Local adaptation to salinity in the three-spined stickleback?

Different lines of evidence suggest that the occurrence and extent of local adaptation in high gene flow marine environments - even in mobile and long-lived vertebrates with complex life cycles - may be more widespread than earlier thought. We conducted a common garden experiment to test for local adaptation to salinity in Baltic Sea sticklebacks (Gasterosteus aculeatus). Fish from three different native salinity regimes (high, mid and low) were subjected to three salinity treatments (high, mid and low) in a full-factorial experimental design. Irrespective of their origin, fish subjected to low (and mid) salinity treatments exhibited higher juvenile survival, grew to largest sizes and were in better condition than fish subjected to the high salinity treatment. However, a significant interaction between native and treatment salinities - resulting mainly from the poor performance of fish native to low salinity in the high salinity treatment - provided clear cut evidence for adaptation to local variation in salinity. Additional support for this inference was provided by the fact that the results concur with an earlier demonstration of significant differentiation in a number of genes with osmoregulatory functions across the same populations and that the population-specific responses to salinity treatments exceeded that to be expected by random genetic drift.

Evidence for adaptive phenotypic differentiation in Baltic Sea sticklebacks.

The evidence for adaptive phenotypic differentiation in mobile marine species remains scarce, partly due to the difficulty of obtaining quantitative genetic data to demonstrate the genetic basis of the observed phenotypic differentiation. Using a combination of phenotypic and molecular genetic approaches, we elucidated the relative roles of natural selection and genetic drift in explaining lateral plate number differentiation in threespine sticklebacks (Gasterosteus aculeatus) across the entire Baltic Sea basin (approximately 392 000 km(2) ). We found that phenotypic differentiation (PST  = 0.213) in plate number exceeded that in neutral markers (FST  = 0.008), suggesting an adaptive basis for the observed differentiation. Because a close correspondence was found between plate phenotype and genotype at a quantitative trait loci (QTL; STN381) tightly linked to the gene (Ectodysplasin) underlying plate variation, the evidence for adaptive differentiation was confirmed by comparison of FST at the QTL (FSTQ  = 0.089) with FST at neutral marker loci. Hence, the results provide a comprehensive demonstration of adaptive phenotypic differentiation in a high-gene-flow marine environment with direct, rather than inferred, verification for the genetic basis of this differentiation. In general, the results illustrate the utility of PST -FST -FSTQ comparisons in uncovering footprints of natural selection and evolution and add to the growing evidence for adaptive genetic differentiation in high-gene-flow marine environments, including that of the relatively young Baltic Sea.

Phylogeography of isolated freshwater three-spined stickleback Gasterosteus aculeatus populations in the Adriatic Sea basin.

Analyses of mitochondrial (mt) DNA and microsatellite variation were carried out to examine the relationships between 10 freshwater populations of three-spined sticklebacks Gasterosteus aculeatus along the eastern coast of the Adriatic Sea. Partial sequences of the mtDNA control region and cytochrome b gene, in addition to 15 microsatellite loci, were used to analyse populations from four isolated river catchments. Results uncovered an Adriatic lineage that was clearly divergent from the European lineage, and confirmed that the most divergent and ancient populations are located within the Adriatic lineage as compared with other European populations. Two northern Adriatic populations formed independent clades within the European mitochondrial lineage, suggesting different colonization histories of the different Adriatic populations. Nuclear marker analyses also indicated deep divergence between Adriatic and European populations, albeit with some discordance between the mtDNA phylogeny of the northern Adriatic populations, further highlighting the strong differentiation among the Adriatic populations. The southern populations within the Adriatic lineage were further organized into distinct clades corresponding to respective river catchments and sub-clades corresponding to river tributaries, reflecting a high degree of population structuring within a small geographic region, concurrent with suggestions of existence of several microrefugia within the Balkan Peninsula. The highly divergent clades and haplotypes unique to the southern Adriatic populations further suggest, in accordance with an earlier, more limited survey, that southern Adriatic populations represent an important reservoir for ancient genetic diversity of G. aculeatus.

Idiopathic pulmonary hemosiderosis with cystic lesions: a rare presentation.

This report describes a case of a 49-year-old man with cough, recurrent hemoptysis, and dyspnea during 18 months, presenting with radiological findings of alveolar infiltrate and cystic lesions in left upper lobe. Laboratory studies revealed normocytic hypochromic anemia and normal coagulation tests. C-reactive protein and mucoproteins were negative. Serum protein electrophoresis and complement, urinalysis, serum creatinine, creatinine clearance, and 24-hour urine protein were normal. Tests for antineutrophil cytoplasmic antibodies and anti-glomerular-basement membrane antibodies were negative. Tests for connective tissue diseases were all negative. Histological findings were consistent with those of idiopathic pulmonary hemosiderosis. Radiological findings are discussed.

Effect of high laryngeal mask airway intracuff pressure on the laryngopharyngeal mucosa of dogs.

OBJECTIVES: To evaluate the effect of increased of laryngeal mask airway (LMA) intracuff pressures on the laryngopharyngeal mucosa. STUDY DESIGN: Animal model. METHODS: Sixteen mixed-breed dogs were randomly allocated to two groups, G1 (intracuff volume, 30 mL; n = 8) and G2 (intracuff volume, 54 mL; n = 8), to produce, respectively, high or very high intracuff pressures. Anesthesia was induced and maintained with pentobarbital. Intracuff pressures were measured immediately after insertion and inflation of a No. 4 laryngeal mask airway (LMA) and 30, 60, 90, and 120 minutes thereafter. The dogs were euthanized, and biopsy specimens from eight predetermined areas of the laryngopharynx in contact with LMA cuff were collected for light microscopic (LM) and scanning electron microscopic (SEM) examination. RESULTS: Initial LMA cuff inflation in G1 and G2 resulted in intracuff pressures of 119 mm Hg +/- 4 mm Hg and 235 mm Hg +/- 13 mm Hg, respectively. Over a 2-hour period, the intracuff pressure decreased significantly in G1 (P < .001) and G2 (P < .01), and there was a significant difference between the groups over time (P < .001). The LM study of laryngopharyngeal mucosa in both groups showed mild congestion in the subepithelial layer. There were no differences between the groups (P > .10) or among the areas sampled (P > .10). In some areas of G2, the SEM study showed epithelial desquamation that was more intense than that in G1. CONCLUSIONS: The increase in LMA intracuff pressure caused only mild alterations in the laryngopharyngeal mucosa of the dog.

Experimental paracoccidioidomycosis of the Syrian hamster: fungicidal activity and production of inflammatory cytokines by macrophages.

Phagocytic cells play an important role in nonspecific resistance to fungal infection by mediating an inflammatory response and by a direct fungicidal action. In this study, the functional activity of peritoneal macrophages obtained from hamsters experimentally infected with strain Pb18 of Paracoccidioides brasiliensis was evaluated during 16 weeks of infection. The results showed that macrophages had a higher spreading ability associated with increased production of tumor necrosis factor alpha (TNF-alpha) and enhanced fungicidal activity during the early periods of infection. TNF-alpha levels remained elevated during all periods studied, while low levels of interleukin-1 beta (IL-1 beta) were produced during the infection. A necrotic area with dead fungi was observed at the inoculation site and the infection disseminated only to liver and lymph nodes in a few animals. These results suggest that during the early stages of infection with P. brasiliensis, macrophage activation by the high levels of TNF-alpha limited fungal dissemination. In contrast, in the later stages of infection, high levels of TNF-alpha were observed while the fungicidal activity of macrophages was lower and the animals presented loss of vitality resulting in their death. These observations suggest a complex role of TNF-alpha in experimental paracoccidioidomycosis of Syrian hamsters, involving not only resistance but also pathogenesis.

Study of pulmonary experimental paracoccidioidomycosis by analysis of bronchoalveolar lavage cells: resistant vs. susceptible mice.

Adult Swiss (susceptible) and BALB/c (non-susceptible) mice were inoculated by the intravenous route with 1 x 10(6) yeast cells of Paracoccidioides brasiliensis, strain 18. Immunologic parameters, histopathology and features of the bronchoalveolar lavage (BAL) were evaluated at week 2, 4, 8 and 16 post-infection. The pulmonary infection was progressive in Swiss mice and regressive in Balb/c mice. The numbers of total cells, lymphocytes and polymorphonuclear neutrophils increased in BAL, as well as the percentages of giant cells, and CD4 and CD8 positive cells. The ultrastructural study of BAL cells revealed a predominance of macrophages and a frequency of 13.2% of type II pneumocytes. As the infection progressed, the number of fungal cells and spreading macrophages, as well as the stimulated release of H2O2 by macrophages, increased. The animals exhibited an exacerbation of the humoral immune response and a depression of cellular immunity during the infection. There was a good correlation between the intensity and the pattern of the pulmonary histopathology and the cellular findings in the BAL. The present model reproduces some anatomoclinical patterns of the human disease and shows that BAL may be a useful tool in monitoring the pulmonary infection caused by P. brasiliensis.

Pathogenicities and GP43kDa gene of three Paracoccidioides brasiliensis isolates originated from a nine-banded armadillo (Dasypus novemcinctus).

We studied three different isolates of Paracoccidioides brasiliensis obtained from the mesenteric lymph node (D3LY1), the spleen (D3S1) and the liver (D3LIV1) of the same armadillo (Dasypus novemcinctus). Pulmonal inflammatory area was evaluated by intravenous inoculation of 10(6) yeast cells of each isolates in young, male, ddY mice. Moreover, the partial sequence of GP43kDa gene of P. brasiliensis was analyzed. The lung inflammatory area was greater in animals inoculated with isolate D3S1. The partial sequence of GP43kDa gene indicated that isolate D3S1 is different from isolates D3LY1 and D3LIV1. This study suggested that the same armadillo might be susceptible to multiple P. brasiliensis isolates simultaneously.

Seminoma de mediastino relato de um caso e revisäo da literatura / Mediastinal seminoma case report and review of the literature

Oa autores relatam caso de seminoma primário de mediastino em criança de 13 anos, do sexo masculino, sem sintomas clínicos. Exame tomográfico revelou massa cística no mediatino anterior de aproximadamente 10cm de diâmetro. Foi realizad cirurgia , com ressecçäo de tumor medindo 13x7x6cm, a maior parte representada por área cística unilocular. Microscopicamente, tratava-se de seminoma clássico, que, à semelhança dos originados nos testículos, apresentava hiperplasia linfóide e granulomas epitelióides; ao lado disso, havia formaçäo de corpúsculos de Hassal abortivos associados a intensa hiperplasia epitelial, cuja intensidade näo é relatada na literatura. Estes últimos aspectos sugerem fortemente que o seminoma tenha tido origem intratímica. Os autores chamam ainda a atençäo para a dificuldade de diagnóstico destes casos na biópsia de congelaçäo, podendo ser confundidos com timoma

Pulmonary paracoccidioidomycosis in immunized mice.

Paracoccidioidomycosis was induced in immunized (IM) and non-immunized (NI) mice. The histopathology, the number of fungi in the lungs, the cellular (footpad test--FPT and macrophage inhibition factor assay--MIF) and humoral (immunodiffusion test) immune response were investigated serially post-infection. In the IM mice, at days 1 and 3, there was intense and predominant macrophagic-lymphocytic alveolitis with loose granulomatous reaction; at day 30, inflammation was mild. In the NI group, up to day 3, the lesions were focal; later there was formation of extensive epithelioid granuloma. The number of fungi in IM mice were always smaller than those of NI group. Immunization alone induced positive FPT and MIF indices with low titer of antibody. After infection, there was a significant decrease of the FPT indices in the IM group, which we interpreted as desensitization due to trapping of sensitized lymphocytes in the lungs. In conclusion, (1) The lesional pattern of pulmonary paracoccidioidomycosis in IM mice was similar to that of a hypersensitivity pneumonitis. This reaction was probably effective in reducing the extension of the infection and decrease the number of fungi. (2) In this model, pulmonary resistance against P. brasiliensis seems to be related to local and systemic delayed-type hypersensitivity reaction.

Immunohistopathology of murine pulmonary histoplasmosis during normal and hypersensitive conditions.

Pulmonary histoplasmosis was induced in nonimmunized and immunized Balb-C nu/+ mice. The lung tissue burden of H. capsulatum, histopathology, the size of the inflammatory area, and the numbers of total T lymphocytes and subtypes in situ were evaluated serially after challenge. Over 3 days previously immunized mice developed a large lymphocyte/macrophage inflammatory response. This rapidly decreased in the next 2 wk. In contrast, the nonimmunized control mice developed a predominantly polymorphonuclear infiltrate that evolved more slowly over the first week of infection. This initial response was nonspecific but, after the first week, shifted to lymphocytes and granuloma formation. The lymphocyte infiltration in both immunized and nonimmunized mice was predominantly CD4. Previously immunized mice had a rapid decrease in tissue counts of H. capsulatum after Day 3, but nonimmunized mice continued to have increased counts through Day 7 after infection. These studies reproduce in the mouse model a host response similar to acute reinfection histoplasmosis in the human. In this condition an intense cell-mediated inflammatory response is thought to be elicited by fungal antigens and to represent host reaction rather than fungal replication. Our experimental model added new information relevant to the understanding of the pathogenesis of this process.

Pulmonary aspergillosis in mice: treatment with a new triazole SCH39304.

The triazole SCH39304 was compared with amphotericin B and fluconazole for the treatment of pulmonary aspergillosis in corticoid-immunosuppressed mice intranasally challenged with 5 x 10(6) conidia of Aspergillus fumigatus. In vitro, the minimum inhibitory concentration (MIC) for fluconazole was greater than 80 micrograms/ml, for SCH39304 40 micrograms/ml, and for amphotericin B 0.29 micrograms/ml. Beginning 1 day after challenge, groups of 10 mice were treated orally, twice daily, for 15 days with Noble agar (control), SCH39304, fluconazole, or amphotericin B at various doses. For lung tissue counts of A. fumigatus, mice were similarly challenged and treated only for 5 days with SCH39304, fluconazole, or amphotericin B. Only SCH39304 significantly reduced the number of A. fumigatus in the lung. SCH39304 at doses of 5 mg/kg or higher significantly prolonged the survival of mice, as did amphotericin B at 3 mg/kg. Fluconazole did not significantly prolong survival at doses of 15 or 30 mg/kg. SCH39304 appears to be as effective as amphotericin B in murine pulmonary aspergillosis and warrants further evaluation for aspergillosis in humans.

Treatment of chronic murine chromoblastomycosis with the triazole SCH39304.

Congenitally athymic (nu/nu) mice were inoculated sc with 10(5) conidia of Fonsecaea pedrosoi and treated orally from the 1st to the 16th week of infection with either a new triazole, SCH39304, or itraconazole at doses of 20 or 60 mg/kg/day. The volumes of the lesions were measured with calipers at 4 week intervals and compared statistically by the Wilcoxon test. At the end of the experiment, mice were killed and samples of the lesions were examined histopathologically and by electron microscopy. Treatment with itraconazole or SCH39304 significantly reduced lesion sizes as compared with controls. There were no differences between the 2 drugs at the dosages used. Histopathologically, lesions of mice treated with either drug had less inflammation with fewer fungi and more diffuse fibrosis than controls. Electron microscopy showed damage to the fungal cell walls in mice treated with itraconazole or SCH39304, characterized by gaps, fragmentation, and delamination. These studies confirm clinical observations that itraconazole is effective in chromoblastomycosis and suggest that SCH39304 should be considered for clinical evaluation.

Treatment of murine coccidioidal meningitis with SCH39304.

The triazole SCH39304 was compared with itraconazole and fluconazole for treatment of murine coccidioidal meningitis. Mice were treated for 30 days with 1, 5, 10, or 30 mg of each drug per kg of body weight. Survival and brain tissue counts were measured. At equivalent doses, SCH39304 was more effective than the other triazoles.

Hypersensitivity pneumonitis to Paracoccidioides brasiliensis antigens in mice.

Hypersensitivity pneumonitis was induced in mice immunized with Paracoccidioides brasiliensis and challenged, one week later, with soluble (SPbAg) or particulate (PPbAg) antigen (formalin-killed yeast cells), administered by the intratracheal route. Between 24 and 48 h post-challenge, animals developed an interstitial and intra-alveolar pneumonitis. Macrophages and lymphocytes arranged focally into loose or mature granulomata were observed by light and electron microscopy. The distribution and fate of antigens was studied by immunofluorescence. Three hours after challenge with SPbAg the lungs showed linear fluorescent deposits, whereas after challenge with PPbAg the pattern was globular, corresponding to the particulate antigen. After 24 and 48 h, the pattern was diffuse and finely granular in both groups, with a decreasing number of animals showing detectable fluorescence. Immunization induced a positive footpad swelling test (FPT) in all animals. After pulmonary challenge, there was a significant decrease in FPT indices, interpreted as desensitization due to trapping of specifically sensitized lymphocytes in the lungs. In conclusion, immunization induced a marked cellular immune response, the inflammatory pattern and the tempo of the induced pneumonitis being compatible with delayed hypersensitivity in the lungs. Immunized mice also cleared the injected antigens rapidly. These data suggest that hypersensitivity pneumonitis may be an expression of pulmonary resistance to infection with P. brasiliensis.

Histological and ultrastructural study of the inflammation evoked by Paracoccidioides brasiliensis antigen in previously immunized mice.

Bentonite particles uncoated and coated with soluble antigen of Paracoccidioides brasiliensis (Pb) were intravenously injected into mice with and without previous immunization with Pb antigen. The inflammatory reaction around the bentonite emboli in small lung vessels was quantitated and morphologically studied by light and electron (EM) microscopy, 2 to 8 days after challenge. In control nonimmunized animals, coated and uncoated bentonite particles caused mild and nonspecific inflammation made up by macrophages. By EM, they formed loosely aggregated clusters with cytoplasm containing few organelles and borders without interdigitation. In immunized mice injected with coated bentonite particles, the inflammatory area was significantly greater than that in nonimmunized animals in all periods of study with maximum difference at day 2. The inflammatory process at days 2 and 4 was characterized as mature granulomata, composed of macrophages with great number of organelles in the cytoplasm, large euchromatic nuclei and prominent nucleoli. Altogether these findings indicated a lesion with high metabolic activity, compatible with a granulomatous hypersensitivity reaction. At days 6 and 8, there was a change from mature to epithelioid granulomata, well demonstrated by EM which showed macrophages with characteristically interdigitated cytoplasmic borders. The results strengthen the importance of cellular immunity in the genesis of epithelioid granuloma in paracoccidioidomycosis and reinforce the usefulness of the present model in studies of the inflammatory cellular sequency and events in this mycosis.

Skin and pulmonary models using coated bentonite particles for the study of the inflammation evoked by Paracoccidioides brasiliensis antigens in previously immunized mice.

Bentonite particles coated with polysaccharide antigen or crude soluble antigen of Paracoccidioides brasiliensis were injected intradermally or intravenously in mice. In control animals that were not pre-immunized with P. brasiliensis antigens, coated and uncoated bentonite caused minimal and nonspecific inflammation around the cutaneous injection site or around the bentonite thrombi in small lung vessels after intravenous injection. However, in mice previously immunized with P. brasiliensis antigens, the coated bentonite particles boosted the humoral and cellular immune responses to P. brasiliensis and evoked intense inflammatory reactions. Twelve days after intradermal injection, the inflammatory reaction around the bentonite was rich in neutrophils, macrophages, lymphocytes and plasma cells associated with young granulation tissue. In intravenously injected mice, the pulmonary inflammation was maximal at day 2, and was characterized by a florid neutrophilic and macrophagic cellular infiltration around bentonite thrombi; in some foci, there was incipient organization to mature granuloma. However, in both models, there was no formation of epithelioid granulomata, demonstrating that in paracoccidioidomycosis cellular immunity alone, without the presence of intact micro-organisms, may not be enough for the development of this type of granuloma.

Experimental pulmonary paracoccidioidomycosis in mice: morphology and correlation of lesions with humoral and cellular immune response.

The present paper describes a murine model for pulmonary paracoccidioidomycosis injecting 6 X 10(5) yeast forms of Paracoccidioides brasiliensis (Pb) by the direct intratracheal route. The sequential histopathology of lung and dissemination lesions together with humoral (immunodiffusion test) and cellular immune response (footpad test and macrophage inhibition factor assay - MIF assay) were investigated since the 1st to the 360th day after infection. All infected animal showed pulmonary Pbmycosis up to Day 30; onwards the lesions subsided being found only in one mouse at Day 360. Dissemination lesions were observed in paratracheal and cervical lymph nodes in 9 out of 68 infected animals. Histologically early lesions were rich in polymorphonuclear cells and evolved to a macrophage desquamative pneumonitis at Day 15 and to typical epithelioid granulomata from Day 30 up to Day 360. Specific precipitating antibodies were first detected 15 days after infection, peaked from Day 30 to 60 and were not observed at Day 360. Significant cell-mediated immunity to Pb was noted at Day 15 with the peak reaction at Day 60 and 90. The intratracheal route represents a highly effective way of infecting mouse with Pb. This experimental pulmonary Pbmycosis is a granulomatous inflammation which courses with specific humoral and cellular immune response. It may be a good tool for further investigation in the pathogenesis and natural history of the disease.