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.

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.

Regulatory role of OX22high T cells in mercury-induced autoimmunity in the brown Norway rat.

The monoclonal antibody OX22 defines a functional split within CD4+ T cells in the rat, with OX22high cells mainly producing interleukin 2 (IL-2) and interferon gamma and responsible for delayed-type hypersensitivity responses, and OX22low cells mainly producing IL-4 and -5 and responsible for providing B cell help. There are reciprocal interactions between OX22high and OX22low cells, and it has been suggested that the OX22low subset has a role in the prevention of autoimmunity. We have used OX22 in vivo to define the role of these subsets in mercuric chloride-induced autoimmunity in the Brown Norway rat. In this model, there is polyclonal B cell activation and animals develop widespread tissue injury. Treatment of thymectomized animals with OX22 led to a profound reduction in the number of OX22high T cells in the peripheral blood. OX22-treated animals consistently developed more severe tissue injury than controls given an irrelevant antibody of the same isotype. Control animals pretreated with broad spectrum antimicrobial drugs showed milder tissue injury, but this protective effect of antimicrobials was lost in OX22-treated animals. Transfer of naive T cells to OX22-treated animals provided protection, but if T cells were depleted in vitro of OX22high cells before transfer, this effect was lost. These data provide evidence for a protective immunoregulatory role for OX22high T cells in mercuric chloride-induced autoimmunity.

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.

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.

Structural analysis of two HLA-DR-presented autoantigenic epitopes: crucial role of peripheral but not central peptide residues for T-cell receptor recognition.

Specific and major histocompatibility complex (MHC)-restricted T-cell recognition of antigenic peptides is based on interactions of the T-cell receptor (TCR) with the MHC alpha helices and solvent exposed peptide residues termed TCR contacts. In the case of MHC class II-presented peptides, the latter are located in the positions p2/3, p5 and p7/8 between MHC anchor residues. For numerous epitopes, peptide substitution studies have identified the central residue p5 as primary TCR contact characterized by very low permissiveness for peptide substitution, while the more peripheral positions generally represent auxiliary TCR contacts. In structural studies of TCR/peptide/MHC complexes, this has been shown to be due to intimate contact between the TCR complementarity determining region (CDR) three loops and the central peptide residue. We asked whether this model also applied to two HLA-DR presented epitopes derived from an antigen targeted in type 1 diabetes. Large panels of epitope variants with mainly conservative single substitutions were tested for human leukocyte antigen (HLA) class II binding affinity and T cell stimulation. Both epitopes bind with high affinity to the presenting HLA-DR molecules. However, in striking contrast to the standard distribution of TCR contacts, recognition of the central p5 residue displayed high permissiveness even for non-conservative substitutions, while the more peripheral p2 and p8 TCR contacts showed very low permissiveness for substitution. This suggests that intimate TCR interaction with the central peptide residue is not always required for specific antigen recognition and can be compensated by interactions with positions normally acting as auxiliary contacts.

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.

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.

Bulk immunopurification and development of a radioimmunoassay for human and murine F liver protein.

By taking advantage of the cross-reaction for human F liver protein (F antigen) of a monoclonal antibody raised to mouse F antigen, human F antigen has been immunopurified in bulk. The molecular weight is 44 000. Using this material as a standard, affinity-purified polyclonal anti-F antibody on a solid phase and radiolabelled monoclonal anti-F antibody to detect bound protein, a capture immunoradiometric assay capable of detecting down to 1 ng X ml-1 of F antigen has been developed. Using this assay, the average level of F antigen in normal mouse serum (CBA strain) is 16 ng X ml-1 (360 pM) and in human serum 10 ng X ml-1 (250 pM).

Serum F protein estimation in liver allograft recipients with graft dysfunction.

Serum levels of F protein, a 44 kD cytoplasmic protein mainly found in hepatocytes, became elevated during episodes of graft dysfunction following orthotopic liver transplantation. In a study of 27 liver transplant recipients, the rise in F protein did not precede rises in the other conventional biochemical indices of hepatic dysfunction. Serum F protein concentration significantly correlated with serum levels of aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase, and bilirubin (all P less than 0.001) and also with the prothrombin time (P = 0.048). Despite its high concentration in liver cells, this marker does not provide any additional benefit in the diagnosis of graft dysfunction or in monitoring liver allograft function following transplantation.

Anti-CD45 monoclonal antibody perfusion of human renal allografts prior to transplantation. A safety and immunohistological study. CD45 Study Group.

The perfusion of kidneys with anti-CD45 monoclonal antibodies prior to transplantation offers a means of targeting passenger antigen-presenting cells with the aim of reducing the subsequent incidence of rejection episodes. A safety study was performed in humans of such pretreatment in 40 unsensitized recipients of first cadaveric renal grafts, who were followed for 3 months after transplantation. A 50-ml solution containing 2 mg of each of the rat anti-CD45 mAbs YTH 24.5 and YTH 54.12 was injected into the allograft renal artery ex vivo and just before transplantation while the renal vein was kept clamped. No patients died, but 4 grafts were lost. Two were lost due to primary nonfunction, 1 was lost because of late renal artery thrombosis, and 1 was lost to rejection. There were no cases of renal vein thrombosis and 1 trivial renal artery stenosis, and only 2 patients produced human anti-rat antibodies. Between 63.5% and 100% (median 96.4%) of CD45+ cells in the postperfusion biopsies were coated with anti-CD45 as determined by double-immunolabeling. The number of patients experiencing rejection episodes was inversely associated with this "antibody uptake": 75% of the low uptake group (< 95%) had at least 1 rejection episode, compared with 22% of the high uptake group (> or = 95%) (P = 0.001). The complement components C3 and C5b-9 colocalized with perfused anti-CD45 in 32/33 (97.0%) and 11/33 (33.3%) of the biopsy specimens, respectively. We conclude that: (1) this technique appears free of adverse effects, (2) high antibody uptake within the kidney is associated with a lower incidence of rejection, and (3) the antibodies used fix and activate complement in vivo.

Serum F protein: a new sensitive and specific test of hepatocellular damage.

F protein is a 44-kDa protein which is found mostly in the liver and circulates at much lower concentrations in the serum. Serum F protein has been measured, using a recently developed radioimmunoassay, in a variety of diseases and was appreciably raised only in patients with hepatocellular damage. The serum F protein concentration was a more sensitive and specific marker of liver damage than conventional liver function tests and showed a close correlation with the histological assessment of liver damage (r = 0.86, P less than 0.001). This new marker may be of value in the diagnosis and treatment of liver disease.

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.

Further characterization of an animal model of systemic vasculitis.

The demonstration of vasculitis and anti-myeloperoxidase antibodies in the mercuric chloride treated Brown Norway rat provides a useful, though limited, animal model of systemic vasculitis. We describe some preliminary experiments on the effect of transfer of serum from mercuric chloride treated rats and of two forms of immunotherapy: intravenous immunoglobulin and an anti-CD4 antibody. Transfer of serum did not lead to tissue injury and neither of the two forms of therapy proved beneficial in this model.

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.

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.