Peptide specificity and HLA restriction do not dictate lymphokine production by allergen-specific T-lymphocyte clones.

Human and murine CD4+ T lymphocytes can be subdivided into distinct subsets [T-helper type 0 (Th0), Th1 or Th2], based on their lymphokine production profiles. Not much is known about the factors that determine these restricted lymphokine secretion profiles. Peptide specificity and human leucocyte antigen (HLA) restriction may be such factors. As it is well established that allergen-specific T lymphocytes from atopic individuals and non-atopic controls differ in their lymphokine secretion profile, we studied two allergen-specific T-lymphocyte clones (TLC) with identical peptide specificity and HLA restriction that were generated from the peripheral blood of an atopic donor and a non-atopic control donor. The two CD4+ TLC recognize the same epitope (20-33) of the house dust mite Dermatophagoides pteronyssinus major allergen Der p II. Both TLC recognize the epitope in an HLA-DQB1*0602-restricted manner. However, the lymphokine production profiles of these TLC show clear differences after allergen-specific or polyclonal activation. As expected, TLC JBD4 from the atopic donor produced high levels of interleukin-4 (IL-4) without detectable interferon-gamma (IFN-gamma), whereas TLC PBA1 from the non-atopic donor produced both IFN-gamma and IL-4 upon allergen-specific or polyclonal activation. Inasmuch as both TLC recognized the same epitope of Der p II in association with the same HLA-DQ molecule, these data suggest that peptide specificity and HLA restriction of human allergen-specific TLC do not dictate their lymphokine secretion profile.     

Treating Eating Disorders at Higher Levels of Care: Overview and Challenges

Higher levels of care (HLC)—including inpatient hospitalization, residential treatment, partial hospitalization, and intensive outpatient treatment—are frequently utilized within routine care for eating disorders. Despite widespread use, there is limited research evaluating the efficacy of HLC, as well as clinical issues related to care in these settings. This review describes the different levels of care for eating disorders and briefly reviews the most up-to-date guidelines and research regarding how to choose a level of care. In addition, as HLC approaches for ED continue to be developed and refined, pragmatic and conceptual challenges have emerged that provide barriers to clinical efficacy and the execution of high-quality treatment research. This review includes a discussion of various issues specific to HLC, as well as a summary of recent literature addressing them.     

Purging of acute myeloid leukaemia cells from stem cell grafts by hyperthermia: enhancement of the therapeutic index by the tetrapeptide AcSDKP and the alkyl-lysophospholipid ET-18-OCH(3)

Hyperthermia has been shown to be a potential purging modality in autologous stem cell transplantation settings owing to its selective toxicity towards leukaemic cells. We describe two approaches to further increase the therapeutic index of the hyperthermic purging modality by using normal murine bone marrow cells and a murine model for acute myeloid leukaemia. First, the tetrapeptide AcSDKP was used to protect the normal haematopoietic progenitor cells against hyperthermic damage. Pretreatment for 8 h at 37 degrees C with 1 x 10(-9) mol/l AcSDKP resulted in a decrease in hyperthermic sensitivity of only normal haematopoietic progenitor cells. This combined treatment protocol revealed a therapeutic index (ratio of surviving fractions of normal vs. leukaemic cells) of > 500, which was considered to be sufficient for purging. This was confirmed in vivo by the survival of lethally irradiated recipients transplanted with purged simulated remission bone marrow (1 x 10(6) normal bone marrow cells and 5 x 10(4) leukaemic cells). A further increase of the therapeutic index cells was achieved by the alkyl-lysophospholipid ET-18-OCH(3). An incubation for 4 h at 37 degrees C with 25 microg/ml in the presence of 5% fetal calf serum preferentially enhanced the cytotoxic effect towards the leukaemic stem cell. The combination of AcSDKP and ET-18-OCH(3) with hyperthermia resulted in a therapeutic index of > 5000. This enabled a reduction of the hyperthermic treatment and will further minimize the toxicity to normal haematopoietic stem cell subsets, while a therapeutic index far above the required value is achieved. This tripartite purging treatment therefore offers a safe and fast purging protocol for the elimination of residual leukaemic cells in autografts.     

Induction of globin mRNA transcription by erythropoietin in differentiating erythroid precursor cells

The effects of erythropoietin (epo) on the proliferation of late erythroid progenitor cells (CFU-E) and on the formation of hemoglobin and of globin mRNA in these cells are described. CFU-E were isolated from thiamphenicol-pretreated anemic mice by elutriation and Percoll density gradient methods. These CFU-E are restricted in their capacity to proliferate in vitro without added epo. The epo dependence in vitro was not absolute. With no epo in the culture medium the first cell division was unimpaired, whereas the third division was only 1%-2% of the control. In the absence of epo the synthesis of hemoglobin is very low in CFU-E, but is increased significantly after about 5 h of incubation with epo present. In epo deprived cells there was considerable hemoglobin formed at about 14 h, but not earlier. The presence as detected by the Northern blot technique of globin mRNA, isolated from CFU-E, was variable, probably depending on the presence of some more mature erythroid cells. By an extrapolation method we show evidence that pure CFU-E would have virtually no detectable globin mRNA. The production of globin mRNA is rapidly (2 h) induced in cells incubated with epo. We conclude that epo, besides having a mitogenic effect on CFU-E, induces the rapid expression of the globin genes.     

Regional hyperperfusion in older adults with objectively-defined subtle cognitive decline

Although cerebral blood flow (CBF) alterations are associated with Alzheimer’s disease (AD), CBF patterns across prodromal stages of AD remain unclear. Therefore, we investigated patterns of regional CBF in 162 Alzheimer’s Disease Neuroimaging Initiative participants characterized as cognitively unimpaired (CU; n = 80), objectively-defined subtle cognitive decline (Obj-SCD; n = 31), or mild cognitive impairment (MCI; n = 51). Arterial spin labeling MRI quantified regional CBF in a priori regions of interest: hippocampus, inferior temporal gyrus, inferior parietal lobe, medial orbitofrontal cortex, and rostral middle frontal gyrus. Obj-SCD participants had increased hippocampal and inferior parietal CBF relative to CU and MCI participants and increased inferior temporal CBF relative to MCI participants. CU and MCI groups did not differ in hippocampal or inferior parietal CBF, but CU participants had increased inferior temporal CBF relative to MCI participants. There were no CBF group differences in the two frontal regions. Thus, we found an inverted-U pattern of CBF signal across prodromal AD stages in regions susceptible to early AD pathology. Hippocampal and inferior parietal hyperperfusion in Obj-SCD may reflect early neurovascular dysregulation, whereby higher CBF is needed to maintain cognitive functioning relative to MCI participants, yet is also reflective of early cognitive inefficiencies that distinguish Obj-SCD from CU participants.     

Naturalistic outcomes for a day‐hospital programme in a mixed diagnostic sample of adolescents with eating disorders

Despite initial data suggesting positive treatment outcomes for adolescent eating disorder day‐hospital programmes (DHPs), existing studies have included limited follow‐up, small samples, and a focus on restricting‐type eating disorders. To address these gaps, we explored naturalistic outcomes for an adolescent eating disorders DHP. Adolescent participants (N = 265) completed measurements at treatment admission, discharge (n = 170), and various lengths of follow‐up (n = 126; M_{follow up} = 278.87 days). Results from multilevel models indicated significant increases in body weight for the anorexia nervosa group throughout treatment and maintenance of increased body weight from discharge to follow‐up. In bulimic spectrum disorders, binge eating and purging significantly decreased from intake to discharge and did not change from discharge to follow‐up. Across the entire sample, eating disorder symptoms decreased from intake to discharge and did not change from discharge to follow‐up. Further, anxiety and depression decreased over the course of treatment and continued to decrease over the follow‐up period. The current investigation represents the first study to explore longitudinal DHP outcomes within adolescent bulimic spectrum eating disorders. Our findings also highlight many challenges inherent in conducting naturalistic research; it is critical that the field continue to develop solutions to the barriers inherent in conducting longitudinal research on eating disorder treatment.     

Sterilization analysis of contaminated healing abutments and impression copings

This study investigated sterilization of used implant impression copings and healing abutments. Components were analyzed after contamination with Enterococcus foecalis, followed by multiple rounds of sterilization by both steam autoclave and Chemiclave protocols. The authors' results demonstrated that used components showed sterility equal to new components without any visible distortion. These data suggest that component resterilization and reuse may be justified or at least considered in clinical practice. Also, implications for cost savings in the placement of implants are advanced.     

Cross-linkers at growing microtubule ends generate forces that drive actin transport

The actin and microtubule cytoskeletons form active networks in the cell that can contract and remodel, resulting in vital cellular processes such as cell division and motility. Motor proteins play an important role in generating the forces required for these processes, but more recently the concept of passive cross-linkers being able to generate forces has emerged. So far, these passive cross-linkers have been studied in the context of separate actin and microtubule systems. Here, we show that cross-linkers also allow actin and microtubules to exert forces on each other. More specifically, we study single actin filaments that are cross-linked to growing microtubule ends, using in vitro reconstitution, computer simulations, and a minimal theoretical model. We show that microtubules can transport actin filaments over large (micrometer-range) distances and find that this transport results from two antagonistic forces arising from the binding of cross-linkers to the overlap between the actin and microtubule filaments. The cross-linkers attempt to maximize the overlap between the actin and the tip of the growing microtubules, creating an affinity-driven forward condensation force, and simultaneously create a competing friction force along the microtubule lattice. We predict and verify experimentally how the average transport time depends on the actin filament length and the microtubule growth velocity, confirming the competition between a forward condensation force and a backward friction force. In addition, we theoretically predict and experimentally verify that the condensation force is of the order of 0.1 pN. Thus, our results reveal an active mechanism for local actin remodeling by growing microtubules that relies on passive cross-linkers.

Vital cellular processes such as cell division and motility are driven by contraction and remodeling of active networks within the cell: the actin and microtubule cytoskeletons. These contraction and remodeling processes require the generation of forces and relative movement of filaments. It is well known that motor proteins can cross-link filaments and organize the network by driving the relative movements of the filaments (1). Besides the well-appreciated role of motor proteins, also polymerization dynamics have been shown to generate forces required for self-organization and remodeling (2–4). In addition, the importance of passive (nonmotor) cross-linkers has recently been increasingly recognized. It is now clear that passive cross-linkers can generate driving forces too, as has been shown for anillin in the contractile ring (5) and Ase1 in the spindle (6). These forces are generated via the entropy associated with the cross-linker’s diffusion within the overlap region (6) or via the condensation (or preferential binding) of cross-linkers to the overlap between filaments (5, 6), therefore named “condensation force.” In addition, passive cross-linkers create frictional forces (6, 7), which not only affect the speed at which cytoskeletal structures are remodeled, but can even be essential for their stability by opposing the motor proteins (8, 9).Prior studies on force generation by passive cross-linkers have focused on individual cytoskeletal systems. However, passive cross-linkers could also be a way for two systems to exert forces on each other. In particular, there is a growing body of work showing the importance of microtubule/actin crosstalk in vital cellular functions (10). Besides crosstalk via signaling pathways, it has been shown that crosstalk via motor proteins such as myosin-X (Myo10) is among the drivers of remodeling the microtubule spindle network during cell division (11, 12). In addition, various passive cross-linking proteins have been identified to enable crosstalk, such as the family of spectraplakins, which are proteins that can bind both actin and microtubules. The most prominent and best-studied member is ACF7 (MACF1) and its Drosophila homolog Short stop (Shot), which contain both microtubule lattice- and end-binding activities. These ubiquitous and conserved cross-linkers (traced back to lower metazoans; ref. 13) play a crucial role in a number of cellular processes, such as cell migration, cell–cell connections, vesicular transport, cell polarity, and cell division (10, 14). For example in the context of cell migration, ACF7 is shown to anchor microtubules at the cell’s leading edge (15) and to guide microtubules along actin bundles toward focal adhesions, where ACF7 depletion resulted in a phenotype with lost coalignment and failure of microtubules to target focal adhesions (16). In addition to ACF7, also other passive cross-linking proteins such as tau and Gas2Like1 have been shown to result in coalignment and stabilization of the actin and microtubule networks (16–19). Another example of a microtubule end-binding cross-linker that links actin to microtubule ends via EB3-mediated interactions is drebrin, which is required for sprouting neurites from a neuronal cell (20).Recent experiments suggest that the driving condensation forces and friction forces associated with passive cross-linkers can also couple to the growth dynamics of filaments and thereby allow passive cross-linkers to play a central role not only in contraction but also in transport. It was shown that an engineered passive cross-linker called TipAct can mediate transport of actin filaments by binding to the tips of growing microtubules (17). The microtubule tip region is chemically different from the microtubule lattice region, due to delayed hydrolysis of guanosine triphosphate (GTP) that is associated with tubulin subunits that incorporate into growing microtubules. This region is recognized by microtubule end-binding (EB) proteins (21–23) as well as EB partners such as TipAct. However, the processivity of the actin transport mechanism and its dependency on relevant parameters such as actin filament length and microtubule growth velocity, as well as the magnitude of the generated forces, are currently unresolved.Here, we investigate the conditions necessary for microtubule-mediated actin transport and elucidate the cross-linker–dependent mechanism. We combine biochemical reconstitution experiments with coarse-grained computer simulations and a minimal theoretical model and show that in the presence of passive cross-linkers, microtubules can transport actin filaments over large (micrometer-range) distances during periods that last up to several minutes. We propose and test a mechanism to explain this movement, in which actin transport is the result of a competition between a forward force that tends to maximize the overlap of the actin filament with the plus end of a growing microtubule and a backward force caused by the friction between the actin and microtubule lattice. These two antagonistic forces are both caused by the binding of cross-linkers. The preferential binding of cross-linkers at the interface between two objects (such as filaments) creates a driving force that tends to maximize the overlap region. This type of force, which, following earlier work (6, 7), we call condensation force, can drive the movement of intracellular cargos (2, 24–26) and the contraction of filament bundles (5, 6, 27, 28) in various biological contexts. However, the combination of a processive transport of cargo by a cross-linker–based condensation force toward a chemically distinct region that is simultaneously hindered by a friction force caused by the same cross-linkers is specific to filament transport and has to our knowledge not been studied before. The active transport of filaments by growing microtubule plus ends thus provides a minimal mechanism by which two cytoskeletal systems can interact, which is distinct from cytoskeletal crosstalk that is driven by motor proteins (11, 29–32). We expect this mechanism to be especially relevant in migrating cells, where growing microtubules nucleate actin filaments and encounter membrane-bound actin arrays.