21 (Becton Dickinson) A total of 10 000 cells per tube were cou

2.1 (Becton Dickinson). A total of 10 000 cells per tube were counted and the positive proportion of total PBMCs or PBMC subpopulations was assessed from the quadrant statistic of the dot plots. The members of the cysteine aspartic acid-specific protease (caspase) family play key roles in apoptosis. Caspase Cell Cycle inhibitor 3 and 7 are downstream effectors directly executing

apoptosis and are activated by the initiator caspases 8 and 9. The death receptor-associated caspase 8 is activated by extrinsic apoptosis signals, and caspase 9 is activated by intrinsic, mitochondrial-dependent apoptosis signals. Levels of activated caspase 3/7, 8 and 9 were determined in total PBMCs using the Caspase-Glo luminescent assays as directed by the manufacturer (Promega GmbH, Mannheim, Germany). A total of 10 000 cells (2000 for caspase 3/7) were incubated in 100 μL of Dulbecco’s Modified Eagle Medium (Gibco, Invitrogen GmbH, Karlsruhe, Germany) in the dark for 60 min at 22°C and luminescence was measured every 1 s for 10 s in a Berthold Sirius luminometer (Berthold Technologies,

Bad Wildbad, Germany). Baseline luminescence-corrected data were expressed as relative light units per selleck second (RLU/s). For evaluation of mitochondrial metabolic function in PBMCs, the production of lactate and pyruvate, the final products of anaerobic and aerobic metabolism, respectively, from glucose was determined. The severity of mitochondrial dysfunction is expressed by the lactate-to-pyruvate ratio. This assay is based on the previously described ex vivo method [14, 15]. For our purposes, quantification was optimized by establishing a specific liquid chromatography − tandem mass spectrometry (LC-MS-MS) method. Briefly, 500 000 cells were incubated in 300 μL of HEPES-modified Krebs buffer supplemented with 10.0 mmol/L glucose for

120 min at 37°C under constant agitation. The reaction was stopped by snap-freezing in liquid nitrogen. Supernatants were quantified by LC-MS/MS on a TSQ Quantum (Thermo Fisher, Dreieich, Germany) Aspartate operating in negative electrospray ionization mode by single reaction monitoring (SRM) of the precursor ion [M-H]– product ion transition for lactate (m/z 89 43 at 10 eV) and pyruvate (m/z 87 43 at 10 eV) with ethylgallate (10 μM; m/z 197 169 at 25 eV) as internal standard. Chromatographic separation was performed onto a 5-μm Aquasil C18 column (100 × 3 mm; Thermo Fisher) and isocratic elution at a flow rate of 300 μL/min with 30% (v/v) acetonitrile/0.1% formic acid and 70% (v/v) deionized water/0.1% formic acid. An increased lactate-to-pyruvate ratio indicated a dysfunction of the mitochondrial respiratory chain complex. Of 159 patients recruited to the Cologne HIV cohort, eight patients on a PI-based regimen and eight patients on an NNRTI-based regimen with a treatment period of 7 years were eligible for analysis in our study (Fig. 2).

Stimulus presentation and randomization were controlled using Presentation® software (Neurobehavioral Systems Inc, Albany, CA) running on a personal computer. Inter-trial timing was determined manually by the experimenter. To maintain the subject’s attention across the study, participants were instructed to decide whether the two stimuli in the pair were physically the ‘Same’ or ‘Different’, regardless of the self/other identity, by pressing two response buttons with the index finger of the left hand (Keenan et al., 2000a). Electromyographic (EMG) recordings were made click here from the first dorsal interosseous (FDI) muscle of the left

hand using a single differential surface EMG electrode, placed over the muscle belly. The ground electrode was placed over the left elbow. The EMG signal was amplified 1000 times with a BagnoliTM

System, band-filtered (25–250 Hz) with a sampling rate of 2 kHz and digitized using a BioPac MP100 system (http://www.biopac.com) and stored for off-line analysis. A MagStim Rapid2 stimulator (The Magstim Company, Carmarthenshire, Wales, UK) was used with a standard figure-of-eight, 70-mm-diameter selleck screening library TMS coil. First, the individual optimal scalp position over the hand motor area of each subject was found by determining the scalp positioning at which the lower stimulation evoked the largest MEP. The intensity of single-pulse TMS was then adjusted to evoke MEPs with a mean peak-to-peak amplitude of ∼0.5 mV in a series of

ten consecutive pulses in the relaxed left FDI (baseline). To stimulate primary motor cortex, the coil was always placed tangentially to the scalp at a 45° angle to the midline to induce a posterior–anterior current flow across the central sulcus. Throughout the experimental session, the TMS coil was held in place by a mechanical arm fixed on an adjustable tripod, and one experimenter stood directly behind the subject and continuously monitored the coil position, correcting the position of the subjects’ head in case L-NAME HCl of involuntary small head displacements. Based on results from a pilot study, magnetic pulses were randomly delivered at 300, 600 or 900 ms after the onset of the first picture in the pair and were triggered by the program used for stimuli presentation. The precise timing of stimulus onset and TMS triggering pulse were checked by means of an oscilloscope. Two baselines (ten pulses each) were acquired for each experimental block. The mean MEP amplitude of the baselines (i.e. before and after presentation of blocks) did not differ and were thus averaged to normalize MEP amplitude. Two baselines (ten pulses each) were acquired, one before and one after, for each experimental block. The mean of the baselines was calculated and used to normalize MEP amplitude. For each trial, MEP amplitude was expressed as a percentage of the mean peak-to-peak amplitude of the averaged baseline.

In the ongoing UK PIVOT study, detailed neuropsychological testin

In the ongoing UK PIVOT study, detailed neuropsychological testing is being assessed prospectively in subjects on PI monotherapy vs. standard therapy, the results of which

will be of great interest to this field. Given the above theoretical concerns regarding the CNS activity of PI monotherapy, and for the majority of HIV-positive subjects Ganetespib mouse it may be possible to select other ARV regimens, we suggest this approach is currently avoided in neurologically symptomatic subjects. In patients with ongoing or worsening NC impairment despite ART, we recommend the following best practice management (GPP): Reassessment for confounding conditions. Assessment of CSF HIV RNA, CSF HIV genotropism and genotyping of CSF HIV RNA. In subjects with detectable CSF HIV RNA, modifications to ART should be based on plasma and CSF genotypic and genotropism results. Several published randomized

controlled studies, assessing both intensification of ART with a new ARV agent [131] and with adjunctive therapies [132-135] have been published. Unfortunately, NVP-BKM120 none of these studies describe improvements in cognition subsequent to the study interventions. Without evidence-based interventions, the Writing Group outlines below a best practice approach based on the current literature. As HIV-associated NC disorders are a diagnosis of exclusion, re-evaluation of subjects with ongoing NC impairment despite ART for confounding conditions, with expert input from other clinical specialties such as psychiatry, neurology and neuropsychology, is recommended and, where possible, input from an HIV neurology service. Assessment of CSF HIV RNA, CSF HIV genotropism and genotypic analysis of CSF RNA may be useful tools in the management of subjects with ongoing NC for the following reasons. First, data from cohorts of untreated HIV-positive subjects would suggest

CSF HIV RNA to be greater in subjects with HIV-associated dementia and cognitive decline [136, 137] and therefore suppression of CSF HIV RNA may be beneficial for cognitive function. Secondly, in subjects with ongoing NC impairment, higher degrees of genetic diversity between HIV viral strains in the CSF and plasma compartment may exist [138], even in subjects with undetectable plasma HIV RNA [139]. Therefore, Edoxaban assessment for CSF HIV resistance may be worthwhile to tailor ART. We recommend patients with HIVAN start ART immediately irrespective of CD4 cell count (1C). We recommend patients with end-stage kidney disease who are suitable candidates for renal transplantation start ART irrespective of CD4 cell count (1C). Proportion of patients with HIVAN started on ART within 2 weeks of diagnosis of CKD. The use of ART has been associated with a decline in the incidence of HIVAN in HIV cohort studies [140], with renal histological improvement in case reports [141, 142], and with delayed progression to end-stage kidney disease in case series [143, 144].

Temporal attention tasks, instead, have been more often shown to

Temporal attention tasks, instead, have been more often shown to lead to activation in the middle temporal gyrus, the superior occipital gyrus and the cerebellum (Coull & Nobre,

1998; Davranche et al., 2011; Li et al., 2012). The neuroimaging findings discussed above, obtained with various methods, indicate similarities but also profound differences in the neural mechanisms underlying temporal and spatial attention. This must in part be due to the dramatic differences between encoding the dimensions of space and time. Temporal attention usually involves processing of time-shifted events, while spatial attention involves competition between (possible) events occurring at about the same time. In other words, during spatial attention a person usually has to focus attention on one out of several isochronous potential events, which are all competing for processing resources at the same time (Desimone & Duncan, 1995). In contrast, GSI-IX while focusing attention in time, potentially relevant events are anisochronous. Depending on the temporal difference between two events, temporal attention can allocate resources flexibly and dynamically to adapt efficiently towards task demands. In the light of this framework, it seems only logical

that temporal and spatial attention may share some similarities Transmembrane Transproters modulator but also display very different outcomes at the behavioural level. While in spatial attention the isochrony of possible events tends to create cross-modal linkage to optimize resources, in temporal attention

events can be cross-modally decoupled as they are anisochronous and resources can be allocated dynamically. Within the present study, we manipulated the participants’ attention through different target probabilities, in terms of its onset times and modality. For example, a more likely modality is also more relevant for participants and therefore it will necessarily drive their endogenous attention. On the other hand, different target probabilities lead also to different target predictabilities and therefore modulate the participants’ expectations (Lange, 2013). Thus, as in most other temporal attention studies, we are well aware that for the RANTES moment these findings must be attributed to a combination of attention and expectation effects. Although attention and expectation can be functionally distinguishable and lead to different effects (Summerfield & Egner, 2009), it is not the goal of this study to measure their different contributions. This study addressed whether orienting attention in time leads to synergistic behavioural cross-modal effects, as shown previously for spatial attention (i.e., Spence & Driver, 1996) and more recently suggested for temporal attention (Lange & Röder, 2006). We found that processing of a likely (primary) modality is enhanced at its expected (most likely overall) time point. This is an expected result.

, 1993) After ingestion of the crystal toxins by the susceptible

, 1993). After ingestion of the crystal toxins by the susceptible larvae, crystalline inclusions are dissolved due to

the alkaline pH of the larval midgut. Then the 51- and 42-kDa protoxins are activated by midgut proteases to form the active proteins, of approximately 43 and 39 kDa, respectively (Broadwell & Baumann, 1987; Nicolas et al., 1990). This is then followed by the binding of the activated binary toxin to a specific receptor presented on the surface of midgut epithelium cells of susceptible larvae (Davidson, 1988; Silva-Filha et al., 1997). The binary toxin receptor has been identified as a 60-kDa α-glucosidase (Cpm1), which is attached to the cell membrane by a glycosyl-phosphatidyl inositol anchor (Silva-Filha et al., 1999; Darboux et selleck compound al., 2001). Using N- and C-terminal deletion

constructs of both BinA and BinB in in vivo gut binding studies, it has been proposed that the C-terminus of BinA is important for larvicidal toxicity, whereas both N- and C-terminal fragments of BinA are required for interaction with BinB. In addition, it has been proposed that the N-terminus of BinB is crucial for binding to the receptor in gut epithelial cells (Oei et al., 1992). Even though BinB has been shown to play a role in receptor recognition, its binding mechanism is still unknown. Because of the lack of structural information for the binary toxin, this website functional studies have been based mainly on its primary amino acid sequence and OSBPL9 secondary structure prediction (Broadwell et al., 1990; Berry et al., 1993; Shanmugavelu et al., 1998; Elangovan et al., 2000; Yuan et al., 2001; Promdonkoy et al., 2008; Sanitt et al., 2008). Interestingly, the amino acid sequences of BinA or BinB are not similar to other bacterial toxins. They

are, however, homologous to each other, with a 25% amino acid identity and a 40% similarity, which suggests a similar 3D structure (Promdonkoy et al., 2008). Despite their homology, the two proteins have distinct functions: BinB is responsible for receptor binding, whereas BinA acts as a toxic component (Oei et al., 1992; Charles et al., 1997; Shanmugavelu et al., 1998; Elangovan et al., 2000). It is thus possible that the different functions of these two proteins are contributed by the nonhomologous segments. For example, an amino acid sequence alignment shows that two regions in BinB are absent in BinA (Fig. 1). These regions are located in the N-terminal part of BinB. It is possible that some amino acids in these regions confer distinct functionality to BinB. To identify these possible functional elements, we have performed amino acid substitutions at residues spanning positions 111–117 and 143–150. Our results demonstrate that the aromaticity of F149 and Y150 plays a crucial role in larvicidal activity, with these residues possibly being involved in interaction with the epithelial membrane and receptor. Escherichia coli K-12 JM109 was used as a host strain for mutagenesis.

Both afferents converge onto dendritic spines, the critical site for synaptic integration in MSNs. In advanced PD there is a marked atrophy of dendrites and spines in these neurons, Ganetespib molecular weight indicative of dysfunctional signal integration in the striatofugal pathway. Similar pathology, triggered by a dysregulation of intraspine Cav1.3 L-type Ca2+ channels (Day et al., 2006), has been observed in rodent and primate models of

PD (Day et al., 2006; Neely et al., 2007; Scholz et al., 2008). The significance of such dendritic atrophy and spine pruning for the symptoms and the treatment of PD has remained poorly understood. However, there is increasing awareness that these morphological alterations represent a major obstacle for therapeutic approaches

to enhance striatal function (Schuster et al., 2009). Most notably, the efficacy of dopamine cell replacement strategies, designed to restore nigrostriatal connectivity, may be hampered by striatal dendritic and spine buy Compound Library atrophy. In order for grafted dopamine neurons to re-establish functional connections, the morphological target of such reinnervation would need to be preserved or reestablished. In this issue of EJN, Soderstrom et al. (2010) report the results of a study on the impact of dendritic spine preservation in MSNs upon both anti-parkinsonian and prodyskinetic effect of fetal mesencephalic cell grafts. The authors elegantly and convincingly Edoxaban show that administration of the L-type Ca2+ channel blocker nimodipine prevented loss of spines in MSNs in unilaterally lesioned rats that were grafted with embryonic ventral midbrain cells. Nimodipine treatment also resulted in improved therapeutic benefit and reduced graft-induced behavioral abnormalities of these hemi-parkinsonian rats. Specifically, the results indicate

that graft-mediated anti-parkinsonian efficacy was not potentiated by the prevention of spine loss; however, the impact of the graft- and levodopa-induced side-effects was greatly diminished by nimodipine treatment. Interestingly, these effects were not due to increased survival of grafted cells but correlated with a greater reinnervation of the affected striatum. These results underscore the importance of prevention (or reversal) of spine loss in striatofugal neurons for effective therapy based on dopamine cell replacement. They extend a previous report of reduced levodopa-induced dyskinesia by prior treatment with L-type Ca2+ channel antagonists (Schuster et al., 2009). The results described in Soderstrom et al. (2010) suggest that unless MSN spine loss and dendritic atrophy are reversed by appropriate pharmacological treatment, therapeutic interventions may be of limited efficacy or even cause unwarranted outcome. The findings and conclusions from the study by Soderstrom et al.

, 1987; Cardinale & Clark, 2005 and references cited therein). A possible exception to this statement is the

report that Salmonella within macrophages might be exposed to up to 10 μM NO (Raines et al., 2006). However, nitrite was a more effective inducer of Phcp expression than growth-inhibitory concentrations of 10 or 20 μM NO added repeatedly at 30 min intervals. The smaller and slower response to NO was not due to the rapid decomposition of NO by oxygen because separate experiments with an NO-sensitive electrode confirmed that NO was stable under the anaerobic conditions used. Note that the high pKa value of nitrous acid means that at physiological pH, nitrous acid diffuses across the cytoplasmic membrane, and nitrite can be transported by at least three mechanisms, NarK,

NarU and NirC (see, e.g. Jia et al., 2009). Three of the Small molecule library price obvious possible explanations for the minimal response of the hcp promoter to external NO are that derepression of NsrR was counter-balanced 3-MA by loss of transcription activation by FNR; that derepression of the NsrR regulon resulted in sufficient capacity to repair nitrosative damage to FNR as rapidly as it occurred or that the capacity of the bacteria to reduce NO was sufficient to prevent its cytoplasmic accumulation. Control experiments with the Nsr-independent promoter, FF-37.5::lacZ, eliminated the first possibility and hence, by inference also, the second explanation (Table 2). The results of these experiments also challenged claims that FNR can function as a physiologically relevant sensor of NO (Cruz-Ramos et al., 2002; Corker & Roole, 2003; Pullan et al., 2007). Although the periplasmic

nitrite reductase, NrfAB, was the obvious candidate to provide protection against externally added NO by catalysing its reduction to ammonia in the periplasm (as proposed by van Wonderen et al., 2008), externally added NO still did not induce Phcp::lacZ transcription in a nrfAB deletion mutant as effectively as nitrite. The 10-fold higher rates of NO reduction than nitrite reduction by strains defective in both NirB and NrfA suggest that E. coli has a greater capacity to reduce NO than to produce it from nitrite. We recently reported that even in the absence of all currently characterized mafosfamide NO reductase activities, anaerobic cultures of E. coli still reduce NO rapidly (Vine & Cole, 2011). The data in the current study therefore reinforce our previous conclusion that a significant NO reduction activity remains to be characterized. We favour the explanation that this activity prevents significant damage to cytoplasmic proteins by concentrations of externally generated NO relevant to pathogenicity. We thank Merve Yasa for help with some of the control experiments. ”
“Institute of Microbiology, AS ČR, Praha 4-Krč, Czech Republic SpoIISAB is a toxin–antitoxin module encoded on the chromosomes of Bacillus subtilis and related Bacilli species.

It has been strongly suggested that physical and/or MP of a movem

It has been strongly suggested that physical and/or MP of a movement sequence improves performance and induces plasticity in the cerebellum (Jenkins et al., 1994; Toni et al., 1998; Lacourse et al., 2004). Strangely, anodal tDCS over the right cerebellar hemisphere impaired the motor performance. Similarly, a former study using anodal tDCS over the cerebellum showed that anodal tDCS impaired the practice-dependent proficiency increase in working memory (Ferrucci et al., 2008). Galea et al. (2009) found that anodal tDCS over the right cerebellar cortex Kinase Inhibitor Library chemical structure can increase the inhibitory tone that the cerebellum exerts over the M1. The inhibition of the M1 after

cerebellar tDCS could be one explanation for the impairment of handwriting legibility observed in our study. Potential PLX3397 limiting aspects of the study should be mentioned. (i) In principle, motor practice alone of the handwriting task with the non-dominant hand over six experimental sessions could have had an impact on motor performance and it might have somewhat compromised the interpretation of the results. However, this is improbable in our opinion as the experimental session order was counterbalanced among subjects and baseline writing performance on the experimental first day did not differ from that on the last day, (ii) It cannot

be ruled out that additional cortical areas may have been influenced by tDCS due to the relatively poor spatial resolution of the technique (Nitsche et al., 2008; Datta et al., 2009). Although we cannot almost completely rule out this possibility, it should be noted that other studies using tDCS successfully modulated close cortical areas in different ways (Nitsche & Paulus, 2000; Nitsche et al., 2003b; Vollmann et al., 2012). (iii) Some studies have reported gender differences in responses to tDCS (Knops et al., 2006; Boggio et al., 2008; Chaieb et al., 2008). In the present study, as the most of subjects were women, it is possible that sex hormones somewhat influenced the results of our study. It is necessary to replicate the study using male participants in future research to investigate

a potential gender influence on the results. In conclusion, our results suggest that MP-induced effects in improving motor performance can be successfully consolidated by excitatory non-invasive brain stimulation on the M1 and left DLPFC. Although this finding is novel, further investigation is needed to understand how motor performance improvement is consolidated after mental training and whether it can be extended to other populations such as patients with neurological pathologies. If so, tDCS could be effectively used as a complementary method to increase the mental training effects. Moreover, our findings may help to improve to understanding about the specific role of each area involved in the MP effects on motor learning. However, a better understanding of the action mechanisms is essential for MP to be used effectively as a therapeutic tool.

Our task was similar to directed forgetting designs (Baddeley et 

Our task was similar to directed forgetting designs (Baddeley et al., 2009) when memory for ‘to-be-forgotten’ items is weaker, but regularly above chance level. The above-chance level of performance for distractor letters suggests reliable

responses (i.e. extreme below-chance performance might suggest that participants intentionally did not report distractor letters that they remembered). One may assume that participants simply knew that the distractor will be asked and thus attempted to remember it better and with it they encoded the scenes. However, our results do not indicate that participants attempted to remember the distractor-associated scenes better because scene recognition performance was at the chance level in this condition, which was similar to the case when scenes were presented INCB018424 order alone. Moreover, in the dopamine replacement condition, a boosting effect was observed for the recognition of distractor-associated scenes but not for the recall of distractor letters, which indicates an intriguing dissociation between the recall of the central stimulus (letters) and the recognition of the background information (scenes).

A possible explanation may be that the short-term memory systems responsible for maintaining the letters and the neural systems responsible for the attentional boost are not equally affected by PD and dopaminergic medications, and that medicated patients with PD have less control LDE225 over distracting items (e.g. Moustafa et al., 2008). The BCKDHA neuronal correlates of attentional boost and its pharmacological modulation need to be investigated using functional neuroimaging methods. Swallow et al. (2012) provided evidence

that responding to target stimuli at behaviorally relevant points of time enhanced activity in early visual cortical areas, but it is not clear how it affects memory for contextual background images. Although our current results and data from individuals with hippocampal atrophy (Szamosi et al., 2013) suggest the relevance of midbrain dopaminergic–hippocampal interactions in attentional boost (Shohamy & Wagner, 2008; Wimmer et al., 2012), this hypothesis should be directly tested. This study was supported by the National Development Agency (TÁMOP-4.2.2.A-11/1/KONV-2012-0052). Abbreviations ABT attentional boost test ANT attention network test BIS-11 Barratt Impulsiveness Scale-11 HAM-D Hamilton Depression Rating Scale HSD Honestly Significant Difference L-DOPA l-3,4-dihydroxyphenylalanine LED levodopa equivalent dose MIDI Minnesota Impulsive Disorders Interview PD Parkinson’s disease SOGS South Oaks Gambling Screen UPDRS Unified Parkinson’s Disease Rating Scale The authors (S.K., H.N., E.L.G., O.K.) declare no conflict of interest. ”
“Several studies have already shown that transcranial direct current stimulation (tDCS) is a useful tool for enhancing recovery in aphasia. However, all tDCS studies have previously investigated the effects using unihemisperic stimulation.

When the passaged cells reached 80% confluence they were used for

When the passaged cells reached 80% confluence they were used for growing raft cultures. Raft cultures were grown as previously described [19, 20]. Briefly, mouse fibroblast 3T3 J2 cells were trypsinized and re-suspended to a concentration of 2.5 × 105 cells/mL in 1% reconstitution buffer, 10% 10× DMEM (Life Technologies, Gaithersburg, MD), 2.4 μL of 10 M NaOH and 80% collagen (Dickinson, Franklin Lakes, NJ) on ice. The mixture was then aliquoted into six-well plates, each well containing 2.5 mL of the mixture. The plates were incubated at 37°C for

NVP-BGJ398 2 h to allow the collagen matrices to solidify. Two millilitres of E-medium was then added to each well so that the matrix could equilibrate. Human gingival epithelial keratinocytes were trypsinized and re-suspended in E-medium plus 5 ng/mL epidermal growth factor (EGF) at a concentration of 1 × 106 cells/mL and 1 mL of the suspension was added to the top of each collagen matrix. Epithelial cells were allowed to attach to the collagen for 2–4 h before the medium was removed and the matrices

were lifted onto stainless steel grids. The grids rested at the air–liquid interface and the raft cultures were fed by diffusion from below with E-medium supplemented with ZDV. ZDV capsules (Aurobindo Pharma, Cranberry, NJ, USA) were purchased from the pharmacy at The Milton S. Hershey Selleck 3-deazaneplanocin A Medical Center, Penn State University. The contents of either a 100-mg or 300-mg capsule were removed and re-suspended in sterile PBS. Serial dilutions were made directly in E-medium to obtain the correct concentration. The maximum

level of ZDV reached in the blood of patients, or Cmax, is 2 μg/mL [21-23]. Two additional concentrations on either Exoribonuclease side of the Cmax were also used. In the first set of experiments, the rafts were treated with ZDV at concentrations of 0.5, 1, 2, 4 and 6 μg/mL from day 0. Control rafts were fed with E-medium only. In the second set of experiments the same concentrations of ZDV were used but the rafts were treated on day 8. All rafts were fed every other day and harvested at the time-points indicated in Figure 1. Raft cultures were fixed in 10% buffered formalin, and embedded in paraffin. Four-micrometre sections were cut and stained with haematoxylin and eosin as described previously [19]. Immunostaining was performed with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA) [19]. Briefly, a vacuum oven was used to bake slides at 55°C for 1 h. Tissue sections were then dehydrated in xylene and rehydrated in alcohol gradients. Endogenous peroxide activity was neutralized by incubating the slides in a 3% H2O2 solution. Tissue sections were blocked for 1 h with 3% normal horse serum and 20% normal goat serum for primary mouse antibodies and rabbit antibodies, respectively. Next, slides were incubated in primary antibodies for 1 h.