A new acridone with antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans.

AIM: The increase in the number of fungal infections worldwide, coupled with the limitations of current antifungal chemotherapy, demand the development of safe and effective new antifungals. Here, we presented the synthesis of a novel acridone (M14) and its antifungal properties against Candida and dermatophytes species. METHODS AND RESULTS: A series of 17 acridones was designed, synthesized and tested for its antifungal activity. The minimum inhibitory concentration (MIC) was determined by the broth microdilution method. Only the acridone M14 showed growth-inhibitory activity against reference strains and clinical isolates of Candida and dermatophytes, with MIC range of 7·81-31·25 µg ml-1 . Moreover, M14 exhibited fungicidal activity and prevented biofilm formation by C. albicans as well as reduced the viability of preformed biofilms, even at sub-MICs. The confocal laser scanning microscopy analysis revealed that C. albicans hyphal growth was completely inhibited in the presence of M14. Similarly, there was a severe inhibition on hyphal growth of Trichophyton rubrum. We also found that M14 has relatively low toxicity to human fibroblasts. CONCLUSIONS: The new acridone M14 has antifungal properties against Candida spp. and dermatophytes, and antibiofilm activity against C. albicans. In addition, M14 is relatively selective to fungal cells compared to human normal cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of its in vitro antifungal activity, anti-Candida biofilm effect and moderate cytotoxicity towards normal human cell, M14 may serve as a valuable lead compound to develop a new antifungal agent.

Detection and Molecular Characterization of Yellow Fever Virus, 2017, Brazil.

At the end of 2016, Brazil experienced an unprecedented yellow fever (YF) outbreak. Clinical, molecular and ecological aspects of human and non-human primate (NHP) samples collected at the beginning of the outbreak are described in this study. Spatial distribution analyses demonstrated a strong overlap between human and NHP cases. Through molecular analyses, we showed that the outbreak had a sylvatic origin, caused by the South American genotype 1 YFV, which has already been shown to circulate in Brazil. As expected, the clusters of cases were identified in regions with a low vaccination coverage. Our findings highlight the importance of the synchronization of animal surveillance and health services to identify emerging YF cases, thereby promoting a better response to the vulnerable population.

First fatal case of CNS infection caused by Enterovirus A in Brazil.

We describe what is to our knowledge the first fatal case of central nervous system Enterovirus infection in Brazil. Molecular and phylogenetic characterization revealed that Enterovirus A was the aetiologic agent of this case.

Outbreak of herpangina in the Brazilian Amazon in 2009 caused by Enterovirus B.

In October 2009, our laboratory was contacted by a Brazilian Public Health organization regarding a severe community outbreak of an acute exanthematic and febrile disease in the Brazilian Amazon that primarily affected children. A total of 44 patients with febrile disease were identified by the local public health system, 37 of whom were children between 1 and 9 years of age. Molecular virological and phylogenetic characterization revealed that enterovirus B was the etiological agent of this outbreak, which was characterized by a clinical presentation known as herpangina.

Identification and antifungal susceptibility of fungi isolated from dermatomycoses.

BACKGROUND: Dermatomycoses are superficial fungal infections of the skin, hair and nails that affect more than 20-25% of the people worldwide. These infections can be caused by yeasts, dermatophytes and non-dermatophyte filamentous fungi (NDFF) and are considered a public health problem. Despite this, few studies have investigated the prevalence and antifungal susceptibility of causative agents of dermatomycoses in the developing world. OBJECTIVES: The aims of this study were to identify and determine the antifungal susceptibility profile of yeast and filamentous fungi isolated from dermatomycoses in Uberaba, Minas Gerais, Brazil. METHODS: Specimens were obtained from patients with clinically diagnosed and laboratory confirmed dermatomycosis between July 2009 and July 2011. Fungal identification was based on classical methods and antifungal susceptibility testing was performed by broth microdilution method. RESULTS: Of the 216 fungal isolates, 116 (53.8%) were yeasts, 70 (32.4%) dermatophytes and 30 (13.8%) NDFF. Onychomycosis was the most common clinical condition. Candida parapsilosis (24.1%) and Trichophyton rubrum (17.1%) were the fungi most frequently isolated. Voriconazole, ketoconazole and itraconazole were the most potent antifungal agents against yeast, whereas terbinafine, voriconazole and itraconazole had a high in vitro activity against dermatophytes. Overall, the antifungal agents had little or no activity against NDFF and the highest minimum inhibitory concentrations were those against Fusarium spp. CONCLUSION: Yeasts, particularly C. parapsilosis, play an important role as causative agents of dermatomycosis in our region. Our results suggest that the antifungal susceptibility testing coupled with proper identification of the fungi may be useful to assist clinicians in determining the appropriate therapy for dermatomycoses.

Epidemiological and molecular characterization of rubella virus isolated in São Paulo, Brazil during 1997-2004.

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997-2004 were isolated in cell culture and genotyped. Twenty-eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil.

Hospital-acquired human bocavirus in infants.

Human bocavirus (HBoV) is a respiratory pathogen that affects young children. We screened 511 nasopharyngeal aspirates for hospital-acquired HBoV from infants hospitalised with respiratory infection from January to December 2008. Among 55 children with HBoV infection, 10 cases were hospital-acquired. Compared with the community-acquired cases, coinfection with other respiratory viruses in these patients was uncommon. HBoV should be considered for inclusion in screening protocols for nosocomial childhood respiratory infections, especially in intensive care units.

In vitro anti-rotavirus activity of some medicinal plants used in Brazil against diarrhea.

Acute diarrhea, especially in children, is a very common disease with worldwide distribution and with a significant public health impact. Rotaviruses have been recognized as the major agents of diarrhea in infants and young children in developed as well as developing countries. In Brazil, diarrhea is one of the principal causes of death, mainly in the infant population. To fight diarrhea, traditional Brazilian medicine uses a great variety of plants. In this work, 12 medicinal plant species were screened for simian (SA-11) and human (HCR3) rotaviruses inhibition in vitro. At non-cytotoxic concentrations, the extracts from Artocarpus integrifolia L. (Moraceae) bark (480 microg/ml) and Spondias lutea L. (Anacardiaceae) leaves (160 microg/ml) had antiviral activity against both viruses. They showed inhibition of 99.2% and 97%, respectively, for human rotavirus, and 96.4% and 96.2% for simian rotavirus. The extracts from Myristica fragrans Houtt (Myristicaceae) seeds (160 microg/ml) and Spongias lutea bark (40 microg/ml) inhibited human rotavirus (90% and 82.2% inhibition, respectively), whereas the extracts from Anacardium occidentale L. (Anacardiaceae) leaves (4 microg/ml) and Psidium guajava L. (Myrtaceae) leaves (8 microg/ml) showed activity only against simian rotavirus (82.2% and 93.8% inhibition, respectively). Our results indicate that the extracts of Artocarpus integrifolia, Myristica fragrans and Spongias lutea can be useful in the treatment of human diarrhea if the etiologic agent is a rotavirus.

A role for alphabeta T cells in the resistant phase of the Brown Norway rat model of vasculitis.

Administration of mercuric chloride (HgCl(2)) to Brown Norway rats causes Th2 dominated autoimmunity including a caecal vasculitis. Disease peaks 14 days after starting HgCl(2) after which animals immunoregulate spontaneously. In a third phase, if animals are rechallenged with HgCl(2) 6 weeks later they appear resistant, developing only attenuated disease. Previous studies suggested a role for CD8(+) cells as partial mediators of resistance but no groups had studied the role of alphabeta T cells, gammadelta T cells or natural killer (NK) cells in resistance. We used adoptive transfer and in vitro cell depletion to show that alphabeta T cells are also partially responsible for resistance. Donor animals were treated with HgCl(2) or saline and killed 21 days later. Cells from donor spleens were transferred into recipient animals which were challenged with HgCl(2) and killed 14 days later. Test recipients received spleen cells from HgCl(2)-treated donors after in vitro depletion of one subset of cells. Recipients receiving spleen cells from saline-treated donors remained susceptible to HgCl(2)-induced vasculitis; those receiving spleen cells from HgCl(2)-treated donors were resistant. Animals receiving alphabeta T-cell-depleted spleen cells from HgCl(2)-treated donors showed partial reversal of resistance. Our results suggest a role for alphabeta T cells in the resistant phase of the Brown Norway rat model of vasculitis.

Resistance to re-challenge in the Brown Norway rat model of vasculitis is not always complete and may reveal separate effector and regulatory populations.

Administration of mercuric chloride to Brown Norway rats results in T helper type 2 (Th2)- dominated autoimmunity characterized by high immunoglobulin E (IgE) concentrations, the production of multiple IgG autoantibodies, including those to glomerular basement membrane (GBM), arthritis and caecal vasculitis. After 14 days animals immunoregulate and auto-immunity resolves even if mercuric chloride injections are continued. In a third phase, if animals are re-challenged with mercuric chloride 6 weeks later, they show only attenuated autoimmunity with lower anti-GBM antibody concentrations and arthritis scores. Resistance to the induction of anti-GBM antibodies can also be achieved following an initial challenge with low-dose (one-tenth standard dose) mercuric chloride. We have now studied this resistant phase in more detail. We have shown, first, that following an initial full-dose mercuric chloride challenge, resistance also affects susceptibility to caecal vasculitis. Second, following an initial full-dose mercuric chloride challenge, the IgE response upon re-challenge is initially accelerated but subsequently enters a resistant phase and third, following an initial challenge with low-dose mercuric chloride, resistance is also seen to the induction of caecal vasculitis but is not seen in IgE serology (where results suggest competing effector and regulatory cell populations). Studying such regulatory phases in animal models of autoimmunity may be of benefit in the future in designing new therapies for human vasculitis.

Detecção do gene da nucleoproteína do vírus da cinomose canina por RT-PCR em urina de cães com sinais clínicos de cinomose / Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

A presença do vírus da cinomose canina (CDV) foi avaliada pela reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR), em 87 amostras de urina de cães que apresentavam sinais clínicos sugestivos de cinomose. Os animais foram distribuídos em três grupos. No grupo A foram incluídos 41 cães com alterações sistêmicas; no grupo B, 37 cães com alterações neurológicas; e no grupo C, nove cães com alterações sistêmicas e neurológicas simultâneas. O grupo D (controle) foi composto por 20 cães assintomáticos. Os resultados da RT-PCR foram correlacionados com a forma clínica da infecção e com as alterações hematológicas encontradas. Foi possível a amplificação parcial do gene da nucleoproteína do CDV em 41 (47,1 por cento) das 87 amostras de urina provenientes de cães com sinais clínicos sugestivos de cinomose. Todas as amostras obtidas de animais assintomáticos foram negativas na RT-PCR. Amostras positivas foram encontradas nos três grupos de animais com sinais clínicos na proporção de 51,2 por cento (24/41), 29 por cento (11/37) e 100 por cento (9/9) para os grupos A, B e C, respectivamente. A leucocitose foi a alteração hematológica mais freqüente nos três grupos de cães com sinais clínicos porém, não foi possível estabelecer correlação entre o resultado da RT-PCR e as alterações hematológicas. Os resultados demonstraram que, independente da forma de apresentação clínica, a técnica da RT-PCR realizada em urina pode ser utilizada no diagnóstico ante mortem da infecção pelo CDV.

Reactive oxygen species in the initiation of IL-4 driven autoimmunity as a potential therapeutic target.

Helper T-lymphocytes have been shown to differentiate into two mutually regulatory subsets. Cells primarily secreting interleukin-2 (IL-2) and interferon-gamma are known as Th1 cells and mediate classical cell-mediated immune responses such as delayed-type hypersensitivity. Cells secreting interleukin-4 (IL-4) are known as Th2 cells and promote humoral immune responses, in particular the production of IgE and IgG4 (human) or IgG1 (rodents). Over-activity of either cell type can result in tissue-damaging autoimmune disease. A number of human diseases including asthma and some kidney diseases are thought to be caused by a Th-2 type autoimmune response. Study of an animal model of Th2-driven autoimmunity (mercuric chloride-induced autoimmunity in Brown Norway rats) has yielded insights into a possible role for oxidant stress in the generation of Th-2 driven autoimmune responses. Mercuric chloride probably causes oxidant stress by the generation of free-radicals, activating NK-kappaB, a transcription factor for the IL-4 gene. Treatment with the antioxidants N-acetlcysteine and desferrioxamine has been shown to suppress vasculitis and IgE production in this model. These findings suggest a possible clinical role for antioxidants in the therapy of human autoimmune disease.

Desferrioxamine modulates chemically induced T helper 2-mediated autoimmunity in the rat.

A rise in interleukin (IL) 4-dependent immunoglobulin E (IgE) is a hallmark of the mercuric chloride (HgCl2)-induced Th2-mediated autoimmune syndrome in the Brown Norway (BN) rat, and one of the mediators in allergic asthma in human. Oxidative stress, a potential factor related to the pathogenesis of allergy and asthma, has been shown to up-regulate IL-4 in mast cells and predispose to degranulation in vitro. However, it remains unknown whether oxidative/antioxidative imbalance plays a role in this Th2-driven model of autoimmunity in the rat. Here we show that administration of the non-sulphydryl-containing antioxidant desferrioxamine i.p. and s.c. to BN rats reduces HgCl2-enhanced IL-4 gene expression and inhibits HgCl2-induced Th2-mediated autoimmunity. Desferrioxamine treatment suppresses significantly IgE production and lymphoproliferation, and reduces tissue injury in the form of caecal vasculitis in the HgCl2-induced autoimmune syndrome. These results support a role for oxidative stress in the pathogenesis of the HgCl2-induced Th2-dominated autoimmune syndrome. This finding might have implications for understanding the mechanisms involved in Th2 cell responses as seen in allergy and asthma and thereby aid the development of new therapeutic strategies for these diseases.

Preliminary in vitro studies on the Marsypianthes chamaedrys (boia-caá) extracts at fibrinoclotting induced by snake venoms.

The extract of Marsypianthes chamaedrys, a plant used against snakebites, in the present study was shown to inhibit fibrinoclotting induced by several Brazilian snake venoms or thrombin. These data indicate that this extract affected thrombin-like enzymes. In this first report we determine some features of the components present in the extract regarding the antifibrinoclotting action. Our results show that active components responsible for those effects are thermo-resistant and are concentrated in the methanolic fraction.

Acute glomerulonephritis.

Glomerulonephritis is an important cause of renal failure thought to be caused by autoimmune damage to the kidney. While each type of glomerulonephritis begins with a unique initiating stimulus, subsequent common inflammatory and fibrotic events lead to a final pathway of progressive renal damage. In this article the different forms of inflammatory glomerulonephritis and their diagnosis are discussed. In a review of therapy both immediate life saving treatment given when glomerulonephritis causes acute renal failure and more specific treatments designed to modify the underlying mechanisms of renal injury are considered.

The immunopathogenesis of membranous nephropathy.

Membranous nephropathy is an important disease: it is one of the leading primary causes of the nephrotic syndrome in adults, and, in up to a third of patients, causes progressive renal impairment resulting in end stage renal failure. Ever since histological techniques demonstrated the presence of glomerular immunoglobulin deposits in this disease the immune system has been implicated in pathogenesis. Initial ideas focussed on the deposition of circulating immune complexes, but the development of an animal model (Heymann nephritis) suggested the alternative mechanism of antibody reacting with an intrinsic glomerular antigen. However, attempts to find evidence for this Heymann type mechanism in the human disease have, in general, been unsuccessful. This article briefly reviews the development of ideas about the pathogenesis of membranous nephropathy, and proposes the hypothesis that the disease is caused by formation of low affinity non-complement fixing IgG4-containing immune complexes.

The Th2-response in mercuric chloride-induced autoimmunity requires continuing costimulation via CD28.

Mercuric chloride (HgCl2)-induced autoimmunity in Brown Norway (BN) rats is a highly polarized polyclonal Th2-driven autoimmune response with increased IgE production, lymphoproliferation, vasculitis and proteinuria. The increase in serum IgE concentration is clearly measurable by day 4 after the first HgCl2 injection and peaks between days 15 and 20. Treatment with CD80 and CD86 antibodies prior to administration of HgCl2 completely suppresses the autoimmune process. To determine whether interruption of CD28 signalling after initial stimulation of the Th2-response would be suppressive, antibody treatment was delayed. BN rats were given 5 doses of HgCl2 subcutaneously on alternate days. CD80 and CD86 antibodies, or an isotype control, were given daily for 3 days and then on alternate days until day 12 commencing either on the day of the first HgCl2 injection (day 0) or on days 4 or 8. Treatment from day 0 reduced serum IgE concentrations to below baseline (median 9.34 microg/ml on day 0 versus 4.6 microg/ml, on day 5, P = 0.03) suggesting that ongoing costimulation via CD28 is required to maintain basal serum IgE production. Delaying treatment until day 4 or day 8 after the first HgCl2 injection resulted in significant inhibition of IgE secretion, lymphoproliferation, and vasculitis, although less markedly than when treatment was commenced on day 0. These data indicate that CD28-mediated costimulation is not only required for the initiation of the Th2-response but is required for maintenance of a maximal response, making this an attractive therapeutic target for antibody-mediated autoimmune diseases.

CD80(B7.1) and CD86(B7.2) do not have distinct roles in setting the Th1/Th2 balance in autoimmunity in rats.

Some data suggest that the interaction between CD28 and CD80 (B7.1) stimulates Th1-responses and that CD28 and CD86 (B7.2) stimulates Th2-responses, however this is controversial. We addressed this issue by using mercuric chloride (HgCl2)-induced autoimmunity in Brown Norway (BN) rats as a highly polarized Th2 model and experimental autoimmune encephalomyelitis (EAE) in Lewis rats as a highly polarized Th1 model. Monoclonal antibodies (MoAbs) to CD80 and CD86, given singly, had little effect in either model, however when given together they almost completely suppressed the HgCl2-induced autoimmunity: the peak immunoglobulin (Ig)E concentration was 3.25 microg/ml in treated animals versus 2770 microg/ml in controls (P < 0.0001); caecal vasculitis was suppressed with a median vasculitis score of 0 in treated animals versus 6 in controls (P < 0.0001); and new germinal centre formation was significantly suppressed. A combination of the antibodies also markedly reduced the severity of clinical EAE; from a median aggregate clinical score of 9 to 3 (P = 0.02) and delayed the onset from a median of 12.5 days to 16 days after immunization (P = 0.006). We have demonstrated profound suppression of both Th1 and Th2-driven autoimmunity in rats by a combination of anti-CD80 and CD86, but have been unable to demonstrate any clear differential effects.

IL-4 gene expression up-regulated by mercury in rat mast cells: a role of oxidant stress in IL-4 transcription.

In the Brown Norway (BN) rat, chemical compounds [mercuric chloride (HgCl2), D-penicillamine or gold salts] induce a T(h)2-dominated autoimmune syndrome with tissue injury in the form of a vasculitis and arthritis. An early phase of vasculitis in the model occurs within 24 h of an injection of HgCl2, is alphabeta T cell independent and involves the mast cell. In addition, HgCl2 induces IL-4 mRNA in mast cells from BN rats. Our recent work has demonstrated that the balance of oxidative/antioxidative influences plays an important role in the modulation of mast cell function (degranulation) in chemically induced autoimmunity. The aim of this study was to determine, in mast cells, whether oxidative status influences IL-4 transcription and translation, which is required for the development of a T(h)2 response. Exposure of the mast cell line RBL-2H3 to HgCl2 enhanced both IL-4 mRNA and its promoter activity. Oxidative stress by hydrogen peroxide mimicked the effects of HgCl2 in enhancing IL-4 promoter activity. The enhancement of IL-4 gene expression by HgCl2 was significantly reduced by antioxidants (both sulphydryl and non-sulphydryl containing). The same pattern of regulation was also observed on IL-4 protein expression in the mast cells. These data suggest a novel mechanism of IL-4 transcriptional up-regulation by oxidative stress. Our results provide evidence to support our hypothesis that alterations in intracellular reactive oxygen species production modulate both IL-4 gene expression and mast cell function.

Immunosuppressant effect of gold on IgG subclasses and IgE; evidence for sparing of Th2 responses.

We set out to examine the effect of gold treatment on the Th2-dependent antibodies IgG4 and IgE in relation to other IgG subclasses in patients with rheumatoid arthritis (RA). Eighty-five gold-treated RA patients and 82 RA controls were studied. Serum IgG subclass concentrations were measured by ELISA, IgE was measured by automated enzyme immunoassay. Samples were studied serially in 13 gold-treated patients and in 11 patients with gold-induced adverse events. There was a significant reduction in the concentration of IgG1, IgG2 and IgG3 in gold-treated RA patients compared with RA controls (P 0.004-0.019), whereas IgG4 was less significantly reduced in gold-treated patients (P = 0.044) and there was no difference in IgE. In serial samples there was a significant fall in the concentration of IgG1 (P = 0.001), IgG2 (P = 0.001) and IgG3 (P = 0.026) with time but no change in IgG4 and IgE. The development of gold-induced adverse events was not associated with any change in the concentration of each IgG subclass or IgE. Deficiencies of IgG subclasses were found in 30% of gold-treated RA patients and 8.5% of RA controls, and were associated in gold-treated patients with a longer disease duration (P = 0.003) and with erosive disease (P = 0. 03). IgG2 was affected most frequently and in the majority of these cases subnormal specific IgG2 binding to widespread polysaccharide antigens (Pneumovax II) was found. Gold induces an overall immunosuppressant effect on IgG subclasses, with a deficiency in 21. 5%, adjusted for controls. The effect on the Th2-dependent antibodies IgG4 and IgE is less marked, suggesting a sparing of Th2 responses.