Molecular typing and in vitro resistance of Cryptococcus neoformans clinical isolates obtained in Germany between 2011 and 2017

Cryptococcosis is a fungal infection of the central nervous system predominantly caused by Cryptococcus neoformans in immunocompromised patients. In several countries worldwide, up to 50% of isolates show in vitro resistance to clinically used antifungals including fluconazole. No prospective data on susceptibility to antifungal drugs are available for Germany. In this study, we characterised all C. neoformans isolates collected from individual patients’ samples at the German reference laboratory for cryptococcosis 2011 and 2017 (n = 133) by multi-locus sequence typing and phenotypic drug susceptibility testing. We identified serotype A/genotype VNI isolates belonging to clonal complexes previously described from Europe, Africa, Asia and South America as the most prevalent agents of cryptococcosis in Germany. Overall, we observed minimal inhibitory concentrations (MICs) above the epidemiological cut-offs (ECVs) in 1.6% of isolates regarding fluconazole and 2.3% of isolates regarding 5-flucytosine. Here, two C. neoformans var. grubii isolates displayed decreased drug susceptibility to fluconazole, one of them additionally to 5-flucytosine. We also found 5-flucytosine MICs above the ECV for two C. neoformans var. neoformans isolates. We identified a novel mutation in the ERG11 gene which might be associated with the elevated fluconazole MIC in one of the isolates. The clinical importance of the detected in vitro resistance is documented by patient histories showing relapsed infection or primary fatal disease. Of note, sertraline demonstrated antifungal activity comparable to previous reports. Systematic collection of susceptibility data in combination with molecular typing of C. neoformans is important to comprehensively assess the spread of isolates and to understand their drug resistance patterns.     

Management of venous thromboembolism in high-grade glioma: Does low molecular weight heparin increase intracranial bleeding risk?

BackgroundVenous thromboembolism (VTE) occurs in up to 30% of patients with high-grade glioma (HGG). Concern for increased risk of intracranial hemorrhage (ICH) with therapeutic anticoagulation (AC) complicates VTE treatment. Some retrospective studies have reported an increased risk of ICH associated with therapeutic AC; however, effective alternatives to AC are lacking. The aim of our study is to assess the risk of ICH in HGG patients with VTE on low molecular weight heparin (LMWH).MethodsWe performed a retrospective matched cohort study of HGG patients from January 2005 to August 2016. Blinded review of neuroimaging for ICH was performed. For analysis of the primary endpoint, estimates of cumulative incidence (CI) of ICH were calculated using competing risk analysis with death as competing risk; significance testing was performed using the Gray’s test. Median survival was estimated using the Kaplan-Meier method.ResultsTwo hundred twenty patients were included, 88 (40%) with VTE treated with LMWH, 22 (10%) with VTE, not on AC, and 110 (50%) without VTE. A total of 43 measurable ICH was recorded: 19 (26%) in LMWH, 3 (14%) in VTE not on AC, and 21 (19%) in non-VTE cohort. No significant difference was observed in the 1-year CI of ICH in the LMWH cohort and non-AC with VTE group (17% vs 9%; Gray’s test, P = .36). Among patients without VTE, the 1-year CI of ICH was 13%. Median survival was similar among all 3 cohorts.ConclusionsOur data suggest that therapeutic LMWH is not associated with substantially increased risk of ICH in HGG patients.     

Effect of chronic treatment with bovine recombinant growth hormone on cardiac dysfunction and lesion progression in UM-X7.1 cardiomyopathic hamsters

In view of the potentially beneficial effect of GH on ventricular function of humans suffering from idiopathic dilated cardiomyopathy, we undertook a study to evaluate the optimal time to initiate treatment with GH and its duration in UM-X7.1 cardiomyopathic hamsters (CMH). GH (1 mg/kg.d) therapy was initiated either in the early or late (30 and 160 d old, respectively) phases of the disease and continued until death at 240 d of age. Age- and sex-matched Golden Syrian hamsters (GSH) were used as controls. Basal IGF-1 levels in serum were reduced by nearly half in CMH compared with GSH but were increased within a physiological range in male hamsters. In contrast, female hamsters presented elevated basal serum IGF-1 levels that were not further elevated by GH administration, as reported in experimental models and humans. Accordingly, the present study will focus on the effects of GH therapy on cardiac performance in male hamsters. GH did not improve ventricular function when starting at a late stage of the disease compared with CMH controls. Maximum rate of left ventricular pressure development decreased by approximately 64% in CMH treated early with recombinant bovine GH. Ventricular dysfunction was associated with morphologic indices of hypertrophy, ventricular dilatation, and extensive fibrosis. Mortality was strikingly increased in GH-treated CMH for 210 d (four males and eight females), as opposed to four females (and no male) in the vehicle-treated group. These results suggest that chronic treatment with recombinant bovine GH in CMH, starting at an early stage of lesion development, is associated with a reduced cardiac performance at the terminal stage of the disease.     

Chronic AMPK activation evokes the slow, oxidative myogenic program and triggers beneficial adaptations in mdx mouse skeletal muscle

A therapeutic approach for Duchenne muscular dystrophy (DMD) is to up-regulate utrophin in skeletal muscle in an effort to compensate for the lack of dystrophin. We previously hypothesized that promotion of the slow, oxidative myogenic program, which triggers utrophin up-regulation, can attenuate the dystrophic pathology in mdx animals. Since treatment of healthy mice with the AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) enhances oxidative capacity and elicits a fast-to-slow fiber-type transition, we evaluated the effects of chronic AMPK stimulation on skeletal muscle phenotype and utrophin expression in mdx mice. Daily AICAR administration (500 mg/kg/day, 30 days) of 5-7-week-old mdx animals induced an elevation in mitochondrial cytochrome c oxidase enzyme activity, an increase in myosin heavy-chain type IIa-positive fibers and slower twitch contraction kinetics in the fast, glycolytic extensor digitorum longus muscle. Utrophin expression was significantly enhanced in response to AICAR, which occurred coincident with an elevated β-dystroglycan expression along the sarcolemma. These adaptations were associated with an increase in sarcolemmal structural integrity under basal conditions, as well as during damaging eccentric contractions ex vivo. Notably, peroxisome proliferator-activated receptor γ co-activator-1α (PGC-1α) and silent information regulator two ortholog 1 protein contents were significantly higher in muscle from mdx mice compared with wild-type littermates and AICAR further increased PGC-1α expression. Our data show that AICAR-evoked muscle plasticity results in beneficial phenotypic adaptations in mdx mice and suggest that the contextually novel application of this compound for muscular dystrophy warrants further study.     

Satellite glial cell proliferation in the trigeminal ganglia after chronic constriction injury of the infraorbital nerve

We have examined satellite glial cell (SGC) proliferation in trigeminal ganglia following chronic constriction injury of the infraorbital nerve. Using BrdU labeling combined with immunohistochemistry for SGC specific proteins we positively confirmed proliferating cells to be SGCs. Proliferation peaks at approximately 4 days after injury and dividing SGCs are preferentially located around neurons that are immunopositive for ATF‐3, a marker of nerve injury. After nerve injury there is an increase GFAP expression in SGCs associated with both ATF‐3 immunopositive and immunonegative neurons throughout the ganglia. SGCs also express the non‐glial proteins, CD45 and CD163, which label resident macrophages and circulating leukocytes, respectively. In addition to SGCs, we found some Schwann cells, endothelial cells, resident macrophages, and circulating leukocytes were BrdU immunopositive. GLIA 2013;61:2000–2008     

Essential Role of Osteopontin in Smoking-Related Interstitial Lung Diseases

Smoking-related interstitial lung diseases are characterized by the accumulation of macrophages and Langerhans cells, and fibrotic remodeling, which are linked to osteopontin (OPN) expression. Therefore, OPN levels were investigated in bronchoalveolar lavage (BAL) cells in 11 patients with pulmonary Langerhans cell histiocytosis (PLCH), 15 patients with desquamative interstitial pneumonitis (DIP), 10 patients with idiopathic pulmonary fibrosis, 5 patients with sarcoidosis, 13 otherwise healthy smokers, and 19 non-smoking controls. Furthermore, OPN overexpression was examined in rat lungs using adenoviral gene transfer. We found that BAL cells from patients with either PLCH or DIP spontaneously produced abundant amounts of OPN. BAL cells from healthy smokers produced 15-fold less OPN, and those cells from non-smoking healthy volunteers produced no OPN. BAL cells from patients with either idiopathic pulmonary fibrosis or sarcoidosis produced significantly less OPN, as compared with patients with PLCH. These data were confirmed by immunochemistry. Nicotine stimulation increased production of both OPN and granulocyte-macrophage colony stimulating factor by alveolar macrophages from smokers. Nicotinic acetylcholine receptor expression resembled the pattern of spontaneous OPN production and was dramatically increased in both PLCH and DIP. OPN overexpression in rat lungs induced lesions similar to PLCH with marked alveolar and interstitial accumulation of Langerhans cells. Our findings suggest a pathogenetic role of increased OPN production in both PLCH and DIP by promoting the accumulation of macrophages and Langerhans cells.Cigarette smoke is linked to a variety of lung diseases including chronic obstructive pulmonary disease, lung cancer, and interstitial lung diseases. Respiratory bronchiolar interstitial lung disease, desquamative interstitial pneumonitis (DIP), and pulmonary Langerhans cell histiocytosis (PLCH) belong to the group of smoking-related interstitial lung diseases.^1,2,3 Cigarette smoke is a complex mixture of more than 4000 compounds and is known to cause systemic and pulmonary effects.⁴ However, the underlying mechanisms as to how cigarette smoking leads to the changes observed in smoking-related interstitial lung diseases are largely unknown.^1,2,3Cigarette smoke induces inflammation, oxidative stress, and tissue injury, and has an important effect on the number, distribution, and activation state of macrophages and Langerhans cells.^5,6 There is a strong epidemiological link between PLCH and smoking. PLCH is characterized by the accumulation of activated Langerhans cells originating from the distal bronchiole walls.^1,2,3,7 The accumulations of Langerhans cells are poorly demarcated and extend to the adjacent alveoli, which often contain an abundance of pigmented macrophages. These areas show morphological changes similar to DIP.^7,8 In DIP, the predominant feature is the accumulation of alveolar macrophages, densely filling the alveolar lumen, combined with moderate fibrotic interstitial remodeling.^1,2As measured by bronchoalveolar lavage (BAL) in healthy individuals, cigarette smoking induces a 5- to 10-fold increase in alveolar macrophages in a dose-response curve.^9,10,11 It was shown that concentrations of granulocyte-macrophage colony stimulating factor (GMCSF) in patients with PLCH are increased,¹² but the mechanisms that lead to the expansion of the pulmonary macrophage pool and fibrosis in smokers are poorly understood.^1,2,3 Based on the findings of a microarray study, Woodruff et al¹³ have recently proposed that alveolar macrophages from smokers exhibit a distinctive macrophage activation state that is accompanied by increased OPN expression. Osteopontin is a glycoprotein found in the extracellular matrix of bone.¹⁴ However, multiple studies have reported cytokine properties of OPN in cell-mediated immunity.¹⁴ Further, OPN exhibits a strong chemotactic activity for macrophages, monocytes, Langerhans cells, and dendritic cells.^15,16,17In the context of these findings we speculated that OPN might be involved in the pathogenesis of smoking-related lung interstitial diseases. We found abundant OPN production by alveolar macrophages from patients with PLCH and DIP. Alveolar macrophages from both healthy smokers and patients with DIP and PLCH show up-regulated nicotine receptor expression as a sign of chronic nicotine stimulation. Further, nicotine directly induced OPN and GMCSF in alveolar macrophages. Our data provides evidence for a role of osteopontin in the pathogenesis of smoking- related interstitial lung diseases.     

TRF1 is degraded by ubiquitin-mediated proteolysis after release from telomeres

Mammalian telomeres are coated by the sequence-specific, DNA-binding protein, TRF1, a negative regulator of telomere length. Previous results showed that ADP-ribosylation of TRF1 by tankyrase 1 released TRF1 from telomeres and promoted telomere elongation. We now show that loss of TRF1 from telomeres results in ubiquitination and degradation of TRF1 by the proteasome and that degradation is required to keep TRF1 off telomeres. Ubiquitination of TRF1 is regulated by its telomere-binding status; only the telomere-unbound form of TRF1 is ubiquitinated. Our findings suggest a novel mechanism of sequential post translational modification of TRF1 (ADP-ribosylation and ubiquitination) for regulating access of telomerase to telomeres.     

Early effector maturation of naïve human CD8+ T cells requires mitochondrial biogenesis

The role of mitochondrial biogenesis during naïve to effector differentiation of CD8⁺ T cells remains ill explored. In this study, we describe a critical role for early mitochondrial biogenesis in supporting cytokine production of nascent activated human naïve CD8⁺ T cells. Specifically, we found that prior to the first round of cell division activated naïve CD8⁺ T cells rapidly increase mitochondrial mass, mitochondrial respiration, and mitochondrial reactive oxygen species (mROS) generation, which were all inter‐linked and important for CD8⁺ T cell effector maturation. Inhibition of early mitochondrial biogenesis diminished mROS dependent IL‐2 production – as well as subsequent IL‐2 dependent TNF, IFN‐γ, perforin, and granzyme B production. Together, these findings point to the importance of mitochondrial biogenesis during early effector maturation of CD8⁺ T cells.