The chemokines CCL19 and CCL21 and their role in T and dendritic cell biology

Summary : The chemokines CCL19 and CCL21 and their common receptor CCR7 attract antigenpresenting dendritic cells (DCs) and naive T cells into the T zone of secondary lymphoid organs (SLO) and are therefore critically involved in homeostatic T cell recirculation and the initiation of adaptive immune responses. In addition. CCR7 ligands were proposed to mediate T cell exit from neonatal thymus, allowing colonization of T zones in SLOB. The relative contribution of CCL19 and CCL21 to these processes has remained unclear, as they were studied in mouse models lacking either CCR7 or both ligands. The aim of my thesis was to characterize Cc119-' mice and thereby investigate the relative roles of the two CCR7 ligands in development, homeostasis and immune response. The first study addressed the role of CCR7 ligands in DC biology. We found that CCL19 is dispensable for DC migration to lymph nodes and their localization to T zones. Furthermore, a CCL19-deficient environment did not lead to a defect in DC maturation or T cell priming. Therefore, CCL21 is sufficient to mediate CCR7-dependent processes during the initiation of adaptive immune responses. In the second study we investigated how the two CCR7 ligands affect CCR7 expression and function on naive T cells. We found that in SLOB CCR7 is constantly occupied with CCL19 and CCL21, eventually leading to its internalization. The reduced level of free CCR7 on these cells led to diminished ligand sensitivity and consequently impaired chemotactic responses. This effect was reversible by passage through aCCR7 ligand-free environment like the blood circulation. We propose that the different states of ligand sensitivity in SLOB and blood are important to allow for proper T cell recirculation. In the third study the role of CCL19 in neonatal thymus and spleen was analyzed. While neonatal Cc119-!- mice had no defect in thymic egress, we observed reduced T cell accumulation in the spleen but not lymph nodes. We identified reticular stromal cells in the developing white pulp (WP) as the major CCL 19 source. The development of these WP stromal cells as well as their CCL19 expression were dependent on LTalß2+ B cells. In conclusion, we have found that CCL21 can mostly compensate for lack of CCL19 in homeostasis and immunity. In contrast, during development. CCL19 has anon-redundant function for T cell colonization of the spleen.

Generation and function of mouse central memory and effector memory T cells

1. SUMMARY Based on functional and homing properties, two subsets of memory T lymphocytes have been defined both in humans and in mice. Central memory T cells (TCM cells) express the lymph node homing receptors CD62L and CCR7, have poor effector function and proliferate efficiently upon antigenic stimulation. Effector memory T cells (TEM cells) do not express CCR7, are mostly CD62L negative and therefore are excluded from lymph nodes, but are able to migrate to sites of inflammation where they exert immediate effector function by producing inflammatory cytokines and cytotoxic mediators. In the present work we have addressed two questions that emerged since the definition of TCM and TEM cells. Firstly, what are the priming conditions for generation of TCM and TEM and, secondly, what is the migratory capacity of TCM and TEM cells in inflammatory conditions. By using naive TCR-transgenic OT-I CD8+ T cells and OT-II CD4+ T cells and ovalbumin pulsed-mature dendritic cells (DCs) we set up an in vitro system in which the strength of T cell stimulation is controlled by varying the ratio of T cells and DCs and the duration of DC-T cell interaction. Using this system we found that precursors of TCM and TEM cells are generated at different strength of stimulation and that T cells capable of persisting in vivo in the absence of antigen and of mounting recall responses is optimally induced by intermediate stimulatory strength. In addition, we found that lymph nodes draining sites of mature DC or adjuvant inoculation recruit CD8+ CD62L- CCR7- effector and TEM cells. CD8+ T cell recruitment in reactive lymph nodes requires CXCR3 expression on T cells and occurs through high endothelial venules (HEVs) in concert with HEV lurninal expression of the CXCR3 ligand CXCL9. In reactive lymph nodes, recruited T cells establish stable interactions with and kill antigen-bearing DCs, limiting the ability of these DCs to activate CD4+ and CD8+ T cells. Taken togther these data define conditions for the generation of TCM and TEM cells and define an inflammatory pathway of effector T cell migration in lymph nodes. The inducible recruitment of blood-borne effector and TEM CD8+ cells to lymph nodes may represent a mechanism for terminating primary and limiting secondary immune responses.

Lymphocyte specificity to protein antigens. V. Conformational dependence of activation of cytochrome c-specific T cells.

Variations in the immunogenic and antigenic properties of native and denatured forms of cytochrome c were observed depending on the strain of mouse tested. In C57BL/6 and (C57BL/6 X DBA/2)F1 (BDF1) mice, priming with either native or denatured cytochrome c (apocytochrome c) gave rise to T cell blasts responding in a similar fashion to the two forms of the antigen and to different peptides derived from CNBr cleavage of the protein when tested for proliferation in the presence of C57BL/6 or BDF1 accessory cells. A different pattern of proliferation was observed when apocytochrome c-specific DBA/2 or BDF1 T cell blasts were tested with DBA/2 accessory cells. In this case, no response was obtained to heme peptide 1-65. This was not due to an inability of DBA/2 macrophages to process and present heme peptide 1-65, as they were able to present this antigen to native cytochrome c-specific BDF1 T cell blasts. Thus, it seems that different sets of clones are generated upon priming BDF1 mice with denatured cytochrome c which are able to recognize different sets of peptides depending on the nature of the accessory cells. The results obtained are consistent with the hypothesis that degradation and presentation of native and denatured cytochrome c by macrophages is dependent on the three-dimensional conformation of the protein.

Repeated sprinting on natural grass impairs vertical stiffness but does not alter plantar loading in soccer players.

This study aimed to determine changes in spring-mass model (SMM) characteristics, plantar pressures, and muscle activity induced by the repetition of sprints in soccer-specific conditions; i.e., on natural grass with soccer shoes. Thirteen soccer players performed 6 × 20 m sprints interspersed with 20 s of passive recovery. Plantar pressure distribution was recorded via an insole pressure recorder device divided into nine areas for analysis. Stride temporal parameters allowed to estimate SMM characteristics. Surface electromyographic activity was monitored for vastus lateralis, rectus femoris, and biceps femoris muscles. Sprint time, contact time, and total stride duration lengthened from the first to the last repetition (+6.7, +12.9, and +9.3%; all P < 0.05), while flight time, swing time, and stride length remained constant. Stride frequency decrease across repetitions approached significance (-6.8%; P = 0.07). No main effect of the sprint number or any significant interaction between sprint number and foot region was found for maximal force, mean force, peak pressure and mean pressure (all P > 0.05). Center of mass vertical displacement increased (P < 0.01) with time, together with unchanged (both P > 0.05) peak vertical force and leg compression. Vertical stiffness decreased (-15.9%; P < 0.05) across trials, whereas leg stiffness changes were not significant (-5.9%; P > 0.05). Changes in root mean square activity of the three tested muscles over sprint repetitions were not significant. Although repeated sprinting on natural grass with players wearing soccer boots impairs their leg-spring behavior (vertical stiffness), there is no substantial concomitant alterations in muscle activation levels or plantar pressure patterns.

The role of WNT and mTOR in human memory T cell formation

Cancer is one of the world's leading causes of death with a rising trend in incidence. These epidemiologic observations underline the need for novel treatment strategies. In this regard, a promising approach takes advantage of the adaptive effector mechanisms of the immune system, using T lymphocytes to specifically target and destroy tumour cells. However, whereas current approaches mainly depend on short-lived, terminally differentiated effector T cells, increasing evidence suggests that long lasting and maximum efficient immune responses are mediated by low differentiated memory T cells. These memory T cells should display characteristics of stem cells, such as longevity, self-renewal capacity and the ability to continuously give rise to further differentiated effectors. These stem celllike memory T (TSCM) cells are thought to be of key therapeutic value as they might not only attack differentiated tumour cells, but also eradicate the root cause of cancer, the cancer stem cells themselves. Thus, efforts are made to characterize TSCM cells and to identify the signalling pathways which mediate their induction. Recently, a human TSCM cell subset was described and the activation of the Wnt-ß-catenin signalling pathway by the drug TWS119 during naive CD8+ T (TN) cell priming was suggested to mediate their induction. However, a precise deciphering of the signalling pathways leading to TSCM cell induction and an in-depth characterization of in vitro induced and in vivo occurring TSCM cells remain to be performed. Here, evidence is presented that the induction of human and mouse CD8+ and CD4+ TSCM cells may be triggered by inhibition of mechanistic/mammalian target of rapamycin (mTOR) complex 1 with simultaneously active mTOR complex 2. This molecular mechanism arrests a fraction of activated TN cells in a stem cell-like differentiation state independently of the Wnt-ß-catenin signalling pathway. Of note, TWS119 was found to also inhibit mTORCl, thereby mediating the induction of TSCM cells. Suggesting an immunostimulatory effect, the acquired data broaden the therapeutic range of mTORCl inhibitors like rapamycin, which are, at present, exclusively used due to their immunosuppressive function. Furthermore, by performing broad metabolic analyses, a well-orchestrated interplay between intracellular signalling pathways and the T cells' metabolic programmes could be identified as important regulator of the T cells' differentiation fate. Moreover, in vitro induced CD4+ TSCM cells possess superior functional capacities and share fate-determining key factors with their naturally occurring counterparts, assessed by a first-time full transcriptome analysis of in vivo occurring CD4+ TN cell, TSCM cells and central memory (TCM) cells and in vitro induced CD4+ TSCM cells. Of interest, a group of 56 genes, with a unique expression profile in TSCM cells could be identified. Thus, a pharmacological mechanism allowing to confer sternness to activated TN cells has been found which might be highly relevant for the design of novel T cell-based cancer immunotherapies.

PAR2 Promotes Vaccine-Induced Protection Against Helicobacter Infection in Mice.

BACKGROUND & AIMS: Protective immunization limits Helicobacter infection of mice by undetermined mechanisms. Protease-activated receptor 2 (PAR2) signaling is believed to regulate immune and inflammatory responses. We investigated the role of PAR2 in vaccine-induced immunity against Helicobacter infection. METHODS: Immune responses against Helicobacter infection were compared between vaccinated PAR2(-/-) and wild-type (WT) mice. Bacterial persistence, gastric pathology, and inflammatory and cellular responses were assessed using the rapid urease test (RUT), histologic analyses, quantitative polymerase chain reaction, and flow cytometry, respectively. RESULTS: Following vaccination, PAR2(-/-) mice did not have reductions in Helicobacter felis infection (RUT values were 0.01 ± 0.01 for WT mice and 0.11 ± 0.13 for PAR2(-/-) mice; P < .05). The vaccinated PAR2(-/-) mice had reduced inflammation-induced stomach tissue damage (tissue damage scores were 8.83 ± 1.47 for WT mice and 4.86 ± 1.35 for PAR2(-/-) mice; P < .002) and reduced T-helper (Th)17 responses, based on reduced urease-induced interleukin (IL)-17 secretion by stomach mononuclear cells (5182 ± 1265 pg/mL for WT mice and 350 ± 436 pg/mL for PAR2(-/-) mice; P < .03) and reduced recruitment of CD4(+) IL-17(+) T cells into the gastric mucosa of PAR2(-/-) mice following bacterial challenge (3.7% ± 1.5% for WT mice and 2.6% ± 1.1% for PAR2(-/-) mice; P < .05). In vitro, H felis-stimulated dendritic cells (DCs) from WT mice induced greater secretion of IL-17 by ovalbumin-stimulated OT-II transgenic CD4(+) T cells compared with DCs from PAR2(-/-) mice (4298 ± 347 and 3230 ± 779; P < .04), indicating that PAR2(-/-) DCs are impaired in priming of Th17 cells. Adoptive transfer of PAR2(+/+) DCs into vaccinated PAR2(-/-) mice increased vaccine-induced protection (RUT values were 0.11 ± 0.10 and 0.26 ± 0.15 for injected and noninjected mice, respectively; P < .03). CONCLUSIONS: PAR2 activates DCs to mediate vaccine-induced protection against Helicobacter infection in mice.

Novel anesthetics and other treatment strategies for refractory status epilepticus

Refractory status epilepticus (RSE)-that is, seizures resistant to at least two antiepileptic drugs (AEDs)-is generally managed with barbiturates, propofol, or midazolam, despite a low level of evidence (Rossetti, 2007). When this approach fails, the need for alternative pharmacologic and nonpharmacologic strategies emerges. These have been investigated even less systematically than the aforementioned compounds, and are often used, sometimes in succession, in cases of extreme refractoriness (Robakis & Hirsch, 2006). Several possibilities are reviewed here. In view of the marked heterogeneity of reported information, etiologies, ages, and comedications, it is extremely difficult to evaluate a given method, not to say to compare different strategies among them. Pharmacologic Approaches Isoflurane and desflurane may complete the armamentarium of anesthetics,' and should be employed in a ''close'' environment, in order to prevent intoxication of treating personnel. c-Aminobutyric acid (GABA)A receptor potentiation represents the putative mechanism of action. In an earlier report, isoflurane was used for up to 55 h in nine patients, controlling seizures in all; mortality was, however, 67% (Kofke et al., 1989). More recently, the use of these inhalational anesthetics was described in seven subjects with RSE, for up to 26 days, with an endtidal concentration of 1.2-5%. All patients required vasopressors, and paralytic ileus occurred in three; outcome was fatal in three patients (43%) (Mirsattari et al., 2004). Ketamine, known as an emergency anesthetic because of its favorable hemodynamic profile, is an N-methyl-daspartate (NMDA) antagonist; the interest for its use in RSE derives from animal works showing loss of GABAA efficacy and maintained NMDA sensitivity in prolonged status epilepticus (Mazarati & Wasterlain, 1999). However, to avoid possible neurotoxicity, it appears safer to combine ketamine with GABAergic compounds (Jevtovic-Todorovic et al., 2001; Ubogu et al., 2003), also because of a likely synergistic effect (Martin & Kapur, 2008). There are few reported cases in humans, describing progressive dosages up to 7.5 mg/kg/h for several days (Sheth & Gidal, 1998; Quigg et al., 2002; Pruss & Holtkamp, 2008), with moderate outcomes. Paraldehyde acts through a yet-unidentified mechanism, and appears to be relatively safe in terms of cardiovascular tolerability (Ramsay, 1989; Thulasimani & Ramaswamy, 2002), but because of the risk of crystal formation and its reactivity with plastic, it should be used only as fresh prepared solution in glass devices (Beyenburg et al., 2000). There are virtually no recent reports regarding its use in adults RSE, whereas rectal paraldehyde in children with status epilepticus resistant to benzodiazepines seems less efficacious than intravenous phenytoin (Chin et al., 2008). Etomidate is another anesthetic agent for which the exact mechanism of action is also unknown, which is also relatively favorable regarding cardiovascular side effects, and may be used for rapid sedation. Its use in RSE was reported in eight subjects (Yeoman et al., 1989). After a bolus of 0.3 mg/kg, a drip of up to 7.2 mg/kg/h for up to 12 days was administered, with hypotension occurring in five patients; two patients died. A reversible inhibition of cortisol synthesis represents an important concern, limiting its widespread use and implying a careful hormonal substitution during treatment (Beyenburg et al., 2000). Several nonsedating approaches have been reported. The use of lidocaine in RSE, a class Ib antiarrhythmic agent modulating sodium channels, was reviewed in 1997 (Walker & Slovis, 1997). Initial boluses up to 5 mg/kg and perfusions of up to 6 mg/kg/h have been mentioned; somewhat surprisingly, at times lidocaine seemed to be successful in controlling seizures in patients who were refractory to phenytoin. The aforementioned dosages should not be overshot, in order to keep lidocaine levels under 5 mg/L and avoid seizure induction (Hamano et al., 2006). A recent pediatric retrospective survey on 57 RSE episodes (37 patients) described a response in 36%, and no major adverse events; mortality was not given (Hamano et al., 2006 Verapamil, a calcium-channel blocker, also inhibits P-glycoprotein, a multidrug transporter that may diminish AED availability in the brain (Potschka et al., 2002). Few case reports on its use in humans are available; this medication nevertheless appears relatively safe (under cardiac monitoring) up to dosages of 360 mg/day (Iannetti et al., 2005). Magnesium, a widely used agent for seizures elicited by eclampsia, has also been anecdotally reported in RSE (Fisher et al., 1988; Robakis & Hirsch, 2006), but with scarce results even at serum levels of 14 mm. The rationale may be found in the physiologic blockage of NMDA channels by magnesium ions (Hope & Blumenfeld, 2005). Ketogenic diet has been prescribed for decades, mostly in children, to control refractory seizures. Its use in RSE as ''ultima ratio'' has been occasionally described: three of six children (Francois et al., 2003) and one adult (Bodenant et al., 2008) were responders. This approach displays its effect subacutely over several days to a few weeks. Because ''malignant RSE'' seems at times to be the consequence of immunologic processes (Holtkamp et al., 2005), a course of immunomodulatory treatment is often advocated in this setting, even in the absence of definite autoimmune etiologies (Robakis & Hirsch, 2006); steroids, adrenocorticotropic hormone (ACTH), plasma exchanges, or intravenous immunoglobulins may be used alone or in sequential combination. Nonpharmacologic Approaches These strategies are described somewhat less frequently than pharmacologic approaches. Acute implantation of vagus nerve stimulation (VNS) has been reported in RSE (Winston et al., 2001; Patwardhan et al., 2005; De Herdt et al., 2009). Stimulation was usually initiated in the operation room, and intensity progressively adapted over a few days up to 1.25 mA (with various regimens regarding the other parameters), allowing a subacute seizure control; one transitory episode of bradycardia/asystole has been described (De Herdt et al., 2009). Of course, pending identification of a definite seizure focus, resective surgery may also be considered in selected cases (Lhatoo & Alexopoulos, 2007). Low-frequency (0.5 Hz) transcranial magnetic stimulation (TMS) at 90% of the resting motor threshold has been reported to be successful for about 2 months in a patient with epilepsia partialis continua, but with a weaning effect afterward, implying the need for a repetitive use (Misawa et al., 2005). More recently, TMS was applied in a combination of a short ''priming'' high frequency (up to 100 Hz) and longer runs of low-frequency stimulations (1 Hz) at 90-100% of the motor threshold in seven other patients with simple-partial status, with mixed results (Rotenberg et al., 2009). Paradoxically at first glance, electroconvulsive treatment may be found in cases of extremely resistant RSE. A recent case report illustrates its use in an adult patient with convulsive status, with three sessions (three convulsions each) carried out over 3 days, resulting in a moderate recovery; the mechanism is believed to be related to modification of the synaptic release of neurotransmitters (Cline & Roos, 2007). Therapeutic hypothermia, which is increasingly used in postanoxic patients (Oddo et al., 2008), has been the object of a recent case series in RSE (Corry et al., 2008). Reduction of energy demand, excitatory neurotransmission, and neuroprotective effects may account for the putative mechanism of action. Four adult patients in RSE were cooled to 31_-34_C with an endovascular system for up to 90 h, and then passively rewarmed over 2-50 h. Seizures were controlled in two patients, one of whom died; also one of the other two patients in whom seizures continued subsequently deceased. Possible side effects are related to acid-base and electrolyte disturbances, and coagulation dysfunction including thrombosis, infectious risks, cardiac arrhythmia, and paralytic ileus (Corry et al., 2008; Cereda et al., 2009). Finally, anecdotic evidence suggests that cerebrospinal fluid (CSF)-air exchange may induce some transitory benefit in RSE (Kohrmann et al., 2006); although this approach was already in use in the middle of the twentieth century, the mechanism is unknown. Acknowledgment A wide spectrum of pharmacologic (sedating and nonsedating) and nonpharmacologic (surgical, or involving electrical stimulation) regimens might be applied to attempt RSE control. Their use should be considered only after refractoriness to AED or anesthetics displaying a higher level of evidence. Although it seems unlikely that these uncommon and scarcely studied strategies will influence the RSE outcome in a decisive way, some may be interesting in particular settings. However, because the main prognostic determinant in status epilepticus appears to be related to the underlying etiology rather than to the treatment approach (Rossetti et al., 2005, 2008), the safety issue should always represent a paramount concern for the prescribing physician. Conclusion The author confirms that he has read the Journal's position on issues involved in ethical publication and affirms that this paper is consistent with those guidelines.

The Role of Interleukin-2 in Memory CD8 Cell Differentiation

The current literature on the role of interleukin (IL)-2 in memory CD8(+) T-cell differentiation indicates a significant contribution of IL-2 during primary and also secondary expansion of CD8(+) T cells. IL-2 seems to be responsible for optimal expansion and generation of effector functions following primary antigenic challenge. As the magnitude of T-cell expansion determines the numbers of memory CD8(+) T cells surviving after pathogen elimination, these events influence memory cell generation. Moreover, during the contraction phase of an immune response where most antigen-specific CD8(+) T cells disappear by apoptosis, IL-2 signals are able to rescue CD8(+) T cells from cell death and provide a durable increase in memory CD8(+) T-cell counts. At the memory stage, CD8(+) T-cell frequencies can be boosted by administration of exogenous IL-2. Significantly, only CD8(+) T cells that have received IL-2 signals during initial priming are able to mediate efficient secondary expansion following renewed antigenic challenge. Thus, IL-2 signals during different phases of an immune response are key in optimizing CD8(+) T-cell functions, thereby affecting both primary and secondary responses of these T cells.

A comparison of in vivo 13C MR brain glycogen quantification at 9.4 and 14.1 T.

The high molecular weight and low concentration of brain glycogen render its noninvasive quantification challenging. Therefore, the precision increase of the quantification by localized (13) C MR at 9.4 to 14.1 T was investigated. Signal-to-noise ratio increased by 66%, slightly offset by a T(1) increase of 332 ± 15 to 521 ± 34 ms. Isotopic enrichment after long-term (13) C administration was comparable (≈ 40%) as was the nominal linewidth of glycogen C1 (≈ 50 Hz). Among the factors that contributed to the 66% observed increase in signal-to-noise ratio, the T(1) relaxation time impacted the effective signal-to-noise ratio by only 10% at a repetition time = 1 s. The signal-to-noise ratio increase together with the larger spectral dispersion at 14.1 T resulted in a better defined baseline, which allowed for more accurate fitting. Quantified glycogen concentrations were 5.8 ± 0.9 mM at 9.4 T and 6.0 ± 0.4 mM at 14.1 T; the decreased standard deviation demonstrates the compounded effect of increased magnetization and improved baseline on the precision of glycogen quantification.

CCL17 Promotes Intestinal Inflammation in Mice and Counteracts Regulatory T Cell-Mediated Protection From Colitis.

BACKGROUND & AIMS: Priming of T cells by dendritic cells (DCs) in the intestinal mucosa and associated lymphoid tissues helps maintain mucosal tolerance but also contributes to the development of chronic intestinal inflammation. Chemokines regulate the intestinal immune response and can contribute to pathogenesis of inflammatory bowel diseases. We investigated the role of the chemokine CCL17, which is expressed by conventional DCs in the intestine and is up-regulated during colitis. METHODS: Colitis was induced by administration of dextran sodium sulfate (DSS) to mice or transfer of T cells to lymphopenic mice. Colitis activity was monitored by body weight assessment, histologic scoring, and cytokine profile analysis. The direct effects of CCL17 on DCs and the indirect effects on differentiation of T helper (Th) cells were determined in vitro and ex vivo. RESULTS: Mice that lacked CCL17 (Ccl17(E/E) mice) were protected from induction of severe colitis by DSS or T-cell transfer. Colonic mucosa and mesenteric lymph nodes from Ccl17-deficient mice produced lower levels of proinflammatory cytokines. The population of Foxp3(+) regulatory T cells (Tregs) was expanded in Ccl17(E/E) mice and required for long-term protection from colitis. CCR4 expression by transferred T cells was not required for induction of colitis, but CCR4 expression by the recipients was required. CCL17 promoted Toll-like receptor-induced secretion of interleukin-12 and interleukin-23 by DCs in an autocrine manner, promoted differentiation of Th1 and Th17 cells, and reduced induction of Foxp3(+) Treg cells. CONCLUSIONS: The chemokine CCL17 is required for induction of intestinal inflammation in mice. CCL17 has an autocrine effect on DCs that promotes production of inflammatory cytokines and activation of Th1 and Th17 cells and reduces expansion of Treg cells.

Calpastatin-mediated inhibition of calpains in the mouse brain prevents mutant ataxin 3 proteolysis, nuclear localization and aggregation, relieving Machado-Joseph disease.

Machado-Joseph disease is the most frequently found dominantly-inherited cerebellar ataxia. Over-repetition of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within the ataxin 3 protein, which upon proteolysis may trigger Machado-Joseph disease. We investigated the role of calpains in the generation of toxic ataxin 3 fragments and pathogenesis of Machado-Joseph disease. For this purpose, we inhibited calpain activity in mouse models of Machado-Joseph disease by overexpressing the endogenous calpain-inhibitor calpastatin. Calpain blockage reduced the size and number of mutant ataxin 3 inclusions, neuronal dysfunction and neurodegeneration. By reducing fragmentation of ataxin 3, calpastatin overexpression modified the subcellular localization of mutant ataxin 3 restraining the protein in the cytoplasm, reducing aggregation and nuclear toxicity and overcoming calpastatin depletion observed upon mutant ataxin 3 expression. Our findings are the first in vivo proof that mutant ataxin 3 proteolysis by calpains mediates its translocation to the nucleus, aggregation and toxicity and that inhibition of calpains may provide an effective therapy for Machado-Joseph disease.

Bridge to life: the Lifebridge B2T extracorporeal life support system in an in vitro trial.

Extracorporeal life support systems (ECLS) have become common in cardiothoracic surgery, but are still "Terra Incognita" in other medical fields due to the fact that perfusion units are normally bound to cardiothoracic centres. The Lifebridge B2T is an ECLS that is meant to be used as an easy and fast-track extracorporeal cardiac support to provide short-term perfusion for the transport of a patient to a specialized centre. With the Lifebridge B2T it is now possible to provide extracorporeal bypass for patients in hospitals without a perfusion unit. The Lifebridge B2T was tested on three calves to analyze the handling, performance and security of this system. The Lifebridge B2T safely can be used clinically and can provide full extracorporeal support for patients in cardiac or pulmonary failure. Flows up to 3.9 +/- 0.2l/min were reached, with an inflow pressure of -103 +/- 13mmHg, using a 21Fr. BioMedicus (Medtronic, Minneapolis, MN, USA) venous cannula. The "Plug and Play" philosophy, with semi-automatic priming, integrated check-list, a long battery time of over two hours and instinctively designed user interface, makes this device very interesting for units with high-risk interventions, such as catheterisation labs. If a system is necessary in an emergency unit, the Lifebridge can provide a high security level, even in centres not acquainted with cardiopulmonary bypass.

Munc18-1 mutations that strongly impair SNARE-complex binding support normal synaptic transmission.

Synaptic transmission depends critically on the Sec1p/Munc18 protein Munc18-1, but it is unclear whether Munc18-1 primarily operates as a integral part of the fusion machinery or has a more upstream role in fusion complex assembly. Here, we show that point mutations in Munc18-1 that interfere with binding to the free Syntaxin1a N-terminus and strongly impair binding to assembled SNARE complexes all support normal docking, priming and fusion of synaptic vesicles, and normal synaptic plasticity in munc18-1 null mutant neurons. These data support a prevailing role of Munc18-1 before/during SNARE-complex assembly, while its continued association to assembled SNARE complexes is dispensable for synaptic transmission.

Myeloid-related proteins 8 and 14 contribute to monosodium urate monohydrate crystal-induced inflammation in gout.

OBJECTIVE: Monosodium urate monohydrate (MSU) crystal-induced interleukin-1β (IL-1β) secretion is a critical factor in the pathogenesis of gout. However, without costimulation by a proIL-1β-inducing factor, MSU crystals alone are insufficient to induce IL-1β secretion. The responsible costimulatory factors that act as a priming endogenous signal in vivo are not yet known. We undertook this study to analyze the costimulatory properties of myeloid-related protein 8 (MRP-8) and MRP-14 (endogenous Toll-like receptor 4 [TLR-4] agonists) in MSU crystal-induced IL-1β secretion and their relevance in gout. METHODS: MRP-8/MRP-14 was measured in paired serum and synovial fluid samples by enzyme-linked immunosorbent assay (ELISA) and localized in synovial tissue from gout patients by immunohistochemistry. Serum levels were correlated with disease activity, and MSU crystal-induced release of MRPs from human phagocytes was measured. Costimulatory effects of MRP-8 and MRP-14 on MSU crystal-induced IL-1β secretion from phagocytes were analyzed in vitro by ELISA, Western blotting, and polymerase chain reaction. The impact of MRP was tested in vivo in a murine MSU crystal-induced peritonitis model. RESULTS: MRP-8/MRP-14 levels were elevated in the synovium, tophi, and serum of patients with gout and correlated with disease activity. MRP-8/MRP-14 was released by MSU crystal-activated phagocytes and increased MSU crystal-induced IL-1β secretion in a TLR-4-dependent manner. Targeted deletion of MRP-14 in mice led to a moderately reduced response of MSU crystal-induced inflammation in vivo. CONCLUSION: MRP-8 and MRP-14, which are highly expressed in gout, are enhancers of MSU crystal-induced IL-1β secretion in vitro and in vivo. These endogenous TLR-4 ligands released by activated phagocytes contribute to the maintenance of inflammation in gout.

Immune response to MMTV infection.

Mouse mammary tumor virus (MMTV) has developed a strategy of exploitation of the immune response. It infects dendritic cells and B cells and requires this infection to establish an efficient chronic infection. This allows transmission of infection to the mammary gland, production in milk and infection of the next generation via lactation. The elaborate strategy developed by MMTV utilizes several key elements of the normal immune response. Starting with the infection and activation of dendritic cells and B cells leading to the expression of a viral superantigen followed by professional superantigen-mediated priming of naive polyclonal T cells by dendritic cells and induction of superantigen-mediated T cell B cell collaboration results in long-lasting germinal center formation and production of long-lived B cells that can later carry the virus to the mammary gland epithelium. Later in life it can induce transformation of mammary gland epithelium by integrating close to proto-oncogenes leading to their overexpression. Genes encoding proteins of the Wnt-pathway are preferential targets. This review will put these effects in the context of a normal immune response and summarize important facts on MMTV biology.