Contribution of the National Mycology Laboratory Network to Surveillance of Cryptococcosis in Argentina

Purpose of Review

Cryptococcal meningitis is the most common fungal infection of the central nervous system and the third most frequent neurological complication in AIDS patients. To understand the Argentinean epidemiology of cryptococcosis, several efforts have been made by the National Reference Laboratory in Clinical Mycology.

Recent Findings

In Argentina, reports of distribution and frequency of Cryptococcus neoformans and Cryptococcus gattii isolates are scarce and very little is known about its circulating genotypes and mating types. The National Mycology Laboratory Network and the National Reference Laboratory in Clinical Mycology joined forces to estimate the prevalence of cryptococcosis and to obtain and to analyse epidemiological data of this important fungal infection.

Summary

Data presented here were recovered from 1998 to 2016 and represents an approximation to the actual situation of cryptococcosis in Argentina. These results could be useful to design future investigations.

     

Mdm2 and Mdm4 loss regulates distinct p53 activities

Mutational inactivation of p53 is a hallmark of most human tumors. Loss of p53 function also occurs by overexpression of negative regulators such as MDM2 and MDM4. Deletion of Mdm2 or Mdm4 in mice results in p53-dependent embryo lethality due to constitutive p53 activity. However, Mdm2(-/-) and Mdm4(-/-) embryos display divergent phenotypes, suggesting that Mdm2 and Mdm4 exert distinct control over p53. To explore the interaction between Mdm2 and Mdm4 in p53 regulation, we first generated mice and cells that are triple null for p53, Mdm2, and Mdm4. These mice had identical survival curves and tumor spectrum as p53(-/-) mice, substantiating the principal role of Mdm2 and Mdm4 as negative p53 regulators. We next generated mouse embryo fibroblasts null for p53 with deletions of Mdm2, Mdm4, or both; introduced a retrovirus expressing a temperature-sensitive p53 mutant, p53A135V; and examined p53 stability and activity. In this system, p53 activated distinct target genes, leading to apoptosis in cells lacking Mdm2 and a cell cycle arrest in cells lacking Mdm4. Cells lacking both Mdm2 and Mdm4 had a stable p53 that initiated apoptosis similar to Mdm2-null cells. Additionally, stabilization of p53 in cells lacking Mdm4 with the Mdm2 antagonist nutlin-3 was sufficient to induce a cell death response. These data further differentiate the roles of Mdm2 and Mdm4 in the regulation of p53 activities.     

Aluminum accumulation and its relationship with mineral plant nutrients in 12 pteridophytes from Venezuela

The purpose of this study was to investigate the aluminum (Al) concentration in Lycopodium clavatum, Dicranopteris flexuosa, Sticherus nudus, Anemia villosa, Cyathea gibbosa, Pteridium arachnoideum, Pteris vittata, Thelypteris dentata, Blechnum occidentale, Elaphoglossum sporadolepis, Nephrolepis cordifolia and Polypodium pseudoaureum, species from 11 families with different phylogenetic position, found on soils with a high concentration of Al (up to 13 g kg^?1 dry mass (DM)). When Al concentration and mineral nutrients in aerial organs were considered, pteridophytes were classified into three groups: group one included pteridophytes with Al concentrations over 1000 mg kg^?1 DM in their aerial organs, a ratio between Al and essential plant nutrients such as Ca, Mg and P higher than one and a K/Al ratio between 0.68 and 2.56 mol mol^?1. In group 1 was the well known Al-accumulator L. clavatum (Lycophyte) as well as the Neotropical ferns D. flexuosa, S. nudus (both basal leptosporangiate ferns), and C. gibbosa (core leptosporangiate tree fern). Group 2, ferns which accumulate Al over 1000 mg kg^?1 DM in their fronds, and had an Al/Ca and Al/Mg ratio <1. Species in this group included E. sporadolepis and N. cordifolia (derived polypod ferns). Group 3, ferns classified as Al-excluders, showing Al concentration <782 mg kg^?1 DM in the fronds, had Al/Ca and Al/Mg ratios <1, Al/P ratio ≤1 and a K/Al ratio between 18.10 and 80.36 mol mol^?1. In group 3, were A. villosa (basal leptosporangiate fern) and the derived polypod ferns P. arachnoideum, P. vittata, T. dentata, B. occidentale and P. pseudoaureum. The translocation factor of Al from subterranean to aerial organs was up to 4 in S. nudus, and subterranean organs from E. sporadolepis showed the highest concentration of Al (12 g kg^?1 DM). We coincide with early literature in that other criteria in addition to the Al concentration should be considered to define the Al accumulation, such as its relationship with macronutrients. For example, we propose the inclusion of K/Al ratio. We conclude that out of six Al-excluders five belonged to the derived polypods while two species from Polypodiales showed high Al concentrations. We reconfirm accumulation of Al in L. clavatum and C. gibbosa and discover two new Al-accumulating species in the more ancient ferns: S. nudus and D. flexuosa.     

Galvanic vestibular stimulation improves the results of vestibular rehabilitation

Here, we present findings from a three-step investigation of the effect of galvanic vestibular stimulation (GVS) in normal subjects and in subjects undergoing vestibular rehabilitation (VR). In an initial study, we examined the body sway of 10 normal subjects after one minute of 2 mA GVS. The effect of the stimulation lasted for at least 20 minutes in all subjects and up to two hours in 70% of the subjects. We then compared a group of patients who received conventional VR (40 patients) with a group that received a combination of VR and GVS. Results suggest a significant improvement in the second group. Finally, we attempted to establish the optimal number of GVS sessions and to rule out a placebo effect. Fifteen patients received "systematic" GVS: five sessions, once a week. Five patients received "nonsystematic" galvanic stimulation in a sham protocol, which included two stimulations of the clavicle. These data were analyzed with Fisher's exact test and indicated that the best results were obtained after three sessions of GVS and no placebo effect was observed.     

Joint effects of germ-line <Emphasis Type="Italic">TP53</Emphasis> mutation, <Emphasis Type="Italic">MDM2</Emphasis> SNP309, and gender on cancer risk in family studies of Li–Fraumeni syndrome

Li–Fraumeni syndrome (LFS) is a rare familial cancer syndrome characterized by early cancer onset, diverse tumor types, and multiple primary tumors. Germ-line TP53 mutations have been identified in most LFS families. A high-frequency single-nucleotide polymorphism, SNP309 (rs2279744), in MDM2 was recently confirmed to be a modifier of cancer risk in several case-series studies: substantially earlier cancer onset was observed in SNP309 G-allele carriers than in wild-type individuals by 7–16 years. However, cancer risk analyses that jointly account for measured hereditary TP53 mutations and MDM2 SNP309 have not been systematically investigated in familial cases. Here, we determined the combined effects of measured TP53 mutations, MDM2 SNP309, and gender and their interactions simultaneously in LFS families. We used the method that is designed for extended pedigrees and structured for age-specific risk models based on Cox proportional hazards regression. We analyzed the cancer incidence in 19 extended pedigrees with germ-line TP53 mutations ascertained through the clinical LFS phenotype. The dataset consisted of 463 individuals with 129 TP53 mutation carriers. Our analyses showed that the TP53 germ-line mutation and its interaction with gender were strongly associated with familial cancer incidence and that the association between MDM2 SNP309 and increased cancer risk was modest. In contrast with several case-series studies, the interaction between MDM2 SNP309 and TP53 mutation was not statistically significant in our LFS family cohort. Our results showed that SNP309 G-alleles were associated with accelerated tumor formation in both carriers and non-carriers of germ-line TP53 mutations.     

Effect of acid lactic bacteria isolated from faeces of healthy dogs on growth parameters and aflatoxin B<Subscript>1</Subscript> production by <Emphasis Type="Italic">Aspergillus</Emphasis> species in vitro

The aim of the present study was to evaluate the inhibitory effect of Enterococcus faecium and Lactococcus lactis subsp. lactis isolated from faeces of healthy dogs on (i) lag phase, (ii) growth rate, and (iii) aflatoxin B₁ production by Aspergillus section Flavi on in vitro assays. Thirteen lactic acid bacteria (LAB) isolates were used as antagonist microorganisms. Antagonistic activity was assayed against four potentially aflatoxigenic Aspergillus section Flavi isolates: A. flavus (AF210 and AF281), A. parasiticus (AP245) and A. parasiticus (NRRL 2999). In general, the longest lag phases of Aspergillus isolates were obtained with E. faecium GJ40. Respecting the growth rate, no significant reduction was found in this parameter in the interaction assays with A. flavus and antagonist isolates respecting the control. While in A. parasiticus a significant reduction in growth rate was only observed in the interaction among reference strain and E. faecium MF5 isolate (p < 0.05). In general, AFB₁ production was reduced by most of the LAB isolates assayed, except for E. faecium GJ18, GJ20, MF3 and MF4. This study provides the first data about the antiaflatoxigenic activity of autochthonous LAB isolated from dog faeces.     

A Cys-loop mutation in the Caenorhabditis elegans nicotinic receptor subunit UNC-63 impairs but does not abolish channel function

The nematode Caenorhabditis elegans is an established model organism for studying neurobiology. UNC-63 is a C. elegans nicotinic acetylcholine receptor (nAChR) α-subunit. It is an essential component of the levamisole-sensitive muscle nAChR (L-nAChR) and therefore plays an important role in cholinergic transmission at the nematode neuromuscular junction. Here, we show that worms with the unc-63(x26) allele, with its αC151Y mutation disrupting the Cys-loop, have deficient muscle function reflected by impaired swimming (thrashing). Single-channel recordings from cultured muscle cells from the mutant strain showed a 100-fold reduced frequency of opening events and shorter channel openings of L-nAChRs compared with those of wild-type worms. Anti-UNC-63 antibody staining in both cultured adult muscle and embryonic cells showed that L-nAChRs were expressed at similar levels in the mutant and wild-type cells, suggesting that the functional changes in the receptor, rather than changes in expression, are the predominant effect of the mutation. The kinetic changes mimic those reported in patients with fast-channel congenital myasthenic syndromes. We show that pyridostigmine bromide and 3,4-diaminopyridine, which are drugs used to treat fast-channel congenital myasthenic syndromes, partially rescued the motility defect seen in unc-63(x26). The C. elegans unc-63(x26) mutant may therefore offer a useful model to assist in the development of therapies for syndromes produced by altered function of human nAChRs.     

Mutant p53 disrupts role of ShcA protein in balancing Smad protein-dependent and -independent signaling activity of transforming growth factor-β (TGF-β)

Biomarkers are lacking for identifying the switch of transforming growth factor-β (TGF-β) from tumor-suppressing to tumor-promoting. Mutated p53 (mp53) has been suggested to switch TGF-β to a tumor promoter. However, we found that mp53 does not always promote the oncogenic role of TGF-β. Here, we show that endogenous mp53 knockdown enhanced cell migration and phosphorylation of ERK in DU145 prostate cancer cells. Furthermore, ectopic expression of mp53 in p53-null PC-3 prostate cancer cells enhanced Smad-dependent signaling but inhibited TGF-β-induced cell migration by down-regulating activated ERK. Reactivation of ERK by the expression of its activator, MEK-1, restored TGF-β-induced cell migration. Because TGF-β is known to activate the MAPK/ERK pathway through direct phosphorylation of the adaptor protein ShcA and MAPK/ERK signaling is pivotal to tumor progression, we investigated whether ShcA contributed to mp53-induced ERK inhibition and the conversion of the role of TGF-β during carcinogenesis. We found that mp53 expression led to a decrease of phosphorylated p52ShcA/ERK levels and an increase of phosphorylated Smad levels in a panel of mp53-expressing cancer cell lines and in mammary glands and tumors from mp53 knock-in mice. By manipulating ShcA levels to regulate ERK and Smad signaling in human untransformed and cancer cell lines, we showed that the role of TGF-β in regulating anchorage-dependent and -independent growth and migration can be shifted between growth suppression and migration promotion. Thus, our results for the first time suggest that mp53 disrupts the role of ShcA in balancing the Smad-dependent and -independent signaling activity of TGF-β and that ShcA/ERK signaling is a major pathway regulating the tumor-promoting activity of TGF-β.     

Emergence of Resistance to Fluconazole in <Emphasis Type="Italic">Candida albicans</Emphasis> Isolated From Vaginal Discharge

Purpose of Review

To provide information about the emergence of fluconazole resistance in Candida albicans isolated from vaginal discharge, in a global context, and to update the in vitro susceptibility profile of this species from Argentina.

Recent Findings

Vulvovaginal candidiasis is the second most common vaginal infection after vaginal bacteriosis. C. albicans remains the prevalent etiological yeast species, and despite antifungal treatment, the rate of recurrence remains high, which may be associated to antifungal resistance.

Summary

Data here presented were obtained from the study of C. albicans strains isolated from patients with clinical signs of vulvovaginal candidiasis from 1996 to 2017. Data obtained could represent the susceptibility profile of C. albicans strains circulating in Argentina and could be of potential usefulness to monitor and guide therapy, and also suggests the need for greater surveillance programs to detect fluconazole resistance over time.