Risk factors and outcomes of candidemia caused by biofilm-forming isolates in a tertiary care hospital

Background

Very few data exist on risk factors for developing biofilm-forming Candida bloodstream infection (CBSI) or on variables associated with the outcome of patients treated for this infection.

Methods and Findings

We identified 207 patients with CBSI, from whom 84 biofilm-forming and 123 non biofilm-forming Candida isolates were recovered. A case-case-control study to identify risk factors and a cohort study to analyze outcomes were conducted. In addition, two sub-groups of case patients were analyzed after matching for age, sex, APACHE III score, and receipt of adequate antifungal therapy. Independent predictors of biofilm-forming CBSI were presence of central venous catheter (odds ratio [OR], 6.44; 95% confidence interval [95% CI], 3.21–12.92) or urinary catheter (OR, 2.40; 95% CI, 1.18–4.91), use of total parenteral nutrition (OR, 5.21; 95% CI, 2.59–10.48), and diabetes mellitus (OR, 4.47; 95% CI, 2.03–9.83). Hospital mortality, post-CBSI hospital length of stay (LOS) (calculated only among survivors), and costs of antifungal therapy were significantly greater among patients infected by biofilm-forming isolates than those infected by non-biofilm-forming isolates. Among biofilm-forming CBSI patients receiving adequate antifungal therapy, those treated with highly active anti-biofilm (HAAB) agents (e.g., caspofungin) had significantly shorter post-CBSI hospital LOS than those treated with non-HAAB antifungal agents (e.g., fluconazole); this difference was confirmed when this analysis was conducted only among survivors. After matching, all the outcomes were still favorable for patients with non-biofilm-forming CBSI. Furthermore, the biofilm-forming CBSI was significantly associated with a matched excess risk for hospital death of 1.77 compared to non-biofilm-forming CBSI.

Conclusions

Our data show that biofilm growth by Candida has an adverse impact on clinical and economic outcomes of CBSI. Of note, better outcomes were seen for those CBSI patients who received HAAB antifungal therapy.     

Atrial natriuretic peptide effects on intracellular pH changes and ROS production in HEPG2 cells: role of p38 MAPK and phospholipase D.

AIMS: The present study was performed to evaluate Atrial Natriuretic Peptide (ANP) effects on intracellular pH, phospholipase D and ROS production and the possible relationship among them in HepG2 cells. Cancer extracellular microenvironment is more acidic than normal tissues and the activation of NHE-1, the only system able to regulate pHi homeostasis in this condition, can represent an important event in cell proliferation and malignant transformation. METHODS: The ANP effects on pHi were evaluated by fluorescence spectrometry. The effects on p38 MAPK and ROS production were evaluated by immunoblots and analysis of DCF-DA fluorescence, respectively. RT-PCR analysis and Western blotting were used to determine the ANP effect on mRNA NHE-1 expression and protein levels. PLD-catalyzed conversion of phosphatidylcholine to phosphatydilethanol (PetOH), in the presence of ethanol, was monitored by thin layer chromatography. RESULTS: A significant pHi decrease was observed in ANP-treated HepG2 cells and this effect was paralleled by the enhancement of PLD activity and ROS production. The ANP effect on pHi was coupled to an increased p38 MAPK phosphorylation and a down-regulation of mRNA NHE-1 expression and protein levels. Moreover, the relationship between PLD and ROS production was demonstrated by calphostin-c, a potent inhibitor of PLD. At the same time, all assessed ANP-effects were mediated by NPR-C receptors. CONCLUSION: Our results indicate that ANP recruits a signal pathway associated with p38 MAPK, NHE-1 and PLD responsible for ROS production, suggesting a possible role for ANP as novel modulator of ROS generation in HepG2 cells.     

Protein aggregation and amyloid fibril formation by an SH3 domain probed by limited proteolysis

The SH3 domains are small protein modules of 60-85 amino acid residues that are found in many proteins involved in intracellular signal transduction. The SH3 domain of the p85alpha subunit of bovine phosphatidylinositol 3'-kinase (PI3-SH3) under acidic solution adopts a compact denatured state from which amyloid fibrils are readily formed. This aggregation process has been found to be modulated substantially by solution conditions. Here, we have analyzed the conformational features of the native and acid denatured states of PI3-SH3 by limited proteolysis experiments using proteinase K and pepsin, respectively. Moreover, we have analyzed the propensity of PI3-SH3 to be hydrolyzed by pepsin at different stages in the process of aggregation and amyloid formation at pH 1.2 and 2.0 and compared the sites of proteolysis under these conditions with the conformational features of both native and aggregated PI3-SH3. The results demonstrate that the denatured state of PI3-SH3 formed at low pH is relatively resistant to proteolysis, indicating that it is partially folded. The long loop connecting beta-strands b and c in the native protein is the region in this structure most susceptible to proteolysis. Remarkably, aggregates of PI3-SH3 that are formed initially from this denatured state in acid solution display enhanced susceptibility to proteolysis of the long loop, suggesting that the protein becomes more unfolded in the early stages of aggregation. By contrast, the more defined amyloid fibrils that are formed over longer periods of time are completely resistant to proteolysis. We suggest that the protein aggregates formed initially are relatively dynamic species that are able readily to reorganize their interactions to enable formation of very well ordered fibrillar structures. In addition, the disordered and non-native character of the polypeptide chains in the early aggregates could be important in determining the high cytotoxicity that has been revealed in previous studies of these species.     

Modulation of Quinolinic and Kynurenic Acid Content in the Rat Brain: Effects of Endotoxins and Nicotinylalanine

Quinolinic acid, an endogenous excitotoxin, and kynurenic acid, an antagonist of excitatory amino acid receptors, are believed to be synthesized from tryptophan after the opening of the indole ring. They were measured in the rat brain and other organs using gas chromatography-mass spectrometry or HPLC. The enzyme indoleamine 2,3-dioxygenase, capable of cleaving the indole ring of tryptophan, was induced by administering bacterial endotoxins to rats, which significantly increased the brain content of both quinolinic and kynurenic acids. Nicotinylalanine, an analogue of kynurenine, inhibited this endotoxin-induced accumulation of quinolinic acid while potentiating the accumulation of kynurenic acid. The possibility of significantly increasing brain concentrations of kynurenic acid without a concomitant increase in quinolinic acid may provide a useful approach for studying the role of these electrophysiologically active tryptophan metabolites in brain function and preventing the possible toxic actions of abnormal synthesis of quinolinic acid.     

DNA strand breaks in ejaculated human spermatozoa: comparison of susceptibility to the nick translation and terminal transferase assays

The nick translation and terminal transferase assays have been compared to test their relative efficiency in detecting DNA breakage in ejaculated human spermatozoa. The results have been correlated with the percentage of chromomycin A3 positive sperm, a fluorochrome that is indicative of the protamination state of sperm. Examination of the ejaculated sperm of 30 subjects revealed that the percentage of positivity to the nick translation and terminal transferase assays did not differ, even when using different fixatives. It is concluded that the inability of the two assays to distinguish the type of DNA damage, as is possible in somatic nuclei, is most probably linked to the unique nature of sperm chromatin. It is proposed that the presence of the damaged DNA may be the remnants of an imperfect spermiogenesis, probably related to an inadequate protamine deposition. This is supported by the strong correlation between the presence of DNA damage and underprotamination as evidenced by chromomycin A3. © Chapman & Hall     

Disruption of CD40/CD40L interaction influences the course of Cryptococcus neoformans infection

CD40 signaling has been implicated in various pathogenic processes such as chronic inflammatory disease, graft-versus-host disease, autoimmune disease and cancer. We previously demonstrated in an in vitro system that the CD40/CD40L pathway mediates late interleukin (IL) 12 production in response to Cryptococcus neoformans. The purpose of this study was to examine the course of C. neoformans infection in the absence of CD40/CD40L costimulation. We compared infection in mice genetically lacking CD40L (CD40L(-/-)) and in the wild-type counterpart. The animals were injected intratracheally with C. neoformans and monitored for clearance of the organism and the development of cellular immune response. CD40L(-/-) mice exhibited an exacerbation of infection, evaluated as scarce inflammatory response in the lung, that mirrored an increase of fungal burden. This correlated with impairment of nitrite production and antimicrobial activity by macrophages against C. neoformans and unrelated microorganisms such as Candida albicans. Moreover, IL-12 production by splenic macrophages was diminished in CD40L(-/-) mice and interferon-gamma production by CD4 and CD8 T cells was decreased. CD4 T cells retained the ability to express a costimulatory molecule, CTLA-4, but showed a decrease in CD28 expression. This latter molecule is implicated in a positive effect on proliferation, cytokine production and survival of T cells. Collectively these data demonstrate that absence of CD40L correlates with (i) reduced antimicrobial activity of natural effector cells; (ii) reduction of the magnitude of T cell response; and (iii) increase of fungal growth in the brain. These findings suggest that disruption of CD40/CD40L may be deleterious for development of an efficient immune response to C. neoformans and may identify potential molecular targets for novel immunotherapeutic approaches