decide : either the chromosome is. a system and th genes are not independent, or the genes are independent and the chromosome is not a syntem. What cannot surely exist is a system (the chromosome) formed by independent organs (the genes), as Dobzhansky admits. The parallel made by Dobzhansky between chromosomes and tissues seems to the author to be inadequate because we cannot compare heterogeneous things like a chromosome considered as a system made up by different organs (the genes), with a tissue formed, as we know, by the same organs (the cells) represented many times. The writer considers the chromosome as a true system and therefore gives no credit to the genes as independent elements. Genetists explain position effects in the following way : The products elaborated by the genes react with each other or with substances previously formed in the cell by the action of other gene products. Supposing that of two neighbouring genes A and B, the former reacts with a certain substance of the cellular medium (X) giving a product C which will suffer the action, of the latter (B). it follows that if the gene changes its position to a place far apart from A, the product it elaborates will spend more time for entering into contact with the substance C resulting from the action of A upon X, whose concentration is greater in the proximities of A. In this condition another gene produtc may anticipate the product of B in reacting with C, the normal course of reactions being altered from this time up. Let we see how many incongruencies and contradictions exist in such an explanation. Firstly, it has been established by genetists that the reaction due.to gene activities are specific and develop in a definite order, so that, each reaction prepares the medium for the following. Therefore, if the medium C resulting from the action of A upon x is the specific medium for the activity of B, it follows that no other gene, in consequence of its specificity, can work in this medium. It is only after the interference of B, changing the medium, that a new gene may enter into action. Since the genotype has not been modified by the change of the place of the gene, it is evident that the unique result we have to attend is a little delay without seious consequence in the beginning of the reaction of the product of B With its specific substratum C. This delay would be largely compensated by a greater amount of the substance C which the product of B should found already prepared. Moreover, the explanation did not take into account the fact that the genes work in the resting nucleus and that in this stage the chromosomes, very long and thin, form a network plunged into the nuclear sap. in which they are surely not still, changing from cell to cell and In the same cell from time to time, the distance separating any two genes of the same chromosome or of different ones. The idea that the genes may react directly with each other and not by means of their products, would lead to the concept of Goidschmidt and Piza, in accordance to which the chromosomes function as wholes. Really, if a gene B, accustomed to work between A and C (as for instance in the chromosome ABCDEF), passes to function differently only because an inversion has transferred it to the neighbourhood of F (as in AEDOBF), the gene F must equally be changed since we cannot almH that, of two reacting genes, only one is modified The genes E and A will be altered in the same way due to the change of place-of the former. Assuming that any modification in a gene causes a compensatory modification in its neighbour in order to re-establich the equilibrium of the reactions, we conclude that all the genes are modified in consequence of an inversion. The same would happen by mutations. The transformation of B into B' would changeA and C into A' and C respectively. The latter, reacting withD would transform it into D' and soon the whole chromosome would be modified. A localized change would therefore transform a primitive whole T into a new one T', as Piza pretends. The attraction point-to-point by the chromosomes is denied by the nresent writer. Arguments and facts favouring the view that chromosomes attract one another as wholes are presented. A fact which in the opinion of the author compromises sereously the idea of specific attraction gene-to-gene is found inthe behavior of the mutated gene. As we know, in homozygosis, the spme gene is represented twice in corresponding loci of the chromosomes. A mutation in one of them, sometimes so strong that it is capable of changing one sex into the opposite one or even killing the individual, has, notwithstading that, no effect on the previously existing mutual attraction of the corresponding loci. It seems reasonable to conclude that, if the genes A and A attract one another specifically, the attraction will disappear in consequence of the mutation. But, as in heterozygosis the genes continue to attract in the same way as before, it follows that the attraction is not specific and therefore does not be a gene attribute. Since homologous genes attract one another whatever their constitution, how do we understand the lack cf attraction between non homologous genes or between the genes of the same chromosome ? Cnromosome pairing is considered as being submitted to the same principles which govern gametes copulation or conjugation of Ciliata. Modern researches on the mating types of Ciliata offer a solid ground for such an intepretation. Chromosomes conjugate like Ciliata of the same variety, but of different mating types. In a cell there are n different sorts of chromosomes comparable to the varieties of Ciliata of the same species which do not mate. Of each sort there are in the cell only two chromosomes belonging to different mating types (homologous chromosomes). The chromosomes which will conjugate (belonging to the same "variety" but to different "mating types") produce a gamone-like substance that promotes their union, being without action upon the other chromosomes. In this simple way a single substance brings forth the same result that in the case of point-to-point attraction would be reached through the cooperation of as many different substances as the genes present in the chromosome. The chromosomes like the Ciliata, divide many times before they conjugate. (Gonial chromosomes) Like the Ciliata, when they reach maturity, they copulate. (Cyte chromosomes). Again, like the Ciliata which aggregate into clumps before mating, the chrorrasrmes join together in one side of the nucleus before pairing. (.Synizesis). Like the Ciliata which come out from the clumps paired two by two, the chromosomes leave the synizesis knot also in pairs. (Pachytene) The chromosomes, like the Ciliata, begin pairing at any part of their body. After some time the latter adjust their mouths, the former their kinetochores. During conjugation the Ciliata as well as the chromosomes exchange parts. Finally, the ones as the others separate to initiate a new cycle of divisions. It seems to the author that the analogies are to many to be overlooked. When two chemical compounds react with one another, both are transformed and new products appear at the and of the reaction. In the reaction in which the protoplasm takes place, a sharp difference is to be noted. The protoplasm, contrarily to what happens with the chemical substances, does not enter directly into reaction, but by means of products of its physiological activities. More than that while the compounds with Wich it reacts are changed, it preserves indefinitely its constitution. Here is one of the most important differences in the behavior of living and lifeless matter. Genes, accordingly, do not alter their constitution when they enter into reaction. Genetists contradict themselves when they affirm, on the one hand, that genes are entities which maintain indefinitely their chemical composition, and on the other hand, that mutation is a change in the chemica composition of the genes. They are thus conferring to the genes properties of the living and the lifeless substances. The protoplasm, as we know, without changing its composition, can synthesize different kinds of compounds as enzyms, hormones, and the like. A mutation, in the opinion of the writer would then be a new property acquired by the protoplasm without altering its chemical composition. With regard to the activities of the enzyms In the cells, the author writes : Due to the specificity of the enzyms we have that what determines the order in which they will enter into play is the chemical composition of the substances appearing in the protoplasm. Suppose that a nucleoproteln comes in relation to a protoplasm in which the following enzyms are present: a protease which breaks the nucleoproteln into protein and nucleic acid; a polynucleotidase which fragments the nucleic acid into nucleotids; a nucleotidase which decomposes the nucleotids into nucleoids and phosphoric acid; and, finally, a nucleosidase which attacs the nucleosids with production of sugar and purin or pyramidin bases. Now, it is evident that none of the enzyms which act on the nucleic acid and its products can enter into activity before the decomposition of the nucleoproteln by the protease present in the medium takes place. Leikewise, the nucleosidase cannot works without the nucleotidase previously decomposing the nucleotids, neither the latter can act before the entering into activity of the polynucleotidase for liberating the nucleotids. The number of enzyms which may work at a time depends upon the substances present m the protoplasm. The start and the end of enzym activities, the direction of the reactions toward the decomposition or the synthesis of chemical compounds, the duration of the reactions, all are in the dependence respectively o fthe nature of the substances, of the end products being left in, or retired from the medium, and of the amount of material present. The velocity of the reaction is conditioned by different factors as temperature, pH of the medium, and others. Genetists fall again into contradiction when they say that genes act like enzyms, controlling the reactions in the cells. They do not remember that to cintroll a reaction means to mark its beginning, to determine its direction, to regulate its velocity, and to stop it Enzyms, as we have seen, enjoy none of these properties improperly attributed to them. If, therefore, genes work like enzyms, they do not controll reactions, being, on the contrary, controlled by substances and conditions present in the protoplasm. A gene, like en enzym, cannot go into play, in the absence of the substance to which it is specific. Tne genes are considered as having two roles in the organism one preparing the characters attributed to them and other, preparing the medium for the activities of other genes. At the first glance it seems that only the former is specific. But, if we consider that each gene acts only when the appropriated medium is prepared for it, it follows that the medium is as specific to the gene as the gene to the medium. The author concludes from the analysis of the manner in which genes perform their function, that all the genes work at the same time anywhere in the organism, and that every character results from the activities of all the genes. A gene does therefore not await for a given medium because it is always in the appropriated medium. If the substratum in which it opperates changes, its activity changes correspondingly. Genes are permanently at work. It is true that they attend for an adequate medium to develop a certain actvity. But this does not mean that it is resting while the required cellular environment is being prepared. It never rests. While attending for certain conditions, it opperates in the previous enes It passes from medium to medium, from activity to activity, without stopping anywhere. Genetists are acquainted with situations in which the attended results do not appear. To solve these situations they use to make appeal to the interference of other genes (modifiers, suppressors, activators, intensifiers, dilutors, a. s. o.), nothing else doing in this manner than displacing the problem. To make genetcal systems function genetists confer to their hypothetical entities truly miraculous faculties. To affirm as they do w'th so great a simplicity, that a gene produces an anthocyanin, an enzym, a hormone, or the like, is attribute to the gene activities that onlv very complex structures like cells or glands would be capable of producing Genetists try to avoid this difficulty advancing that the gene works in collaboration with all the other genes as well as with the cytoplasm. Of course, such an affirmation merely means that what works at each time is not the gene, but the whole cell. Consequently, if it is the whole cell which is at work in every situation, it follows that the complete set of genes are permanently in activity, their activity changing in accordance with the part of the organism in which they are working. Transplantation experiments carried out between creeper and normal fowl embryos are discussed in order to show that there is ro local gene action, at least in some cases in which genetists use to recognize such an action. The author thinks that the pleiotropism concept should be applied only to the effects and not to the causes. A pleiotropic gene would be one that in a single actuation upon a more primitive structure were capable of producing by means of secondary influences a multiple effect This definition, however, does not preclude localized gene action, only displacing it. But, if genetics goes back to the egg and puts in it the starting point for all events which in course of development finish by producing the visible characters of the organism, this will signify a great progress. From the analysis of the results of the study of the phenocopies the author concludes that agents other than genes being also capaole of determining the same characters as the genes, these entities lose much of their credit as the unique makers of the organism. Insisting about some points already discussed, the author lays once more stress upon the manner in which the genes exercise their activities, emphasizing that the complete set of genes works jointly in collaboration with the other elements of the cell, and that this work changes with development in the different parts of the organism. To defend this point of view the author starts fron the premiss that a nerve cell is different from a muscle cell. Taking this for granted the author continues saying that those cells have been differentiated as systems, that is all their parts have been changed during development. The nucleus of the nerve cell is therefore different from the nucleus of the muscle cell not only in shape, but also in function. Though fundamentally formed by th same parts, these cells differ integrally from one another by the specialization. Without losing anyone of its essenial properties the protoplasm differentiates itself into distinct kinds of cells, as the living beings differentiate into species. The modified cells within the organism are comparable to the modified organisms within the species. A nervo and a muscle cell of the same organism are therefore like two species originated from a common ancestor : integrally distinct. Like the cytoplasm, the nucleus of a nerve cell differs from the one of a muscle cell in all pecularities and accordingly, nerve cell chromosomes are different from muscle cell chromosomes. We cannot understand differentiation of a part only of a cell. The differentiation must be of the whole cell as a system. When a cell in the course of development becomes a nerve cell or a muscle cell , it undoubtedly acquires nerve cell or muscle cell cytoplasm and nucleus respectively. It is not admissible that the cytoplasm has been changed r.lone, the nucleus remaining the same in both kinds of cells. It is therefore legitimate to conclude that nerve ceil ha.s nerve cell chromosomes and muscle cell, muscle cell chromosomes. Consequently, the genes, representing as they do, specific functions of the chromossomes, are different in different sorts of cells. After having discussed the development of the Amphibian egg on the light of modern researches, the author says : We have seen till now that the development of the egg is almost finished and the larva about to become a free-swimming tadepole and, notwithstanding this, the genes have not yet entered with their specific work. If the haed and tail position is determined without the concourse of the genes; if dorso-ventrality and bilaterality of the embryo are not due to specific gene actions; if the unequal division of the blastula cells, the different speed with which the cells multiply in each hemisphere, and the differential repartition of the substances present in the cytoplasm, all this do not depend on genes; if gastrulation, neurulation. division of the embryo body into morphogenetic fields, definitive determination of primordia, and histological differentiation of the organism go on without the specific cooperation of the genes, it is the case of asking to what then the genes serve ? Based on the mechanism of plant galls formation by gall insects and on the manner in which organizers and their products exercise their activities in the developing organism, the author interprets gene action in the following way : The genes alter structures which have been formed without their specific intervention. Working in one substratum whose existence does not depend o nthem, the genes would be capable of modelling in it the particularities which make it characteristic for a given individual. Thus, the tegument of an animal, as a fundamental structure of the organism, is not due to gene action, but the presence or absence of hair, scales, tubercles, spines, the colour or any other particularities of the skin, may be decided by the genes. The organizer decides whether a primordium will be eye or gill. The details of these organs, however, are left to the genetic potentiality of the tissue which received the induction. For instance, Urodele mouth organizer induces Anura presumptive epidermis to develop into mouth. But, this mouth will be farhioned in the Anura manner. Finalizing the author presents his own concept of the genes. The genes are not independent material particles charged with specific activities, but specific functions of the whole chromosome. To say that a given chromosome has n genes means that this chromonome, in different circumstances, may exercise n distinct activities. Thus, under the influence of a leg evocator the chromosome, as whole, develops its "leg" activity, while wbitm the field of influence of an eye evocator it will develop its "eye" activity. Translocations, deficiencies and inversions will transform more or less deeply a whole into another one, This new whole may continue to produce the same activities it had formerly in addition to those wich may have been induced by the grafted fragment, may lose some functions or acquire entirely new properties, that is, properties that none of them had previously The theoretical possibility of the chromosomes acquiring new genetical properties in consequence of an exchange of parts postulated by the present writer has been experimentally confirmed by Dobzhansky, who verified that, when any two Drosophila pseudoobscura II - chromosomes exchange parts, the chossover chromosomes show new "synthetic" genetical effects.

Nota sobre ácaros (Acari) atacando fruteira-do-conde (Rollinia sp.)

The flowers of this orchard tree presents six fleshy petals, fused two by two, and disposed propeller like. In the center is a cavity which harbors the cone shaped reproductive organs. An eriophyid mite, described by H. H. Keifer as Aculops flechtmann Keifer, 1972, causes considerable bronzing to the petals (Fig. 1-A). Large numbers of this mite attacking the basis of the reproductive organs causes them to dry and drop. On the young fruits, which are squamous, this mite causes some rusting and small necrotic areas (Fig. 1-B). As the fruit grows these small areas remain dark and barky. Very often the necrotic areas are invaded by the false spider mite Brevipalpus phoenicis (Geijskes, 1939), which enlarges the damage. The false spider mite also attacks leaves and green stems; the epidermis of the latter then assumes a barky appearance.

Comparación ontogénica de la frecuencia de muda en Rhinella marina (Anura, Bufonidae)

La muda es un proceso de renovación de la capa externa de la epidermis (estrato corneo) de los anfibios, la cual suministra protección contra daños, patógenos y pérdida de agua. Este trabajo evalúa la frecuencia de muda entre juveniles y adultos de Rhinella marina (Linnaeus, 1758) y la tasa de ocurrencia entre el día y la noche. Para esto, se realizaron dos observaciones diarias (7 am y 7 pm), entre Octubre de 2011 y Marzo de 2012, a tres grupos de individuos clasificados según su tamaño longitud rostro-cloaca, como adultos (promedio=80 mm), juveniles medianos (promedio=19 mm) y juveniles pequeños (promedio=13 mm). Estos animales fueron colocados en terrarios en el laboratorio y marcados en el dorso a través de un punto hecho con un corrector de tinta. La muda se determinó por la pérdida total de la marca y una coloración brillante en el dorso. Se encontró una diferencia significativa (Kruskal-Wallis, H=19.84, p<0.0001) en el periodo de muda entre los tres grupos de estudio: adultos=7.5 días, juveniles medianos=5.4 días, juveniles pequeños=5.3 días. También, en la frecuencia de animales que mudaron en el día y la noche (Ji-cuadrado, χ2=7.891, p=0.019), particularmente en los dos grupos de juveniles, quienes lo hicieron en la noche, ya que en los adultos no hubo una diferencia clara. Posiblemente, la mayor frecuencia de la muda en los juveniles puede relacionarse con su condición ontogénica, de un menor tamaño corporal, alta tasa metabólica y mayor tasa de desarrollo.

Aspectos parasitários observados no local inoculado com esporozoitos de Plasmodium Gallinaceum

The following is a summary of the studies made on the development of Plasmodium gallinaceum sporozoites inoculated into normal chicks. Initially large numbers of laboratory reared Aëdes aegypti were fed on pullets heavily infected with gametocytes. Following the infectious meal the mosquitoes were kept on a diet of sugar and water syrup until the appearance of the sporozoites in the salivary glands. Normal chicks kept in hematophagous arthropod proof cages were then inoculated either by bite of the infected mosquitoes or by subcutaneous inoculations of salivary gland suspensions. By the first method ten mosquitoes fed to engorgement on each normal chick and were then sacrificed immediately afterwards to determine the sporozoite count. By the second method five pairs of salivary glands were dissected out at room temperature, triturated in physiological saline and inoculated subcutaneously. The epidermis and dermis at the site of inoculation were excised from six hours after inoculation to forty eight hours after appearance of the parasites in the blood stream and stretched out on filter paper with the epithelial surface downward. The dermis was then curretted. Slides were made of the scrapings consisting of connective tissue and epithelial cells of the basal layers which were fixed by metyl alcohol and stained with Giemsa for examination under the oil immersion lens. Skin fragments removed from normal chicks and from regions other than the site of inoculation in the infected chicks were used as controls. In these, only the normal histological aspect was ever encountered. In the biopsy made at the earliest period following inoculation clearly defined elongated forms with eight or more chromatin granules arranged in rosary formation were found. The author believes these to be products of the sporozoite evolution. Search for transition stages between these forms and sporozoites is planned in biopsies to be taken immediately following inoculation and at given intervals up to the six hour period. 1.) 6 and 12 hour periods. The bodies referred to above found in the first period in great abundance, apparently in proportion to the large numbers of sporozoites inoculated, were perceptibly reduced in numbers in the second period. 2.) 18 hour period. Only one biopsy was examined. This presented a binuclear body shown in Fig. 1, having a more or less hyaline protoplasm staining an intense blue and a narrow vacuole delimiting the cell boundaries. The two chromatin grains were quite large presenting a clearly defined nuclear texture. 3.) 24 hour period. A similar body to that above (Fig. 2) was seen in the only preparation examined. 4.) 60 hour period. The exoerythrocytic schizonts were found more frequently from this period onward. Several such were found no longer to contain the previously described vacuoles (Fig. 3). 5.) 84 hour period. Cells bearing eight or more schizonts were frequently encountered here. That these are apparently not bodies in process of division may be seen in Fig. 4. From this time onward small violet granules similar to volutine grains appeared constantly in the schizont nucleus and protoplasm. These are definitely not hemozoin. The above observations fell within the incubation period as repeated examinations of the peripheral and visceral blood were negative. Exoery-throcytic parasites also were never encountered in the viscera at this time. Exoerythrocytic schizonts searched for at site of inoculation 1, 24 and 48 hours after the incubation period were present in large number at all three times with apparent tendency to diminish as the number within the blood stream increased. Many of them presented the violet granules mentioned above. The appearance of the chromatin and the intensity of staining of the protoplasm varied from body to body which doubtless corresponds to the evolutionary stage of each. This diversity of aspect may frequently be seen in the parasites of the same host cell (Fig. 5.). These findings lend substance to the theory that the exoerythrocytic forms are the link between the sporozoites and the pigmented parasites of the red blood corpuscles. The explanation of their continued presence in the organism after infection of the blood stream takes place and their presence in cases infected by the inoculation blood does not come within the scope of this work. Large scale observations shortly to be undertaken will be reported in more detail particularly observations on the first evolutionary phases of the sporozoite within the organism of the vertebrate host.

Immunopathology of american cutaneous leishmaniasis. Modulation of MHC class II gene products by Keratinocytes before and after glucantime therapy

Epidermal changes from 32 cutaneous and 3 mucosal American leishmaniasis (ACL) active lesions were studied for HLA-DR, -DP expression, Lanerhans cells and lymphocyte infiltration. In addition to a DR and DQ positivity at the surface of the cells of the inflammatory infiltrate, a strong reaction for DR antigens was detected on keratinocytes. Hyperplasia of Langerhans cells was present in al cutaneous lesions and epidermis was infiltrated by T lymphocytes. When healed lesions of 14 of these subjects were re-biopsied 1 to 12 months after the end of pentavalent antimonial therapy, MHC class antigens could no longer be seen on keratinocytes. Our data represrn evidence for hhe reversibility of the abnormal HLA-DR expression by keratinocytes in ACL after Glucantime therapy or spontaneous scar formation, demonstrating that this expresion is restricted to the period of active lesions. The present findings can be regarded as an indirect evidence that keratinocytes may be involved in the immunopathology of ACL.

Effects of azadirachtin in Rhodnius prolixus: data and hypotheses

The effects of azadirachtin A, a tetranortriterpenoid from the neem tree Azadirachta indica J., on both development and interaction between Trypanosoma cruzi, the causative agent of Chagas' disease, and its vector Rhodnius prolixus were studied. Given through a blood meal, a dose-rsponse relationship of azadirachtin was established using antifeedant effect and ecdysis inhibition as effective parameters. A singlo dose of azadirachtin A was able to block the onset of mitosis in the epidermis and ecdysteroid titers in the hemnolymph, determined by radioimmuneassay, were too low for an induction of ecadysis. The survival of T. cruzi was also studied in R. prolixus treated with the drug. If the trypomastigotes were fed in presence of azadirachtin A the number of parasites drastically decreased. If the drug was applied after infection of the bug with T. cruzi, the parasite was still abolished from the gut. If the insect was pretreated with azadirachtin A before infection the same observation was obtained. A single dose of azadirachtin A was enough for a permanent resistance of the insect host against its reinfection with T. cruzi and for blocking the ecdysis for a long time. The effects of azadirachtin A on the hormonal balance of the host and growth inhibition of the parasite will be discussed on the basis of the present results.

The Histopathology of the Infection of Tilapia rendalli and Hypostomus regani (Osteichthyes) by Lasidium Larvae of Anodontites trapesialis (Mollusca, Bivalvia)

It is described the histopathology of the infection of Tilapia rendalli (Osteichthyes, Perciformes, Cichlidae) and Hypostomus regani (Osteichthyes, Siluriformes, Loricariidae) by lasidium larvae of Anodontites trapesialis (Mollusca, Bivalvia, Mycetopodidae). The larvae were encysted within the epidermis of the host, being surrounded by a thin hyaline membrane, 3-6 µm thick, of parasite origin. A proliferative host cell reaction did not occur. The histopathology of the infection shows that the lesions induced by the parasites are minimal. However, the numerous small lesions produced by the release of the larvae may provide optimal conditions for the infection by opportunistic pathogens, namely fungus, which may eventually cause the death of the host.

Comparative analysis of the tissue inflammatory response in human cutaneous and disseminated leishmaniasis

Cutaneous leishmaniasis (CL) is the most frequent clinical form of tegumentary leishmaniasis and is characterised by a single or a few ulcerated skin lesions that may disseminate into multiple ulcers and papules, which characterise disseminated leishmaniasis (DL). In this study, cells were quantified using immunohistochemistry and haematoxylin and eosin staining (CD4+, CD68+, CD20+, plasma cells and neutrophils) and histopathology was used to determine the level of inflammation in biopsies from patients with early CL, late CL and DL (ulcers and papules). The histopathology showed differences in the epidermis between the papules and ulcers from DL. An analysis of the cells present in the tissues showed similarities between the ulcers from localised CL (LCL) and DL. The papules had fewer CD4+ T cells than the DL ulcers. Although both CD4+ cells and macrophages contribute to inflammation in early CL, macrophages are the primary cell type associated with inflammation intensity in late ulcers. The higher frequency of CD20+ cells and plasma cells in lesions demonstrates the importance of B cells in the pathogenesis of leishmaniasis. The number of neutrophils was the same in all of the analysed groups. A comparison between the ulcers from LCL and DL and the early ulcers and papules shows that few differences between these two clinical forms can be distinguished by observing only the tissue.

Adaptations of two specialist herbivores to movement on the hairy leaf surface of their host, Solanum guaraniticum Hassl (Solanaceae)

Plant trichomes can difficult the attachment and movement of small insects. Here, we examine the hypothesis that the success on the use of densely haired hosts by two cassidine species is determined by differential morphology and behavior. Larvae of Gratiana graminea (Klug, 1829) and Gratiana conformis (Boheman, 1854) move on the leaf surface of their host, Solanum guaraniticum Hassl by anchoring their tarsungulus on the trichome rays or by inserting the tarsungulus tip directly into epidermis. This kind of movement is only possible due to a similar tarsungulus shape among the species. Tarsungulus growth pattern is also similar between species, being relatively small on the posterior aperture, matching the diameter of the host plant trichome rays. The tarsungulus shape associated with differences on ontogenetic growth and attachment pattern allow these two Cassidinae larvae to efficiently move on the pubescent leaf surface of their host.