Cellular responses associated with dibucaine-induced phospholipidosis

A wide range of cationic amphiphilic drugs (CADs) from different therapeutic areas are known to cause phospholipidosis both in vivo and in vitro. Although the relevance of this storage disorder for human health remains uncertain, CADs have been repeatedly associated with clinical side effects, and as a result, phospholipidosis is of major concern for drug development in the pharmaceutical industry. An important unresolved question in this field is whether phospholipidosis is really linked to cellular toxicity. This work was focused on studying cellular responses associated with CAD-induced phospholipidosis in cultured mammalian kidney cells. Dibucaine (2-butoxy-N-[2-diethylaminoethyl]quinoline-4-carboxamide), an amide-type anesthetic with poorly defined cytotoxic effects, was used to induce phospholipidosis in Vero cells. The results from several assays that measure cell viability, proliferation, and morphological changes indicated that dibucaine-induced lysosomal phospholipidosis was accompanied by cellular defense responses such as transient growth arrest and autophagy, under mild stress conditions. Conversely, when tolerance limits were exceeded treated Vero cells underwent extensive and irreparable injury, leading ultimately to cell death. Our data provide additional information that may be of considerable interest for drug safety assessment.     

Increased protein nitration burden in the atherosclerotic lesions and plasma of apolipoprotein A-I deficient mice

Apolipoprotein A-I (apoA-I), the major protein constituent within high-density lipoprotein (HDL), has been associated with antiatherogenic protection by mechanisms that include reverse cholesterol transport and antiinflammatory functions. To evaluate the proposed protective function of apoA-I, proteins modified by nitrating oxidants were evaluated in the aortic tissue and plasma of mice lacking the low-density lipoprotein receptor and apobec (LA) and LA mice with genetic deletion of apoA-I (LA-apoA-I(-/-)). The levels of nitrated proteins in aortic tissue quantified by liquid chromatography with online electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) were 6-fold higher in the LA-apoA-I(-/-) as compared with the LA mice. The quantitative analyses were corroborated by immunohistochemical and high-resolution immunoelectron microscopic evaluation of the lesions, which revealed abundant staining for nitrated proteins in the aortic root lesions of LA-apoA-I(-/-) as compared with the LA mice. Proteomic approaches based on affinity enrichment and site-specific adduct mapping identified unique specific protein targets for nitration in the plasma of LA-apoA-I(-/-) that were not present in the plasma of LA mice. In particular the nitration of fibrinogen was shown to accelerate fibrin clot formation. Another consequence of the augmented levels of nitrated proteins was the induction of humoral responses documented by the increased circulating immunoglobulins that recognize nitrotyrosine in LA-apoA-I(-/-) as compared with the LA mice. These data collectively support a protective function of apoA-I diminishing the burden of nitrative oxidants in these mice models of atherosclerosis.     

Determination of Echinocandin MICs for Candida Species in Less than 8 Hours: Comparison of the Rapid Susceptibility Assay with the Clinical and Laboratory Standards Institute's Broth Microdilution Assay

The echinocandins prevent fungal cell wall synthesis by inhibiting β-1,3-glucan synthesis, a significant glucose-consuming process. Previous studies suggested that echinocandin inhibitory activity is evident within 1 h of exposure. We hypothesized that a susceptibility assay based on glucose consumption may provide clinically useful MICs rapidly. The rapid susceptibility assay (RSA), which provides MICs in less than 8 h, was compared with the standard broth microdilution susceptibility assay (Clinical and Laboratory Standards Institute, document M27-A3, 2008) for 56 Candida species strains. Variables which are known to influence MICs determined by the M27-A3 method were also assessed for their effects on the RSA results. Excellent agreement (>90%) between the results of the RSA and M27-A3 methods was achieved for all three FDA-approved echinocandins (micafungin, caspofungin, and anidulafungin). Candida lusitaniae strains were responsible for most of the discordant results. Assay variables such as the test medium, the age of the inoculum culture, and the presence of human serum affected MIC results from the RSA and the M27-A3 method similarly. The RSA is equivalent to the standard M27-A3 method for determining echinocandin MICs for Candida species. The RSA provides MIC results in less than 8 h and can be applied to old and young yeast colonies. The assay could potentially provide clinically useful MICs on the same day that yeast growth from a specimen is first detected on solid medium.Echinocandins are the newest class of FDA-approved drugs for the treatment of systemic fungal infections and the first approved class of antifungals that target the cell wall, specifically the synthesis of β-1,3-glucan. The spectrum of fungi which are susceptible to echinocandins is narrower than the spectrum of fungi susceptible to the polyenes, despite the presence of β-1,3-glucan in the cell walls of many nonzygomycetous fungi. However, the echinocandins have proven to be excellent agents for the treatment of candidiasis. In 2004, the Infectious Diseases Society of America published a practice guideline in which echinocandins are listed as one of the first-line options for the treatment of candidemia (8).Similar to the actions of β-lactam antibacterial agents, the inhibition of cell wall polysaccharide synthesis by echinocandins potentially results in cell death. Echinocandins inhibit β-1,3-glucan synthase (GS) in the cell membrane by targeting the Fks1p and Fks2p components of GS complexes. As a consequence, β-1,3-glucan, which is the predominant polysaccharide in the cell walls of Candida species, is not produced while cell wall remodeling and cell expansion continue to occur, resulting in weakness in the wall and susceptibility to cell lysis due to overwhelming turgor pressure (6). A potential outcome of the events prior to cell death is diminution of glucose uptake or consumption.Clancy et al. (2) have shown that cells exposed to echinocandins for only 1 h at levels in excess of the known MIC for the strain exhibit growth inhibition, as demonstrated by decreased numbers of CFU compared to those of untreated controls. This observation suggested that it may be possible to determine MICs of echinocandins for different yeast strains on the same day of testing by using the rapid susceptibility assay (RSA), especially because the RSA is based on levels of glucose consumption by cells exposed to the drug relative to those by control cells (4, 11). The RSA is similar to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 broth microdilution assay except that the RSA inoculum concentration is higher, the incubation period is shorter, and the end point is based on glucose uptake measured spectrophotometrically (3, 4, 11).The RSA was originally used by Cutler et al. (4, 11) to provide MICs of fluconazole and amphotericin in less than 24 h. Their results showed that MICs of the rapidly acting antifungal agent amphotericin B can be obtained in 6 h but that the slower-acting triazole fluconazole requires at least 8 to 18 h. The RSA has since been applied to Aspergillus fumigatus to obtain MICs of voriconazole, itraconazole, and amphotericin B (12, 13).Here, we evaluate the ability of the RSA to provide echinocandin MICs equivalent to those obtained by the “gold standard” CLSI M27-A3 method, which requires an incubation period of 24 h, for Candida species on the same day of testing. Further, the RSA and M27-A3 method were compared to determine if the MICs provided are similarly affected by variations in inoculum growth conditions, the test medium, and the presence of human serum. The results demonstrate that the RSA method is comparable to the M27-A3 method but provides MICs in less than 8 h regardless of whether inoculum cells were prepared from 4- or 1-day-old cultures.     

Aberrant IgG galactosylation precedes disease onset,correlates with disease activity,and is prevalent in autoantibodies in rheumatoid arthritis

Objective

To examine the association between aberrant IgG galactosylation and disease parameters in rheumatoid arthritis (RA).

Methods

Analysis of N‐glycan in serum samples from multiple cohorts was performed. The IgG N‐glycan content and the timing of N‐glycan aberrancy relative to disease onset were compared in healthy subjects and in patients with RA. Correlations between aberrant galactosylation and disease activity were assessed in the RA cohorts. The impact of disease activity, sex, age, anti–cyclic citrullinated peptide (anti‐CCP) antibody titer, disease duration, and C‐reactive protein level on aberrant galactosylation was determined using multivariate analysis. The N‐glycan content was also compared between epitope affinity–purified autoantibodies and the remaining IgG repertoire in RA patients.

Results

Our results confirm the aberrant galactosylation of IgG in RA patients as compared with healthy controls (mean ± SD 1.36 ± 0.43 versus 1.01 ± 0.23; P < 0.0001). We observed a significant correlation between levels of aberrant IgG galactosylation and disease activity (Spearman's ρ = 0.37, P < 0.0001). This correlation was higher in women (Spearman's ρ = 0.60, P < 0.0001) than in men (Spearman's ρ = 0.16, P = 0.10). Further, aberrant IgG galactosylation substantially predated the onset of arthritis and the diagnosis of RA (3.5 years) and resided selectively in the anticitrullinated antigen fraction.

Conclusion

Our findings identify aberrant IgG galactosylation as a dysregulated component of the humoral immune response in RA that begins prior to disease onset, associates with disease activity in a sex‐specific manner, and resides preferentially in autoantibodies.

     

Psychosocial issues in adolescents with cancer

Cancer in adolescents is uncommon and when it occurs raises a number of unique challenges for both the patient and their families. Adolescence is a period of time of significant physical and emotional changes and a diagnosis of cancer during this time has a major impact on their psychological and physical development. In this review we will look at the psychosocial issues facing adolescents who have cancer. We will address adolescent development, issues related to informed consent and assent, initial responses to the diagnosis of cancer, quality of life and the experience of the adolescent with cancer, psychological adjustment, support systems, body image issues, sexuality, education, hope, and treatment compliance.     

Haptoglobin genotype determines myocardial infarct size in diabetic mice.

OBJECTIVES: We sought to understand the importance of oxidative stress in explaining why the haptoglobin (Hp) genotype determines myocardial infarction (MI) size in diabetes mellitus (DM). BACKGROUND: Two common alleles (1 and 2) exist at the Hp locus in humans. The Hp 2 allele is associated with increased MI size in individuals with DM. In vitro, the Hp 2 protein is associated with increased generation of oxidatively active iron, whereas the Hp 1 protein is associated with increased production of the antioxidant cytokine interleukin (IL)-10. METHODS: Myocardial infarction was produced by myocardial ischemia-reperfusion (IR) in DM C57BL/6 mice carrying the Hp 1 or Hp 2 allele. Myocardial oxidative stress after IR was assessed using electrospray ionization mass spectrometry. Redox active iron and IL-10 were measured in the serum after IR. RESULTS: Myocardial infarction size was significantly larger in Hp 2 mice as compared with Hp 1 mice (44.3 +/- 9.3% vs. 21.0 +/- 4.0%, p = 0.03), and these larger infarctions were associated with a significant increase in a panel of hydroxyl-eicosatetraenoic acids. Redox active iron was greater in Hp 2 mice (0.45 +/- 0.11 micromol/l vs. 0.14 +/- 0.05 micromol/l, p = 0.02), whereas IL-10 was greater in Hp 1 mice (85.8 +/- 12.9 pg/microl vs. 46.7 +/- 10.8 pg/microl, p = 0.04) after IR. Administration of an antioxidant (BXT-51072) to Hp 2 mice reduced myocardial injury after IR by more than 80% (p = 0.003), but no myocardial protection was provided by the antioxidant to Hp 1 mice. CONCLUSIONS: The increased MI size in DM Hp 2 mice occurring after IR may be due to increased oxidative stress.