Purpureocillium lilacinum an emergent pathogen: antifungal susceptibility of environmental and clinical strains.

Purpureocillium lilacinum is a filamentous and hyaline fungus cosmopolitan, saprophytic, largely used in the biological control of plant-parasitic nematodes and insects, also considered an emerging and opportunistic human pathogen. The standard treatment for hyalohyphomycosis caused by P. lilacinum is not yet defined, since this fungus is resistant to different antifungals, in vitro and in vivo. The aim of this study was to evaluate and compare in vitro antifungal activity against environmental and clinical P. lilacinum isolates and our results demonstrated that these isolates can be resistant to newer generation triazoles, such as voriconazole, and to caspofungin, a drug of the echinocandin class. In summary, we highlight the importance of knowing the different susceptibility profiles of P. lilacinum isolates, and besides that, the emergence of uncommon human and animal opportunistic fungi, such P. lilacinum, especially during COVID-19, highlight the need for antifungal susceptibility testing of isolates since empirical therapy with different treatment schedules failed in great number of patients.

Intoxicação crônica por cobre em ovinos: conduta para o diagnóstico conclusivo / Chronic copper toxicities in sheep: conduct for diagnostic confirmation

Descreveram-se os sinais clínicos e achados anatomopatológicos da intoxicação crônica por cobre em um ovino da raça Texxel e definiu-se a conduta diagnóstica correta para confirmação da enfermidade. Um ovino foi encaminhado ao setor de patologia com histórico de apatia, hemoglobinúria e morte em dois a três dias. No exame necroscópico, observaram-se icterícia e edema subcutâneo, fígado aumentado de volume e amarelado e rins escuros. No exame histológico, observaram-se necrose zonal aleatória e acentuada no fígado, necrose epitelial tubular, gotas hialinas e cilindros marrom-alaranjados em túbulos coletores dos rins. O histórico alimentar, a sensibilidade de espécie/raça, o quadro clínico, as alterações macroscópicas e microscópicas sugeriram o quadro de intoxicação crônica por cobre. A confirmação diagnóstica somente foi possível após a marcação de pigmentos de cobre pela técnica histoquímica de Ulzmann e pela quantificação de cobre em matéria seca de fígado e rins, cujos valores foram mais altos que o normal.

The present work describes the clinical signs and anatomopathological findings of chronic copper toxicities in a Texxel breed sheep and defines the optimal diagnostic procedure for confirmation of the disorder. A sheep was sent to pathology analysis service with a history of apathy, hemoglobinuria and death within two to three days. Necropsy showed jaundice and subcutaneous edema, enlarged yellow liver and dark kidneys. The histologic examination showed random zonal necrosis, marked necrosis in the liver and tubular epithelial and orange-brown spotted hyaline cylinders in the collecting tubules of the kidneys. The dietary history, sensitivity of species/breed, clinical, macroscopic and microscopic alterations suggested the framework of chronic copper poisoning. Diagnostic confirmation was only possible after staining copper pigments trough the Ulzmann technique and quantification of copper in the dry liver and kidney, which were higher than normal levels.

Ação do fungo Beauveria bassiana associado a gel polimerizado de celulose no controle do carrapato Anocentor nitens em teste de campo / Action of the fungus Beauveria bassiana associated with cellulose polymerized gel in the control of the tick Anocentor nitens in a field test

Foram selecionados 20 equídeos naturalmente infestados por Anocentor nitens, dos quais seus pavilhões auriculares foram submetidos a quatro tratamentos distintos: gel associado a Beauveria bassiana, apenas gel, apenas B. bassiana e um grupo-controle. Procederam-se à contagem e ao registro do número total de fêmeas adultas (>3mm) em cada pavilhão auricular, nos dias 0, 1, 4, 7, 11, 14, 18, 21, 25 e 28, para cálculo do percentual de controle. Foram coletadas e mantidas em laboratório 20 fêmeas pertencentes a cada tratamento, para cálculo dos períodos de pré-postura, postura, incubação e eclosão, e peso médio das posturas. No grupo tratado com o composto gel associado a B. bassiana, observou-se percentual de controle maior que 50 por cento, entre o 4º e o 25º dia, assim como decréscimo progressivo do peso médio da postura das fêmeas coletadas no período entre o 7º e 14º dia. Para os outros tratamentos, o percentual de controle manteve-se abaixo de 20 por cento, e o peso da postura não apresentou diferença significativa. Os percentuais de controle, assim como as reduções no peso médio da postura, sugerem que a utilização do composto gel associado a B. bassiana potencializou a virulência do entomopatógeno.

Twenty horses naturally infected by Anocentor nitens were selected and their auricular pinna were submitted to four treatments: gel associated with Beauveria bassiana, gel only, B. bassiana only, and a control group. The count and the register of the total number of grown up tick females (>3mm) were performed in each auricular pinna on days 0, 1, 4, 7, 11, 14, 18, 21, 25, and 28 for the calculus of percentage of control. Twenty females from each treatment were collected and maintained in laboratory, to calculate the periods of pre-oviposition, oviposition, incubation, and hatching and mean weight of oviposition. In the group treated with the gel associated to B. bassiana, it was observed a control percentage higher than 50 percent from the 4º to the 25º day, such as a progressive decrease of mean weight of the ovipositions in the period from the 7º to 14º day. For the other treatments, the control percentage was kept below 20 percent and the oviposition weight showed no significant difference among the treatments. The control percentages, such as reductions in mean weight of oviposition, suggest that the use of gel associated with B. bassiana potentialized the virulence of the entomopathogen.

Synaptic contacts impaired by styrene-7,8-oxide toxicity.

Styrene-7,8-oxide (SO), a chemical compound widely used in industrial applications, is a potential hazard for humans, particularly in occupational settings. Neurobehavioral changes are consistently observed in occupationally exposed individuals and alterations of neurotransmitters associated with neuronal loss have been reported in animal models. Although the toxic effects of styrene have been extensively documented, the molecular mechanisms responsible for SO-induced neurotoxicity are still unclear. A possible dopamine-mediated effect of styrene neurotoxicity has been previously demonstrated, since styrene oxide alters dopamine neurotransmission in the brain. Thus, the present study hypothesizes that styrene neurotoxicity may involve synaptic contacts. Primary striatal neurons were exposed to styrene oxide at different concentrations (0.1-1 mM) for different time periods (8, 16, and 24 h) to evaluate the dose able to induce synaptic impairments. The expression of proteins crucial for synaptic transmission such as Synapsin, Synaptophysin, and RAC-1 were considered. The levels of Synaptophysin and RAC-1 decreased in a dose-dependent manner. Accordingly, morphological alterations, observed at the ultrastructural level, primarily involved the pre-synaptic compartment. In SO-exposed cultures, the biochemical cascade of caspases was activated affecting the cytoskeleton components as their target. Thus the impairments in synaptic contacts observed in SO-exposed cultures might reflect a primarily morphological alteration of neuronal cytoskeleton. In addition, our data support the hypothesis developed by previous authors of reactive oxygen species (ROS) initiating events of SO cytotoxicity.

Isolation of Beauveria bassiana and Metarhizium anisopliae var. anisopliae from Boophilus microplus tick (Canestrini, 1887), in Rio de Janeiro State, Brazil.

The objective of this work were to isolate and identify strains of entomopathogenic fungi from ingurgitated female Boophilus microplus ticks, collected from the soil in the municipality of Paracambi, Rio de Janeiro State, Brazil. The ingurgitated females were inoculated in the selective medium oat dodine agar (oda), where 49 colonies of Beauveria bassiana (71%) and 20 of Metarhizium anisopliae var. anisopliae (29%) were isolated. These isolated strains characterize for the first time in Brazil the natural occurrence of these species of fungi in this tick, and will be used to conduct bioassays to evaluate the pathogenicity and virulence of these strains for ticks of the genus Boophilus microplus.

Fungal flora of the digestive tract of 5 species of triatomines vectors of Trypanosoma cruzi, Chagas 1909.

A study of the mycobiota in the digestive tract of 5 important species of triatomines, Triatoma brasiliensis, T infestans, T. sordida, T. pseudomaculata and T. vitticeps, was made. The digestive tracts of 164 adults and 535 nymphs of those triatomines were studied and 393 fungal strains were isolated. The genera with the greatest number of species were Penicillium (19 species), Aspergillus (17 species) and Acremonium (5 species) and the most frequent species, in decreasing order, were Penicillium corylophilum, Aspergillus niger, Penicillium felluttanum, Cladosporium herbarum, Penicillium waksmanii, Aspergillus awamori and Paecilomyces variotii. Among the isolated fungi, we found species that are recognized as entomopathogenic and pathogenic for humans and animals.

Adoptive immunotherapy of experimental autoimmune encephalomyelitis via T cell delivery of the IL-12 p40 subunit.

CD4+ T cells are believed to play a central role in the initiation and perpetuation of autoimmune diseases such as multiple sclerosis. In the murine model for multiple sclerosis, experimental autoimmune encephalomyelitis, pathogenic T cells exhibit a Th1-like phenotype characterized by heightened expression of proinflammatory cytokines. Systemic administration of "regulatory" cytokines, which serve to counter Th1 effects, has been shown to ameliorate autoimmune responses. However, the inherent problems of nonspecific toxicity limit the usefulness of systemic cytokine delivery as a potential therapy. Therefore, we used the site-specific trafficking properties of autoantigen-reactive CD4+ T cells to develop an adoptive immunotherapy protocol that provided local delivery of a Th1 cytokine antagonist, the p40 subunit of IL-12. In vitro analysis demonstrated that IL-12 p40 suppressed IFN-gamma production in developing and effector Th1 populations, indicating its potential to modulate Th1-promoted inflammation. We have previously demonstrated that transduction of myelin basic protein-specific CD4+ T cells with pGC retroviral vectors can result in efficient and stable transgene expression. Therefore, we adoptively transferred myelin basic protein-specific CD4+ T cells transduced to express IL-12 p40 into mice immunized to develop experimental autoimmune encephalomyelitis and demonstrated a significant reduction in clinical disease. In vivo tracking of bioluminescent lymphocytes, transduced to express luciferase, using low-light imaging cameras demonstrated that transduced CD4+ T cells trafficked to the central nervous system, where histological analysis confirmed long-term transgene expression. These studies have demonstrated that retrovirally transduced autoantigen-specific CD4+ T cells inhibited inflammation and promoted immunotherapy of autoimmune disorders.

Antigen-specific T cell-mediated gene therapy in collagen-induced arthritis.

Autoantigen-specific T cells have tissue-specific homing properties, suggesting that these cells may be ideal vehicles for the local delivery of immunoregulatory molecules. We tested this hypothesis by using type II collagen-specific (CII-specific) CD4(+) T hybridomas or primary CD4(+) T cells after gene transfer, as vehicles to deliver an immunoregulatory protein for the treatment of collagen-induced arthritis (CIA), a mouse model of rheumatoid arthritis (RA). CII-specific T cells or hybridomas were transduced using retroviral vectors to constitutively express the IL-12 antagonist, IL-12 p40. Transfer of engineered CD4(+) T cells after immunization significantly inhibited the development of CIA, while cells transduced with vector control had no effect. The beneficial effect on CIA of IL-12 p40-transduced T cells required TCR specificity against CII, since transfer of T cells specific for another antigen producing equivalent amounts of IL-12 p40 had no effect. In vivo cell detection using bioluminescent labels and RT-PCR showed that transferred CII-reactive T-cell hybridomas accumulated in inflamed joints in mice with CIA. These results indicate that the local delivery of IL-12 p40 by T cells inhibited CIA by suppressing autoimmune responses at the site of inflammation. Modifying antigen-specific T cells by retroviral transduction for local expression of immunoregulatory proteins thus offers a promising strategy for treating RA.

The entomopathogenic potential of Aspergillus spp. in mosquitoes vectors of tropical diseases.

Eleven strains of the most frequent Aspergillus species found in a survey of Brazilian mosquitoes collected in the states of Minas Gerais and Rio de Janeiro, Brazil, were used for bioassays in second-stage larvae of Aedes fluviatilis and Culex quinquefasciatus. Aspergillus ochraceus, A. kanagawaensis and one strain of A. sulphureus were most effective, causing mortality in at least 80% of the larvae of the two mosquito species tested. Variations in entomopathogenic capacity were observed in the experiments with strains of A. sulphureus, A. flavus and A. ochraceus.

A novel transcription factor, T-bet, directs Th1 lineage commitment.

Naive T helper cells differentiate into two subsets, Th1 and Th2, each with distinct functions and cytokine profiles. Here, we report the isolation of T-bet, a Th1-specific T box transcription factor that controls the expression of the hallmark Th1 cytokine, IFNgamma. T-bet expression correlates with IFNgamma expression in Th1 and NK cells. Ectopic expression of T-bet both transactivates the IFNgamma gene and induces endogenous IFNgamma production. Remarkably, retroviral gene transduction of T-bet into polarized Th2 and Tc2 primary T cells redirects them into Th1 and Tc1 cells, respectively, as evidenced by the simultaneous induction of IFNgamma and repression of IL-4 and IL-5. Thus, T-bet initiates Th1 lineage development from naive Thp cells both by activating Th1 genetic programs and by repressing the opposing Th2 programs.

Gene therapy for autoimmune disease.

Autoantigen-specific CD4(+) T lymphocytes have been implicated in the pathogenesis of autoimmune diseases. Tissue-specific homing properties of autoantigen-specific CD4(+) T cells suggested that these cells might be ideal vehicles for delivery of retroviral-encoded regulatory proteins in a site-specific manner as a therapy for autoimmune diseases. Application of retroviral transduction of autoantigen-reactive CD4(+) T cells in gene therapy of autoimmunity must include systems capable of targeting these rare populations of antigen-activated T cells. Studies discussed below suggest that retroviral transduction of autoantigen-specific murine CD4(+) T cells may provide a method to target and isolate nontransformed autoantigen-specific murine CD4(+) T cells and provide a rational approach to gene therapy in animal models of autoimmunity.

Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease.

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.

Pathogenic action of Penicillium species on mosquito vectors of human tropical diseases.

In vitro bioassays were performed in order to assess the pathogenicity of 13 Penicillium strains in 2nd stage larvae of Aedes aegypti, Aedes fluviatilis, Anopheles aquasalis and Culex quinquefasciatus. Mortality rates began in the first 24 hours, ranging from 0 to 100%. P. corylophilum, P. fellutanum, P. implicatum, P. janthinellum, P. viridicatum and P. waksmanii species tested on Aedes aegypti larvae and at different concentrations presented mortality rates from 0 to 6.6%. These species, when tested on Aedes fluviatilis, Anopheles aquasalis and Culex quinquefasciatus larvae, presented a mortality rate of 3.33% to 100%. Susceptibility of Aedes fluviatilis, Aedes aegypti, Anopheles aquasalis and Culex quinquefasciatus mosquitoes to the tested agents, turn P. corylophilum and P. janthinellum into candidates for potential use in biological control of vectors.

Penicillium species in mosquitoes from two Brazilian regions.

Penicillium species were isolated from 1073 adult mosquitoes and larvae of Aedes spp., Anopheles spp., Culex spp. and Mansonia spp. captured in the northern and southeastern regions of Brazil. In a total of 24 collections, 198 Penicillium strains were identified in 13 species: Penicillium canescens, P. chrysogenum, P. citrinum, P. corylophilum, P. decumbens, P. expansum, P. fellutanum, P. implicatum, P. janthinellum, P. oxalicum, P. purpurogenum, P. viridicatum and P. waksmanii. The total isolation frequency of the Penicillium species in the investigated mosquitoes was: Anopheles spp. (51.5%), Aedes spp. (15.1%), Culex spp. (23.7%) and Mansonia spp. (10.1%). The Penicillium species with the highest incidence in the mosquito genera were: P. corylophilum, P. janthinellum, P. fellutanum and P. waksmanii. The highest number of mosquitoes were captured in the state of Rio de Janeiro, followed by Rondônia and Minas Gerais.

Site-directed mutagenesis of double-stranded DNA by the polymerase chain reaction.

We have developed a facile procedure for rapid PCR-based site-directed mutagenesis of double-stranded DNA. Increasing the initial template concentration and decreasing the PCR cycles to 5-10 allows us to reduce the rate of undesired second-site mutations and dramatically increase the time savings. Following PCR, DpnI treatment is used to select against parental DNA molecules. The DpnI (target sequence 5'-Gm6ATC) is specific for methylated and hemimethylated DNA and is used to digest parental DNA and select for mutation-containing amplified DNA. DNA isolated from almost all common Escherichia coli strains is Dam methylated and therefore susceptible to DpnI digestion. Pfu DNA polymerase is used, prior to intramolecular ligation of the linear template, to remove any bases extended onto the 3' ends of the PCR product by Taq DNA polymerase. The recircularized vector DNA incorporating the desired mutations is transformed into E. coli. This method can be used independently of any host strain and vector.

Cloning and analysis of PCR-generated DNA fragments.

Methods are presented for the improved yield and analysis of blunt-ended cloning of PCR-generated DNA fragments. We show that Pfu DNA polymerase polishing of Taq DNA polymerase-generated fragments increases the yield and efficiency of cloning. Using a triple primer set consisting of two outside, asymmetrically distanced primers and one fragment-specific primer, both the presence and orientation of cloned inserts can be determined. Application of these methods allows the generation and cloning of a fragment in 1 day and the analysis of putative clones the next, thereby saving a substantial amount of both time and effort.