Emerging issue of fluconazole-resistant candidemia in a tertiary care hospital of southern italy: time for antifungal stewardship program

An increased number of patients is at risk of Candida spp. bloodstream infection (CBSI) in modern medicine. Moreover, the rising of antifungal resistance (AR) was recently reported. All consecutive CBSI occurred in our Hospital (consisting of 1,370 beds) between 2015 and 2018, were reviewed. For each case, Candida species, AR pattern, ward involved and demographic data of patients were recorded. Overall, 304 episodes of CBSI occurred, with a median (q1:first-,q3:third quartile) of 77 (71-82) CBSI/year. Over the years, a significant increase of CBSI due to C. albicans compared to non-albicans strains was recorded in medical wards (from 65% to 71%, p=0.030), while this ratio remained stable in others. An increase of resistant strains to multiple antifungals such as C. guillermondii was noticed in recent years (from 0% to 9.8%, p=0.008). Additionally, from 2015 to 2018 an increase in fluconazole-resistance was recorded in our Hospital (from 7.4% to 17.4%, p=0.025) and a slight increase in voriconazole-resistance (from 0% to 7% in 2018, p=0.161) was observed, while resistance to echinocandin and amphotericin B remained firmly below 2%.This study suggests a rapid spread of antifungal resistance in our Hospital; therefore, an appropriate antifungal stewardship programs is urgently warranted.     

Dupilumab therapy for patients with refractory eosinophilic otitis media associated with bronchial asthma

ObjectivesEosinophilic otitis media (EOM) is an intractable otitis media mostly associated with bronchial asthma. Dupilumab, an anti-interleukin (IL)-4 receptor (R)α, is effective and has been approved for use in patients with moderate to severe bronchial asthma, atopic dermatitis and chronic rhinosinusitis with nasal polyposis, whose diseases are not controlled by previous treatments including other molecular targeted drugs. We aimed to assess efficacy of dupilumab in three EOM patients with associated bronchial asthma, who were poor responders to previous topical and systemic corticosteroid therapy and molecular targeted therapies.Patients and methodsThree patients with severe, refractory EOM (two with a granulation type) associated with bronchial asthma received dupilumab as add-on therapy for at least 6 months. The efficacy of dupilumab therapy was evaluated using severity scores, symptom scores, hearing acuities, temporal bone computed tomography (CT) scores, and surrogate markers before and after therapy.ResultsSeverity scores in all patients were dramatically reduced to 2 points or less (full score: 16 points) after initiation of therapy. Air conduction hearing levels were improved in all patients. Temporal bone CT scores in two patients were reduced, and serum IgE levels in all three patients also decreased following therapy.ConclusionWe provide the first report that add-on dupilumab therapy was effective in patients with severe, refractory EOM who did not respond to the treatments including other molecular targeted therapy. Patients with severe middle ear mucosal change may benefit particularly from dupilumab therapy.     

A specific basal body linker protein provides the connection function for basal body inheritance in trypanosomes

Centrioles and basal bodies (CBBs) are found in physically linked pairs, and in mammalian cells intercentriole connections (G1–G2 tether and S–M linker) regulate centriole duplication and function. In trypanosomes BBs are not associated with the spindle and function in flagellum/cilia nucleation with an additional role in mitochondrial genome (kinetoplast DNA [kDNA]) segregation. Here, we describe BBLP, a BB/pro-BB (pBB) linker protein in Trypanosoma brucei predicted to be a large coiled-coil protein conserved in the kinetoplastida. Colocalization with the centriole marker SAS6 showed that BBLP localizes between the BB/pBB pair, throughout the cell cycle, with a stronger signal in the old flagellum BB/pBB pair. Importantly, RNA interference (RNAi) depletion of BBLP leads to a conspicuous splitting of the BB/pBB pair associated only with the new flagellum. BBLP RNAi is lethal in the bloodstream form of the parasite and perturbs mitochondrial kDNA inheritance. Immunogold labeling confirmed that BBLP is localized to a cytoskeletal component of the BB/pBB linker, and tagged protein induction showed that BBLP is incorporated de novo in both new and old flagella BB pairs of dividing cells. We show that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent but separable events in evolutionarily diverse cells and we provide a unifying model explaining centriole/BB linkage differences between such cells.

Centrioles and basal bodies (CBBs) are microtubule structures found in many major eukaryotic groups; where present, they are vital for cilia and flagella formation (1) and play important roles in cell division and developmental events. CBB assembly pathways share a common set of key regulatory proteins, indicating that these structures are variations of a common pattern (2).Faithful centriole duplication and segregation in proliferative eukaryotic cells is a well-orchestrated process (albeit with variations of pattern in different cell types across evolution) under strict temporal and spatial control and usually involve “templating”’ from a previously formed CBB (3). Two particular conceptual themes, a linker and a tether (4), have been rehearsed to explain number control, inheritance patterns, and centriole properties in mammalian cells. In interphase G1, each cell has a single centriole pair, and the duplication cycle starts in the G1/S transition and is very well described in its temporal sequence (5). During the centriole duplication and segregation cycle, centrioles are connected by the two different types of structures—the “tether” and “linker”—whose presence and disassembly at specific stages of the cell cycle are important for faithful cell-cycle progression (4, 6). The tether connects the proximal ends of the two parental centrioles from G1 to late G2 and appears important in providing a single cytoplasmic microtubule organizing center in organisms with a centrosomal architecture. Some significant studies have revealed essential components of the tether, for example CNAP and Rootletin (7, 8), Cep68 (9), LRRC45 (10), Centlein (11), and CCDC102B (12). The linker forms during S phase and connects the proximal end of the nascent procentriole to the side of the parental centriole in the orthogonal orientation. In the centriole cycle this link is described as being removed in late M phase when centriole disengagement occurs. There are two iconic features of centriole disengagement: a reorientation resulting in the loss of the original orthogonal orientation of the two paired centrioles and, second, an ability to transiently move apart (4). There is an expectation that there will be molecular components specific to each structure, but other components of the centrosome as a wider concept might play a role in both structures (4). The literature has seen a variety of terms used to describe these conceptual structures: centriole linker, centrosome linker, and so on. Here, for clarity in discussing cross-evolutionary fundamental concepts we will use the simple terms “tether” and “linker” as defined by Nigg and Stearns (4).Current knowledge on the composition of the linker is limited, but studies in Drosophila suggest that the SAS6–ANA2 complex may play a role in centriole engagement (13). Interestingly, linker cleavage in disengagement in human cells requires the activity of the polo-like kinases and of separase, the protease responsible for sister chromatid separation (14, 15).Many eukaryotic cells do not exhibit a centrosomally organized cytoplasm—particularly those that proliferate with assembled flagella or cilia. Although essential for cilia/flagella assembly, in such systems CBBs are often not directly involved in mitotic spindle architecture since mitosis is closed (i.e., without nuclear envelope disassembly), and anaphase and CBB separation are not concurrent. Further, in systems such as trypanosomes (16) and Leishmania (17) CBBs perform a central role in the segregation of the single mitochondrial DNA network (the kinetoplast) (18). Cell division in these organisms, where microtubule organizing centers are dispersed and do not cluster into a centrosome, involves the coordinated duplication and segregation of the nucleus (N) and the kinetoplast (K) (16). Trypanosome cells in G1 have a “1K1N” configuration, characterized by the presence of a single nucleus and a single kinetoplast, physically associated with a BB pair containing a mature BB, which subtends the flagellar axoneme, and a probasal body (pBB). At the start of S phase the pBB matures and elongates forming the new flagellum, and a new pBB forms next to each mature BB (19). The kinetoplast is divided during S/G2 (19, 20) via movement apart of the BB pairs, resulting in a characteristic “2K1N” cell, with two kinetoplasts and one nucleus. The intranuclear mitotic spindle then forms and mitosis gives rise to a cell with a “2K2N” configuration with widely separated BB pairs and associated new and old flagella. Subsequently, a mainly longitudinal cleavage furrow separates the nascent cell daughters (19).In this study, we describe the BB linker protein (BBLP) that localizes between the BB and the pBB throughout the cell cycle. BBLP is a critical component of the linker connection between the BB and pBB, marking its initial construction at S phase and its modulation throughout that and subsequent cell cycles. This functional linker connection is compromised when RNA interference (RNAi) ablation of BBLP expression is induced and, in such knockdowns, the newly matured BB (subtending the new flagellum) becomes detached from its newly formed pBB. BBLP represents an initial insight into components that provide a cell-cycle-modulated connection between a paired mature and an immature CBB. We have analyzed our results in the light of the linker concept in mammalian cells and show that the two aspects of CBB disengagement—loss of orthogonal orientation and ability to separate and move apart—are consistent but separable events in evolutionarily diverse cells. Further, we provide a unifying model explaining linkage differences between the two distinct centriole/BB pairs in proliferating eukaryotic cells.     

Metalloproteinase Inhibition Protects against Reductions in Circulating Adrenomedullin during Lead‐induced Acute Hypertension

Intoxication with lead (Pb) results in increased blood pressure by mechanisms involving matrix metalloproteinases (MMPs). Recent findings have revealed that MMP type two (MMP‐2) seems to cleave vasoactive peptides. This study examined whether MMP‐2 and MMP‐9 levels/activities increase after acute intoxication with low lead concentrations and whether these changes were associated with increases in blood pressure and circulating endothelin‐1 or with reductions in circulating adrenomedullin and calcitonin gene‐related peptide (CGRP). Here, we expand previous findings and examine whether doxycycline (a MMPs inhibitor) affects these alterations. Wistar rats received intraperitoneally (i.p.) 1st dose 8 μg/100 g of lead (or sodium) acetate, a subsequent dose of 0.1 μg/100 g to cover daily loss and treatment with doxycycline (30 mg/kg/day) or water by gavage for 7 days. Similar whole‐blood lead levels (9 μg/dL) were found in lead‐exposed rats treated with either doxycycline or water. Lead‐induced increases in systolic blood pressure (from 143 ± 2 to 167 ± 3 mmHg) and gelatin zymography of plasma samples showed that lead increased MMP‐9 (but not MMP‐2) levels. Both lead‐induced increased MMP‐9 activity and hypertension were blunted by doxycycline. Doxycycline also prevented lead‐induced reductions in circulating adrenomedullin. No significant changes in plasma levels of endothelin‐1 or CGRP were found. Lead‐induced decreases in nitric oxide markers and antioxidant status were not prevented by doxycycline. In conclusion, acute lead exposure increases blood pressure and MMP‐9 activity, which were blunted by doxycycline. These findings suggest that MMP‐9 may contribute with lead‐induced hypertension by cleaving the vasodilatory peptide adrenomedullin, thereby inhibiting adrenomedullin‐dependent lowering of blood pressure.