Cigarette Smoke Causes Caspase-Independent Apoptosis of Bronchial Epithelial Cells from Asthmatic Donors

Background

Epidemiologic studies have demonstrated important links between air pollution and asthma. Amongst these pollutants, environmental cigarette smoke is a risk factor both for asthma pathogenesis and exacerbation. As the barrier to the inhaled environment, the bronchial epithelium is a key structure that is exposed to cigarette smoke.

Objectives

Since primary bronchial epithelial cells (PBECs) from asthmatic donors are more susceptible to oxidant-induced apoptosis, we hypothesized that they would be susceptible to cigarette smoke-induced cell death.

Methods

PBECs from normal and asthmatic donors were exposed to cigarette smoke extract (CSE); cell survival and apoptosis were assessed by fluorescence-activated cell sorting, and protective effects of antioxidants evaluated. The mechanism of cell death was evaluated using caspase inhibitors and immunofluorescent staining for apoptosis-inducing factor (AIF).

Results

Exposure of PBEC cultures to CSE resulted in a dose-dependent increase in cell death. At 20% CSE, PBECs from asthmatic donors exhibited significantly more apoptosis than cells from non-asthmatic controls. Reduced glutathione (GSH), but not ascorbic acid (AA), protected against CSE-induced apoptosis. To investigate mechanisms of CSE-induced apoptosis, caspase-3 or -9 inhibitors were tested, but these failed to prevent apoptosis; in contrast, CSE promoted nuclear translocation of AIF from the mitochondria. GSH reduced the number of nuclear-AIF positive cells whereas AA was ineffective.

Conclusion

Our results show that PBECs from asthmatic donors are more susceptible to CSE-induced apoptosis. This response involves AIF, which has been implicated in DNA damage and ROS-mediated cell-death. Epithelial susceptibility to CSE may contribute to the impact of environmental tobacco smoke in asthma.     

Effects of Mechanical Force on Cytoskeleton Structure and Calpain-Induced Apoptosis in Rat Dorsal Root Ganglion Neurons In Vitro

Background

A sudden mechanical insult to the spinal cord is usually caused by changing pressure on the surface of the spinal cord. Most of these insults are mechanical force injuries, and their mechanism of injury to the spinal cord is largely unknown.

Methods

Using a compression-driven instrument to simulate mechanical force, we applied mechanical pressure of 0.5 MPa to rat dorsal root ganglion (DRG) neurons for 10 min to investigate cytoskeletal alterations and calpain-induced apoptosis after the mechanical force injury.

Results

The results indicated that mechanical forces affect the structure of the cytoskeleton and cell viability, induce early apoptosis, and affect the cell cycle of DRG neurons. In addition, the calpain inhibitor PD150606 reduced cytoskeletal degradation and the rate of apoptosis after mechanical force injury.

Conclusion

Thus, calpain may play an important role in DRG neurons in the regulation of apoptosis and cytoskeletal alterations induced by mechanical force. Moreover, cytoskeletal alterations may be substantially involved in the mechanotransduction process in DRG neurons after mechanical injury and may be induced by activated calpain. To our knowledge, this is the first report to demonstrate a relationship between cytoskeletal degradation and apoptosis in DRG neurons.     

Activation of Bcl-2-Caspase-9 Apoptosis Pathway in the Testis of Asthmatic Mice

Background

Apoptosis plays a critical role in controlling the proliferation and differentiation of germ cells during spermatogenesis. Dysregulation of the fine-tuned balance may lead to the onset of testicular diseases. In this study, we investigated the activation status of apoptosis pathways in the testicular tissues under the background of an asthmatic mouse model.

Methods

Ten BALB/c mice were divided into two groups: the acute asthma group and the control group. In the acute asthma group, ovalbumin (OVA)-sensitized mice were challenged with aerosolized OVA for 7 days, while the control group was treated with physiological saline. After that, both epididymis and testis were collected to determine the sperm count and motility. Apoptosis in the testis was evaluated by DNA ladder, immunochemistry and further by PCR array of apoptosis-related genes. Finally, the cleavage of caspase-3 and poly ADP-ribose polymerase (PARP) was determined by western blot and the enzymatic activities of caspase-9 and 3/7 were assessed using Caspase-Glo kits.

Results

Compared with control mice, significant decreases in the body weight, testis weight, sperm count and motility were seen in the experimental group. DNA ladder and immunochemistry showed significant increase in apoptotic index of the asthmatic testis, whereas a decrease in mRNA expression of Bcl-2 and increases in Bax, BNIP3, caspase-9, and AIF were observed in the asthma group. Furthermore, protein levels of AIF were significantly upregulated, while the translational expression of Bcl-2 was downregulated markedly. Consistently, caspase-9 activity in the testis of asthma mice was significantly higher than that of the control group.

Conclusion

Collectively, these results showed that Bcl-2-caspase-9 apoptosis pathway was clearly activated in the testis of asthmatic mice with the increased expression of apoptosis-related genes and proteins. To our knowledge, this is the first report demonstrating that asthma could lead to the activation of the mitochondrial apoptosis signaling pathway in the mouse testis.     

Neuronal Survival,Morphology and Outgrowth of Spiral Ganglion Neurons Using a Defined Growth Factor Combination

Objectives

The functionality of cochlear implants (CI) depends, among others, on the number and excitability of surviving spiral ganglion neurons (SGN). The spatial separation between the SGN, located in the bony axis of the inner ear, and the CI, which is inserted in the scala tympani, results in suboptimal performance of CI patients and may be decreased by attracting the SGN neurites towards the electrode contacts. Neurotrophic factors (NTFs) can support neuronal survival and neurite outgrowth.

Methods

Since brain-derived neurotrophic factor (BDNF) is well known for its neuroprotective effect and ciliary neurotrophic factor (CNTF) increases neurite outgrowth, we evaluated if the combination of BDNF and CNTF leads to an enhanced neuronal survival with extended neurite outgrowth. Both NTFs were added in effective high concentrations (BDNF 50ng/ml, CNTF 100ng/ml), alone and in combination, to cultured dissociated SGN of neonatal rats for 48 hours.

Results

The neuronal survival and neurite outgrowth were significantly higher in SGN treated with the combination of the two NTFs compared to treatment with each factor alone. Additionally, with respect to the morphology, the combination of BDNF and CNTF leads to a significantly higher number of bipolar neurons and a decreased number of neurons without neurites in culture.

Conclusion

The combination of BDNF and CNTF shows a great potential to increase the neuronal survival and the number of bipolar neurons in vitro and to regenerate retracted nerve fibers.     

Involvement of VDAC,Bax and Ceramides in the Efflux of AIF from Mitochondria during Curcumin-Induced Apoptosis

Background

We previously identified curcumin as a potent inducer of fibroblast apoptosis, which could be used to treat hypertrophic scar formation. Here we investigated the underlying mechanism of this process.

Principal Findings

Curcumin-induced apoptosis could not be blocked by caspase-inhibitors and we could not detect any caspase-3/7 activity. Curcumin predominantly induced mitochondria-mediated ROS formation and stimulated the expression of the redox-sensitive pro-apoptotic factor p53. Inhibition of the pro-apoptotic signaling enzyme glycogen synthase kinase-3β (GSK-3β) blocked curcumin-induced apoptosis. Apoptosis was associated with high molecular weight DNA damage, a possible indicator of apoptosis-inducing factor (AIF) activity. Indeed, curcumin caused nuclear translocation of AIF, which could be blocked by the antioxidant N-acetyl cysteine. We next investigated how AIF is effluxed from mitochondria in more detail. The permeability transition pore complex (PTPC), of which the voltage-dependent anion channel (VDAC) is a component, could be involved since the VDAC-inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid) efficiently blocked AIF translocation. However, PTPC is not involved in AIF release since cyclosporine A, a specific inhibitor of the complex did not block apoptosis. Alternatively, the pro-apoptotic protein Bax could have formed mitochondrial channels and interacted with VDAC. Curcumin caused mitochondrial translocation of Bax, which was blocked by DIDS, suggesting a Bax-VDAC interaction. Interestingly, ceramide channels can also release apoptogenic factors from mitochondria and we found that addition of ceramide induced caspase-independent apoptosis. Surprisingly, this process could also be blocked by DIDS, suggesting the concerted action of Bax, VDAC and ceramide in the efflux of AIF from the mitochondrion.

Conclusions

Curcumin-induced fibroblast apoptosis is totally caspase-independent and relies on the mitochondrial formation of ROS and the subsequent nuclear translocation of AIF, which is released from a mitochondrial pore that involves VDAC, Bax and possibly ceramides. The composition of the AIF-releasing channel seems to be much more complex than previously thought.     

Poly (ADP-ribose) polymerase plays an important role in intermittent hypoxia-induced cell death in rat cerebellar granule cells

Background

Episodic cessation of airflow during sleep in patients with sleep apnea syndrome results in intermittent hypoxia (IH). Our aim was to investigate the effects of IH on cerebellar granule cells and to identify the mechanism of IH-induced cell death.

Methods

Cerebellar granule cells were freshly prepared from neonatal Sprague-Dawley rats. IH was created by culturing the cerebellar granule cells in the incubators with oscillating O₂ concentration at 20% and 5% every 30 min for 1-4 days. The results of this study are based on image analysis using a confocal microscope and associated software. Cellular oxidative stress increased with increase in IH. In addition, the occurrence of cell death (apoptosis and necrosis) increased as the duration of IH increased, but decreased in the presence of an iron chelator (phenanthroline) or poly (ADP-ribose) polymerase (PARP) inhibitors [3-aminobenzamide (3-AB) and DPQ]. The fluorescence of caspase-3 remained the same regardless of the duration of IH, and Western blots did not detect activation of caspase-3. However, IH increased the ratio of apoptosis-inducing factor (AIF) translocation to the nucleus, while PARP inhibitors (3-AB) reduced this ratio.

Results

According to our findings, IH increased oxidative stress and subsequently leading to cell death. This effect was at least partially mediated by PARP activation, resulting in ATP depletion, calpain activation leading to AIF translocation to the nucleus.

Conclusions

We suggest that IH induces cell death in rat primary cerebellar granule cells by stimulating oxidative stress PARP-mediated calpain and AIF activation.     

CD36 Mediated Fatty Acid-Induced Podocyte Apoptosis via Oxidative Stress

Background

Hyperlipidemia-induced apoptosis mediated by fatty acid translocase CD36 is associated with increased uptake of ox-LDL or fatty acid in macrophages, hepatocytes and proximal tubular epithelial cells, leading to atherosclerosis, liver damage and fibrosis in obese patients, and diabetic nephropathy (DN), respectively. However, the specific role of CD36 in podocyte apoptosis in DN with hyperlipidemia remains poorly investigated.

Methods

The expression of CD36 was measured in paraffin-embedded kidney tissue samples (Ctr = 18, DN = 20) by immunohistochemistry and immunofluorescence staining. We cultured conditionally immortalized mouse podocytes (MPC5) and treated cells with palmitic acid, and measured CD36 expression by real-time PCR, Western blot analysis and immunofluorescence; lipid uptake by Oil red O staining and BODIPY staining; apoptosis by flow cytometry assay, TUNEL assay and Western blot analysis; and ROS production by DCFH-DA fluorescence staining. All statistical analyses were performed using SPSS 21.0 statistical software.

Results

CD36 expression was increased in kidney tissue from DN patients with hyperlipidemia. Palmitic acid upregulated CD36 expression and promoted its translocation from cytoplasm to plasma membrane in podocytes. Furthermore, palmitic acid increased lipid uptake, ROS production and apoptosis in podocytes, Sulfo-N-succinimidyloleate (SSO), the specific inhibitor of the fatty acid binding site on CD36, decreased palmitic acid-induced fatty acid accumulation, ROS production, and apoptosis in podocytes. Antioxidant 4-hydroxy-2,2,6,6- tetramethylpiperidine -1-oxyl (tempol) inhibited the overproduction of ROS and apoptosis in podocytes induced by palmitic acid.

Conclusions

CD36 mediated fatty acid-induced podocyte apoptosis via oxidative stress might participate in the process of DN.     

Effect of Immunosuppressive Agents on Hepatocyte Apoptosis Post-Liver Transplantation

Introduction

Immunosuppressants are used ubiquitously post-liver transplantation to prevent allograft rejection. However their effects on hepatocytes are unknown. Experimental data from non-liver cells indicate that immunosuppressants may promote cell death thereby driving an inflammatory response that promotes fibrosis and raises concerns that a similar effect may occur within the liver. We evaluated apoptosis within the liver tissue of post-liver transplant patients and correlated these findings with in vitro experiments investigating the effects of immunosuppressants on apoptosis in primary hepatocytes.

Methods

Hepatocyte apoptosis was assessed using immunohistochemistry for M30 CytoDEATH and cleaved PARP in human liver tissue. Primary mouse hepatocytes were treated with various combinations of cyclosporine, tacrolimus, sirolimus, or MMF. Cell viability and apoptosis were evaluated using crystal violet assays and Western immunoblots probed for cleaved PARP and cleaved caspase 3.

Results

Post-liver transplant patients had a 4.9-fold and 1.7-fold increase in M30 CytoDEATH and cleaved PARP compared to normal subjects. Cyclosporine and tacrolimus at therapeutic concentrations did not affect hepatocyte apoptosis, however when they were combined with MMF, cell death was significantly enhanced. Cell viability was reduced by 46% and 41%, cleaved PARP was increased 2.6-fold and 2.2-fold, and cleaved caspase 3 increased 2.2-fold and 1.8-fold following treatment with Cyclosporine/MMF and Tacrolimus/MMF respectively. By contrast, the sirolimus/MMF combination did not significantly reduce hepatocyte viability or promote apoptosis.

Conclusion

Commonly used immunosuppressive drug regimens employed after liver transplantation enhance hepatocyte cell death and may thus contribute to the increased liver fibrosis that occurs in a proportion of liver transplant recipients.     

Decoy Receptor 3 Improves Survival in Experimental Sepsis by Suppressing the Inflammatory Response and Lymphocyte Apoptosis

Purpose

Unbalanced inflammatory response and lymphocyte apoptosis is associated with high mortality in septic patients. Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, is an anti-inflammatory and anti-apoptotic factor. Recently, DcR3 expression was found to be increased in septic patients. This study evaluated the therapeutic effect and mechanisms of DcR3 on cecal ligation and puncture (CLP)-induced sepsis in mice.

Methods

C57BL/6 mice were subjected to CLP-induced polymicrobial sepsis. DcR3 Fc was intravenously injected 30 min before and 6 h after CLP. Bacterial clearance, cytokine production, histology, lymphocyte apoptosis and survival were evaluated. Furthermore, we investigated the systemic effects of DcR3 in in vitro lymphocyte apoptosis regulation.

Results

Our results demonstrated that DcR3 protein treatments significantly improved survival in septic mice (p <0.05). Treatment with DcR3 protein significantly reduced the inflammatory response and decreased lymphocyte apoptosis in the thymus and spleen. Histopathological findings of the lung and liver showed milder impairment after DcR3 administration. In vitro experiments showed that DcR3 Fc inhibited Fas-FasL mediated lymphocyte apoptosis.

Conclusions

Treatment with the DcR3 protein protects mice from sepsis by suppressing the inflammatory response and lymphocyte apoptosis. DcR3 protein may be useful in treatment of sepsis.     

N-Acetyl Cysteine Depletes Reactive Oxygen Species and Prevents Dental Monomer-Induced Intrinsic Mitochondrial Apoptosis In Vitro in Human Dental Pulp Cells

Purpose

To investigate the involvement of intrinsic mitochondrial apoptosis in dental monomer-induced cytotoxicity and the influences of N-acetyl cysteine (NAC) on this process.

Methods

Human dental pulp cells (hDPCs) were exposed to several dental monomers in the absence or presence of NAC, and cell viability, intracellular redox balance, morphology and function of mitochondria and key indicators of intrinsic mitochondrial apoptosis were evaluated using various commercial kits.

Results

Dental monomers exerted dose-dependent cytotoxic effects on hDPCs. Concomitant to the over-production of reactive oxygen species (ROS) and depletion of glutathione (GSH), differential changes in activities of superoxide dismutase, glutathione peroxidase, and catalase were detected. Apoptosis, as indicated by positive Annexin V/propidium iodide (PI) staining and activation of caspase-3, was observed after dental monomer treatment. Dental monomers impaired the morphology and function of mitochondria, and induced intrinsic mitochondrial apoptosis in hDPCs via up-regulation of p53, Bax and cleaved caspase-3, and down-regulation of Bcl-2. NAC restored cell viability, relieved oxidative stress and blocked the apoptotic effects of dental monomers.

Conclusions

Dental monomers induced oxidative stress and mitochondrial intrinsic apoptosis in hDPCs. NAC could reduce the oxidative stress and thus protect hDPCs against dental monomer-induced apoptosis.     

Metformin Induces Apoptosis and Downregulates Pyruvate Kinase M2 in Breast Cancer Cells Only When Grown in Nutrient-Poor Conditions

Introduction

Metformin is proposed as adjuvant therapy in cancer treatment because of its ability to limit cancer incidence by negatively modulating the PI3K/AKT/mTOR pathway. In vitro, in addition to inhibiting cancer cell proliferation, metformin can also induce apoptosis. The molecular mechanism underlying this second effect is still poorly characterized and published data are often contrasting. We investigated how nutrient availability can modulate metformin-induced apoptosis in three breast cancer cell lines.

Material and Methods

MCF7, SKBR3 and MDA-MB-231 cells were plated in MEM medium supplemented with increasing glucose concentrations or in DMEM medium and treated with 10 mM metformin. Cell viability was monitored by Trypan Blue assay and treatment effects on Akt/mTOR pathway and on apoptosis were analysed by Western Blot. Moreover, we determined the level of expression of pyruvate kinase M2 (PKM2), a well-known glycolytic enzyme expressed in cancer cells.

Results

Our results showed that metformin can induce apoptosis in breast cancer cells when cultured at physiological glucose concentrations and that the pro-apoptotic effect was completely abolished when cells were grown in high glucose/high amino acid medium. Induction of apoptosis was found to be dependent on AMPK activation but, at least partially, independent of TORC1 inactivation. Finally, we showed that, in nutrient-poor conditions, metformin was able to modulate the intracellular glycolytic equilibrium by downregulating PKM2 expression and that this mechanism was mediated by AMPK activation.

Conclusion

We demonstrated that metformin induces breast cancer cell apoptosis and PKM2 downregulation only in nutrient-poor conditions. Not only glucose levels but also amino acid concentration can influence the observed metformin inhibitory effect on the mTOR pathway as well as its pro-apoptotic effect. These data demonstrate that the reduction of nutrient supply in tumors can increase metformin efficacy and that modulation of PKM2 expression/activity could be a promising strategy to boost metformin anti-cancer effect.     

Protective Effects of Astaxanthin on ConA-Induced Autoimmune Hepatitis by the JNK/p-JNK Pathway-Mediated Inhibition of Autophagy and Apoptosis

Objective

Astaxanthin, a potent antioxidant, exhibits a wide range of biological activities, including antioxidant, atherosclerosis and antitumor activities. However, its effect on concanavalin A (ConA)-induced autoimmune hepatitis remains unclear. The aim of this study was to investigate the protective effects of astaxanthin on ConA-induced hepatitis in mice, and to elucidate the mechanisms of regulation.

Materials and Methods

Autoimmune hepatitis was induced in in Balb/C mice using ConA (25 mg/kg), and astaxanthin was orally administered daily at two doses (20 mg/kg and 40 mg/kg) for 14 days before ConA injection. Levels of serum liver enzymes and the histopathology of inflammatory cytokines and other maker proteins were determined at three time points (2, 8 and 24 h). Primary hepatocytes were pretreated with astaxanthin (80 μM) in vitro 24 h before stimulation with TNF-α (10 ng/ml). The apoptosis rate and related protein expression were determined 24 h after the administration of TNF-α.

Results

Astaxanthin attenuated serum liver enzymes and pathological damage by reducing the release of inflammatory factors. It performed anti-apoptotic effects via the descending phosphorylation of Bcl-2 through the down-regulation of the JNK/p-JNK pathway.

Conclusion

This research firstly expounded that astaxanthin reduced immune liver injury in ConA-induced autoimmune hepatitis. The mode of action appears to be downregulation of JNK/p-JNK-mediated apoptosis and autophagy.     

Nicotine-Induced Apoptosis in Human Renal Proximal Tubular Epithelial Cells

Background

Nicotine is, to a large extent, responsible for smoking-mediated renal dysfunction. This study investigated nicotine’s effects on renal tubular epithelial cell apoptosis in vitro and it explored the mechanisms underlying its effects.

Methods

Human proximal tubular epithelial (HK-2) cells were treated with nicotine. Cell viability was examined by using the WST-1 assay. Intracellular levels of reactive oxygen species (ROS) and the expression of mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB) proteins were determined. The messenger ribonucleic acid and the protein expression associated with the nicotine acetylcholine receptors (nAChRs) in HK-2 cells was examined, and apoptosis was detected using flow cytometry, cell cycle analysis, and immunoblot analysis.

Results

The HK-2 cells were endowed with nAChRs. Nicotine treatment reduced cell viability dose dependently, increased ROS levels, and increased extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK expression. Nicotine increased NF-κB activation, which was attenuated by N-acetyl-L-cysteine, and ERK and JNK inhibitors, but was not affected by a p38 MAPK inhibitor. Nicotine increased the Bax/Bcl-2 ratio, which was attenuated by N-acetyl-L-cysteine, the NF-κB inhibitor, Bay 11–7082, and hexamethonium, a non-specific nAChR blocker. Flow cytometry revealed nicotine-induced G2/M phase arrest. While nicotine treatment increased the expression of phosphorylated cdc2 and histone H3, a marker of G2/M phase arrest, hexamethonium and Bay 11–7082 pretreatment reduced their expression.

Conclusions

Nicotine caused apoptosis in HK-2 cells by inducing ROS generation that activated the NF-κB signaling pathway via the MAPK pathway and it arrested the cell cycle at the G2/M phase. Nicotine-induced apoptosis in HK-2 cells involves the nAChRs.     

Simultaneous Increases in Proliferation and Apoptosis of Vascular Smooth Muscle Cells Accelerate Diabetic Mouse Venous Atherosclerosis

Aims

This study was designed to demonstrate simultaneous increases in proliferation and apoptosis of vascular smooth muscle cells (VSMCs) leading to accelerated vein graft remodeling and to explore the underlying mechanisms.

Methods

Vein grafts were performed in non-diabetic and diabetic mice. The cultured quiescent VSMCs were subjected to mechanical stretch stress (SS) and/or advanced glycosylation end products (AGEs). Harvested vein grafts and treated VSMCs were used to detect cell proliferation, apoptosis, mitogen-activated protein kinases (MAPKs) activation and SM-α-actin expression.

Results

Significantly thicker vessel walls and greater increases in proliferation and apoptosis were observed in diabetic vein grafts than those in non-diabetic. Both SS and AGEs were found to induce different activation of three members of MAPKs and simultaneous increases in proliferation and apoptosis of VSMCs, and combined treatment with both had a synergistic effect. VSMCs with strong SM-α-actin expression represented more activated JNKs or p38MAPK, and cell apoptosis, while the cells with weak SM-α-actin expression demonstrated preferential activation of ERKs and cell proliferation. In contrast, inhibition of MAPKs signals triggered significant decreases in VSMC proliferation, and apoptosis. Treatment of the cells with RNA interference of receptor of AGEs (RAGE) also resulted in significant decreases in both proliferation and apoptosis.

Conclusions

Increased pressure-induced SS triggers simultaneous increases in proliferation and apoptosis of VSMCs in the vein grafts leading to vein arterializations, which can be synergistically accelerated by high glucose-induced AGEs resulting in vein graft atherosclerosis. Either SS or AGEs and their combination induce simultaneous increases in proliferation and apoptosis of VSMCs via different activation of three members of MAPKs resulting from different VSMC subtypes classified by SM-α-actin expression levels.     

Distinctive Features of NREM Parasomnia Behaviors in Parkinson’s Disease and Multiple System Atrophy

Objective

To characterize parasomnia behaviors on arousal from NREM sleep in Parkinson’s Disease (PD) and Multiple System Atrophy (MSA).

Methods

From 30 patients with PD, Dementia with Lewy Bodies/Dementia associated with PD, or MSA undergoing nocturnal video-polysomnography for presumed dream enactment behavior, we were able to select 2 PD and 2 MSA patients featuring NREM Parasomnia Behviors (NPBs). We identified episodes during which the subjects seemed to enact dreams or presumed dream-like mentation (NPB arousals) versus episodes with physiological movements (no-NPB arousals). A time-frequency analysis (Morlet Wavelet Transform) of the scalp EEG signals around each NPB and no- NPB arousal onset was performed, and the amplitudes of the spectral frequencies were compared between NPB and no-NPB arousals.

Results

19 NPBs were identified, 12 of which consisting of ‘elementary’ NPBs while 7 resembling confusional arousals. With quantitative EEG analysis, we found an amplitude reduction in the 5-6 Hz band 40 seconds before NPBs arousal as compared to no-NPB arousals at F4 and C4 derivations (p<0.01).

Conclusions

Many PD and MSA patients feature various NREM sleep-related behaviors, with clinical and electrophysiological differences and similarities with arousal parasomnias in the general population.

Significance

This study help bring to attention an overlooked phenomenon in neurodegenerative diseases.     

The Computerized Table Setting Test for Detecting Unilateral Neglect

Background

Patients with unilateral neglect fail to respond normally to stimuli on the left side. To facilitate the evaluation of unilateral spatial neglect, we developed a new application that runs on a tablet device and investigated its feasibility in stroke patients.

Methods

We made the computerized table setting test (CTST) to run on the tablet computer. Forty acute ischemic stroke patients (20 patients with right hemispheric infarction with neglect, 10 patients with right hemispheric infarction without neglect, and 10 patients with left hemispheric infarction) and 10 healthy controls were prospectively enrolled to validate the CTST. The test requires subjects to set a table by dragging 12 dishes located below the table on the tablet screen. The horizontal deviation of the 12 dishes from the midline of the table, the selection tendency measured by the sequence of the dish selection, and the elapsed time for table setting were calculated automatically.

Results

Parameters measured by the CTST were correlated with the results of conventional neglect tests. The horizontal deviation was significantly higher in patients with right hemispheric infarction with neglect compared with the other groups. The selection tendency and elapsed time also were significantly different in patients with right hemispheric infarction with neglect compared with the left hemispheric infarction and control groups, but were similar to those with right hemispheric infarction without neglect.

Conclusions

The CTST is feasible to administer and comparable with conventional neglect tests. This new application may be useful for the initial diagnosis and follow-up of neglect patients.     

Inhibition of NOS-NO System Prevents Autoimmune Orchitis Development in Rats: Relevance of NO Released by Testicular Macrophages in Germ Cell Apoptosis and Testosterone Secretion

Background

Although the testis is considered an immunoprivileged organ it can orchestrate immune responses against pathological insults such as infection and trauma. Experimental autoimmune orchitis (EAO) is a model of chronic inflammation whose main histopathological features it shares with human orchitis. In EAO an increased number of macrophages infiltrate the interstitium concomitantly with progressive germ cell degeneration and impaired steroidogenesis. Up-regulation of nitric oxide (NO)-NO synthase (NOS) system occurs, macrophages being the main producers of NO.

Objective

The aim of our study was to evaluate the role of NO-NOS system in orchitis development and determine the involvement of NO released by testicular macrophages on germ cell apoptosis and testosterone secretion.

Method and Results

EAO was induced in rats by immunization with testicular homogenate and adjuvants (E group) and a group of untreated normal rats (N) was also studied. Blockage of NOS by i.p. injection of E rats with a competitive inhibitor of NOS, L-NAME (8mg/kg), significantly reduced the incidence and severity of orchitis and lowered testicular nitrite content. L-NAME reduced germ cell apoptosis and restored intratesticular testosterone levels, without variations in serum LH. Co-culture of N testicular fragments with testicular macrophages obtained from EAO rats significantly increased germ cell apoptosis and testosterone secretion, whereas addition of L-NAME lowered both effects and reduced nitrite content. Incubation of testicular fragments from N rats with a NO donor DETA-NOnoate (DETA-NO) induced germ cell apoptosis through external and internal apoptotic pathways, an effect prevented by N-acetyl-L-cysteine (NAC). DETA-NO inhibited testosterone released from Leydig cells, whereas NAC (from 2.5 to 15 mM) did not prevent this effect.

Conclusions

We demonstrated that NO-NOS system is involved in the impairment of testicular function in orchitis. NO secreted mainly by testicular macrophages could promote oxidative stress inducing ST damage and interfering in Leydig cell function.     

Vitamin D Status Is an Independent Risk Factor for Global Cognitive Impairment in Peritoneal Dialysis Patients

Objective

Vitamin D (VD) deficiency is an independent risk factor for cognitive impairment (CI) in the general population, but VD status in peritoneal dialysis (PD) patients has not been investigated. In this study, we aimed to investigate the relationship between serum VD levels and global and specific cognitive functions in PD patients.

Design and Setting

Cross-sectional study, simultaneously conducted at two PD centers.

Patients

Clinically stable patients (n = 273) undergoing PD for at least 3 months were enrolled over a period of one year.

Main outcome Measures

Demographic and comorbidity data were recorded, and routine biochemical parameters and serum 25-hydroxyvitamin D (25(OH) D) levels of overnight fasted patients were determined. Global cognitive function was assessed by the Modified Mini-Mental State Examination (3MS) score; executive function, by the trail making tests (Trails A and B); and immediate memory, delayed memory, and language ability by the Repeatable Battery for the Assessment of Neuropsychological Status (RBANS) sub-tests.

Results

In the univariate analysis, serum 25(OH) D levels significantly correlated with 3MS scores (r = -0.139; P = 0.02), and Trail A (r = -0.188; P = 0.002) and B (r = -0.154; P = 0.01) completion times. In the multivariate analysis, 25(OH) D was found to be independently associated with global CI, but not with executive dysfunction. Serum 25(OH) D could not predict scores of immediate/delayed memory and language ability.

Conclusions

VD deficiency is highly prevalent in PD patients and is an independent risk factor for global CI in this patient cohort.