A antagonist (bicuculline methiodine) delivered multiple times per day from postnatal day (p) 21 to p25, we showed that rat pups experiencing frequent, focal, recurrent epileptiform activity in the form of interictal spikes during neurodevelopment have significant long-term deficits in attention and sociability that persist into adulthood. To determine if treatment with ACTH, a drug widely used to treat early-life seizures, altered outcome we administered ACTH once per day subcutaneously during the time of the induced interictal spike activity. We show a modest amelioration of the attention deficit seen in animals with a history of early life interictal spikes with ACTH, in the absence of alteration of interictal spike activity. These results suggest that pharmacological intervention that is not targeted to the interictal spike activity is worthy of future study as it may be beneficial for preventing or ameliorating adverse cognitive outcomes.Children with epilepsy often present with pervasive cognitive and behavioral comorbidities including working memory impairments, attention deficit hyperactivity disorder (ADHD) and autism spectrum disorder. These non-seizure characteristics are severely detrimental to overall quality of life. Some of these children, particularly those with epilepsies classified as Landau-Kleffner Syndrome or continuous spike and wave during sleep, have infrequent seizure activity but frequent focal epileptiform activity. This frequent epileptiform activity is thought to be detrimental to cognitive development; however, it is also possible that these IIS events initiate pathophysiological pathways in the developing brain that may be independently associated with cognitive deficits. These hypotheses are difficult to address due to the previous lack of an appropriate animal model. To this end, we have recently developed a rat model to test the role of frequent focal epileptiform activity in the prefrontal cortex. Using microinjections of a GABA It is widely believed that frequent epileptiform events seen in children with epilepsy are capable of causing deleterious alterations in developing brain networks and are therefore associated with the high incidence of cognitive deficits and psychiatric comorbidities in these patients We have recently shown selective deficits in measures of attention and sociability in adult animals with a history of interictal spikes (IIS) in the prefrontal cortex (PFC) during development. The PFC was chosen based on the preponderance of PFC-related comorbidities in children with pediatric epilepsy. We hypothesized that pharmacological treatment could improve the IIS activity and that this treatment could also ameliorate these associated behavioral deficits.hypothalamic-pituitary-adrenal (HPA) axis and leads to downstream release of cortisol in humans. ACTH is commonly used in epilepsies with frequent IIS and has been shown to decrease seizure frequency and normalize the EEG The drug H.P. Acthar gel was chosen to test these hypotheses. The primary active compound in Acthar gel is adrenocorticotrophic hormone (ACTH), which is part of the All experiments were performed in strict accordance with the guidelines set down by the National Institutes of Health and the Geisel School of Medicine at Dartmouth for the humane treatment of animals. The animal protocol was approved by the Institutional Animal Care and Use Committee of Dartmouth College (IACUC protocol number 09-08-01). All surgeries were performed under isoflurane anesthesia (2–3% in oxygen) and 5 mg/kg ketoprofen was used as an analgesic post-operatively. All efforts were made to minimize animal pain and utilize the smallest number of animals necessary to achieve the goal of the study.ad libitum unless otherwise noted. Behavioral data presented herein are pooled from three experimental cohorts of animals. The first cohort, containing only a control (N = 5) and an untreated IIS group (N = 5) and an additional two cohorts contain a control group (N = 4), an untreated IIS group (N = 4) and a treated IIS group (N = 8).Animals were housed 2 animals per cage on a 12∶12 light: dark cycle with access to standard rat chow and water A antagonist (bicuculline methiodine) was infused through the cannula multiple times per day for five consecutive days during development and local EEG was recorded at the time of infusion. Bicuculline-injected animals developed interictal spike (IIS)-like events on the EEG, with minimal seizure involvement. The long-term effect of treatment with ACTH on later PFC-related cognition was examined.See Sprague-Dawley male rat pups  age p17, p18 or p19  were impAfter a 2–4 day recovery period, animals were injected intracortically with 0.5–0.7 µL of 200 µM bicuculline methiodine (BMI) using a pump that delivered fluid unilaterally to the right PFC at 0.2 µL/minute. From p21–p25, animals were injected a total of 19 times. Three intracerebral infusions were made on the first day, with four infusions made per day on each of the four days thereafter. Injections were separated by approximately 1 hour. Littermate controls were also chronically implanted with an electrode/cannula and injected with sterile saline at the same time as BMI in the IIS group . Continu2  was injected. Subcutaneous injection was chosen to maximize duration of release of the drug. Body surface area (BSA) was estimated as follows: surface area (in cm2) = 9.1× (mass in grams)2/32 were multiplied by a factor of.0001 to obtain BSA in m2. BSA measurements (in m2) were then multiplied by the 150 IU/m2 to obtain a daily dose in IU. Acthar Gel was provided in 5 mL vials containing 80 IU of ACTH per mL. N = 8 animals were treated with ACTH, N = 8 animals were treated with vehicle.IIS and littermate control animals were injected subcutaneously with either vehicle or H.P. Acthar Gel . Vehicle control gel was also obtained from Questcor pharmaceuticals and had the same composition as Acthar diluent. ACTH injections were made once per day, 1 hr before BMI injections began. A dose of 150 IU/mAn operant box  enclosed in a dimly lit, sound-attenuating chamber was used for delayed non-match to sample (DNMS) experiments. Inside the box, two retractable levers were located on one wall separated by a pellet dispenser. The opposite wall contained a nose-poke hole. Stimulus lights were located above each lever, the food cup and the nose-poke hole. Food pellets were used as rewards . Animals were food deprived to approximately 85–90% of predicted weight according to the growth chart provided by Charles River Laboratories for Sprague-Dawley rats. During this time, each animal received 14.5 g of food per day, in addition to any food pellets rewarded during the task. DNMS sessions were done one session per day, 7 days a week at approximately the same time each day.The DNMS task involved several training steps before a full trial could be executed. Rats were initially trained to associate both freely available levers with food reward (labeled “PR1”). They were then trained to press only one extended lever (“Lever Press”), and subsequently trained to place their snout in the nose hole when the light above it was illuminated. This last training part was first performed while the operant box lights were switched off entirely to encourage the rat to follow the illuminated light about the nosepoke hole (“Nosepoke Dark”) and then in a dimly-lit operant box (“Nosepoke Light”). Criteria for advancing through these training sessions were as follows; bar presses: >25 rewards received, with both levers pressed at least once each; nose-poke dark: >25 rewards received, nose poke light: >10 rewards. If <10 rewards were given in a session, rats were demoted to the previous session stage.A final training session was a forced lever-nosepoke-opposite lever session (“L-NP-L”). The rat was presented with one lever (sample phase), had to press the lever, break the nose-poke beam in the opposite wall, and was then presented only the lever (test phase) that was not presented before (non-match lever). A successful press of the test lever in less than 10 s was associated with a food reward, or else the trial would reset.Once animals were able to perform this type of forced trial with an 80% response rate , they advanced to “DNMS training” trials, which contained the full DNMS paradigm with a 0 s delay. Here rats were presented with both test levers and had to press the one that was not presented in the sample phase. During this training paradigm, an error during one trial  resulted in a subsequent forced L-NP-L as in the previous training step. Once criterion  was achieved within the DNMS training session, full DNMS sessions began.DNMS was done in blocks with increasing delay times during the nosepoke phase; block 0 had a 0 s delay, block 1 had trials with randomly assigned 0–5 s delays, and block 2 had trials with randomly assigned 0–10 s delays. Criteria to pass to the next block include accuracy of 80% or more  during the session and no clear lever preference. Lever preference or less than 60% accuracy  resulted in regression to the previous block or phase of DNMS training. Behavioral software (Med Associates) gave output measures on correct and incorrect trials, as well as errors of omission: the former relating to the working memory component of the task and the latter relating to attention during the task. An error of omission was counted when a response was expected from the animal and the animal failed to respond within 10 s.The sociability experiment was based on a previously described mouse sociability task Statistics were done using Prism  and SPSS v.20 . We carried out multivariable repeated measures regression analysis using generalized estimating equations in order to assume the correct distribution for the data. IIS (bicuclline administration) and ACTH administration were both modeled as predictors. The effects of litter and seizures were also tested and were not significant in any of our experiments; the effect of session number was modeled in our DNMS data set and was not significant. The effect of ACTH on IIS-width and average spike rate and all histological data was analyzed using a two-tailed Student’s t-test, the effect of ACTH on IIS rate over time was assessed using a repeated-measures ANOVA.IIS activity began immediately after BMI microinjection and lasted for 10–20 mins post-injection while the animal was awake . Saline-Brief seizures lasting less than 15 s (average length 14.9±0.3 s) occurred in 8 of 17 BMI-exposed animals. There was no difference in seizure activity between groups exposed to the ACTH and vehicle-treated IIS animals. Overall frequency was 0.22±0.10 seizures per day. Presence or absence of seizures was modeled into all statistics and there was no significant effect of seizures in any of our behavioral outcomes.To determine whether or not behavioral deficits seen in animals with a history of IIS could be ameliorated with pharmacological treatment, animals were injected subcutaneously with ACTH one hour before beginning BMI injection. ACTH injection did not alter spike width, the frequency or duration of IIS as measured during the first 20 mins post-injection EEG recording period . QualitaA delayed non-match to sample (DNMS) task was chosen in order to test for the presence of deficits in working memory and attention. During the DNMS task, all animals were fed 14.5 g of food per day; weights did not significantly differ between the treated and untreated IIS groups during the DNMS experiment . Additionally, number of rewards eaten on the first day of the task, when rewards are freely available with the press of either of both continuously extended levers during a fixed 20-min session, were not different between the groups . Taken together, all of these data indicate that rats in all groups were equally motivated to complete the task. We found no significant difference in number of sessions to block completion , or in aHowever, animals with a history of IIS did make significantly more errors of omission than control animals (1.3±0.4 in controls vs. 8.3±1.7 in IIS). Treatment with ACTH decreased the number of omissions errors IIS animals made during the DNMS task. Errors of omission have previously been interpreted as a deficit in attention during the task.On day one of the DNMS task, one animal in the ACTH+IIS group made 82 total omissions in one session, four times more omissions than were ever observed in any other session from any animal in the cohort. This session is significantly influencing the data and is a statistical outlier  control for secondary effects of IIS on the PFC through connections from other brain regions (2) control for global brain injury incurred during generalized seizures. While this model may not speak directly to pediatric epilepsies with global IIS activity or frequent generalized seizures, our goal was to understand the long-term cognitive consequence of frequent GABA signaling disruption and resultant epileptiform activity on a single developing brain region whose dysfunction underlies cognitive deficits seen in patients with pediatric epilepsies. We have tailored the time of exposure roughly to the time period of development of the PFC itself. Thus in modeling early life IIS experimentally, we hoped to understand specific cognitive deficits seen in patients with pediatric epilepsies such as Landau Kleffner where seizures are infrequent but epileptiform activity is prevalent. Our subsequent goals, addressed in this study, were to ameliorate some of these cognitive deficits using a pharmacological agent.This experimental model of early life IIS was developed to assess the effect of any improvement in cognitive outcome has been shown in the context of pediatric epilepsy in the absence of any alteration in epileptiform activity.To this end, we have examined the role of ACTH treatment on IIS-associated deficits in behavior and found a modest but significant decrease in the number of omissions made during a DNMS task, suggesting ACTH may have a role in improving an attention deficit associated with early life epileptiform activity. Since ACTH is one of the few drugs that has been shown to not only treat the symptoms of epilepsy but also normalize the EEG A receptors, can reduce neuroinflammation ACTH has many targets in the brain and peripheral tissues. Melanocortin receptors that bind ACTH directly are located on a variety of different cell types throughout the CNS . This selectivity may be due to a difference in the nature of the sociability versus the DNMS task. The sociability task is a single session task, where there is one outcome measure per animal. This makes the task less sensitive than the DNMS attention measure, which has 5 or more sessions per animal. ACTH does ameliorate the attention deficit, but it does not bring it back to control levels, so it is possible that the change in sociability may be subtle as well. The decreased sensitivity of the sociability task suggests that subtle changes are not measureable with this task.The selectivity of the ACTH effect on attention over sociability was an unexpected but interesting resultThe suggestion that treating the cognitive deficits may be possible without treating the overt aberrant electrical activity has important implications for the way epilepsies are treated in the pediatric population ACTH administration to animals experiencing IIS does not change the features of the IIS. However, ACTH treatment appears to produce some alteration in attention deficits in these animals. This result is deserving of future study in order to determine the extent to which ACTH can alter deleterious cognitive outcomes, but is both surprising and exciting as it suggests that we may be able to treat a subset of cognitive deficits associated with abnormal EEG activity, even when we are unsuccessful at eliminating the EEG abnormalities. Important future directions should focus on understanding full extent to which ACTH can alter cognitive outcomes in this and other animal models, and subsequently understanding the mechanism by which ACTH exerts its effects in order to identify other potentially useful treatments for cognitive deficits in the pediatric epilepsy.Figure S1Improvements in attention after ACTH administration. Working memory as measured by cumulative number of sessions to task completion (A) and accuracy in the training session block (B) in the DNMS task were not different after IIS, or with treatment with ACTH. However, IIS animals (red) made significantly more omissions per session in the task than their control (blue) counterparts, and this was significantly improved with ACTH treatment (orange) (p<0.05) (C). Inset in (C) shows the presence of an outlier (circled) in the ACTH+IIS (orange) group. The number of omissions errors per day did not vary based on the difficulty in the working memory component of the task (D). Data points on all graphs represent mean ± SEM, N = 4 Control, N = 4 IIS, N = 8 IIS+ACTH.(TIF)Click here for additional data file.Figure S2Deficits in sociability are not significantly ameliorated by treatment with ACTH. Sociability as defined by the relative amount of time spent with a novel rat (bars labeled “R”) over a novel object (bars labeled “O”), was also altered by early life IIS. The group by time effect can be seen as a reduction in the ratio of the amount of time spent with the rat compared to the amount of time spent with the object. Control animals (blue) spent a significantly greater proportion of time with the novel rat than the novel object compared to IIS rats (red), and the IIS rats treated with ACTH (orange). There are no significant differences between the IIS and IIS+ACTH groups. Data points on all graphs represent mean ± SEM, N = 4 Control, N = 4 IIS, N = 8 IIS+ACTH.(TIF)Click here for additional data file.


High glucose can induce apoptosis in vascular endothelial cells, which may contribute to the development of vascular complications in diabetes. We evaluated the role of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in human coronary artery endothelial cells (HCAECs).HCAECs were treated with media containing 5.6, 11.1, and 16.7 mM of glucose for 24 h in the presence or absence of tumor necrosis factor (TNF)-α. For detection of apoptosis, DNA fragmentation assay was used. HCAEC expression of death receptors were analyzed by the PCR and flow cytometry methods. Also, using immunohistochemical techniques, coronary expression of death receptors was assessed in streptozotocin-nicotinamide-induced type 2 diabetic mice.Exposure of HCAECs to high glucose resulted in a significant increase in TNF-R1 and Fas expression, compared with normal glucose. High glucose increased TNF-α production by HCAECs and exogenous TNF-α up-regulated TNF-R1 and Fas expression in HCAECs. High glucose-induced up-regulation of TNF-R1 and Fas expression was undetectable in the presence of TNF-α. Treatment with TNF-R1 neutralizing peptides significantly inhibited high glucose-induced endothelial cell apoptosis. Type 2 diabetic mice displayed appreciable expression of TNF-R1 and Fas in coronary vessels.In association with increased TNF-α levels, the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions, which could in turn play a role in high glucose-induced endothelial cell apoptosis. A wide variety of studies suggest that endothelial dysfunction and damage present pathophysiological early steps for the development of vascular complications ,2. A groinopathy ,12. Moreinopathy . Severalinopathy -10,14,15inopathy ,17, are inopathy . Thus, tc into the cytoplasm, where it can bind to apoptotic protease activating factor (APAF)-1 and activate caspase networks that induce apoptosis [c in human umbilical vein endothelial cells [c, in human aortic endothelial cells [Apoptotic cell death in mammals can proceed by two distinct pathways that ultimately converge into a common pathway causing the activation of the effector enzymes caspases. The intrinsic pathway feeds cell death signals through the mitochondrion, which appears to act as a genetic damage sensor and monitor of metabolic status. Thus, injured mitochondria can release cytochrome poptosis ,20. Receal cells . Furtheral cells . Proapopal cells ,24. One al cells , and seval cells ,27, althal cells ,29. NeveIn the present study, we initially examined whether high ambient glucose up-regulates expression of death receptors in cultured human coronary artery endothelial cells (HCAECs). We found that gene expression levels of the death receptors, TNF-R1 and Fas, were strikingly increased in endothelial cells under high-glucose conditions. Next, we investigated the possible involvement of the death receptor pathway of apoptotic signaling in high glucose-induced apoptosis in endothelial cells. Finally, we evaluated the possibility that coronary arteriolar endothelium may significantly express TNF-R1 and Fas in type 2 diabetic mice.in vitro cell aging. Cells were grown in endothelial growth media-2  supplemented with growth factors and 5% (v/v) fetal bovine serum to 70-80% confluency. Cells were cultured in a humidified incubator at 37°C with a 5% CO2 atmosphere and the medium was changed every second day. To determine the effect of high glucose or TNF-α on cell apoptosis and death receptor expression, cells were incubated with both zero growth factors and zero hydrocortisone for 30 h, and they were then cultured for 24 h under different concentrations of glucose .HCAECs were purchased from Cambrex Bioscience . Cells were used between passages 6 and 7 for experiments to avoid effects of ® III  and 0.5 μg oligo(dT)12-18 primer  at 42°C for 1 h, followed by 51°C for 30 min. The primer pairs for TNF-R1 , for Fas , for DR4 , and for DR5  yielded a 218-, 207-, 154-, and 221-bp PCR product, respectively. Human β-actin  was used as a reference gene. PCR was performed using an iCycler . The reaction mixture consisted of template cDNA, 0.2 μM of each primer, 300 μM dNTPs, 3 mM MgCl2, and 0.05 U/μl ExTaq DNA polymerase . PCR was performed for 32 cycles for each gene with denaturation at 95°C for 15 s, annealing at 61°C for 15 s, and extension at 72°C for 20 s. PCR products were quantified using NIH Image.Total RNA was isolated from cells using standard procedure, including ISOGEN . The RNA was reverse-transcribed in a 20-μl reaction mixture that contained 5 U ReverScriptReal-time PCR was performed in a final volume of 20 μl containing cDNA template and primers using Takara RNA PCR kit (Takara Shuzo) as described in the manufacture's manual. PCR amplification was carried out on the genomic DNA using the following primers: TNF-R1, forward 5'-AAC AGA ACA CCG TGT GCA CCT-3' and reverse 5'-AGT CCT CAG TGC CCT TAA CAT TCT C-3'; FAS, forward 5'-AGA GTA AAT GCA GTG GCA TGC TAA G-3' and reverse 5'-GGG TTA GCC TGT GGA TAG ACA TTT G-3'; DR4, forward 5'-GGA ACA CAG CAT GTC AGT GCA A-3' and reverse 5'-TGT CAC TCC AGG GCG TAC AAT C-3'; DR5, forward 5'-CAT CTA TGG ACA GGC TGG GAC A-3' and reverse 5'-CCC AAA CAG GGC TCA AGT TCA-3'; and actin, forward 5'-TGG CAC CCA GCA CAA TGA A-3' and reverse 5'-CTA AGT CAT AGT CCG CCT AGA AGC A-3'. The PCR program consisted of 95°C for 30 s for initial denaturation of DNA, followed by 40 cycles of 95°C for 5 s, 60°C for 34 s for annealing of primers, and 95°C for 1 min for elongation. The PCR product size for TNF-R1, FAS, DR4, DR5, and actin was 141, 150, 130, 96, and 186 bp, respectively; they were visualized after agarose gel electrodes and ethidium bromide staining. Actin served as an internal control for normalization.Griffonia simplicifolia isolectin-B4  overnight at 4°C, followed by an overnight incubation at 4°C with streptavidin . The nucleus was counterstained with Hoechst 33258 . Immunofluorescent images were observed under a Leica TCS-SP5 confocal sytem.After being fixed with 4% paraformaldehyde for 15 min at room temperature, cells were kept in PBS at 4°C until staining. Unspecific binding sites were blocked in PBS containing 10% normal goat sera. The samples were treated with rabbit anti-human von Willebrand factor (vWF) antibody  at 1:100 dilution overnight at 4°C, carefully washed and stained with Alexa goat anti-rabbit secondary antibody (Invitrogen) at 1:500 dilution overnight at 4°C. For visualization of endothelial cells, cells were incubated with biotinylated in situ cell death detection kit  was used according to the manufacturer's instructions. The fluorosein-labeled cells undergoing apoptosis were recognized as a green fluorescent nucleus. The samples were analyzed using a fluorescence microscope.The terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique was conducted to detect apoptotic endothelial cells. The fluorescein g for 10 min. Subsequently, each DNA sample in the supernatant and the pellet was precipitated in 12.5% trichloroacetic acid at 4°C and quantified using a diphenylamine reagent after hydrolysis in 5% trichloroacetic acid at 90°C for 20 min. After the absorbance of the supernatant and the pellet was determined spectrophotometrically at a wavelength of 600 nm, the percentage of fragmented DNA in each sample was calculated as the amount of DNA in the supernatant divided by the total DNA for that sample (supernatant plus pellet). The average percentage of fragmented DNA in control samples was 2.7%.To determine the extent of apoptosis, DNA fragmentation was assessed following the method by Arai et al. . In brie3), stained with FITC-labeled anti-human TNF-R1 antibody  for 30 min at 4°C, followed by phycoerythrin-labeled goat polyclonal anti-mouse IgG . Propidium Iodide  was used to discriminate between dead and live cells. Flow cytometric analysis was performed using FACSCalibur and Cell QuestPro software version 6.0 . TNF-R1 expression was determined using mean fluorescence intensity (MFI), with vehicle-treated cells as the baseline for all comparisons.Cells were washed with FACS buffer  . Sensitivity of the assay was 4 pg/ml for TNF-α. No cross reactivity was observed with other human inflammatory cytokines including various interleukins. Optical density was measured at 450 nm by use of a microplate reader. Plasma levels of TNF-α were measured by the use of a commercially available enzyme-linked assay kit  according to the manufacture's instructions.The animal study was carried out as approved by the Animal Care and Use Committee of University of Toyama. Male ICR mice, 5-weeks old, received an intraperitoneal injection of 1.5 g/kg body weight of nicotinamide dissolved in saline 15 min before a tail-vein injection of streptozotocin  dissolved in a citrate buffer solution , following the protocol previously reported ,32. ContMouse hearts were harvested, fixed with 4% buffered formalin solution, immersed in sucrose solutions, dipped into OCT compound , and frozen at -20°C. The embedded tissues were then sectioned at a thickness of 30 μm and air dried. For immunohistochemical detection of the target molecules, the tissue sections were exposed to the fluorescent secondary antibody after incubation with the suitable primary antibody according to the method in our previous study with minor modification . Thus, rP < 0.05.All experiments were performed at least three times. Data are presented as means ± SE. Statistical assessment of the data was made by one- or two-way ANOVA, and then differences among groups were analyzed by Turkey's multiple comparison test, with level of significance set at The blood clotting protein vWF is synthesized and secreted by endothelial cells. It is a useful maker for endothelial cells, but appears to represent the presence of vascular, especially endothelial, damage and play an important role in thrombosis and inflammation . Immunofin situ TUNEL assay to detect apoptotic cells, exposure to high glucose resulted in a striking appearance of TUNEL-positive cells .To determine how high glucose environment regulates gene expression of death receptors in human endothelial cells, the mRNA expression levels of TNF-R1, Fas, DR4, and DR5 in HCAECs cultured for 24 h under high glucose conditions were evaluated by PCR analysis Figure . High glTreatment with TNF-α 0.3 nM) strikingly up-regulated mRNA expression levels of all death receptors , TNF-α levels were increased about 3.5- and 5.5-fold, respectively, compared to normal glucose medium Figure .When TNF-R1 neutralizing peptides (80 nM) were given during exposure to high glucose, DNA fragmentation caused by high glucose was significantly inhibited by 23% Figure . We confp < 0.05). Plasma TNF-α levels were much higher in diabetic than in control mice . Immunofluorescence staining for TNF-R1 and Fas in the left ventricular cross-sections is shown in Figure We generated type 2 diabetic mice which were administered streptozotocin and partially protected with nicotinamide. The non-fasting plasma glucose levels were 149 ± 28 mg/dl (n = 4) and 583 ± 90 mg/dl (n = 5) for control and diabetic mice, respectively  by which TNF-α can increase gene expression of these TRAIL receptors awaits further study.A potential role of the TNF-α system in high glucose-induced human endothelial cell apoptosis was tested with TNF-R1 neutralizing peptides when given at a concentration of 80 nM. TNF-R1 neutralizing peptides showed a significant inhibition of high glucose-induced DNA fragmentation in HCAECs. This suggests that the TNF-α system is involved in human endothelial cell apoptosis induction under high glucose conditions. However, the inhibitory effect of TNF-R1 neutralizing peptides was less than 40%. It should be noted that the dose of TNF-R1 neutralizing peptides employed in this study caused >50% inhibition of 0.3 nM TNF-α-induced DNA fragmentation in HCAECs. Alternatively, the Fas/Fas ligand system as well as the intrinsic mitochondrial-mediated apoptotic signaling pathway may be additional mechanisms for the remaining effect of high ambient glucose to induce human endothelial cell apoptosis. The involvement of the Fas/Fas ligand system in high glucose-induced apoptosis of human islets has been demonstrated using the antagonistic anti-Fas antibody ZB4 . It may Exposure of endothelial cells to excess glucose has been reported to induce inflammation ,44. The Finally, our immunofluorescence study found that chronic type 2 diabetic mice showed appreciable expression of TNF-R1 and Fas in the intimal surface of coronary arterioles. No positive staining for these death receptors was obtained in age-matched control mice. Circulating TNF-α levels were elevated in diabetic mice as reported in diabetic patients as well as in streptozotocin-induced diabetic rats ,47. AlsoIn conclusion, the present study showed that the death receptors, TNF-R1 and Fas, are up-regulated in HCAECs under high glucose conditions. This up-regulation of death receptor expression, coupled with increased TNF-α secretion, could promote endothelial cell apoptosis, which is likely to contribute to coronary arterial endothelial dysfunction and the development of ischemic heart disease in diabetes.The authors declare that they have no competing interests.SK, HY, NM, and YH designed research; SK, HY, KT, NK-Y, SY, and RY performed research; SK, HY, and KT analyzed data; SK, HY, and YH wrote the paper. All authors have read and approved submission of the final manuscript.


Timp3/−− involuting glands. Crossing in Tnf deficiency abrogates caspase 3 activation, but heightens macrophage and T-cell influx into Timp3/−− glands. The data indicate that TIMP3 differentially impacts apoptosis and inflammatory cell influx, based on involvement of TNF, during the process of mammary involution. An understanding of the molecular factors and wound healing microenvironment of the postpartum mammary gland may have implications for understanding pregnancy-associated breast cancer risk.Post-lactation mammary involution is a homeostatic process requiring epithelial apoptosis and clearance. Given that the deficiency of the extracellular metalloproteinase inhibitor TIMP3 impacts epithelial apoptosis and heightens inflammatory response, we investigated whether TIMP3 regulates these distinct processes during the phases of mammary gland involution in the mouse. Here we show that TIMP3 deficiency leads to TNF dysregulation, earlier caspase activation and onset of mitochondrial apoptosis. This accelerated first phase of involution includes faster loss of initiating signals  concurrent with immediate luminal deconstruction through E-cadherin fragmentation. Epithelial apoptosis is followed by accelerated adipogenesis and a greater macrophage and T-cell infiltration in  Tissue involution is a postnatal process that offers a unique window to study physiological cell death. Involution occurs in the thymus lpr mice and FasL-deficient gld mice do not show apoptotic cells until the later stages of involution Mammary gland involution in the mouse has been described as a two-step process defined by the reversibility of involution The second irreversible phase of involution occurs around day 3 and involves widespread apoptosis and tissue remodeling, with extracellular matrix turnover and structural reorganization of the gland Interestingly, microarray and histological studies have revealed that inflammatory mediators are present during mammary gland involution Timp3/−− mice. TIMP3 deficiency leads to a loss of the survival signal provided by E-cadherin-mediated cell to cell contact as well as an accelerated first phase of involution. A far greater and sustained influx of macrophages also occurs, which is coupled with an infiltration of T-cells in Timp3/−− involuting glands. Combining the TIMP3 and TNF deficiencies, we further dissect the TNF dependency of these molecular and cellular events. Our results show that TIMP3 is a critical physiological regulator for both apoptosis and inflammatory cell recruitment during murine mammary gland involution.Both gain- and loss-of-function studies underscore the complex apoptotic role of TIMP3 Timp3/−− mice have increased TNF levels and signaling owing to increased TACE activity in several systems Timp3/−− mammary gland also displayed two distinct periods of TNF increase. It was 4-fold higher than wild-type at 10L, reappeared earlier and stayed higher than in wild-type tissue (Timp3/−− glands throughout involution (data not shown). Death receptor-mediated apoptosis induced by TNF occurs through activation of the initiator caspase, caspase 8 Timp3/−− glands, the cleaved form of caspase 8 was detectable at 10L and elevated earlier than wild-type  e tissue . Fas andild-type .Timp3/−− mammary glands , but absent in wild-type tissue. Caspases 3 and 9 were also clearly activated earlier in TIMP3 null tissue as indicated by the presence of their cleaved forms  expression to be earlier than wild-type in Timp3/−− involuting glands  by this manipulation . In contrast, macrophage numbers were comparable during lactation, but increased up to 4-fold (P<0.01) in Timp3/−− involuting glands at early stage involution (1i and 2i) when compared to wild-type glands, and rapidly declined thereafter at 3i  have described neutrophil and macrophage infiltration during the process of mammary gland involution er at 3i . The maxTimp3/−− livers timp3/−− glands throughout involution. The number of T-cells in involuting Timp3-deficient glands peaked at 3i and was sustained until 5i. The wild-type tissue displayed similar kinetics but a smaller increase of these cells. The T-cell infiltration exhibited different kinetics compared to the macrophage influx, temporally occurring subsequent to macrophage .The presence of lymphocytes has been indicated in the involuting mammary glands of ruminants crophage . B cellsTimp3/−− mice was dependent on increased TNF bioactivity, by combining the Timp3 and Tnf deficiencies. For these experiments we used Timp3 mice that we backcrossed seven times into the C57BL/6 background to match the Tnf deficient mice, whereas the data in Timp3/−− mice. Activation of caspase 3 is a measure of apoptosis downstream of death receptor signaling by TNF, and its positivity has been previously reported in regressing mammary glands Timp3/−− or Tnf/−−) and compound (Timp3/−−/Tnf/−−) knockouts in the C57BL/6 background at 1i and 3i  when compared to Timp3/−− glands (P<0.05) at 1i following compound removal of TIMP3 and TNF .Timp3/−− mice were in FVB/N background. For the compound knockout studies with TNF, wild-type, Timp3/−−, Tnf/−−, and Timp3/−−/Tnf/−− mice were all in C57/BL6 background. Forced experimental involution was performed as previously described For the majority of the analyses, wild-type and The antibodies used were: Apaf-1 (Upstate); caspase 9 and activated caspase 3 ; E-cadherin, β-catenin, cytochrome c, SMAC, and Mitotracker CD3, F4/80, B220 (BD Biosciences); anti-neutrophil antibody (Serotec); AIF (gift from Dr. J. Penninger); caspase 8 (gift from Dr. R. Hakem). Recombinant human TIMP3 was prepared according to the manufacturer's specifications (R&D Systems).Mammary gland collection and lysate preparation were performed as previously described 50 µg of recombinant human TIMP3 (R&D Systems) were loaded onto pellets as per manufacturer's instructions. Implantation of pellets was done as previously described Two photon confocal microscopy was used for all immunofluorescence and immunocytochemistry analyses. Exposure time, gain, and offset parameters were first empirically determined and then kept constant throughout. Preparation of mammary glands for immunofluorescence and immunocytochemistry were performed as previously described Preparation of mammary glands for immunohistochemistry were performed as previously described Timp3/−−). One-way ANOVA was performed for statistical comparison between four groups , followed by Tukey test for pair-wise statistical analysis.Statistical analyses were performed for observations that have at least three mice per group. Student's t test was performed for statistical analyses between two groups (WT and


Mechanisms that expand the otherwise narrow neutralization capacity observed during early HIV-1 infection are currently undefined, but multiple lines of evidence suggest that the ability to elicit broad and potent neutralizing antibodies (nAbs) via vaccination could increase the protective efficacy of immunization.Here we characterized the initial nAb response in a subtype A HIV-1-infected Rwandan seroconverter and investigated how consequent immune events influenced the downstream development of cross-clade breadth. Autologous envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants were utilized to define the neutralization targets of autologous plasma and monoclonal antibodies (mAbs), the latter of which were also examined genetically and structurally through crystallization. Heterologous Env glycoproteins from nine cross-clade variants were used to determine neutralization breadth.Initially, nAbs targeted a single region of gp120 at the base of V3 involving the alpha2 helix. A single amino acid change at one of three positions conferred early escape from plasma nAbs. Then, two autologous mAbs, revealed to have flat epitope contact surfaces, typified the second wave of nAb pressure and neutralized escape Envs carrying the defined V3/alpha2 helix substitutions in a manner dependent on immunoglobulin light chain variable domain modifications. Subsequent mAb resistance arose in later Envs through alteration of two glycan motifs previously implicated in the development of nAb breadth. Finally, three-year autologous plasma displayed moderate neutralization breadth and most potently neutralized heterologous Envs containing the altered glycan motifs.Our data demonstrate that the V3-proximal nAb epitope originally recognized in this individual elicited strain-specific mAbs and that glycan-mediated escape from these mAbs likely initiated the development of heterologous neutralization breadth. These findings suggest that epitope localization and the resultant routes of viral immune evasion, which include exposure to a specific sequence of nAb escape variants, drive humoral immune responses toward cross-clade viral recognition.


Patients with colorectal cancer (CRC) often develop liver metastases, in which case surgery is considered the only potentially curative treatment option. However, liver surgery is associated with a risk of ischemia-reperfusion (IR) injury, which is thought to promote the growth of colorectal liver metastases. The influence of IR-induced tumor necrosis factor alpha (TNF-α) elevation in the process still is unknown. To investigate the role of TNF-α in the growth of pre-existing micrometastases in the liver following IR, we used a mouse model of colorectal liver metastases. In this model, mice received IR treatment seven days after intrasplenic injections of colorectal CT26 cells. Prior to IR treatment, either TNF-α blocker Enbrel or low-dose TNF-α, which could inhibit IR-induced TNF-α elevation, was administered by intraperitoneal injection.Hepatic IR treatment significantly promoted CT26 tumor growth in the liver, but either Enbrel or low-dose TNF-α pretreatment reversed this trend. Further studies showed that the CT26 + IR group prominently increased the levels of ALT and AST, liver necrosis, inflammatory infiltration and the expressions of hepatic IL-6, MMP9 and E-selectin compared to those of CT26 group. Inhibition of TNF-α elevation remarkably attenuated the increases of these liver inflammatory damage indicators and tumor-promoting factors.These findings suggested that inhibition of TNF-α elevation delayed the IR-enhanced outgrowth of colorectal liver metastases by reducing IR-induced inflammatory damage and the formation of tumor-promoting microenvironments. Both Enbrel and low-dose TNF-α represented the potential therapeutic approaches for the protection of colorectal liver metastatic patients against IR injury-induced growth of liver micrometastases foci. Colorectal cancer (CRC) is the third most frequently diagnosed cancer in men and the second most frequently diagnosed cancer in women worldwide. A signiLiver surgery entails the occlusion of hepatic blood vessels and as a result is associated with ischemia-reperfusion (IR) injury. After IR injury, the damaged liver tissue becomes infiltrated with inflammatory cells, and the associated release of inflammatory mediators is thought to promote the development of metastatic foci. Indeed,Enbrel (etanercept) is a genetically engineered, soluble, systemic TNF-α blocker that competitively binds to and neutralizes both soluble and transmembrane forms of TNF-α,14. The Male wild-type BALB/C mice  were purchased from the Shanghai Experimental Center of the Chinese Science Academy, Shanghai, and housed under pathogen-free conditions. All animal experiments were carried out in accordance with animal experimentation protocols approved by the Animal Care Committee of Shanghai Jiaotong University.2. Confluent cultures were harvested by brief trypsinization (0.05% trypsin in 0.02% EDTA), and after centrifugation, single cell suspensions were prepared in physiological saline (106 cells/100 μl). Then, 100 μl of the cell suspension (106 cells) was injected into the parenchyma of the spleens of the animals. After 3 min, the spleens were removed to prevent intrasplenic tumor growth. Animals were reared for a further 5 days to allow sufficient time for liver metastasis to develop.CT26 (a murine colon carcinoma cell line) cells were cultured in Dulbecco’s Modified Eagle Medium  containing 10% fetal bovine serum (GIBCO), penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified atmosphere containing 5% COThe mice were anesthetized with pentobarbital , and partial hepatic IR was induced by clamping the left hepatic artery, portal vein, and bile duct to the left and middle lobes of the liver for 15 min. Surgical procedures were performed under aseptic conditions, and to prevent dehydration, a small amount of saline was left in the abdominal cavity, which was covered with gauze. The animals were kept on a heated table to maintain a body temperature of 37°C.The mice were divided into 5 treatment groups: (1) sham group (n = 5), intraperitoneal injection of saline only, followed by a sham surgical procedure; (2) CT26 group (n = 5), injection of CT26 cells, but no surgery for induction of IR injury; (3) CT26 + IR group (n = 5) injection of CT26 cells followed by surgery for induction of IR injury; (4) CT26 + IR + Enbrel group (n = 5), injection of CT26 cells, surgery for induction of IR injury, and pretreatment with the TNF-α blocker Enbrel ; and (5) CT26 + IR + TNF-α group (n = 5), injection of CT26 cells, surgery for induction of IR injury, and pretreatment with recombinant murine TNF-α .The mice were euthanized at 0, 30, 90, 180, and 360 min after surgery for IR induction, and the levels of serum TNF-α and hepatic TNF-α in liver homogenates were quantified using a mouse-specific TNF-α ELISA kit  according to the manufacturer’s instructions.3) = (long diameter × short diameter2)/2] and visible tumor numbers per liver. The tumor load of each excised liver was quantified by a hepatic replacement area (HRA) score, which was was the percentage of tumor tissue in the whole hepatic tissue. On 3 nonsequential H&E stained sections per liver, 30 random fields (objective magnification 100×) were selected and were used to calculate the ratio of tumor cells versus normal hepatocytes plus necrotic cells. The average percentage of tumor tissue of all the fields was expressed by HRA.The mice were euthanized at 7 days following surgery for IR induction. The livers were calculated total tumor volume per liver [volume  and aspartate aminotransferase (AST) were examined using a Fuji DRICHEM 55500 V  according to the manufacturer’s instructions.The livers were fixed using 10% neutral-buffered formalin and embedded in paraffin. Sections of the livers were stained with hematoxylin and eosin (H&E) stain and examined for evidence of morphological changes. The livers were examined for signs of tissue necrosis at 360 min after reperfusion by using a myeloperoxidase (MPO) assay kit  to measure liver homogenate MPO levels, a marker of neutrophil infiltration.The mice were harvested at 360 min after IR. Then total RNA of their liver tissues were isolated and purified using Trizol Reagent  and RNase-free DNase . Oligo dT18-primers and MMLV reverse transcriptase (Promega) were used to prepared complementary DNA. qPCR were performed using a LightCycler 480 system . Gene mRNA expressions were analyzed by specific primers as follows: IL6, Forward-5′-GTCAACTCCATCTGCCCTTC-3′, Reverse-5′-CTTGGTCCTTAGCCACTCCT-3′; MMP9, Forward-5′-CAATCCTTGCAATGTGGATG-3′, Reverse-5′-TAAGGAAGGGGCCCTGTA AT-3′; E-selectin, Forward-5′-CTCCTGCGAAGAAGGATT TGA-3′, Reverse-5′-CCCCTC TTGGACCACACTGA-3′. Endogenous β-actin was used as an internal control to determined the fold change of gene expression, which primer as follows: Forward-5′-AGATGTGGATC AGCAAGC AG-3′, Reverse-5′-GCGCAAGTTAGGTTTTGTCA-3′.P < 0.05 was considered statistically significant. Statistical analyses were performed using GraphPad Prism 5.0 software .All data are presented as means ± SEM, which are in each case averaged from 3 independent experiments. Observed differences between the treatment groups were analyzed using the Student’s t-test and one-way ANOVA to test for statistical significance; Compared to the Sham and CT26 only groups, the CT26 + IR group showed significantly elevated serum TNF-α level Figure A and hep3 vs. 58.7 ± 15.5 mm3), and a 2-fold lower tumor number (15.3 ± 2.5 vs. 7.7 ± 1.5) (Figure IR treatment led to a significant increase in tumor growth; however, both Enbrel and low-dose TNF-α pretreatment tended to attenuate this increase Figure A. ComparCompared to the CT26 group, the CT26 + IR group showed significantly elevated serum levels of ALT Figure A and ASTMicroscopy examination revealed that compared to the liver tissues of the CT26 and sham groups, the liver tissues of the CT26 + IR group had significant cytoplasmic vacuolization at 180 min after IR Figure A, and exRT-PCR results showed that liver tissues mRNA expressions of IL-6 Figure A, MMP9 B and E-sThe findings of this study demonstrated that both Enbrel and low-dose TNF-α pretreatment before IR, remarkably decreased serum and hepatic TNF-α levels, reduced tumor growth, decreased serum ALT and AST levels, reduced hepatic tissue injury and cytoplasmic vacuolization of cells, and reduced hepatic cellular necrosis and infiltration of inflammatory cells. These findings suggest that TNF-α might play an important role in IR-accelerated outgrowth of colorectal liver metastases, through the production of an inflammatory response and a microenvironment that is conducive to tumor growth.A previous study reported that surgical resection for liver metastases of CRC often leads to IR injury. BalkwilTo explore the effect of TNF-α on IR-induced growth of colorectal liver metastases, we examined tumor growth in the excised livers. Our findings showed that compared to the CT26 group, the CT26 + IR group showed significantly increased tumor load whereas the CT26 + IR + Enbrel and CT26 + IR + TNF-α groups showed markedly reduced tumor loads. These results indicated that TNF-α might play a role in IR-induced growth of pre-existing colorectal liver metastases.Studies in rodent models have shown that pretreatment with an anti-TNF-α antibody, or low doses of TNF-α and pentoxifylline, a methylxanthine inhibitor of TNF-α, prior to hepatic IR, can significantly reduce hepatic injury,20-22. ISeveral studies have demonstrated that IR-related liver injury results from an severe inflammatory response involving the release of TNF-α by Kupffer cells, which alone can further intensify the inflammation reaction via the production of vascular adhesion molecules and neutrophil-attracting chemokines-27. Our Taken together, our results suggest inhibition of TNF-α in the tumor microenvironment through a reduction in inflammatory cell infiltration, following liver IR, and our findings are consistent with that of previous research, for example, the inhibition or neutralization of TNF-α reduces the infiltration of inflammatory cells into hepatic tissue, and reduces liver IR injury,22. StudTaken together our findings suggest that TNF-α could up-regulate the inflammatory response following IR, and possibly produce a microenvironment that promotes tumor growth. Other research supports this, for example, in a TNF-α KO mouse model, hepatic IR injury was attenuated. This study also demonstrated that JNK-1 and NF-κB are activated in both the early and late inflammatory phases of hepatic IR injury, and that TNF-α is main agent for triggering these 2 pathways. GiannanIn conclusion, our results demonstrated that TNF-α plays an important role in IR-induced outgrowth of colorectal liver metastases by enhancing inflammatory cell infiltration and the formation of the microenvironment that facilitates tumor progression. The finding that pretreatment with both Enbrel and low-dose TNF-α prior to IR protects against liver injury and prevents the growth of liver metastases suggests that these treatments may have the potential for protecting patients undergoing resection for colorectal liver metastases.The authors have declared that no competing interests exist.SFJ and KS contributed equally to this work. SFJ, KS, LXW, XMK and YJL participated in the design and performance of this study. SFJ and XJC carried out the mice experiment. XZ carried out cell culture. SFJ and NC carried out Biochemical analysis and ELISA experiment. SFJ and LMX carried out HE experiment. LXW and XMK conceived of the study and participated in the design and coordination. The manuscript was drafted by SFJ and KS, and reviewed by all authors. All authors approved the final version of the manuscript to be published.


HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine.chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen.HIV-1 Gag VLPs encapsidating reporter HIV-1 Gag VLPs produced had significantly high levels of Gp64 (~1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/μg Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold.Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice. Inert virus-like particles (VLPs) made from virus structural protein(s) are an ideal substitute for live, attenuated and peptide-based virus vaccines, as they present epitopes in an immunologically relevant context and lack any replicative nucleic acid. Several VLP-based vaccines have been developed against human viruses, such as the recently-released human papillomavirus (HPV) vaccines, and the well-established hepatitis B virus (HBV) vaccines -3. Gag pDuring virion assembly, HIV Gag encapsidates two copies of the viral genomic RNA displaying a HIV packaging signal called the ψ-site -17. The The primary mode of HIV entry into cells was assumed to be by Gp120/Gp41-and CD4+ host cell CD4 + CCR5/CXCR4 receptor-mediated plasma membrane fusion ,35. MoreHIV-1 Gag VLPs are often produced in baculovirus-based insect cell expression systems, which provide an ideal eukaryotic environment for the production of biologically active recombinant proteins as they enable post-translational modifications such as proper protein folding, disulphide bond formation, oligomerisation and proteolytic cleavage . VLPs thin vivo in mouse nasal epithelia, mouse skeletal muscle cells, rabbit endothelial cells, and in vitro in various mammalian cell lines including HeLa, Vero, BHK-21, 293 T-cells, rat hepatocytes and human hemocytes and hepatocytes [in vivo.Gp64-mediated baculovirus uptake is not limited to insect cells. Several studies have demonstrated the ability of recombinant baculoviruses to enter host mammalian cells, and subsequent gene expression to occur within these cells. Their uptake has been demonstrated atocytes -61. Lentatocytes -64. In aatocytes  and SinnWhile endosomal degradation of non-pseudotyped (Gp160) VLPs subsequent to cellular entry should effectively prevent the transmission and expression of VLP-contained RNAs, the presence of baculovirus Gp64 on the surface of Gag VLPs could enable expression of these RNAs within eventual mammalian vaccine recipients. The potential delivery and expression of insect cell-derived nucleic acids by VLPs within a vaccinated individual is regarded as a problematic trait by vaccine regulatory bodies such as the United States Food and Drug Administration (FDA). The FDA specifically requires vaccine developers to show that VLPs do not encapsidate "specific" nucleic acid sequences from the expression system, and especially those encoding VLPs components.In this study we tested the ability of insect cell-produced HIV Gag VLPs to function as a vector for RNA uptake in several mammalian cell lines. Also, as the amount of Gp64 on the surface of insect cell-produced HIV-1 Gag VLPs has not been determined, this study initially estimated the relative levels of Gp64 per VLP. This study is the first, to our knowledge, to analyse the potential transfer and successful expression of encapsidated reporter CAT RNAs from non-replicating Gag VLPs in mammalian cell lines: previous studies have all been conducted on more complex live lentivirus models that carried reporter genes as part of the replicative viral genome ,63. VLPsgag gene encoding the Gag sequence with a myristoylation signal, derived from the Du422 clinical isolate [Spodoptera frugiperda-derived Sf21 cell line (Invitrogen) [Sf21 cells were maintained as a monolayer at 27°C in TC-100 insect medium (Sigma) supplemented with 10% (v/v) foetal bovine serum (FBS), 50 μg/ml neomycin, 69.2 μg/ml penicillin G and 100 μg/ml streptomycin. Sf21 cells (1 × 106 cells/ml) were infected with the recombinant pFBDGagCAT and control pFBDGag baculovirus at a multiplicity of infection (MOI) of 2-10, and VLPs were harvested from 100 ml of culture supernatant 72 h post infection by means of cross-flow filtration using a cross-flow filter unit  with a 300 kDa cut-off filter cartridge according to the manufacturer's protocol. VLPs were pelleted by centrifugation at 83, 000 × g and resuspended in 300 μl of 1 × sterile Dulbecco's phosphate buffered saline  and incubated with 7.5 u RNase A and 1.33 u RNase T1 for 2 h at 37°C [g and resuspended in PBS containing 15% trehalose to maintain VLP stability.A Pr55  isolate ,66 was hitrogen) ,42,67-70 at 37°C  to removGagCAT VLPs were visualized using a LEO 912 transmission electron microscope after adsorption onto 200 mesh carbon-coated copper grids stained with 2% uranyl acetate.The purity of VLPs were assessed and Gag concentration of the GagCAT VLP preparation was measured by electrophoresis of purified VLPs through a 10% denaturing SDS polyacrylamide gel and western blotting . To quanFor use in cell-uptake experiments, GagCAT VLP samples were heated at 62°C for 20 minutes  based on the method used for heat-inactivating live HIV particles .2 with 50 pmol of the primers designed to yield a 350 bp product. The forward and reverse primer sequences used were 5' GCAATGAAAGACGGTGAGC 3' and 5' ATGAACCTGAATCGCCAGC 3', respectively. In vitro-transcribed CAT RNA was used as a positive control . The cycle profile was as follows: 45°C, 45 min; 94°C, 2 min; 25 cycles of 94°C, 30 sec; 57°C, 30 sec; 72°C, 1 min.RT-PCR  of GagCAT VLPs and these VLPs after heat treatment (hVLPs) contained 5 μl of the VLP preparation  in 100 μl and 1 mM MgClin vitro-transcribed CAT RNA as a standard or 10 μl of the RNA extracted from the GagCAT VLP preparation, 50 mM MgCl2 and 50 pmol of forward and reverse primers to amplify a 150 bp DNA fragment. The forward and reverse primer sequences used were 5' AGATGTGGCGTGTTACGGT 3' and 5' ATGAACCTGAATCGCCAGC 3', respectively. The reaction profile used was as follows: 49°C, 45 min; 95°C, 10 min; 30 cycles of 95°C, 15 sec, 57°C, 15 sec and 72°C, 15 sec. All real-time RT-PCR data was analysed using the Rotor-gene 6, Version 6.0 (Build 27) software (Corbett Research).RNA was extracted from GagCAT VLPs after heat treatment using the RTP Virus DNA-RNA Isolation Kit (Invitek). Each RNA extraction was carried out using 5 μl of the VLP preparation  and the RNA was eluted in 50 μl of extraction kit elution buffer after which 10 μl was used for real-time RT-PCR. These reactions were performed using a SensiMix One-Step real-time RT-PCR kit (Quantace) and a Rotor-Gene RG-3000A real-time PCR machine (Corbett Research). Reactions (25 μl) contained 2. RAW 264.7 cells were cultured in RPMI  and BHK-21 cells, HEK 293 and HeLa cells were cultured in DMEM . These media were supplemented with 10% (v/v) foetal calf serum (Gibco), 1% penicillin G/streptomycin and 1% Fungin (supplemented culture medium). Twenty four hours prior to GagCAT VLP uptake the RAW 264.7, BHK-21 and HeLa cell cultures were seeded in 6-well plates (Nunc™) in 2 ml supplemented culture medium at a concentration of 0.5 × 105 cells/ml per well, while HEK 293 cells were seeded in 6-well plates at 0.2 × 105 cells/ml supplemented culture medium per well. Cells were washed twice with 2 ml PBS prior to the addition of GagCAT VLPs in a volume of 1 ml unsupplemented culture medium and incubated at 37°C for 2 h, which was then replaced with 2 ml of supplemented culture medium and incubated for 24 h at 37°C after which cell lysates were prepared and CAT protein content analysed by ELISA (Roche CAT ELISA kit). Each cell-type transfection was carried out with the same VLP and heated VLP preparations and the entire experiment was carried out in triplicate. ELISA plates were analysed using a PowerWave™ XS ELISA plate reader (BioTek®) and results analysed using the KCL™ (version 3.4) program (BioTek®). Mean values of triplicate assays for a cell lysate did not vary by more than 5%. Data is presented as mean responses for 6 replicate uptake experiments.Murine macrophages (RAW 264.7), baby hamster kidney cells (BHK-21), human embryonic kidney cells (HEK 293) and HeLa cells were maintained as continuous cultures at 37°C in 5% CO6 splenocytes ± SD.Female BALB/c mice (8-10 weeks old) were used to compare immune responses of GagCAT VLPs and heated GagCAT VLPs (hGagCAT VLPs) when given alone. The ability of these VLPs to boost a primary response induced by a matched HIV-1 subtype C DNA vaccine, pTHGagC  was alsoSf21 insect cells with pFBDGagCAT yielded VLPs that could be harvested by cross-flow filtration from insect cell culture supernatant. Transmission electron microscopy of the purified VLPs showed numerous doughnut-shaped particles with diameters ranging in size from 120 to 150 nm . To exclude the possibility that DNA fragments could have resulted from amplification of contaminating CAT DNA co-purified from the insect-cell preparation, GagCAT VLPs were subjected to PCR using CAT-specific primers, which failed to amplify any product  (data not shown).The amount of CAT RNA encapsidated by 1 μg of Gag VLPs was quantitated by real-time RT-PCR using in vitro-transcribed CAT RNA. CAT RNA transcription and translation was confirmed by the detection of CAT in cell lysates (data not shown). To investigate whether CAT RNA can be expressed in mammalian cells after uptake of GagCAT VLPs, VLPs containing 10 ng of CAT RNA were added to 4 different mammalian cell types and CAT protein expression levels in cell lysates quantified by CAT ELISA after 24 hours. The cell lines were selected on the basis of availability in the laboratory, as well as the fact that at least 3 of them are known to facilitate entry of Gp64-pseudotyped viral particles by means of a Gp64 receptor that is present on their outer cell wall [To test whether CAT RNA could indeed be transcribed and translated into protein in mammalian cells, we determined the presence of CAT protein in BHK-21 cells after transfection with ell wall ,58,74. Cell wall  did not The cellular uptake and subsequent transcription of GagCAT RNA encapsidated by the VLPs has implications for their use as a vaccine. It is important to limit transmission and expression of RNA encapsidated by VLPs to ensure no production of a protein that may cause ill effects when VLPs are administered as a vaccine. We tested whether heating the VLPs  woul6 splenocytes in the IFN-γ ELISPOT assay while a heat treated GagCAT VLP only inoculation induced a low response of 70 sfu/106 splenocytes to the Gag CD4(13) peptide  was compared to that of two vaccinations of pTHGagC. T cell responses to a Gag CD8 peptide and two Gag CD4 peptides (Gag CD4 (13) and Gag CD4 (17)) were measured using IFN-γ and IL-2 ELISPOT assays as the measure of the immune response Figure . A VLP oe Figure . No respe Figure . Both VLe Figure . A cumuls Figure . The higy Figure 6 splenocytes and 93 sfu/106 splenocytes respectively . We concluded from this that the inhibition of CAT expression was most likely as a result of Gp64 damage on the GagCAT VLP surface. In the HEK 293 cells, CAT protein levels were relatively unaffected when higher levels of encapsidated RNA were used in the cell uptake assay. It is possible this is a result of the HEK-293 cells employing an additional or alternate VLP cell uptake pathway to that used by the BHK-21 and HeLa cell lines. This variation in sensitivity to heat may be cause for concern considering that a mixed population of cell types may be present in the area of inoculation when used as a vaccine in mammalian subjects. UV irradiation of VLPs has also been tested in this laboratory, but this had little effect on abrogating or even decreasing GagCAT transcription and translation (data not shown).The VLP heat inactivation experiments showed that heat treatment at 62°C reduced CAT protein levels in lysates of two of the cell types tested (BHK-21 and HeLa) to almost undetectable levels Figure . Heating+ T-cells and drive them towards a Th-1 response [With regard to particle immunogenicity, a literature review has suggested that pseudotyping Gag VLPs with functional/fusion competent envelope glycoproteins may be of paramount importance in stimulating both MHC-I and MHC-I Gag processing pathways ,82-85. Iresponse . Monkeysresponse ,85,87-95The cellular immune response is partly dependent on the successful entry of viruses into the cytoplasm of the target cell resulting in viral replication rather than viral particle degradation which stimulates the Th1 immune response. This kind of entry would be facilitated by receptor-mediated Gp64 interactions. If heat treatment disrupts Gp64 on the Gag VLPs, successful entry of VLPs into the target cell would not occur, thus preventing a Th1 immune response to be stimulated. However, we showed that VLP heat inactivation did not appear to affect induction of subsequent immune responses Figure . GagCAT In summary, BEVS-produced HIV-1 Gag VLPs were shown to be able to transfer and express foreign expression system-derived RNAs in mammalian cell lines. Transfection of mammalian cells with heat-treated VLPs did not prevent RNA expression in all cell lines, but reduced the range of cell lines that the VLPs could enter and thus the range of cells into which RNA was taken up. These results highlight the need to control the nucleic acid content of assembled Gag particles notwithstanding their need for random RNA encapsidation, crucial for particle assembly. A possible alternative to limit RNA encapsidation and hence transmission may lie with creating chimeric gag particles that do not require RNA for assembly, as has been shown by Crist et al., 2009 . ChimeriThe authors declare that they have no competing interests.ZV-O produced and characterised the VLPs, carried out the PCR and RT-PCR and tested expression of the RNA in cell lines and drafted the manuscript. ES and her group were responsible for carrying out the mouse experiments and performing the ELISPOT assays. AM and EPR were involved in supervision of the work, critically revising the manuscript for important intellectual content, and together with A-LW, conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.


Neuroinflammation is associated with virtually all major neurodegenerative disorders, including Alzheimer’s disease (AD). Although it remains unclear whether neuroinflammation is the driving force behind these disorders, compelling evidence implicates its role in exacerbating disease progression, with a key player being the potent proinflammatory cytokine TNF-α. Elevated TNF-α levels are commonly detected in the clinic and animal models of AD.1–42 challenge, and biochemical and behavioral assessment of 3xTg-AD mice following chronic 3,6′-dithiothaliodmide.The potential benefits of a novel TNF-α-lowering agent, 3,6′-dithiothalidomide, were investigated in cellular and rodent models of neuroinflammation with a specific focus on AD. These included central and systemic inflammation induced by lipopolysaccharide (LPS) and Aβ1–42 peptide, prior systemic 3,6′-dithiothalidomide suppressed Aβ-induced memory dysfunction, microglial activation and neuronal degeneration. Chronic 3,6′-dithiothalidomide administration to an elderly symptomatic cohort of 3xTg-AD mice reduced multiple hallmark features of AD, including phosphorylated tau protein, APP, Aβ peptide and Aβ-plaque number along with deficits in memory function to levels present in younger adult cognitively unimpaired 3xTg-AD mice. Levels of the synaptic proteins, SNAP25 and synaptophysin, were found to be elevated in older symptomatic drug-treated 3xTg-AD mice compared to vehicle-treated ones, indicative of a preservation of synaptic function during drug treatment.3,6′-Dithiothaliodmide lowered TNF-α, nitrite (an indicator of oxidative damage) and secreted amyloid precursor protein (sAPP) levels in LPS-activated macrophage-like cells (RAW 264.7 cells). This translated into reduced central and systemic TNF-α production in acute LPS-challenged rats, and to a reduction of neuroinflammatory markers and restoration of neuronal plasticity following chronic central challenge of LPS. In mice centrally challenged with AβOur data suggest a strong beneficial effect of 3,6′-dithiothalidomide in the setting of neuroinflammation and AD, supporting a role for neuroinflammation and TNF-α in disease progression and their targeting as a means of clinical management. Neuroinflammation is a critical and invariable component of many neurodegenerative diseases. Indeed, the co-occurrence of inflammatory markers in plasma, cerebral spinal fluid (CSF) and post-mortem brain tissue has become a hallmark feature of Alzheimer’s disease (AD) and Parkinson’s disease (PD) -4. This In human post-mortem AD brain, a dramatic loss in neurons has been observed in the medial temporal lobe . Over time, AD patients develop classical patterns of brain pathology, as described by Braak and Braak , and corIt is plausible that the dysfunction of viable neurons during early stages of AD may not represent a permanent state and may thus be amenable to rescue, potentially providing improved outcomes for long-term nerve cell survival and function. Transgenic animal models are commonly used to investigate pathological processes of neurodegenerative diseases; one such model is the 3xTg-AD mouse model . Data frHerein, to further define the role of TNF-α in neuroinflammation, neuronal dysfunction and cognitive impairment, we utilized a TNF-α synthesis-lowering agent, 3,6′-dithiothalidomide, developed within our laboratory . This agEscherichia coli (E coli) serotype 055:B5 (Sigma-Aldrich). Aβ1–42 or Aβ42–1 peptides were from American Peptide, Sunnyvale, CA.3,6′-Dithiothalidomide was prepared in 100% tissue culture grade dimethyl sulfoxide  for cell culture experiments, or as a suspension in a 1% carboxy methyl cellulose/saline solution (Fluka 21901). A concentration of 56 mg/kg body weight of drug was used for animal work unless stated otherwise. Lipopolysaccharide (LPS) was obtained from 2. Cells were propagated as described by ATCC guidelines. RAW 264.7 cells were cultured as has been previously described , plasma was generated from blood by conventional means. After 240 min the CNS was harvested, and all samples were immediately frozen to −70°C and stored for analyses.n = 4); (2) aCSF plus 3,6′-dithiothalidomide ; (3) LPS plus drug vehicle  and (4) LPS plus 3,6′-dithiothalidomide . The LPS or aCSF were infused directly into the brain via an intracerebroventricular (i.c.v.) catheter  into the lateral ventricle as previously described [The rodents used for these experiments where male Fisher 344 rats (3 months of age). Four study groups were utilized: (1) artificial cerebrospinal fluid (aCSF) plus drug vehicle  and aggregated by incubation at 37°C for 7 days prior to administration. Aβ1–42 and Aβ42–1 (400 pmol) were then infused i.c.v. into the lateral ventricle as previously described [Adult male C57BL/6 mice (3 months of age) were utilized in this study. Mice received 3,6′-dithiothalidomide  or vehicle daily for 14 days; after 7 days of treatment with drug animals were challenged with Aβ peptide. Aβescribed .1–42, total tissue APP, tau and phospho-tau protein, and the presynaptic proteins: SNAP25 and synaptophysin.Adult and old male 3xTg-AD mice (10 and 17 months of age) were maintained on a 12-h light/dark cycle with free access to water and food. Animals received administration of either 3,6′-dithiothalidomide (42 mg/kg) or the vehicle i.p., daily for 6 weeks. Four to five weeks after the initiation of drug treatment, the animals were tested in the Morris Water Maze to assess acquisition (learning) and retention of spatial memory. After the completion of the Morris Water Maze assessment, the animals were euthanized by decapitation while under isoflurane anesthesia. The brain was immediately removed, and specific regions were excised and instantly frozen to allow later quantification of levels of various cortical proteins of interest: soluble AβAll animal studies were undertaken according to protocols approved by the respective Institutional Animal Care and Use Committee’s of the Intramural Research Program, National Institute on Aging , and the University of California (AN082537-03A), in compliance with the guidelines for animal experimentation of the National Institutes of Health .Total mRNA was isolated from the hippocampus using the RNeasy RNA isolation kit . Various concentrations of total mRNA extracted from rat brain were prepared for the generation of absolute and relative standard curves. The RNA samples and serial dilutions for standard curves were reverse-transcribed (Applied Biosystems), and PCR reactions were carried out using primers and probe sets purchased from Applied Biosystems : the TNF-α primers and probe set recognize exon 2–3 of TNF-α , and the GAPDH primers and probe set recognize exon 3 . The signals from the amplified PCR products were detected using the ABI Prism 7700 Sequence Detection System (Applied Biosystems), and obtained Ct values were calculated to the relative amount of RNA from the standard curves for each RNA transcript.1–42, levels were measured by use of a human Aβ42 ELISA kit from Invitrogen. For all ELISA measurements, samples were assayed in duplicate, and the appropriate procedures were followed according to the manufacturer’s instructions.TNF-α levels were measured by species-specific ELISAs; Mouse RAW 264.7 cell culture media TNF-α protein were measured with a mouse ELISA, BioLegend, Mouse TNF-α ELISA MAX™ Deluxe. Rat plasma and CNS protein was measured with a rat ELISA, BD OptEIA Rat TNF ELISA Kit II, BD Biosciences or an Ultrasensitive rat TNF-α, Invitrogen, respectively. Soluble human AβThe brains of animals that received i.c.v. administered LPS or Aβ peptides were processed as previously described for the appropriate procedure ,21,29,32Spatial learning and memory were assessed using the Morris Water Maze. The target platform was submerged 1 cm below the water surface and placed at the midpoint of one quadrant. Visual cues were placed around the tank to orient the mice. The acquisition training sessions took place over 4 days  or over 6 days . The memory retention assessments were performed at 24 h (Aβ peptide-injected mice) or at 4 and 24 h (3xTg-AD mice) after the last training session. The variables of interest were mouse reference memory, the time spent in the platform area and the number of platform crossings. These events were recorded and analyzed to determine age- and drug-related differences between the groups.Secreted APP levels were measured by Western blotting of equal volumes of harvested culture media after separation in a 10% Bis-Tris protein gel and probed with an antibody that recognizes all forms of APP . For 3xTg-AD mouse studies protein from each sample was separated by electrophoresis in Criterion gels (BioRad), then the transferred proteins were probed for APP , phospho and non-phospho tau ; SNAP 25 (Millipore), and synaptophysin . Protein signals were obtained by chemiluminescence substrate methods, and signals were normalized to β-actin (GE/Amersham) ,36.p < 0.001), individual between-group comparisons were performed with Bonferroni’s post hoc tests to correct for multiple comparisons. Statistical analyses are provided in each figure legend and, where appropriate, involved one- or two-tailed t-tests for specific comparisons.GraphPad Prism software was used to perform the statistical analysis of the following variables: plasma and CNS TNF-α levels; hippocampus TNF-α mRNA; Morris Water Maze platform escape latency; time spent in quadrants and platform crossings. A one-way ANOVA was performed on each group followed by a Bonferroni’s post test or a Fisher’s post-hoc test, where appropriate. For two-group comparisons, unpaired Student’s t-tests were carried out. One-way ANOVA was performed for variables measured from RAW 264.7 cells, TNF-α, nitrite and sAPP, and were followed by Bonferroni’s post hoc tests, where appropriate. For the immunostaining analysis, the control and experimental groups were the independent variable, and the percentages of neurons expressing Arc or activated microglia from various categories were the dependent variables. When an ANOVA was significant , TNF-α synthesis-lowering agent 3,6′-dithiothalidomide  was utilThe potential benefits of lowering TNF-α levels on neuroinflammation-induced altered neuroplasticity were assessed. To recreate the hostile brain microenvironment associated with chronic neuroinflammation, we utilized the properties associated with direct chronic CNS infusion of a small quantity of LPS in rats. This infusion, with and without systemic 3,6′-dithiothalidomide treatment, was well tolerated. When animals were allowed to explore an open field arena for 10 min, no differences were observed in either speed or exploration pattern Figure , indicat1–42 was undertaken 7 days after the initiation of a daily schedule of systemic (i.p.) 3,6′-dithiothaliomide, utilizing a dose determined to be effective to ameliorate LPS-induced CNS elevations in TNF-α in the prior studies. Control animals were administered reverse peptide . As illustrated in Figures 1–42 alone induced a marked deficit in the learning ability of mice in the Morris Water Maze paradigm (for both acquisition and retention). This was accompanied by neuronal degeneration (assessed by Fluoro-Jade B staining) and an increased presence of CD11b-positive staining microglial cells within the dentate gyrus (DG) of the same animals  mice, was undertaken to emulate the inflammatory microenvironment of the AD brain. In our study, a single i.c.v. administration of Aβs Figure . TreatmeSwe + PS1M146V + tauP301L) associated with AD [Mice containing three transgenes  was evident when comparing adult vehicle with old vehicle animals Figure . 3,6′-Dit Figure , with olt Figure . Total tt Figure . Howeverp Figure . Interesp Figure .Immunohistochemical staining of insoluble Aβ plaques indicated marked plaque formation only in the old 3xTg-AD mice; this Aβ plaque staining was dramatically reduced by drug treatment Figure . Memory The levels of CD68-positive microglial cells within the subiculum and CA1 brain region were quantified as a marker of the inflammatory microenvironment in the hippocampus, as these regions are among those showing the highest concentration of Aβ plaque staining Figure . CD68-poHere we demonstrate that the TNF-α-lowering agent, 3,6′-dithiothalidomide, ameliorates key aspects of neuroinflammation in multiple acute and longer term CNS rodent models. Importantly, several of these models emulate specific cardinal characteristics of AD, and highlight the complex cyclic interaction among the synthesis of TNF-α, the development of neuroinflammation and impact on disease progression, inducing its advancement. Our data suggest that breaking this cycle by lowering TNF-α generation and neuroinflammation can favorably impact AD, as assessed at both a behavioral and biochemical level, even late during the disease course. Our studies hence reinforce the significant role of neuroinflammation in AD and other degenerative neurological disorders, and highlight the potential for targeting TNF-α.TNF-α has been implicated in the pathogenesis of a wide number of neurological disorders, including AD, PD, stroke and head trauma -12. IndeProtein-based TNF-α inhibitors (etanercept and infliximab) that can effectively regulate circulating TNF-α levels by binding them  have proExogenous signals, as arise from exposure to bacterial proteins, potently induce inflammatory responses within the CNS in a manner mimicked by RAW 264.7 cells challenged with LPS . The resAdministered to brain, LPS reliably induces chronic neuroinflammation associated with the activation of microglia, which is allied to impaired hippocampal-dependent spatial cognitive function ,18,21. IAβ, particularly in the form of soluble oligomeric assemblies or Aβ-derived diffusible ligands (ADDLs) -69, has 1–42 and phosphorylated tau levels, a decline in total tau and a trend towards elevation of APP levels  are driven in the 3xTg-AD model by the unnatural mouse Thy1.2 regulatory element [A caveat with this 3xTg-AD mouse model, like all such models, is that it provides a partial model of the human disease. APP and tau expressions  and TNFR2 (p75) . TNFR1, 5 and TNFUnder appropriate conditions TNF-α signaling, primarily via TNFR2, can mediate homeostatic actions, epitomized by its role in AMPA receptor surface expression and synaptic scaling to impact LTP , as wellIn synopsis, the present data demonstrate the key role of TNF-α in acute and chronic neuroinflammatory models that have relevance to neurodegeneration and, in particular, to AD and its progression. The TNF-α synthesis inhibitor 3,6′-dithiothalidomide, administered at doses that compare favorably to those of thalidomide in human studies , appearAREs, adenylate-uridylate-rich elements; AD, Alzheimer’s disease; ADDLs, amyloid-β-derived diffusible ligands; Aβ, amyloid-β peptide; aCSF, artificial cerebrospinal fluid; CSF, cerebral spinal fluid; CNS, central nervous system; DG, dentate gyrus; DMSO, dimethyl sulfoxide; GFAP, glial fibrillary acidic protein; IEG, immediate early gene; IL, interleukin; IP, interperitoneal; ICV, intracerebroventricularly; JNK, c-Jun N-terminal kinase; LPS, lipopolysaccharide; LTP, long-term potentiation; MCI, mild cognitive impairment; MW, molecular weight; NFκB, nuclear factor kappa B; PD, Parkinson’s disease; RBPs, RNA-binding proteins; sAPP, secreted amyloid precursor protein; TNF-α, tumor necrosis factor-alpha; TNFR1, TNF-α receptor 1; TNFR2, TNF-α receptor 2; TLR, Toll-like receptors; WT, wild type.Debomoy K Lahiri Francesca Bosetti, Nigel H Greig and Susanna Rosi equal contributorsNHG and HWH declare that they are co-inventors of the original 3,6′-dithiothalidomide patent. Having assigned all their rights to the US government, they declare that they have no ownership, financial interest or any other competing interests. All other authors declare no competing interests.DT contributed to the design of the study, and undertook experimental studies and biochemical analyses in Figures


Tumor necrosis factor alpha (TNF-α) is a pro-inflammatory cytokine produced by macrophages and T-cells. It plays an important role both in inflammation and apoptosis. In the eye, TNF-α appears to have a role in the pathogenesis of inflammatory, edematous, neovascular and neurodegenerative disorders. Several TNF-blocking drugs have been developed and approved, and are in clinical use for inflammatory diseases such as rheumatoid arthritis, psoriasis and ankylosing spondylitis. TNF-α blockers are widely used in ophthalmology as an off-label alternative to “traditional” immunosuppressive and immune-modulatory treatments in noninfectious uveitis. Preliminary studies suggest a positive effect of intravenously administered TNF-α blockers, mainly infliximab, for treating refractory diabetic macular edema and neovascular age-related macular degeneration. Unfortunately, much of the current data raises considerable safety concerns for intravitreal use of TNF-α inhibitors, in particular, intraocular inflammatory responses have been reported after intravitreal injection of infliximab. Results of dose-finding studies and humanized antibody or antibody fragments  are anticipated in the coming years; these will shed light on potential benefits and risks of local and systemic TNF-α blockers used for treatment of diseases of the retina and choroid. The tumor necrosis factor alpha (TNF-α) belongs to the group of pro-inflammatory cytokines produced by macrophages and T-cells and plays an important role in inflammation and apoptosis.in vivo animal model of retinal injury, Berger et al showed that TNF-α plays a deleterious role in ischemia-reperfusion injury. Direct neutralization of this cytokine partially preserved retinal function.10In the eye, TNF-α appears to participate in the pathogenesis of inflammatory, edematous, neovascular, and neurodegenerative diseases.In the 1990’s, pharmaceutical companies succeeded in developing drugs that neutralize TNF-α, i.e. TNF-α blockers. These agents were expected to have a positive effect on reducing symptoms of various diseases associated with increased TNF-α activity.Currently available TNF-α blockers on the market include the following:®), a chimeric human immunoglobulin G1 (IgG1) with a mouse variable fragment (Fv) having high TNF-α affinity and neutralizing capacity.Infliximab (Remicade®), a fully humanized antibody against TNF-α.Adalimumab (Humira®), a recombinant dimeric protein generated by fusion of the ligand-binding portion of human type 2 TNF receptor (TNFR-2) linked to the fragment crystallizable (Fc) portion of human IgG1.Etanercept (Enbrel®), a PEGylated humanized anti-TNF fragment antigen binding (Fab).Certolizumab pegol (Cimzia®), a human anti-TNF-α IgG1κ monoclonal antibody.Golimumab  with chronic, refractory, noninfectious uveitis receiving systemic anti-TNF-α therapy as single or adjunctive therapy.In a comparative study of patients with retinal vasculitis due to Behçet’s disease, the infliximab treatment group (n=10) showed a significant decrease in inflammation, improvement in visual acuity and reduced ocular complications compared to the conventional therapy group (n=33).Diaz-Llopis et al presented outcomes of therapy in 19 patients with refractory uveitis including 33 eyes with cystoid macular edema (CME).®) is by intravenous infusion and is usually started with a loading dose at initiation of treatment and two weeks thereafter. Maintenance treatment is administered at 4 to 6 week intervals. Inflammatory activity usually diminishes within 12 weeks after initiating treatment, otherwise such treatment should be reconsidered.Systemic administration of infliximab  is administered by subcutaneous injections with single 40 mg doses at biweekly intervals. A loading dose may be given up to 160 mg (4 injections on the first day of treatment), when inflammatory disease activity must be quickly reduced. Should the response be insufficient, treatment may be increased to weekly injections.Etanercept (Enbrel®) is usually injected subcutaneously at a 25 mg dose twice a week or as a single subcutaneous injection 50 mg per week.Certolizumab pegol (Cimzia®) is available in preloaded syringes containing 200 mg for subcutaneous injection. A loading dose of 400 mg (i.e. two injections on the same day) is recommended at weeks 0, 2 and 4. Injections of 200 mg are administered biweekly thereafter.Golimumab (Simponi®) is also available in preloaded syringes for subcutaneous injection. These syringes contain 50 mg of the agent and injections are given once monthly. In patients weighing over 100 kg and unresponsive to treatment after 3 to 4 months, the dosage may be increased to 100 mg (two syringes on a single day) once a month.Since the immune system may produce antibodies against any administered TNF-α inhibitor, its therapeutic efficacy may diminish over time. When that happens, switching to another TNF-α blocker may be considered, however, the efficacy and side-effects of the second drug must be monitored meticulously.The most severe side effects of TNF blockers are due to increased susceptibility to infectious diseases, most notably tuberculosis and other respiratory tract infections and gastrointestinal infections, e.g. hepatitis B reactivation.Immune-mediated side effects may also occur such as atopic dermatitis, rash, hypersensitivity types I, II, III, lupus-like reactions and exacerbation of autoimmune diseases (e.g. multiple sclerosis). Other reported side effects include thromboembolic disorders, congestive heart failure, and malignancies, especially lymphomas and skin tumors.In 2005, Markomichelakis et al reported three cases of CNV regression secondary to age-related macular degeneration in patients under systemic administration of infliximab for inflammatory arthritis.These observations, together with the hypothesis that TNF-α may play a decisive pathogenetic role in the development of neovascularization in the eye, are leading to the development of animal models and human studies on intravitreal application of anti-TNF drugs in vascular and neovascular diseases of the retina and choroid.Systemic TNF-α inhibitors are established drugs for therapy of rheumatic and auto-immune diseases, and positive effects have been observed in concurrent retinal and inflammatory eye diseases. The particular nature of ocular diseases, i.e. being restricted to ocular tissues, allows use of the vitreous cavity as a drug reservoir. Thus attempts to inject TNF-α inhibitors intravitreally (as routinely performed with anti-VEGF agents) is the next step. Anticipated advantages include bypassing the notorious side effects of systemic TNF-α inhibitors and achieving higher local concentrations.Two current studies investigated the human vitreous for inflammatory mediators. Yoshimura et al collected vitreous specimens from 345 eyes with several retinal diseases such as diabetic retinopathy (DR), central retinal vein occlusion (CRVO), rhegmatogenous retinal detachment, and diabetic macular edema; they did not observe an elevated concentration of TNF-α in any of these disorders as compared to the control group (patients with idiopathic macular hole or macular pucker)In light of these results, it is not surprising that results of intravitreally administered TNF-α inhibitors are so heterogeneous. Wu and coworkers performed an interventional, multicenter retrospective study on diabetic macular edema.Giganti et al treated two patients with neovascular AMD and two patients with diabetic macular edema with low-dose intravitreal infliximab (0.5 mg/0.05 ml).There is only one report by Theodossiadis and coworkers describing three patients demonstrating improved VA after intravitreal infliximab for neovascular AMD. Their first patient had already received three injections of ranibizumab before undergoing an injection of 1 mg infliximab; VA improved from 20/200 to 20/100.Current knowledge suggests that intravitreal TNF-α inhibitors have no permanent effect on macular edema in neovascular AMD or diabetic maculopathy. The three aforementioned patients showing visual improvement had short follow-up of only two months and markedly less pre-treatment than other patients and subjects studied by Wu et al. TNF-α inhibitors have been mainly used in chronic, long-lasting refractory disease where visual improvement is less likely. Dosage seems to be critical. A “safe” dosage of infliximab might be 1 mg/0.05 ml, as no inflammatory reactions were reported using this concentration, while lower or higher concentrations lead to frequent severe inflammatory reactions. Adalimumab 2 mg was well tolerated. Despite local application, systemic antibodies against non-human parts of the inhibitors have been detected, compromising treatment success. Thus, fully-humanized antibodies may solve this crucial problem.Current data do not support the routine use of intravitreal TNF-α inhibitors in any retinal disease outside controlled trials. Further investigations, particularly dose-finding studies, will shed light on the potential therapeutic benefits of intravitreal or systemic anti-TNF therapy for vascular and neovascular diseases of the choroid and retina. We await the results of intravitreally administered humanized antibodies such as adalimumab in near future.Although the systemic use of TNF-α inhibitors is an appropriate off-label alternative to “traditional” immunosuppressive and immune-modulatory treatments in noninfectious uveitis, its use in vascular and neovascular retinal diseases has not yet been thoroughly examined. While preliminary results for intravitreal treatment of neovascular AMD were promising, subsequent case reports have raised considerable safety concerns, particularly the occurrence of intraocular inflammatory responses after intravitreal injection of infliximab. Dose-finding studies and the use of humanized antibodies are probably crucial for the success of intravitreal application. Further investigations will determine the role of TNF-α blockers in treating diseases of the choroid, retina and macula.Literature selection for this review was based on a MEDLINE database search in June 2010 and again in June 2011 using the terms tumor necrosis factor, TNF, retinal disease, macular disease, intravitreal injection, uveitis. All publication years were searched, and relevant articles related to intravitreal injections of anti-TNF substances were included. With regards to systemic use of anti-TNF drugs selected articles were included for this manuscript. Articles in foreign languages with abstracts in English were included as well.


The RV144 vaccine trial of canarypox vCP1521 (ALVAC-HIV) prime and bivalent HIV-1 envelop gp120 protein subtype B/CRF01_AE boost (AIDSVAX B/E) demonstrated a significant effect in preventing HIV-1 infection. A case-control analysis suggested that variable loops 1 and 2 (V2) of gp120 may have contributed to protection against HIV-1 acquisition. Two other vaccine trials using gp120 only– VAX003 (AIDSVAX B/E) and VAX004 (AIDSVAX B/B) failed to show protection.Binding antibody responses induced by the RV144, VAX003 and VAX004 vaccine regimens were compared using ELISA. Recombinant gp120 envelope proteins MN (subtype B), 92TH023 (CRF01_AE), A244 (CRF01_AE) and cyclic V2 peptides were used as capture antigens.After two protein injections, VAX004 had the highest geometric mean titers (GMT) against MN , VAX003 against A244  and RV144 against 92TH023 . Antibody responses against V2 (CRF01_AE) were detected in plasma samples from RV144 and VAX003 with GMTs of 972 and 1100, respectively. However, VAX004 failed to generate antibodies against CRF01_AE V2. None of the three vaccines generated antibodies against MN V2 after two protein immunizations.Compared to VAX004, VAX003 had higher antibody responses against all three recombinant proteins: 2-fold (MN), 4-fold (A244) and 4-fold (92TH023) when two additional protein injections were administered. Two additional protein inoculations in the VAX trials failed to increase antibody titers against, CRF01_AE V2, but generated a small response against MN V2  in VAX003.Antibody responses against V2 were induced by CRF01_A E recombinant proteins as there were no responses induced by the AIDSVAX B/B vaccine regimen. Repeated protein immunization increased the magnitude of responses against recombinant proteins in VAX003 but failed to increase titers against CRF01_AE V2. If antibodies against V2 are protective against HIV-1 acquisition, designing antigens with greater V2 antigenicity would be critical.