In vitro and in vivo studies of the trypanocidal activity of a diarylthiophene diamidine against Trypanosoma cruzi

Aromatic diamidines are DNA minor groove-binding ligands that display excellent antimicrobial activity against fungi, bacteria, and protozoa. Due to the currently unsatisfactory chemotherapy for Chagas’ disease and in view of our previous reports regarding the effect of diamidines and analogues against both in vitro and in vivo Trypanosoma cruzi infection, this study evaluated the effects of a diarylthiophene diamidine (DB1362) against both amastigotes and bloodstream trypomastigotes of T. cruzi, the etiological agent of Chagas’ disease. The data show the potent in vitro activity of DB1362 against both parasite forms that are relevant for mammalian infection at doses which do not exhibit cytotoxicity. Ultrastructural analysis and flow cytometry studies show striking alterations in the nuclei and mitochondria of the bloodstream parasites. In vivo studies were performed at two different drug concentrations (25 and 50 mg/kg/day) using a 2-day or a 10-day regimen. The best results were obtained when acutely infected mice were treated with two doses at the lower concentration, resulting in 100% survival, compared to the infected and untreated mice. Although it did not display higher efficacy than benznidazole, DB1362 reduced both cardiac parasitism and inflammation, and in addition, it protected against the cardiac alterations (determined by measurements) common in T. cruzi infection. These results support further investigation of diamidines and related compounds as potential agents against Chagas’ disease.     

Mild cognitive impairment and Alzheimer patients display different levels of redox-active CSF iron

Oxidative stress constitutes a hallmark of Alzheimer's disease (AD). Recent studies also point to redox active metals such as iron, copper and zinc in mediating oxidative stress in AD pathogenesis. However, the reactivity of cerebrospinal fluid (CSF) iron and its possible correlation with the severity of cognitive decline in both Alzheimer's patients and subjects with mild cognitive impairment (MCI) is still unknown. Here we show that different stages of cognitive and functional impairment are associated with changes in CSF reactive iron. In this work, we compared CSF samples from 56 elders, classified into 4 groups according to their scores on the Clinical Dementia Rating scale (CDR). Total CSF iron was analyzed by atomic absorption spectrometry. Redox-active iron was analyzed by a novel fluorimetric assay. One-way ANOVA was used to test differences in mean values, and Newman-Keuls Multiple Comparison Test was used for multi group comparisons. No difference in total CSF iron was found between different groups. Significant amounts of redox-active iron were found in CSF and their levels correlated with the extent of cognitive impairment. Redox-active CSF iron levels increased with the degree of cognitive impairment from normal to MCI subjects, while AD patients showed an abrupt decrease to levels close to zero. Given the relevance of oxidative damage in neurodegeneration, it might be possible to associate the development of cognitive and functional decline with the presence of redox-active iron in the CSF. The decrease in redox-active iron found in AD patients may represent a terminal situation, whereby the central nervous system attempts to minimize iron-associated toxicity.     

The damage signals hypothesis of Alzheimer's disease pathogenesis

Virtually none of the hypotheses on Alzheimer's disease (AD) pathogenesis address the earliest events that trigger the molecular alterations that precede cerebral degeneration and account for the diversity of risk factors that converge on a well-defined disease phenotype. We propose that long-term activation of the innate immune system by an individual array of risk factors constitutes a unifying mechanism leading to the triggering of an inflammatory cascade that converges in cytoskeletal alterations (tau aggregation, paired helical filament formation) as a previously hypothesized final common pathway in AD. The key pathogenic phenomena consist in the long-term, maladaptive activation of innate immunity-triggering receptors--such as the toll-like and advanced glycation end-products receptors, and others located in the microglial membrane--by seemingly heterogeneous risk factors such as hyperlipidemia, hyperglycemia, oxidative stress, head injury, amyloid oligomers, etc. Our hypothesis provides a unifying mechanism that explains both the diversity of risk factors acting over long periods of time and the individual response to such insults. This formulation is amenable to both empirical testing and implementation into therapeutic strategies that may lead to effective prevention of AD as well as other disorders in which impaired regulation of the innate immunity is the unifying cause of the condition.     

Treatment of the intraabdominal abscesses through percutaneous ultrasound-guided drainage in oncological patients: Clinical and microbiological data

Aim of the study Oncological patients are particularly prone to the onset of septic complications such as abdominal abscesses. The aim of our study was to analyze clinical and microbiological data in a population of oncological patients, submitted to percutaneous ultrasound-guided drainage (PUD) for postoperative abdominal abscesses. Patients and methods Data from 24 patients operated on for neoplastic pathologies and treated with PUD for abdominal abscesses during the postoperative period were reviewed. In all cases cultural examination with antibiogram was performed. Results In 5 out of 24 patients (20.8%), the abdominal abscesses appeared after the discharge, with a mean hospital stay of 34.2 ± 24.9 days. In six out of 24 patients (25%) there were multiple abscesses localizations. The cultural examination was positive in 23 patients and negative only in one patient. Abscesses localized only in the upper abdominal regions had a significant prevalence of monomicrobial cultural examinations (57.1%) with respect to the results for abscesses placed in the lower abdominal regions, that were polymicrobial in 88.8% of cases (p = 0.027). An antibiogram demonstrated a stronger activity of beta-lactamines, chinolones, and glycopeptides with respect to aminogycosides, cephalosporins, and metronidazole. Conclusions In oncological patients, the planning of the empiric antibiotic therapy should be based on the anatomotopographic localization of the abdominal abscess and on the typology of the operation performed giving preference to beta-lactamines, chinolones and glycopeptides.     

Newborn hearing screening test with multiple auditory steady-state responses

IntroductionThe techniques most frequently used within a screening context (otoacoustic emissions and click auditory brainstem response) have well-known limitations in hearing loss detection.ObjectiveThis study examines the feasibility of a semi-automated multiple auditory steady-state responses (MSSR) system designed for neonatal hearing screening.MethodsA sample of 50 newborns without risk factors (well-babies) was tested within two weeks of birth. All had detectable auditory brainstem responses to clicks down to 40 dB nHL in both ears. Two amplitude modulated carrier tones of 500 and 2,000 Hz were mixed together and presented simultaneously. Each infant (and ear) was screened with the MSSR system; to simulate a hearing loss, a recording without stimulation was also obtained.ResultsMean auditory thresholds were 42.5 ± 7 dB HL at 500 Hz and 35.5 ± 6 dB HL at 2,000 Hz. The average duration of the MSSR recording was 2.6 ± 1.6 minutes for each tested ear and the overall duration of the screening procedure (including electrode fitting and infant preparation) was 17.8 ± 3.7 minutes. The diagnostic sensibility and the positive predictive values of the MSSR semi-automatic screening system was 100% and 96% respectively, with specificity of 96% and negative predictive values of 100%.ConclusionsAlthough the diagnostic efficiency of the semi-automated MSSR system was found adequate, further technological improvements are still necessary to facilitate its use in the context of universal newborn hearing screening program.     

RIPK1 activates distinct gasdermins in macrophages and neutrophils upon pathogen blockade of innate immune signaling

Injection of effector proteins to block host innate immune signaling is a common strategy used by many pathogenic organisms to establish an infection. For example, pathogenic Yersinia species inject the acetyltransferase YopJ into target cells to inhibit NF-κB and MAPK signaling. To counteract this, detection of YopJ activity in myeloid cells promotes the assembly of a RIPK1–caspase-8 death–inducing platform that confers antibacterial defense. While recent studies revealed that caspase-8 cleaves the pore-forming protein gasdermin D to trigger pyroptosis in macrophages, whether RIPK1 activates additional substrates downstream of caspase-8 to promote host defense is unclear. Here, we report that the related gasdermin family member gasdermin E (GSDME) is activated upon detection of YopJ activity in a RIPK1 kinase–dependent manner. Specifically, GSDME promotes neutrophil pyroptosis and IL-1β release, which is critical for anti-Yersinia defense. During in vivo infection, IL-1β neutralization increases bacterial burden in wild-type but not Gsdme-deficient mice. Thus, our study establishes GSDME as an important mediator that counteracts pathogen blockade of innate immune signaling.

Gasdermins are a family of recently described pore-forming proteins and are emerging as key drivers of cell death and inflammation. Gasdermins comprise a cytotoxic N-terminal domain connected to an inhibitory carboxyl-terminal domain and are activated upon proteolytic cleavage (1, 2). This cleavage event releases the cytotoxic N-terminal fragment, which creates membrane pores and triggers a form of lytic cell death called pyroptosis (3–6). Gasdermin D (GSDMD) is arguably the best characterized family member to date and is activated upon proteolysis by caspase-1, 4, 5, 8, and 11 and serine proteases (7–14). Active GSDMD promotes host defense by eliminating the replicating niche of intracellular pathogens (15) and inducing the extrusion of antimicrobial neutrophil extracellular traps (NETs) (16). In addition, GSDMD pores act as a conduit for bioactive IL-1β release (17–19), a potent proinflammatory cytokine that similarly requires proteolytic cleavage by caspase-1 or -8 to gain biological activity (20). By contrast, gasdermin E (GSMDE [also known as DFNA5]) is activated by apoptotic caspase-3 and 7 and granzyme B, which drives tumor cell pyroptosis and anti-tumor immunity (21–23). The physiological function of GSDME in primary immune cells and its potential role in host defense remain unresolved and have not been reported.Pathogenic Yersinia are a group of Gram-negative extracellular bacteria that causes disease ranging from gastroenteritis (Yersinia pseudotuberculosis) to plague (Y. pestis). A major mechanism by which pathogenic Yersinia establish systemic infection is by injecting the effector protein YopJ, an acetyltransferase that blocks transforming growth factor beta-activated kinase 1 (TAK1), to inhibit host innate immune signaling and proinflammatory cytokine production (24). To counteract this, detection of YopJ activity by myeloid cells induces the assembly of a cytoplasmic death–inducing complex that comprises receptor-interacting serine/threonine protein kinase 1 (RIPK1), fas-associated protein with death domain, and caspase-8 (24–26). During in vivo infection, RIPK1/caspase-8–dependent cell death in myeloid cells restricts bacterial dissemination and replication at distal sites by inducing proinflammatory cytokine production from uninfected bystander cells (24). More recently, GSDMD was identified as a caspase-8 substrate during Yersinia infection that drives antimicrobial defense in vivo (11, 12, 27). However, whether RIPK1 activates additional substrates to restrict Yersinia infection is unclear and is a focus of this study. Here, we identify GSDME as a substrate activated downstream of RIPK1 that confers host resistance against Yersinia. Gsdme-deficient mice failed to control bacterial replication in the spleen and liver and consequently are more susceptible to Yersinia infection than wild-type (WT) animals. Mechanistically, our data reveal that RIPK1 promotes caspase-3–dependent GSDME activation and IL-1β release in neutrophils, but not macrophages. Neutralization of IL-1β impaired bacterial clearance in WT, but not Gsdme^−/−, animals, indicating that IL-1β is mainly secreted through GSDME pores during Yersinia challenge in vivo.