The in vitro susceptibilities of the reference strain Leishmania donovani MHOM/ET/67/L82 to sodium stibogluconate, amphotericin B, miltefosine, and the experimental compound PX-6518 were determined for extracellular log-phase promastigotes, established axenic amastigotes, fresh spleen-derived amastigotes, and intracellular amastigotes in primary mouse peritoneal macrophages. Susceptibility to amphotericin B did not differ across the various axenic models (50% inhibitory concentrations [IC ₅₀ ], 0.6 to 0.7 μM), and amphotericin B showed slightly higher potency against intracellular amastigotes (IC ₅₀ , 0.1 to 0.4 μM). A similar trend was observed for miltefosine, with comparable efficacies against the extracellular (IC ₅₀ , 0.4 to 3.8 μM) and intracellular (IC ₅₀ , 0.9 to 4.3 μM) stages. Sodium stibogluconate, used either as Pentostam or as a crystalline substance, was inactive against all axenic stages (IC ₅₀ , >64 μg Sb ^V /ml) but showed good efficacy against intracellular amastigotes (IC ₅₀ , 22 to 28 μg Sb ^V /ml); the crystalline substance was about two to three times more potent (IC ₅₀ , 9 to 11 μg Sb ^V /ml). The activity profile of PX-6518 was comparable to that of sodium stibogluconate, but at a much higher potency (IC ₅₀ , 0.1 μg/ml). In conclusion, the differential susceptibility determines which in vitro models are appropriate for either drug screening or resistance monitoring of clinical field isolates. Despite the more complex and labor-intensive protocol, the current results support the intracellular amastigote model as the gold standard for in vitro Leishmania drug discovery research and for evaluation of the resistance of field strains, since it also includes host cell-mediated effects. Axenic systems can be recommended only for compounds for which no cellular mechanisms are involved, for example, amphotericin B and miltefosine.

This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.

Context: Responses to critical illness, such as excessive inflammation and hyperglycemia, may trigger detrimental chain reactions that damage cellular proteins and organelles. Such responses to illness contribute to the risk of (nonresolving) multiple organ dysfunction and adverse outcome. Objective: We studied autophagy as a bulk degradation pathway able to remove toxic protein aggregates and damaged organelles and how these are affected by preventing hyperglycemia with insulin during critical illness. Design and Setting: Patients participated in a randomized study, conducted at a university hospital surgical/medical intensive care unit. Patients: We studied adult prolonged critically ill patients vs. controls. Interventions: Tolerating excessive hyperglycemia was compared with intensive insulin therapy targeting normoglycemia. Main Outcome Measures: We quantified (ultra)structural abnormalities and hepatic and skeletal muscle protein levels of key players in autophagy. Results: Morphologically, both liver and muscle revealed an autophagy-deficiency phenotype. Proteins involved in initiation and elongation steps of autophagy were induced 1.3- to 6.5-fold by critical illness (P ≤ 0.01), but mature autophagic vacuole formation was 62% impaired (P = 0.05) and proteins normally degraded by autophagy accumulated up to 97-fold (P ≤ 0.03). Mitophagy markers were unaltered or down-regulated (P = 0.05). Although insulin preserved hepatocytic mitochondrial integrity (P = 0.05), it further reduced the number of autophagic vacuoles by 80% (P = 0.05). Conclusions: Insufficient autophagy in prolonged critical illness may cause inadequate removal of damaged proteins and mitochondria. Such incomplete clearance of cellular damage, inflicted by illness and aggravated by hyperglycemia, could explain lack of recovery from organ failure in prolonged critically ill patients. These data open perspectives for therapies that activate autophagy during critical illness.