Åsa Holm

Leishmania donovani lipophosphoglycan causes periphagosomal actin accumulation: correlation with impaired translocation of PKCα and defective phagosome maturation

Lipophosphoglycan (LPG) is the major surface glycoconjugate of Leishmania donovani promastigotes. The repeating disaccharide–phosphate units of LPG are crucial for promastigote survival inside macrophages and establishment of infection. LPG has a number of effects on the host cell, including inhibition of PKC activity, inhibition of nitric oxide production and altered expression of cytokines. LPG also inhibits phagosomal maturation, a process requiring depolymerization of periphagosomal F‐actin. In the present study, we have characterized the dynamics of F‐actin during the phagocytosis of L. donovani promastigotes in J774 macrophages. We observed that F‐actin accumulated progressively around phagosomes containing wild‐type L. donovani promastigotes during the first hour of phagocytosis. Using LPG‐defective mutants and yeast particles coated with purified LPG, we obtained evidence that this effect could be attributed to the repeating units of LPG. LPG also disturbed cortical actin turnover during phagocytosis. The LPG‐dependent accumulation of periphagosomal F‐actin correlated with an impaired recruitment of the lysosomal marker LAMP1 and PKCα to the phagosome. Accumulation of periphagosomal F‐actin during phagocytosis of L. donovani promastigotes may contribute to the inhibition of phagosomal maturation by physically preventing vesicular trafficking to and from the phagosome.

Mechanical manipulation of bone and cartilage cells with 'optical tweezers'.

The single beam optical gradient trap (optical tweezers) uses a single beam of laser light to non‐invasively manipulate microscopic particles. Optical tweezers exerting a force of approximately 7 pN were applied to single bone and cartilage derived cells in culture and changes in intracellular calcium levels were observed using Fluo‐3 labelling. Human derived osteoblasts responded to optical tweezers with an immediate increase in [Ca2+]i that was inhibited by the addition of a calcium channel blocker nifedipine. Force applied to different regions of cells resulted in a variable response. [Ca2+]i elevation in response to load was lower in rat femur derived osteoblasts, and not apparent in primary chondrocytes and the osteocytic cell line (MLO Y4).

p56Lck anchors CD4 to distinct microdomains on microvilli

Cell-surface microvilli play a central role in adhesion, fusion, and signaling processes. Some adhesion and signaling receptors segregate on microvilli but the determinants of this localization remain mostly unknown. In this study, we considered CD4, a receptor involved in immune response and HIV infection, and p56Lck, a CD4-associated tyrosine kinase. Analysis of CD4 trafficking reveals that p56Lck binds tightly to CD4 independently of its activation state and inhibits CD4 internalization. Electron microscopy analysis established that p56Lck mediates CD4 association with microvilli whereas biochemical data indicate that p56Lck expression renders CD4 insoluble by the nonionic detergent Triton X-100. In addition, cytoskeleton-disrupting agent increased CD4 solubility, suggesting the involvement of cytoskeletal elements in CD4 anchoring to microvilli. This concept was supported further by the observation that the lateral mobility of CD4 within the plasma membrane was decreased in cells expressing p56Lck. Finally, isolation of detergent-resistant membranes revealed that the complex CD4-p56Lck is enriched within these domains as opposed to conditions in which CD4 does not interact with p56Lck. In conclusion, our results show that p56Lck targets CD4 to specialized lipid microdomains preferentially localized on microvilli. This localization, which prevents CD4 internalization, might facilitate CD4-mediated adhesion processes and could correspond to the signaling site of the receptor.

Leishmania donovani lipophosphoglycan inhibits phagosomal maturation via action on membrane rafts.

Lipophosphoglycan (LPG), the major surface glycoconjugate on Leishmania donovani promastigotes, is crucial for the establishment of infection inside macrophages. LPG comprises a polymer of repeating Galbeta1,4Manalpha-PO(4) attached to a lysophosphatidylinositol membrane anchor. LPG is transferred from the parasite to the host macrophage membrane during phagocytosis and induces periphagosomal F-actin accumulation correlating with an inhibition of phagosomal maturation. The biophysical properties of LPG suggest that it may be intercalated into membrane rafts of the host-cell membrane. The aim of this study was to investigate if the effects of LPG on phagosomal maturation are mediated via action on membrane rafts. We show that LPG accumulates in rafts during phagocytosis of L. donovani and that disruption of membrane rafts abolished the effects of LPG on periphagosomal F-actin and phagosomal maturation, indicating that LPG requires intact membrane rafts to manipulate host-cell functions. We conclude that LPG associates with membrane rafts in the host cell and exert its actions on host-cell actin and phagosomal maturation through subversion of raft function.

Role of protein kinase C α for uptake of unopsonized prey and phagosomal maturation in macrophages

Protein kinase C alpha (PKC alpha) participates in F-actin remodeling during phagocytosis and phagosomal maturation in macrophages. Leishmania donovani promastigotes, which inhibit phagosomal maturation, cause accumulation of periphagosomal F-actin instead of the disassembly observed around other prey [Cell. Microbiol. 7 (2001) 439]. This accumulation is induced by promastigote lipophosphoglycan (LPG), which has several effects on macrophages including inhibition of PKC alpha. To investigate a possible connection between PKC alpha and LPGs effects on actin dynamics, we utilized RAW264.7 macrophages overexpressing dominant-negative PCK alpha (DN PKC alpha). We found increased cortical F-actin and decreased phagocytic capacity, as well as defective periphagosomal F-actin breakdown and inhibited phagosomal maturation in the DN PKC alpha-overexpressing cells, effects similar to those seen in controls subjected to LPG-coated prey. The results indicate that PKC alpha is involved in F-actin turnover in macrophages and that PKC alpha-dependent breakdown of periphagosomal F-actin is required for phagosomal maturation, and endorse the hypothesis that intracellular survival of L. donovani involves inhibition of PKC alpha by LPG.

Leishmania donovani Requires Functional Cdc42 and Rac1 To Prevent Phagosomal Maturation

ABSTRACT Leishmania donovani promastigotes survive inside macrophage phagosomes by inhibiting phagosomal maturation. The main surface glycoconjugate on promastigotes, lipophosphoglycan (LPG), is crucial for survival and mediates the formation of a protective shell of F-actin around the phagosome. Previous studies have demonstrated that this effect involves inhibition of protein kinase Cα. The present study shows that functional Cdc42 and Rac1 are required for the formation of F-actin around L. donovani phagosomes. Moreover, we present data showing that phagosomes containing LPG-defective L. donovani, which is unable to induce F-actin accumulation, display both elevated levels of periphagosomal F-actin and impaired phagosomal maturation in macrophages with permanently active forms of Cdc42 and Rac1. We conclude that L. donovani engages Cdc42 and Rac1 to build up a protective coat of F-actin around its phagosome to prevent phagosomal maturation.

Impact of Using Linear Optimization Models in Dose Planning for HDR Brachytherapy

PURPOSE Dose plans generated with optimization models hitherto used in high-dose-rate (HDR) brachytherapy have shown a tendency to yield longer dwell times than manually optimized plans. Concern has been raised for the corresponding undesired hot spots, and various methods to mitigate these have been developed. The hypotheses upon this work is based are (a) that one cause for the long dwell times is the use of objective functions comprising simple linear penalties and (b) that alternative penalties, as these are piecewise linear, would lead to reduced length of individual dwell times. METHODS The characteristics of the linear penalties and the piecewise linear penalties are analyzed mathematically. Experimental comparisons between the two types of penalties are carried out retrospectively for a set of prostate cancer patients. RESULTS When the two types of penalties are compared, significant changes can be seen in the dwell times, while most dose-volume parameters do not differ significantly. On average, total dwell times were reduced by 4.2%, with a reduction of maximum dwell times by 25%, when the alternative penalties were used. CONCLUSIONS The use of linear penalties in optimization models for HDR brachytherapy is one cause for the undesired long dwell times that arise in mathematically optimized plans. By introducing alternative penalties, a significant reduction in dwell times can be achieved for HDR brachytherapy dose plans. Although various measures for mitigating the long dwell times are already available, the observation that linear penalties contribute to their appearance is of fundamental interest.

Mechanical manipulation of polymorphonuclear leukocyte plasma membranes with optical tweezers causes influx of extracellular calcium through membrane channels

Optical tweezers are used mechanically to manipulate the plasma membrane of polymophonuclear leukocytes attached to the bottom of a glass manipulation chamber. The laser trapping beam is dragged across the membrane of cells in calcium-containing and calcium-depleted extracellular medium. This treatment causes a significant rise in the intracellular calcium concentration compared with controls, in cells in calcium-containing medium (239.8±49.0% against 75.4±16.4%, respectively), but not in cells in calcium-depeleted medium (69.1±9.6% against 83.4±18.5%, respectively), indicating that the calcium rise is caused by an influx of calcium from the environment. The rise in calcium concentration is blocked (23.5±7.1% against 17.1±4.1%, respectively) by the addition of lansoprazole, indicating that the influx is not due to unspecific membrane damage caused by the mechanical manipulation of the cell. It can therefore be concluded that mechanical manipulation of the neutrophil membrane, in the piconewton force range exerted by the optical tweezer, does not damage the plasma membrane but stimulates a mechanically inducible, membrane channel-mediated influx of extracellular calcium.

SU‐E‐T‐583: Optimizing Dosimetric Indices for HDR Brachytherapy Using CVaR

PURPOSE To develop a new model for automated optimization of HDR brachytherapy (BT) dose distributions that operates on the dose volume indices used in plan evaluation. The model can be solved to optimality, with constraints that are easy to interpret and modify for the clinical user. Recent research has shown that the optimization model hitherto used corresponds only weakly to such dosimetric indices. Alternative models that include such dosimetric indices have been presented; however, the explicit inclusion of dosimetric indices yields intractable models. METHODS An approach for optimizing HDR BT dose distributions is presented where dosimetric indices are taken into account through surrogates based on the conditional value-at-risk concept (CVaR). This yields a linear optimization model that is easy to solve, and has the additional advantage that the constraints are easy to interpret and modify to obtain satisfactory dose distributions. RESULTS Experimental comparisons have shown that our proposed model corresponds well with constraining dosimetric indices. All modifications of the parameters in our model yield the expected Result. Both our approach and those presented earlier for optimization of dosimetric indices include an approximation, earlier models solve the models approximately, and we use a model based on an approximation. This gives us better control of the optimization. The dose distributions generated by the proposed optimization model are comparable to those generated by the standard model with respect to dosimetric indices. CONCLUSION Our new model is a viable surrogate to optimizing BT dosimetric indices that quickly and easily yield high-quality dose distributions. New optimization models that are capable of optimizing dosimetric indices will become of increasing importance in new applications involving more degrees of freedom than conventional HDR 192 Ir BT such as electronic BT.

Mechanical manipulation of human osteoblast and chondrocyte cells with 'optical tweezers'

The single beam optical gradient trap, known less formally as optical tweezers, uses a single beam of laser light to noninvasively manipulate microscopic particles. In this study, optical tweezers were used to apply force to single human bone and cartilage derived cells in vitro. Effects of various forces applied were monitored by observing intracellular calcium fluctuation during application. In all cell types, human and rat bone derived osteoblasts and human chondrocytes, an increase in fluorescent intensity of calcium indicating fluo-3 was observed within seconds in response to force applied with the optical tweezers, suggesting a rise in [Ca/sup 2+/]i. The greatest response was observed in the human derived osteoblasts and a minimal responses recorded in the dedifferentiated chondrocytes in low density culture and rat derived osteoblasts. Force applied to different regions of cells identified variations in responses with the greatest response observed when the force was transmitted through the body of the cell. This preliminary study highlights a variation in calcium response to a force applied via optical tweezers on individual connective tissue cells. These results suggest a possible role for optical tweezers as a method for application of mechanical strain without additional fluid flow on single cells in culture. This technique will assist one in modelling the mechanical environment in vitro to enable one to identify mechanical effects in connective tissue cell types that are important when designing artificial materials for tissue repair.