Sports is a multi-billion dollar business — and growing. The Wharton School of the University of Pennsylvania and the Wharton Sports Business Initiative (WSBI) present the Wharton Sports Business Academy (WSBA), imparting key insights to rising high school juniors and seniors interested in sports business leadership careers.
Students have a rare chance to engage with industry leaders and discuss the essentials of a successful sports organization. Guest speakers include world-renowned Wharton faculty as well as sports agents, broadcast network executives, brand marketers and professional team executives.
The Wharton Sports Business Academy encourages leadership development through collaborative teamwork. Teams tackle case studies and create business plans for a new sports-focused business — all under the leadership of Penn’s finest.
“I enjoyed the experience along with the academic challenge, the new connections I made and the welcoming environment that JKCP has created! This has been the best 4 weeks of my life!”
“I wanted to thank you, for all the summers of guidance and friendship, along with the tennis, golf and now Wharton Business Sports program, that you have provided for my children.”
“This has been the best four weeks of my life without a shadow of a doubt. When I arrived I was scared but everyone was so cool and friendly. I will never forget this life changing experience.”
Mornings will be spent attending classes at the Wharton School. You will be learning from professors and other leading sports business academics who are known for their ability to condense difficult concepts into easily understandable ones with enthusiasm, making the material come to life.
Afternoons will be spent learning from and meeting with some of the most respected leaders in the sports industry in both the classroom and at sports facilities.
Hear from sports agents, broadcast network executives, league and team executives and brand marketers.
Be introduced to core business disciplines such as management, law, negotiation, marketing and leadership as they intersect with the sports industry.
Develop and enhance leadership skills through collaborative teamwork in a small team of talented and diverse students.
The Wharton School is the business school at the University of Pennsylvania, a private Ivy League university located in Philadelphia. Wharton was established in 1881 and was the first business school in the United States. Wharton is widely regarded as one of the world’s top institutions for business education and, currently, U.S. News & World Report ranks Wharton’s undergraduate program #1.
Essay (required, Topic: “In 300 – 450 words, tell us why you are interested in learning about the business of sports as a possible area for study and professional career path.”)
Essay (required, Topic: “Because this is a leadership based program, please provide an example(s) of where you have exhibited leadership outside of the classroom. Do not exceed 450 words.”)
Hosted by the Wharton School of the University of Pennsylvania and the Wharton Sports Business Initiative (WSBI), the Moneyball Academy welcomes talented high school juniors and seniors intrigued.
Valet Mode can be used to prevent damage to the audio system from unwanted or unauthorized use when your vehicle is being serviced, valet parked, etc. Due to the covert nature of this function, we recommend concealing the location and/or method of its activation. This can be accomplished with a hidden toggle switch (not supplied) or other disguised mechanism to apply negative ground to this connection.
I often get questions about where to find past tests for the new ‘N’ tests. You see before 2010, the JEES published old tests a couple of months after they were administered. This was a great advantage because you could just take an old test to see if you could pass and then go and take the real test.
It was so useful actually, that too many people went about studying this way and became really good at taking the test. This was a problem because well, the test is suppose to measure your level of Japanese not your ability to take a test. So, they revised the test to be more practical, and stopped publishing previous exams.
Initially, to help people study and get a feel for the test, they made a JLPT N4 Practice Test available for free. This was pretty handy because it gave you at least two examples of every question, but it wasn’t a full test. You couldn’t really get your timing down or anything.
They just recently released what they are calling Official Workbooks though. I’ve already released a version of the JLPT N5 Official Workbook that has some notes I made to help you get a better feel for the exam, but I thought I would release the N4 Official Workbook, so that those of you taking it this December could get a chance to practice with it before the big day.
The best part of these workbooks is that they are made up of previous test questions so should be pretty dead on for the level of the exam.
If you don’t have the time to do a full test, remember, I still have the much shorter JLPT N4 Practice Test (that is also free) you can take instead.
Or, if you are taking the N5, pick up the official workbook from the previous blog post. Or if you are taking N3, N2, or N1, pick up the official workbooks at the main JEES site.
Again, I separated out the 4 large audio files into separate CD tracks for each question, so that it is a lot easier to review. I even included the muzak break!
Also, if you prefer a hard copy, so that you can doodle on it and take notes, you can pick up the printed copy of the Official JLPT N4 Workbook here.
Go forth and take this practice test. Download it and give it a whirl, you really don’t have anything to lose. Let me know how you did in the comments.
This practice test is totally “間に合う” for my study sessions in the next week. Thank you so much, Mac!
Thanks Mac, this will be really helpful, I’m not going to do N4 until July but this gives me a good headstart on studying! Keep up the good work, really appreciate it.
Hello Mac, just finished the N4 test downloaded from here and realized that my grammar is quite weak (a bit more than 55%) while vocabulary and listening was almost 100% (thanks to your Memrise N4 Vocab course, I successfully completed). So there are 2 weeks until test, I would like to improve my grammar. Could you recommend some way how to make it more effectively and better focused for N4 level? Thank you for all your efforts and help!
Well, that is a tough question to answer. With just two weeks to go, it is time to go back over and really review what you are weak at whether that’s particles or conjugations, which are really difficult to master at the N5 and N4 levels. Anyway, 55% is not REALLY bad. You can still pass with that, especially if your vocabulary and listening is close to 100%.
Hi Mac, Thank you so much for the study materials and I have passed N5 exam. And preparing for N4 exam for Dec…
wow. thanks. They are selling this on Amazon.co.jp for 1500 yen. I almost bought it today. glad I didnt.
Yeah, they are a bit hard to find on the official site. So I thought I would make them a little more prominent. Hope they help!
Hey Mac, thanks for everything! Taking the next N4, I have two questions for you, if you don’t mind:
1. Is the structure of the 3 subtests always the same? (For example in the first test: First choosing a reading, than choosing kanji, etc)
2. I read on wikipedia that the questions are unequally scored, but that the weighting is not published. Do you have an impression which もんだい are more important? (Maybe it’s roughly weighted by the time you gave in the workbook?)
1. You mean the different sections of the test? Yes, they are always the same, same number of questions per part and section. Each level has a different number of questions for each section though, and some levels have different kinds of questions that aren’t on other levels.
2. I have a section on this in the study guide kit that estimates the value of each type of question for on each of the levels. In summary though, the more difficult the question is to answer the more points it is going to be worth. I know that sounds a little obvious, but that is the basic idea without going into specific numbers.
Thanks so much for the free dls!!! I was already stressing out on where to get the cash to buy those workbooks and BOOM! you have the VERY WELL-HIDDEN links and (even better!) workbooks with notes here~ I thank you from the bottom of my heart! I plan to take at least N3 this December, I will definitely come back here and post my results~
Thanks u very much sir. I don’t know where can i search for my studay that JLPT N-4.I don’t attend japanese class to answer JLPT test. So i need many facts n book relative that JLPT test. Now i can download related book n material also listening.
thanks mac it really helped me with preparing for the N4 test , unfortunately doesn’t work for apple mac book , can you find any solution for it :3 ?
by ‘not working’ what do you mean? It won’t open on your computer? It is a regular pdf (except the package that includes everything), so you should be able to open it as long as you have Adobe Acrobat installed.
Hi, Thank you for the information. I have the book but I don’t know how to check if my answers are correct. does the book have the answers?
Hey Mac, I’m from the Philippines, and this coming December 5, 2016 is the schedule of JLPT exam here n the Philippines, I have 1 question for you. Mac, is there a possibility that the questioner is the same? Because this is my day 1 of my review for N4 test. Thank you and God bless!!!
The questions are different each time the test is given. They only reuse the questions for practice tests and workbooks.
Is there a way to convert the JLPT raw scores into points like what is doable with SAT official practice tests?
Not really since the test is graded on a curve now. Basically if you score below 60% in an area, you need to study harder for that section, because you are at risk of failing it. If you score above 80%, you are generally okay, and should consider that a pass. Between 60 and 80% means are kind of okay, but should study more.
Novel Aβ peptide immunogens modulate plaque pathology and inflammation in a murine model of Alzheimer's disease
Alzheimer's disease, a common dementia of the elder, is characterized by accumulation of protein amyloid deposits in the brain. Immunization to prevent this accumulation has been proposed as a therapeutic possibility, although adverse inflammatory reactions in human trials indicate the need for novel vaccination strategies.
Here vaccination with novel amyloid peptide immunogens was assessed in a transgenic mouse model displaying age-related accumulation of fibrillar plaques.
Immunization with any conformation of the amyloid peptide initiated at 12 months of age (at which time fibrillar amyloid has just begun to accumulate) showed significant decrease in total and fibrillar amyloid deposits and in glial reactivity relative to control transgenic animals. In contrast, there was no significant decrease in amyloid deposition or glial activation in mice in which vaccination was initiated at 16 months of age, despite the presence of similar levels anti-Aβ antibodies in young and old animals vaccinated with a given immunogen. Interestingly, immunization with an oligomeric conformation of Aβ was equally as effective as other amyloid peptides at reducing plaque accumulation. However, the antibodies generated by immunization with the oligomeric conformation of Aβ have more limited epitope reactivity than those generated by fAβ, and the microglial response was significantly less robust.
These results suggest that a more specific immunogen such as oligomeric Aβ can be designed that achieves the goal of depleting amyloid while reducing potential detrimental inflammatory reactions. In addition, the data show that active immunization of older Tg2576 mice with any amyloid conformation is not as efficient at reducing amyloid accumulation and related pathology as immunization of younger mice, and that serum anti-amyloid antibody levels are not quantitatively related to reduced amyloid-associated pathology.
Alzheimer's disease (AD) is an age-related common dementia or loss of cognitive abilities. Neuronal loss, neurofibrillar tangles and senile plaques, abnormal protein deposits which include cleavage products of the amyloid precursor protein (amyloid β peptides (Aβ)) are pathologic characteristics of the disease. While the mechanism of this neurodegeneration remains to be defined, substantial evidence implicating a significant role for the Aβ peptide (40–42 amino acids) has been reported (reviewed in [1, 2]). As a result, one general therapeutic approach being investigated is the reduction of amyloid peptide accumulation in the brain. Several reports have shown that when mice containing the transgene for human mutant amyloid precursor protein (APP) were immunized with fibrillar Aβ peptide prior to the accumulation of amyloid deposits, Aβ deposition observed at later ages was greatly decreased [3–6]. However, when applied to humans, "immunization" with Aβ resulted in the development of an adverse inflammatory reaction in a fraction of the patients [7–9], which led to a reevaluation of this strategy for AD in humans, particularly at that stage of the disease when substantial fibrillar amyloid deposits have begun to accumulate [10]. It is this stage of the disease that often correlates with appearance of cognitive deficiencies that is a defined point at which potential therapy may be initiated.
Several studies in mouse models have shown that passive immunization, in these cases intracranial or peripheral injection of anti-Aβ antibodies, resulted in relatively rapid clearance of significant amounts of Aβ immunoreactivity, both extracellular deposits as well as intraneuronal Aβ accumulation [11–15]. Furthermore, decreases in amyloid accumulation by either passive or active immunization are accompanied by improvement of cognitive function in these murine models [16, 17] and previous work reviewed in [18]). However, not all anti-amyloid antibodies provide the same degree of protection [19], and there have been at least two reports in which animals with established robust plaque load did not respond to a particular immunogen [3, 20]. Thus, as with other immunological responses, the nature of the immunogen, the adjuvant used for immunization, the age and the genetics of the animals immunized all contribute to defining the immune response that subsequently develops and these differences lead to various degrees of clearance and protection from injury.
Recent reports have defined an oligomeric conformation of the Aβ structure that alters LTP activity [21, 22] and induces neurotoxicity in vitro that can be reversed by addition of anti-oligomeric antibody [23, 24]. Since Aβ oligomers are proposed to be an intermediary conformation prior to fibril formation and it has been proposed that antibodies preventing or reversing amyloid assemblies may be therapeutic [25–27], we tested immunization with a novel immunogen presenting the oligomeric conformation of Aβ [23]. In addition, the Aβ oligomers may be more transient (and present at lower concentrations at any given time) than other conformations, and thus immunization with the oligomeric form of the amyloid peptide may provide benefit with minimal induction of inflammatory cascade. The data obtained demonstrate that immunization with an oligomeric conformation of the peptide is as efficient as immunization with either fibrillar amyloid or a multiple antigen peptide amyloid immunogen in terms of clearing amyloid, and that microglial reactivity is significantly less with oligomers as the immunogen than other amyloid conformations.
The experiments described here were also designed to assess the effect of immunization of animals at an advanced age/stage of pathology on the mitigation of amyloid associated neuropathology in a mouse overexpressing human mutant amyloid precursor protein, and to determine whether differences in complement deposition could be detected on plaques resistant to clearance. Our results, in addition to identifying a novel candidate immunogen, demonstrate that while the level of measured serum antibodies are similar or only slightly different in animals immunized with a given immunogen at different ages, a decrease in the accumulation of both fibrillar and diffuse amyloid plaques occurs only when mice are immunized at early stages of the disease (12–16 months of age). The level of C3 activation fragments associated with plaques was also reduced in animals immunized with any amyloid immunogen, correlating with reduced fibrillar plaque burden. Finally, a new, automated, computer assisted method of quantification of immunoreactivity is described and shown to correlate well with conventional image analysis.
Lyophilized Aβ1–42 peptides were resuspended in 50% acetonitrile in water and re-lyophilized. Soluble oligomers were prepared by dissolving 1.0 mg of peptide in 400 μL hexafluoroisopropanol (HFIP) for 10–20 min at room temperature. 100 μl of the resulting seedless solution was added to 900 μl MilliQ H2O in a siliconized Eppendorf tube. After 10–20 min incubation at room temperature, the samples were centrifuged for 15 min. at 14,000 × G and the supernatant fraction (pH 2.8–3.5) was transferred to a new siliconized tube and subjected to a gentle stream of N2 for 5–10 min to evaporate the HFIP. The samples were then stirred at 500 RPM using a Teflon coated micro stir bar for 24–48 hr at 22°C. Oligomers were validated by atomic force microscopy (AFM), electron microscopy (EM) and size exclusion chromatography (SEC) as described [23]. Fibrils are formed by stirring the same solution for 7 days. Fibrils were sedimented and washed in PBS, and resuspended at 2 mg/ml. Fibrillar β amyloid (fAβ) peptides were stored at -70°C until immunization. For the oligomer antigen (oligo), Aβ oligomer molecular mimic was prepared by conjugating Aβ40 via a carboxyl terminal thioester to 5 nm colloidal gold as previously described [23], and stored at 4°C until used. A multiple antigen peptide (MAP) which contains a core matrix of 4 branching lysines contiguous with the amyloid beta 1–33 peptide (ie.MAPAβ1–33) containing both the native B and T cell epitopes of Aβ was synthesized (Invitrogen Inc., Carlsbad, CA) to increase the response to Aβ. Peptides were resuspended in sterile PBS at 2 mg/ml, vortexed and stored at -70°C.
Tg (HuAPP695.K670N-M671L)2576 mice from K. Hsiao [28] and non-transgenic littermates or B6/SJL wild type mice were used as controls. fAβ or oligomer mimic were emulsified 1:1 (v/v) with complete Freund's adjuvant (CFA) for the first immunization, while MAPAβ1–33 peptides were emulsified 1:1 (v/v) with complete Freund's adjuvant containing 4 mg/ml Mycobacterium tuberculosis (Difco, Voight Global, Kansas City, Mo) [29]. Subsequent immunizations with each immunogen in incomplete Freund's adjuvant (IFA) were performed after 2 weeks, and monthly thereafter for 3 additional injections. Two weeks after the final immunization, animals were bled and perfused as described below. In all immunizations 100 ug peptide was injected subcutaneously per mouse. In addition, at the time of initial immunization with MAPAβ1–33 500 ng of pertussis toxin (PTX) (Sigma, St. Louis, MO) in 200 ul PBS was injected IP, followed by a second injection 24 hours later [29]. Immunization controls for both wild type and transgenic mice included injections of adjuvant with PBS only (no peptide antigen). All experimental procedures were carried out under protocols approved by the University of California Irvine Institutional Animal Care and Use Committee.
Mice were deeply anesthetized with an overdose of pentobarbital (150 mg/kg, IP), blood collected by cardiac puncture, and then animals perfused transcardially with cold phosphate-buffered saline (PBS). After dissection, brain tissue was fixed overnight with 4% paraformaldehyde in PBS, pH 7.4 at 4°C. Thereafter, fixed tissue was stored in PBS/0.02% sodium azide (NaN3) at 4°C until use. Fixed brain tissue was sectioned (40 um) with a vibratome, and coronal sections were collected in PBS (containing 0.02% sodium azide), and stored at 4°C prior to staining.
Immunohistochemistry (IHC) was performed on free-floating brain sections. To stain for Aβ plaques, sections were immersed in 50% formic acid for 5 min. Endogenous peroxidase in tissue was blocked by treating with 3% H2O2 in PBS, 10 min at room temperature. Nonspecific background staining was blocked by1 hour incubation in 2% BSA with 0.3% Triton X-100 (TX) at room temperature. Sections were then incubated with primary antibodies (Table 1) overnight at 4°C, rinsed 3 times with PBS with 0.1% TX and incubated with biotinylated secondary antibody followed by ABC kit reagent (Vector, Burlingame, CA) for 1 hour each at room temperature. Finally, after washing three times, the sections were incubated for approximately 2~5 min with diamino-benzidine (DAB), (Vector). Sections were mounted on slides, dehydrated in a series of graded ethanol, cleared with xylene, and then coverslipped with DePeX (Biomedical Specialities, CA). For fluorescent staining, biotinylated secondary was detected by incubation with Streptavidin-CY3 (Vector) for 1 h at RT. Fibrillar Aβ was visualized by incubating the sections in 1% thioflavine for 30 minutes followed by a 1 minute wash in 50% ethanol, 5 min in deionized distilled water, and 5 min in PBS. Sections incubated in parallel without primary antibody or IgG control did not develop staining.
Immunostaining was observed under a Zeiss Axiovert-200 inverted microscope (Carl Zeiss, Thornwood NY) and images acquired with a Zeiss Axiocam high-resolution digital color camera (1300 × 1030 pixel) using Axiovision 3.1 software. Digital images were analyzed using KS300 analysis program (Zeiss). Percentage of immunostained area (area of immunostaining/total image area × 100) was determined for all the markers studied by averaging % Field Area of several images per section that cover most, or all, of the region of study. Assays were repeated at least twice, with n = 4–7 animals per group per age per marker as noted in legends and text. Quantitative comparisons were performed on sections processed at the same time. Single ANOVA statistical analysis was used to assess the significance of the differences in plaque area, glial and C3 activation products reactivity among the animals groups.
A second method of quantification developed for the ACIS image analysis system (Clarient, Inc., San Juan Capistrano, CA) was utilized to analyze the 6E10 immunoreactivity. Images were acquired automatically. Cortical and hippocampal regions appropriate for analysis were selected and automatically scored using an algorithm that identifies objects based on user-configurable parameters. Object identification was paired with a watershed segmentation algorithm to facilitate separation of touching and overlapping deposits. In this manner large deposits that form contiguous bands of Aβ were separated into individual objects. Because Aβ deposits in these animals can vary markedly in size and shape, the identification of Aβ-positive objects utilized a broad size filter (12–3,000 microns effective diameter) and did not employ rigorous morphometric filters. Data collected for each Region of Interest (ROI) were the area of tissue scored (area of the ROI), the number of Aβ-positive objects identified and the total area of the Aβ-positive objects. These parameters allowed calculation of two different measures of amyloid load: the Aβ-Positive Object Density, which is simply the number of objects per mm2 of tissue scored and is used as an approximation of the number of plaque-like structures per mm2, and secondly, the ratio (as a percent) of the total area of the Aβ-positive objects to the area of tissue scored. It should be noted that, since the numerator of this second ratio contains only the area enclosed within the boundaries of the identified objects and does not incorporate small particles of Aβ-immunoreactivity that are excluded by the size filter (i.e. <12 microns effective diameter), this measure is distinct from the area ratio described in the previous section and will thus be denoted as the Aβ-Positive Object Area Ratio.
Comparisons among experimental groups were based on single values per animal for Aβ-Positive Object Density and Aβ-Positive Object Area Ratio. These were calculated by determining the sum of the numbers of Aβ-positive objects (or the sum of the areas of the Aβ positive objects) for the entire section and dividing by the sum of the areas of all ROIs. This analysis was performed blinded to results from prior conventional quantification as described above.
ELISA assays were performed as previously described [23]. Briefly, 50 ng/100 ul of monomeric, oligomeric or fibrillar Aβ40 was plated on ELISA wells and blocked with BSA. Serum samples were initially diluted 250-fold and then serially diluted two-fold to an end point of 1:64,000. The secondary antibodies used for detection were peroxidase-conjugated AffiniPure Goat Anti-mouse IgG (H+L) (Jackson ImmunoResearch) and peroxidase-conjugated anti-mouse IgM (Zymed/Invitrogen, Carlsbad, CA). For samples where the absorbance exceeded 3 times the background absorbance, the titer was determined from the midpoint of the dilution curve (IC50). For samples that did not exceed this criterion, the titer was assumed to be less than the initial dilution of 1:250.
Immunization initiated at 12 months of age with any Aβ conformation decreased both total and fibrillar Aβ immunostaining in Tg2576 mice
In AD significant cognitive decline is generally correlated with the appearance of "mature" amyloid plaques [26, 30]. These plaques contain fibrillar amyloid peptide and as such can be detected by thioflavine, a reagent that stains proteins in beta sheet conformation [31]. Therefore, the accumulation of thioflavine stained plaques in animals immunized at ages 12–16 months was assessed in hippocampus and cortical regions of control animals or animals immunized from 12–16 months of age with fAβ, oligo Aβ, MAPAβ1–33 or adjuvant alone. Representative photomicrographs presented in Figure 1A, and image analysis using KS300 analysis program (Zeiss) of sections from multiple animals demonstrated that the mean % thioflavine positive area in oligo Aβ and fAβ immunized groups (sacrificed at 16 months of age) was decreased by 56 and 40% respectively, relative to adjuvant control. Similarly, when MAPAβ1–33 was used as an immunogen, fibrillar plaque accumulation was decreased by 53% relative to adjuvant control (Figure 2). There was no significant difference between untreated controls and animals treated with adjuvant only (Figure 1A and 1B) or adjuvant plus pertussis toxin in thioflavine positive plaques (Figure 2A and 2B) or any of the markers tested below.
Thioflavine positive plaques are decreased in TG 2576 mice immunized from 12–16 months with oligomeric or fibrillar Aβ conformations. A. Cortex of Tg2576 at 16 months was stained with thioflavine as described in Materials and Methods (control: untreated, CFA: adjuvant only, Oligo Aβ: colloidal gold conjugated amyloid β, and fAβ1–42: fibrillar amyloid). Scale bar = 100 microns. B. Image analysis of thioflavine in hippocampus and cortex of animals immunized at 12–16 months. Mean of each animal is the average of 2–4 sections (except untreated control which is 1 section per animal) in which most to all of the area of study was analyzed (total 4–8 images per section). Bars represent group mean ± SD of n mice per group: Control n = 9, CFA n = 4, oligo Aβ n = 7, Aβ n = 5. *p < 0.02, **p < 0.005 by ANOVA relative to CFA (adjuvant only) control.
Immunization of animals at 12–16 months with MAPAβ1–33 decreases thioflavine and anti CD45 reactivity. A. Representative photomicrographs of thioflavine (green) or CD45 (brown) staining of brain sections from 16 months Tg2576 untreated (control), or injected with adjuvant alone (SCFA) or MAPAβ1–33 (MAP). Scale bar 100 microns (upper panel) and 50 microns (lower panel). B. Image analysis of thioflavine staining or CD45 immunoreactivity in cortex and hippocampus of animals untreated or immunized at 12–16 months. Mean of each animal is the average of two sections (stained in two independent assays, each of which contained animals from each treatment group) in which most to all the area of study was analyzed (4–8 images per section). Bars represent mean +/- SD of n mice per group. Thioflavine: Control n = 6, SCFA n = 6, MAP n = 5, *p < 0.02; CD45: Control n = 5, SCFA n = 7, MAP n = 5, * p < 0.04.
To assess total amyloid deposits in the APP transgenic mice after the 4 month regime of immunization, the human Aβ specific monoclonal antibody, 6E10, was used as described in Material and Methods. Figure 3 shows representative photomicrographs from the cortex and hippocampal region of control or immunized animals. Image analysis of sections from multiple animals demonstrated that the mean % field stained area (ie. Aβ deposits) in oligo Aβ and fAβ immunized groups was decreased by greater than 44% relative to the CFA/IFA controls (Figure 3). Immunization with MAPAβ1–33 decreased total amyloid deposition (6E10 staining) by 52% and 44% relative to adjuvant and untreated control respectively (data not shown, n = 6 animals per group, p < 0.03).
Immunization initiated at 12 months of age in Tg2576 animals decreases total amyloid deposits. A. Representative photomicrographs of sections from brains of 16 m Tg2576 (Control, CFA, Oligo Aβ and fAβ) that had been immunized as described in Materials & Methods from 12 to 16 months of age immunostained with 6E10 (which reacts with the human amyloid peptide). Scale bar: 100 microns. B. Image analysis (% Field area) of Aβ (6E10 antibody) immunoreactivity in hippocampus and cortex of animals untreated or immunized at 12–16 months. Mean of each animal is the average of 2 sections (except untreated control which is 1 section per animal) assessing 4–8 images per section (most to all of the area of the section was analyzed). Bars represent group mean ± SD of n mice per group: Control n = 6, CFA n = 4, Oligo Aβ n = 7, Aβ n = 4, *p < 0.04, **p < 0.03 by ANOVA.
To further validate the quantification of these immunohistochemical results, sections were also analyzed using an automated digital imaging system (ACIS, CLARiENT). A digital image was acquired for the entirety of each section at a resolution of one pixel per micron. All cortical and hippocampal tissues were then analyzed by object identification and feature extraction algorithms. Since analysis of over 50 sections containing both cortex and hippocampus demonstrated that immunoreactivity was higher in the cortex than in the hippocampus, only sections containing cortex and hippocampus were used in these treatment comparisons. Both oligomeric and fibrillar Aβ conformations, as well as the MAPAβ1–33 immunogen, resulted in significant changes in Aβ deposition, with 2- and 3-fold reductions in both plaque number (Aβ-Positive Object Density) and plaque area (Aβ-Positive Object Area Ratio) relative to the respective adjuvant controls (p < 0.05 by two-tailed t-test). In particular, the extent of the reduction in the novel measure of Object Density by Aβ, oligo or the MAPAβ1–33 immunized groups was essentially identical compared to the adjuvant controls, (ie. reductions in plaque number were 2.73, 2.76 and 2.63-fold respectively, p < 0.01, one tail t-test). The significant decreases in plaque area in Aβ, oligo or the MAPAβ1–33 immunized groups obtained by this method were 1.96, 2.39, and 2.72-fold. These quantitative results correlated well with conventional image analysis of the same specimens (r = 0.84, p < 0.0001 for Aβ Positive Object Density vs. conventional Area Ratio, and r = 0.75, p < 0.0001 for Aβ Positive Object Area Ratio vs. conventional Area Ratio), providing additional validation to the conclusion that immunization with three different forms of the amyloid peptide result in a similar decrease in amyloid plaque accumulation in this murine model when immunized during a period of rapid amyloid deposition.
Characteristic of fibrillar amyloid plaques in both human AD brain and in mouse models of amyloid accumulation is robust association of activated astrocytes, which can be envisioned by the astrocyte cell marker GFAP. GFAP staining of B6/SJL control animals was negligible relative to the APP transgenic animals, whether untreated, immunized with adjuvant only or any Aβ immunogen (data not shown). However, while prominent plaque associated GFAP staining was seen in both untreated and adjuvant only transgenic mouse brain, the mean GFAP % field area after the 4 month immunization scheme described in M & M with oligo Aβ or fAβ was 42 and 41% respectively of CFA control, ie a decrease of 58% and 59% (Figure 4A and 4B). This is consistent with the quantitative correlation between mature, fibrillar amyloid plaque accumulation and astrogliosis. That is, the amount of fibrillar plaque deposition correlates with astrogliosis.
Immunization with oligomeric or fibrillar Aβ at 12–16 months decreases astrocyte activation relative to untreated or adjuvant treated age matched controls. A. GFAP reactivity (red) around fibrillar plaques (thioflavine, green) at 16 months in Tg2576 mice either untreated (Control), or immunized at 12–16 months of age with adjuvant only (CFA), oligo Aβ or fAβ. Scale bar = 50 microns. B. Image analysis of GFAP immunoreactivity in cortex of animals immunized at 12–16 months. Mean of each animal is the average of 2–4 sections (total 4–8 images per section) in which most to all of the area of study was analyzed. Bars represent group mean ± SD of n mice per group: untreated = 4, CFA n = 4, oligo Aβ n = 7, fAβ n = 5. *p < 0.03, **p < 0.01 by ANOVA.
Immunization with oligomeric Aβ at 12–16 months resulted in less microglial immunoreactivity relative to immunization with fAβ
Microglia play a major role in regulating homeostasis in the brain and have the ability to actively phagocytose, to secrete cytokines, and to present antigen to T cells depending on their stimulatory environment [32]. However, some of these same neuroprotective functions can become detrimental if dysregulated [33]. Given the common observation that reactive microglia are characteristically associated with fibrillar amyloid containing plaques in both human AD and animal models, we assessed the effect of immunization on microglial reactivity as measured by CD45 and MAC-1 surface antigens. The photomicrographs from cortical brain areas show a pronounced decrease in MAC-1 in animals immunized with oligo Aβ or fAβ as compared to untreated or adjuvant treated transgenic mice (Figure 5A). Mean % area of MAC-1 staining in oligo Aβ and fAβ immunized groups is 40 and 76% respectively of CFA control (Figure 5B). Results were similar when immunoreactive CD45 was assessed with mean field area % staining in oligo Aβ and fAβ groups at 35 and 60% respectively of the CFA control (Figure 5C and data not shown). Adjuvant only (CFA) animals and untreated animals were indistinguishable. Interestingly, while there is a clear trend to lower microglial activation in the fAβ immunized animals, a statistically significant difference was not established in the group studied relative to the CFA control, perhaps due in part to the variability in the animals. However, the difference in microglial reactivity in those animals immunized with the oligomeric conformation of Aβ was statistically significant relative to either the untreated controls or adjuvant controls (p < 0.01, MAC-1; p < 0.001, CD45). Indeed, CD45 and MAC-1 reactivity in the Oligo Aβ immunized animals is significantly lower than that in the fAβ immunized animals (p < 0.02 and p < 0.05, for MAC-1 and CD45 respectively). These results could reflect a lower variability in the activation of microglia in response to immunization with oligo Aβ than with fAβ and could be related to the mechanisms resulting in the decreased accumulation of amyloid in the immunized animals in each case. Interestingly, immunization with MAPAβ1–33 also showed significantly less CD45 (50% of adjuvant control, p < 0.036) (Figure 2) and MAC-1 (38% of adjuvant control, p < 0.012, 1 experiment, 4–7 animals per group, data not shown).
Immunization with oligomeric Aβ at 12–16 months decreases microglia reactivity to a greater extent than immunization with fAβ. Photomicrographs of immunohistochemical staining of microglial reactivity (MAC-1, brown) in Tg2576 untreated or immunized with CFA, Oligo Aβ and fAβ at 12–16 mos (A). Scale bar = 50 microns Image analysis of MAC-1 (B) and CD45 (C) immunoreactivity in hippocampus and cortex of animals immunized at 12–16 months. Values for each animal were the average of 6–8 images per section which resulted in analysis of most to all of the area of the section. Bars represent group mean ± SD of n mice per group: Control n = 7, CFA n = 4, oligo Aβ n = 6–7, fAβ n = 5. By ANOVA, for MAC, CFA: oligo Aβ *p < 0.01; for CD45, CFA: oligo Aβ *p < 0.001. Data for each marker are from one assay representative of 2 independent assays.
The identical immunization scheme was performed with older Tg2576 and wild type (B6/SJL) animals. Immunization was initiated at 16 months of age, a time at which there was robust total and fibrillar amyloid deposits (see untreated controls in Figures 1, 2, 3, 4, 5) in the transgenic mice, and continued through 20 months of age. Animals were then bled, sacrificed and assessed for each of the markers described above. No differences in thioflavine staining, 6E10, GFAP, MAC-1, or CD45 were seen between any treatment groups regardless of the immunogen (Figure 6 and data not shown for MAC-1 and the MAPAβ1–33 immunized animals).
Immunization of Tg2576 mice at 16 – 20 months provided no significant change in brain pathology. Image analysis of immunoreactivity in sections of cortex/hippocampus of animals immunized at 16–20 months. Animals were immunized and tissue processed and stained for thioflavine (A), 6E10 (B), CD45 (C) and GFAP (D) as described above for the younger animals. Mean of each animal is the average of 2–3 assays/sections per marker, derived from 4 – 8 images per section (including most to all of the area of study). Bars represent group mean ± SD of n mice per group: Control (untreated) = 4–6, CFA n = 6, oligo Aβ n = 6–7, fAβ n = 4–6. No significant differences are seen between groups in any of the markers.
Immunization with oligomeric Aβ conformations induced measurable antibody reactivity to oligomers but not to soluble or fibrillar amyloid
Levels of anti-amyloid antibody induced in all immunized animals differed to some extent among the immunogen and the age of animals at the time of immunization, although all animals responded to immunization. A most notable observation was that immunization of both transgenic and wild type mice with the oligomeric conformation of Aβ resulted in measurable antibody to the oligomeric form of Aβ (Table 2), with trace to no reactivity to fibrillar Aβ or soluble Aβ as assessed by conformation specific ELISA. Immunization with fAβ or Aβ1–33 as a MAP peptide generated significant levels of IgG anti-oligomeric Aβ as detected by ELISA (Table 2), but also induced antibody to fAβ and to soluble Aβ (data not shown). The transgenic animals immunized with fAβ or oligo Aβ at a younger age (12–16 m) had slightly higher antibody titers than those immunized later (16–20 m), although the level of antibody in animals immunized with MAPAβ1–33 was not related to age of immunization. Nontransgenic immunized mice had greater responses to all immunogens relative to the transgenic mice, and those responses did not vary according to the age of immunization. However, the level of anti-oligomeric IgG antibody or any anti Aβ antibody had no detectable correlation with any of the pathologic markers at either age group when individual transgenic animals were compared (data not shown). Furthermore, as would be expected after 4 months of immunization, no appreciable IgM anti oligomeric Aβ or anti fibrillar Aβ was detected (using an ELISA specific for IgM detection). Only very low levels of IgM anti soluble Aβ was observed in sera from animals immunized with fibrillar Aβ.
To determine if the antibodies generated with each immunogen were able to bind amyloid plaques in the APP transgenic mice, dilutions of sera from each group of animals immunized at 12–16 months and animals immunized with fAβ at 16–20 months were screened for the ability to bind to amyloid deposits in brain sections from an unimmunized 16 month APP mouse. Consistent with the ELISA data above, antisera from oligo immunized animals did not stain plaques, whereas antisera from animals immunized at 12–16 months and 16–20 months with fAβ and sera from MAPAβ1–33 animals all stained cortical plaques (data not shown).
As an indication of whether any induced antibody to β-amyloid had access to the brain, sections from brain of immunized animals were tested for immunoreactivity to mouse IgG using two approaches: direct staining with biotinylated anti mouse IgG (Figure 7A) and indirect immunohistochemistry using rabbit anti mouse IgG as a primary antibody and subsequently probing with anti rabbit immunoglobulin (data not shown). Both staining approaches gave comparable results. Deposits of IgG were detected in cortex/hippocampus sections of animals immunized with fAβ at 12 months (Figure 7A) and these deposits of mIgG colocalized with Aβ positive plaques (Figure 7B). All animals immunized with fAβ at 12–16 months that were tested (n = 6) showed these deposits. Of the animals tested immunized with the oligo Aβ (n = 7) only one had slight IgG reactivity (Figure 7A) within the cortex, while no staining was detected on any of the adjuvant controls (Figure 7A, n = 5). Interestingly, none of the animals immunized at 16 months showed any IgG deposited in the brain (data not shown, CFA controls = 3, oligo Aβ n = 5, fAβ n = 5). All the animals immunized with SCFA (n = 4) or MAPAβ1–33(n = 4) at 12–16 months that were tested were negative for plaque-associated mouse IgG. Sections from each animal were tested 2–4 times. Thus, only animals immunized with fAβ at 12–16 months showed any plaque-like IgG deposition in brain tissue, even though the titer of anti fibrils and anti-soluble Aβ, were generally higher in the MAPAβ1–33 immunized animals.
IgG is deposited at detectable levels in Tg 2576 animals immunized at 12 months with fAβ. Representative pictures of cortex sections of mice immunized from 12–16 months with CFA, Oligo Aβ and fAβ. A. Direct immunoperoxidase labeling of mouse IgG using a biotinylated anti mouse IgG and streptavidin-HRP. B. Fluorescent double labeling with horse biotinylated anti mouse IgG (Vector) detected with streptavidin-CY-3 (red) and polyclonal anti Aβ antibody (M-16) detected with FITC conjugated anti rabbit antibody (green). Arrowheads show colocalization of reactivity in some of the plaques. Scale bars: 50 microns.
Complement activation products associated with plaques have been detected in murine models of AD [34, 35]. Since antibody-antigen complexes are known to activate complement, it was hypothesized that complement activation on plaques with associated antibodies (ie. those immunized with fAβ at 12–16 months) may initiate the complement cascade thereby increasing the amount of C3 bound to the plaques (and thereby possibly contributing to greater microglial reactivity). Novel monoclonal antibodies have recently become available that specifically recognize C3 activation products (C3b, iC3b and C3c) [36]. These antibodies (2/16 and 2/11) were used to compare C3 activation products in brain sections from mice immunized with oligo Aβ and fAβ or with adjuvant alone. C3b/iC3b immunoreactivity was detected in all brain sections of Tg2576 mice (Figure 8A). Interestingly, results of image analysis demonstrated that the level of activated C3 correlated with fibrillar Aβ plaque load (Figure 8B). That is, no apparent increase in C3 deposition was detected in those animals immunized with fAβ and shown to have antibody associated with the plaques. Rather, the amount of C3 reactivity reflected the reduced quantity of plaques present in the immunized animals relative to the adjuvant control animals, and was nearly identical to that seen in similar brain sections of animals immunized with oligo Aβ. C3 staining in animals immunized at 16–20 months correlated with thioflavine staining with no increase or decrease in C3 deposition detectable in any of the animals (data not shown).
Anti murine activated C3 is detected in plaque-like structures in Tg2576 and is decreased in animals immunized with either fAβ or oligo Aβ. A. Photomicrographs of immunohistochemical straining with 2/16 anti mouse C3b/iC3b (brown) in Tg2576 untreated or immunized with CFA, Oligo Aβ and fAβ at 12–16 mo. Scale bar = 20 microns. B. Anti C3b/iC3b immunoreactivity in animals immunized at 12–16 months. Values for each animal were the average of 6–8 images per section which resulted in analysis of most to all of the area of the section. Bars represent group mean ± SD of n mice per group: Control n = 4, CFA n = 3, oligo Aβ n = 6, fAβ n = 5. *p < 0.002, **p < 0.001 relative to CFA, by ANOVA.
The data presented here is the first investigation of the effect of active immunization with amyloid beta peptide in a specific oligomeric conformation in a murine mouse model of Alzheimer's Disease. Immunization with this form of the peptide was as efficient at reducing amyloid deposition in Tg2576 animals as either fibrillar Aβ or a multiple Aβ antigen peptide when immunization was initiated at 12 mos. Interestingly, there was significantly less (p < 0.05) activated microglia in the animals immunized with the oligomeric form of Aβ than in those immunized with fAβ. In addition, the antibody response induced by immunization with oligomeric Aβ had a much more restricted epitope response, generating antibodies to oligomeric Aβ, but not to soluble or fibrillar Aβ, similarly to that observed in vaccination of rabbits with the oligomeric conformation of Aβ [23]. The other two immunogens tested, fAβ and MAPAβ1–33, induced antibodies to oligomeric, fibrillar and soluble Aβ. Thus, one could speculate that the lower microglia activation is the result of this restricted induction and that this feature may lead to a lower potential for autoimmune responses and inflammatory responses. Further delineation of the basis for the apparently lower microglial activation is, however, necessary to substantiate this hypothesis. Since we did not stain these sections for T cells, it is unknown if the Oligo Aβ immunogen elicited a strong T cell mediated immune response beyond the induction of anti-oligomeric antibody. While this is a concern since a fraction of patients in the human amyloid immunization trial (AN1792) developed meningoencephalitis [7–9, 37], any reduction of potentially adverse effects of immunization is desirable.
In contrast, no significant decrease was seen in any pathological marker measured in mice vaccinated with any peptide form when vaccination was initiated at 16 months, a time at which there are high levels of accumulated plaques. The lack of efficacy in reduction of plaque associated pathology by immunization of these older mice, similar to that reported by Das, et al [3], was not due to lower anti-Aβ titers of antibodies in the mice as the levels of reactivity were either equivalent or only slightly lower than those of the mice immunized at an earlier age (12 mo) suggesting age related alterations/differences in trafficking of amyloid out of the brain (see below) or antibodies into the CNS via BBB (or to antibody-independent clearance mechanisms). While it has been hypothesized that older plaques are refractory to degradation and clearance, intracranial injections of anti Aβ antibodies in old APP/Tg mice (16–20 month-old) have been reported to clear long established plaques [38]. Interestingly, Dodart and colleagues reported that passive peripheral immunization of 24 month old PDAPP mice had no effect on amyloid deposition in contrast to similar immunization of younger mice. However, in these very old PDAPP mice significant improvement of behavioral tasks were demonstrated [39]. While the results here clearly demonstrate the lack of effect on clearance of amyloid deposits in mice immunized at an older age, it remains to be seen if improvement in learning can be detected in the very old Tg2576 mice actively immunized with specific conformations of Aβ (particularly the oligomeric conformation), and whether effects of human Aβ immunization are similarly influenced by age and/or plaque deposition at the time of immunization.
In this study we used a second, novel method for quantification of immunohistochemical detection, specifically of amyloid deposits. Although image analysis can bring accuracy and objectivity to IHC, certain drawbacks have historically limited its use. Not only are the processes of image acquisition and data management labor-intensive, but the mechanics of analysis itself can introduce some subjectivity. Automated digital imaging systems, such as the one tested here, have been developed to address such drawbacks. Results from analysis of amyloid burden using such an automated platform correlated tightly with those obtained with an accepted more traditional image analysis approach. Thus, this method is shown to accurately assess amyloid burden in transgenic mouse models of AD. In addition, the use of object-based analysis provides data not obtainable through simple computation of % area positive. For example, immunization with MAPAβ1–33 resulted in similar 2.63- and 2.72-fold reductions in Aβ-Positive Object Density (i.e. plaque number) and Aβ-Positive Object Area Ratio (i.e. plaque area), respectively. In contrast, fibrillar Aβ immunization affected these parameters somewhat differently, resulting in a 2.73-fold reduction in Aβ-Positive Object Density but a lower (though still significant) 1.96-fold reduction in Aβ-Positive Object Area Ratio. This raises the intriguing possibility that different immunization paradigms might effect change in amyloid burden preferentially through alteration in either plaque number or in plaque size, although additional study will be required to rigorously test such an assertion.
Interestingly, plaque-associated IgG was detected only in brain of animals immunized with fibrillar Aβ from 12 to 16 months old. While this is consistent with the lack of anti-fibrillar amyloid antibody in the Oligo Aβ immunization scheme, sera of all animals immunized with MAPAβ1–33 contained levels of anti-fibrillar Aβ similar to the fAβ immunized animals, but no IgG was detected on amyloid plaques even in the younger set (immunization initiated at 12 m of age) of animals (n = 6) in which reduction of plaques and associated pathology was comparable to fAβ immunized animals. These data suggest that there may be differences in access of the antibody to brain tissue via blood brain barrier or IgG-transport systems in mice immunized with fAβ (perhaps due to antibody epitope specificity [40]). Multiple injections of pertussis toxin have been reported to result in infiltration of immune cells into the brain [29]. However, while PT was given to the MAPAβ1–33 mice at the time of initial injection and 24 hours later, these animals showed no immunoglobulin associated with the remaining plaques and similar levels of reduction of plaques in an age specific manner was seen with the Oligo Aβ and fAβ immunogens. However, we did not stain these sections for T cells and therefore cannot confirm nor refute the previously published observation of vascular associated T cells by Furlan and colleagues [29].
Finally, the amount of C3 activation fragments deposited on the plaques in all animals was largely correlated with the amount of thioflavine staining detected in each animal. While this would be expected since fibrillar amyloid has been shown to activate complement [41–43], it is somewhat surprising that there is no apparent increase in those plaques with associated IgG (fAβ immunized, 12–16 mo), since immune complexes (here Aβ/anti Aβ) avidly bind C1q and activate complement. However, additional investigations will be necessary to directly determine the extent of complement activation in each animal treatment group. Indeed increased complement activation is predicted to enhance removal of complement coated plaques (via C3 activation fragments, C3b and iC3b) and thus, whether those plaques remaining are less opsonized or display some other distinguishing characteristic remains to be determined.
The mechanism by which immunization schemes decrease neuropathology and prevent or reverse behavior deficits has not yet been defined. A decrease in amyloid deposition as a result of active immunization was seen in APP overexpressing transgenic animals that are FcR gamma chain deficient [44], suggesting that FcR mediated ingestion by phagocytic cells is not a requirement. However, as stated above, complement activation fragments (particularly C3b and iC3b) are also capable of mediating particle ingestion and thus, complement receptors and/or other phagocytic receptors may provide a mechanism for enhanced ingestion in the absence of FcR. Bacskai et al. demonstrated clearance of plaques by passive immunization of Fab anti amyloid antibody fragments [13] which lack the Fc portion of the antibody molecule and therefore can neither engage Fc receptors nor activate complement, thus providing support for multiple alternative clearance mechanisms. Additional possible mechanisms by which both passive and active immunization may be advantageous have been proposed. The "peripheral sink" model suggests amyloid is cleared in the periphery after being transported from the brain across the vasculature into the blood [12, 45]. If therapeutically relevant concentrations of anti Aβ antibodies do not cross the blood brain barrier (BBB), then a mechanism for transporting Aβ out of the brain across the BBB is required [46]. The principle molecule that appears to be involved in transport of Aβ out of the brain across the BBB is the low-density lipoprotein receptor-protein-1 (LRP-1) [47–49], although other molecules such as ApoE and α2- macroglobulin may be involved in this process. Antibodies specific to LRP-1 substantially inhibited the clearance of Aβ40 from the brain supporting the role of LRP-1 as a transporter of Aβ peptide out of the brain [47]. Moreover, APP transgenic mice crossed to receptor-associated protein knockout mice (RAP-/-) mice, which are deficient in LRP-1 function, develop increased extracellular Aβ deposition and neurodegeneration [50]. Interestingly, the LRP-1 expression in the BBB appears to decrease significantly with normal aging and in AD. Whether this occurs in aging mice and contributes to the lack of clearance reported here remains to be tested.
Another possible mechanism proposed is that antibodies inhibit fibrillization of amyloid and/or promote depolymerization of fibrils (reviewed in [51]). Still others suggest antibodies must be reactive with the Aβ intermediates to provide their functional protective effect. Wisniewski and colleagues reported that the induction of a predominant IgM anti Aβ response also resulted in behavioral improvements in the Tg2576 animal which did not necessarily correspond to Aβ load in the brain, although high IgM titers did correlate with low amyloid burden [52]. Active immunization of rats with mixed conformations of Aβ peptides prevented the inhibition of LTP activity when conditioned media containing Aβ oligomers were injected intracerebroventricularly, and this prevention of inhibition of LTP correlated with antibody recognition of the Aβ oligomers [24]. Anti-oligomeric antibody induced here could promote therapeutic clearance and removal of amyloid via any of these mechanisms, and by limiting the immune response, may limit detrimental activation and/or promotion of inflammation.
A hypothesis consistent with the observations in both human AD and transgenic mouse models of AD is that there are at least two, likely overlapping, stages contributing to neuronal dysfunction in AD. Early events involving the intracellular accumulation of Aβ peptides [53] and/or generation of oligomeric Aβ peptides [21, 23] or complexes containing oligomeric Aβ [54] would lead to neuronal alterations and cellular death. Factors arising during aging such as oxidative stress, mitochondrial dysfunction, deficiencies in lysosomal function or regulation of neurotrophic factors that lead to processing outcomes which are harmful to the cell may contribute to, or enhance, susceptibility to stress induced by Aβ [55–57]. Increased extracellular fibrillar Aβ deposits from these processes and/or overload of the phagocytic capacity of the local region provide a nidus for complement activation [41, 42]. The generation of the proinflammatory complement activation products would initiate a secondary phase of inflammatory events that accelerate local neuronal damage, loss, and decline of cognitive function [34]. Neuronal injury at both stages could be avoided or diminished by a decrease in amyloid peptide in the brain, a direct goal of immunotherapy. Providing increased specificity for the immune response however, may also decrease the probability of activating detrimental inflammatory responses, and thus is a potential advantage of the oligomeric amyloid conformation as the immunogen.
In summary, immunization with any amyloid peptide immunogen resulted in significant reduction (45–55%) of plaque pathology and decreased inflammatory microglial cell reactivity (40–65%) only when immunized prior to the massive deposition of large mature plaques. These decreases in pathology did not necessarily correlate with the level of anti Aβ1–42 IgG in sera or with plaque-associated IgG. This first analysis of deposition/accumulation of complement C3 activation within an immunization trial shows association of C3b/iC3b with plaques that correlates with the amount of thioflavine plaques present rather than any association with the antibody response induced. Finally, immunization with oligomeric Aβ1–42 induced a greater decrease in "reactive" microglia relative to immunization with fibrillar Aβ as determined by microglial surface expression of CD45 and MAC-1 antigens. The lower microglial response resulting from immunization with oligomeric Aβ suggests that the goal of depleting plaques can be accomplished while inducing less neuroinflammation, and thus facilitating the application of immunotherapy to treatment and/or prevention of AD in humans.
Charles Glabe and Rakez Kayed are consultants for Kinexis, Inc. and Scott Webster is an employee at Clarient, Inc. The authors declare that they have no other competing interests.
JZ performed all animal treatments, perfusions, immunohistochemistry, image analysis, data analysis and presentation, and guided or performed tissue preparation and serum collection. MF performed immunohistochemistry, image analysis, data analysis and presentation, molecular genetic studies and manuscript preparation; RK prepared immunogens and performed immunoassays, S.W. performed image analysis, data analysis and manuscript preparation; IH performed immunohistochemistry, image analysis, data analysis and presentation and assisted with perfusions and animal care; OY performed immunohistochemistry and image analysis; DC assisted with the design of the study and manuscript preparation; CG contributed to the design of the study and data analysis, and manuscript preparation. AJT developed the design of the study, guided study execution, directed data analysis, interpretation and presentation, and drafted and edited the manuscript. All authors read and approved the final manuscript.
Insight into the molecular mechanisms by which SE transforms a normal brain into an epileptic brain may reveal novel targets for development of preventive therapies. It has been widely hypothesized that the brain-derived neurotrophic factor (BDNF) receptor TrkB is required for SE-induced TLE (Boulle et al., 2012; but see Paradiso et al., 2009); however, off-target effects of TrkB inhibitors together with inadequate temporal control afforded by genetically
modified animals have precluded testing this idea. We therefore sought a method to selectively inhibit TrkB after SE. Here we use a chemical-genetic method (Chen et al., 2005) Tenofovir clinical trial and demonstrate that inhibition of TrkB signaling for 2 weeks after SE prevents development of TLE and ameliorates comorbid anxiety-like behavior and destruction of hippocampal neurons. We first sought to confirm that SE induction enhanced click here activation of TrkB. A major pathway by which SE can be induced in hippocampus and related temporal lobe structures involves activation of neurons in the amygdala by chemical or electrical methods (Goddard et al., 1969 and Mouri et al., 2008). Infusion of the chemical convulsant kainic acid (KA) into the right amygdala of an awake wild-type (WT) mouse
induced SE (Ben-Ari et al., 1980 and Mouri et al., 2008) (Figures S1A, S1B, S3, and S4 available online). Mice were euthanized either immediately (0) or at 3, 6, 24, or 96 hr later. Mice infused with vehicle (PBS) served as controls. Using p-TrkB (pY816 and pY705/706) immunoreactivity as surrogate measures of activation (Segal et al., 1996), we detected increased activation of TrkB in the hippocampus ipsilateral to the infused amygdala immediately upon termination of SE and at each of the subsequent time points relative to the vehicle controls (p < 0.01) (Figure S2A). We next sought to verify that we could selectively inhibit TrkB activation using a chemical-genetic approach. A genetic modification of mice in the TrkB locus (TrkBF616A) in which
alanine is substituted for phenylalanine at residue 616 within kinase subdomain V renders TrkB sensitive to inhibition by a blood-brain and barrier and membrane-permeable, small-molecule, 1-(1, 1-dimethylethyl)-3-(1-naphthalenylmethyl)-1H-pyrazolo[3, 4-d]pyrimidin-4-amine (1NMPP1; henceforth, the terms 1NMPP1 and inhibitor will be used interchangeably). Importantly, in the absence of 1NMPP1, no differences in TrkB kinase activity or overt behavior are detectable in TrkBF616A compared to WT mice ( Chen et al., 2005). We infused the amygdala of TrkBF616A mice either with PBS or KA and then administered vehicle or 1NMPP1, respectively (see Experimental Proceduresand Figure S1B). We detected enhanced p-TrkB (pY816) immunoreactivity in western blots of lysates from the hippocampus ipsilateral to the infused amygdala in vehicle-treated WT (3 hr post-SE, p < 0.001) and TrkBF616A mice (3 hr post-SE, p < 0.001; 24 hr post-SE, p < 0.
sensory cells that grow axons through the lamina cribosa to reconnect with the olfactory bulb, providing there is not much scar tissue from the surgery. The lateral line of fish and amphibians and the inner ear of all nonmammalian vertebrates can regenerate new hair cells that will function nearly as well as those lost through injury. Birds that have recovered from noise damage have thresholds near normal (for review see Bermingham-McDonogh and Rubel, 2003). The same is true for the retina of the fish; visual acuity is nearly restored to normal, though there is some disruption in the details of cone patterning. The fact that function can be restored in these systems is a rather amazing feat, and so it gives us a bit of hope Fluorouracil concentration that stimulation of regeneration in the mammalian retina or inner ear sensory receptor cells might be sufficient to trigger the associated processes that must take place for effective restoration of function. Even in mammals, there is evidence from both the inner ear and
the retina that considerable plasticity remains Sirtuin inhibitor in the cells that synaptically connect with the sensory receptor cells; ectopic hair cells induced form Atoh1 overexpression can be innervated by the ganglion neurons, and rod photoreceptors transplanted into normal adult mice will reconnect to host bipolar cells and appear to function (MacLaren et al., 2006). Lastly, we can conclude that those sensory epithelia SB-3CT that display little or no capacity for regeneration generally do not have ongoing proliferation or addition of new neural cells anywhere in the organ. What is even more striking about most of the systems where regeneration is absent is that the cells in these epithelia don’t respond to damage by increasing proliferation; in both the mammalian inner ear and retina there is very little proliferation of the support cells or Müller glia, respectively, after injury. In addition,
the nonneuronal support/glial cells in these epithelia do not undergo extensive reprogramming after injury but for the most part retain most morphological and gene expression characteristics as they have in the undamaged tissue (though they frequently become “reactive”). So in the specialized sensory epithelia that do not have ongoing new sensory cell addition, like the mammalian inner ear and retina, the tissue may no longer retain the “developmental niche” that is characteristic of the olfactory epithelium, or the retina and inner ear of nonmammalian vertebrates. There are two interesting exceptions to some of the above conclusions: the retina and the cochlea of birds. The avian retina responds to damage with robust Müller glial proliferation, though the reprogramming of these cells to a progenitor pattern of gene expression is much more limited than in the fish, and very few of the BrdU+ Müller cells go on to differentiate into neurons.
While proliferation in the Dlx1/2-cre;ShhF/− mutant’s rostrodorsal MGE appeared normal at E11.5 and E14.0 ( Figures S4 and S5 and data not shown), by E18.5 there was a trend for a reduction in PH3+ cells (∼50%; p = 0.07) ( Figure S6). Furthermore, while the number cortical interneurons in the mutant appeared normal at E14.0, by E18.5, there was a
clear reduction in MGE-derived interneuron numbers ( Figures 7A, 7A′, 7B, 7B′, and S6; Table S3). Increased apoptosis in the mutant’s MGE may have also contributed selleck compound to the reduction in cortical interneurons ( Figures 6 and S5). Thus, we propose that Shh expression in the MGE MZ, by promoting expression of Nkx2-1, Nkx6-2, Lhx6, and Lhx8 in the rostrodorsal MGE ( Figure 4, Figure 5 and Figure 6 and S4–S6), may equally drive production and/or survival of SOM+ and PV+ cortical interneurons. The Dlx1/2-cre;ShhF/− mutant also have reduced numbers of CR+ interneurons; we suggest that these largely correspond to the SOM+;CR+ subtype. On the other hand, we did not detect a change in NPY+ interneuron numbers, consistent with evidence that Lhx6 is not essential in their generation ( Zhao et al., 2008). Finally, Y-27632 manufacturer we propose that Shh expression in neurons of the rostrodorsal MGE and septum
is required for the development of subpallial cell types in the anterior extension of the bed nucleus of
Linifanib (ABT-869) stria terminalis (medial division; STMA), the core of the nucleus accumbens (AcbC), the lateral septum and the diagonal band complex (VDB/HBD), whereas the ventral pallidum, substantia inmoninata, and globus pallidus appeared normal ( Figures 6 and S6). Future studies are needed to determine whether loss of Shh in the MGE MZ affects other aspects of its development such as guidance of axons that project to the pallidum ( Charron et al., 2003). The loss of Shh expression in neurons of the MGE MZ in the Lhx6PLAP/PLAP;Lhx8−/− mutant suggests that these transcription factors could directly regulate the Shh gene expression. We established using EMSA assays that LHX6 and LHX8 bind to a specific site in the SBE3 shh enhancer (ECR3) ( Figure 8); SBE3 is a regulatory element that is specifically active in the MGE MZ ( Jeong et al., 2006). Furthermore, Lhx6 and Lhx8 drive expression from the SBE3 Shh enhancer in MGE neurons ( Figure 8). The transcriptional activation was context specific; while the SBE3 Shh enhancer was activated by Lhx6 and Lhx8 in MGE primary cultures, it was not activated in two tissue culture cell lines (P19 and HEK293T) (data not shown). Currently, we do not have antibodies that are effective for chromatin precipitation, and therefore cannot provide corroborative evidence for in vivo binding of LHX6 and LHX8 to the SBE3 Shh enhancer.
, 2012). Recent studies indicate that GluD2 regulates GluA2 tyrosine 876 and serine 880 phosphorylation (Kohda et al., 2013). We have made steady progress in our understanding of the molecular mechanisms underlying synaptic plasticity in the last 25 years. However, it is clear that we have a lot more to discover. Major accomplishments have been the general acceptance that hippocampal LTP is expressed as a postsynaptic mechanism triggered by activation of CaMKII and downstream signaling pathways that involve Ras,
Rho, and other small G-proteins. Also, it has been recognized that the membrane trafficking of AMPARs is quite dynamic and that increases and decreases in synaptic strength during LTP and LTD, respectively, are mediated by rapid and long-lasting Ivacaftor purchase changes in AMPAR number at synaptic spines. The regulation of the membrane trafficking and synaptic retention of AMPARs is quite complex and involves both recruitment of receptors from intracellular pools such as recycling endosomes and also recruitment of receptors from extrasynaptic pools that laterally diffuse into the synapse (Figure 2). These processes are regulated by a large number of proteins that retain and guide the receptors from these nonsynaptic locations and scaffolding proteins that finally retain receptors at the synapse (Figure 3).
In addition, extracellular transsynaptic interactions of adhesion-like Adenosine molecules have recently been implicated NVP-BGJ398 cell line in the expression of LTP and add a new layer of complexity (Figure 3). Although there is significant evidence that there are subunit specific rules for AMPAR trafficking during plasticity, recent work has suggested that, although distinct subunits may have a competitive advantage to support LTP, and respond differentially to neuromodulators, they are not absolutely required for LTP. All AMPAR subunits and even kainate receptor subunits can be engaged by LTP signaling pathways and
expression mechanisms. This means that, whatever the core mechanism of LTP is, it can act on both AMPARs and kainate receptors. Conceptually, this is hard to explain as these receptors have distinct auxiliary subunits, but they have been reported to have common interacting proteins (Anggono and Huganir, 2012 and Coussen, 2009), suggesting that these shared interactors may be functionally important for LTP. These new results have challenged the field to come up with new ideas on how these receptors can be recruited and captured at synapses. Future work will need to include the further characterization of the complex receptor recycling pathways and the extrasynaptic pools of receptors. We need to better understand the regulation of these pools during LTP and the molecules involved. In addition, further attention to scaffolding and transsynaptic proteins and their specific role in LTP is required.
8% ± 3.5%, n = 14; Cpx KD 78.9% ± 2.5%, n = 14). We also examined whether Cpx KD might affect the proportion of REs containing AMPARs. However, Cpx KD did not affect the percentage of REs containing GluA1 (Figure S3) or the percentage of dendritic GluA1 puncta that colocalized CCI-779 nmr with REs (Figure S3). Cpx KD also did not affect the subcellular localization of REs relative to dendritic spines as defined by simultaneous expression of recombinant TfR fused to mCherry and soluble GFP (Figure S3). Thus, consistent with the lack of
effects of Cpx KD on basal synaptic transmission, these results demonstrate that Cpx KD had no detectable effects on the pool of intracellular AMPARs that are thought to be the source of the AMPARs that are exocytosed during LTP. A final possibility is that the Cpx KD did affect constitutive delivery of AMPARs to synapses but that basal surface expression of AMPARs and thus basal AMPAR EPSCs were not affected because of a compensatory change in the rate of steady state AMPAR endocytosis. To address this possibility, we measured the effect of Cpx KD on constitutive AMPAR endocytosis (Bhattacharyya et al., 2009). There was no detectable effect of Cpx KD on constitutive endocytosis of endogenous surface AMPARs (Figure S3), thus ruling out this hypothesis. Previous work showed
that postsynaptic SNARE-mediated membrane fusion is required for LTP (Kennedy et al., 2010, Lledo et al., 1998 and Lu et al., 2001). However, these experiments focused on SNARE proteins that are ubiquitously involved in both regulated and constitutive membrane fusion selleck products events. Thus, the mechanisms underlying the regulated, calcium-dependent triggering of AMPAR exocytosis during LTP remained unknown. Using in vivo injection of lentiviruses, we molecularly manipulated complexin only in CA1 pyramidal Isotretinoin cells and thus only in the postsynaptic compartment of the excitatory synapses being studied in acute hippocampal slices. The results, which
were confirmed in a neuronal culture model of LTP, provide strong evidence that complexin is a key component of the molecular mechanism by which NMDAR-mediated increases in calcium during LTP induction leads to the exocytosis of AMPARs at the postsynaptic membrane. The importance of postsynaptic complexin in LTP is consistent with immunohistochemical and electron microscopic studies that confirm the presence of complexin in dendritic spines and shafts (McMahon et al., 1995 and Yamada et al., 1999). Our results also suggest that postsynaptic complexin is not required for constitutive delivery of AMPARs and NMDARs into the synaptic plasma membrane, a process that probably occurs on a much slower timescale than the delivery of AMPARs during LTP (Adesnik et al., 2005 and Washbourne et al., 2002). Consistent with this conclusion, Cpx KD had no effects on the intracellular pools of AMPARs found in dendrites or on dendritic REs that have been suggested to be the source of the AMPARs that are exocytosed during LTP (Park et al.
Restoring binocular vision by reopening the deprived eye during the critical period induced a third stage of plasticity, the rapid restoration of both eyes’ responses to baseline levels (Kaneko et al., 2008a). These three stages and their characteristics were similar regardless of which eye was deprived, contralateral or ipsilateral eye (Sato and Stryker, 2008). Collectively, these findings in the mouse are consistent with observations in other species that a decrease
in V1 plays a critical role in ODP. The past decade has seen the creation of transgenic mice in which critical period timing and the development of response properties are normal, but the changes in responses and circuitry during critical period ODP are perturbed. These studies reveal that the three stages of critical period ODP expression are mechanistically distinct (Figure 5): Linsitinib cost (1) The initial reduction in deprived-eye responses relies on a mechanism involving calcium signaling with pharmacology similar to long-term depression (LTD). (2) The later increase in open-eye responses involves both homeostatic and long-term potentiation (LTP)-like mechanisms. (3) The restoration of normal visual responses after opening the deprived eye involves neurotrophic signaling mechanisms. The first stage of critical period ODP, the decrease in deprived-eye responses, is hypothesized to result from a loss of deprived-eye connections or a depression in their synaptic efficacy. Consistent with this idea, blocking ( Bear et al.,
1990) or genetically deleting N-methyl-D-aspartate receptors (NMDARs) ( Roberts et al., 1998), manipulations that block LTD, also prevented a shift in ocular dominance. However, these manipulations can also affect MycoClean Mycoplasma Removal Kit LTP and other forms of plasticity. Viral expression of a peptide that blocks LTD and, specifically, NMDAR-dependent internalization of postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) also blocked the reduction in deprived-eye responses in layer 4, consistent with the operation of LTD in the first stage of critical period ODP ( Yoon et al., 2009). Spike timing-dependent plasticity (STDP) is an alternative mechanism that shares a dependence on NMDARs and calcium signaling and appears, at least in the short term, to be a potential explanation of changes during MD (Yao and Dan, 2005).
One definition of “history of safe use” proposes “significant human consumption of food over several generations and in a large, genetically diverse population for which there exist adequate toxicological and allergenicity data to provide reasonable certainty that no harm will result from consumption of the food” (Health Canada, 2003). In order to evaluate the history of safe use of a microorganism, it is necessary to document not just the occurrence of a microorganism
in a fermented food product, but also to provide evidence whether the presence of the microorganism is beneficial, fortuitous, or undesired. In the United States, selleck products food and substances used in food are regulated according to the Food Drug and Cosmetic Act (1958), in which the status of Generally Recognized Hydroxychloroquine in vivo As Safe (GRAS) was introduced (FDA, 2010). Accordingly, a GRAS substance is generally recognized, among qualified experts, as having been adequately shown to be safe under the conditions of its intended use. A substance recognized for such use prior to 1958 is by default GRAS (like food used in the EU prior to May 15, 1997, not being Novel Food) (Anon, 1997, ILSI Europe Novel Food Task Force, 2003). MFC are an integral part of traditional fermented foods. As a significant number of people have consumed these foods for many centuries before 1958, the fermenting microorganisms of these products can be said to
for all food uses. It is the no use of a substance rather than the substance itself that is GRAS, as the safety determination is always limited to its intended conditions of usage. When microorganisms with a safe history in food are employed for a different use or at a significantly higher dosage, a GRAS determination for these new usages is needed. There are three ways to obtain GRAS status for an MFC: 1. A GRAS notification where a person/company informs FDA of a determination that the usage of a substance is GRAS and followed by the receipt of a no-objection letter from FDA Lists of microorganisms and microbial derived ingredients used in foods can be found at the FDA web site (FDA, 2001). As a result of the different ways to obtain GRAS, the FDA lists of GRAS substances are not expected to include all substances, nor all pre-1958 natural, nutritional substances. For a more comprehensive US regulatory update on MFC, we refer to a recent review by Stevens and O’Brien Nabors (2009). In the European Union, the MFCs are considered ingredients and must satisfy the legal requirements of regulation EC no. 178/2002. Consequently, the responsibility for the safe use of microorganisms in food should be ensured by food manufacturers. In 2007, the European Food Safety Authority (EFSA) introduced “Qualified Presumption of Safety” (QPS) for a premarket safety assessment of microorganisms used in food and feed production.
Neurons were recorded in area V4 in two rhesus macaques. Experimental and surgical procedures have been described previously (Reynolds et al., 1999). All procedures were approved by the Salk Institute Institutional Animal Care and Use Committee and conformed to NIH guidelines. See Supplemental Experimental Procedures for further details. Stimuli were presented on a computer monitor (Sony Trinitron Multiscan, TC, 640 × 480 pixel resolution, 120 Hz) placed 57 cm from the eye. Eye position was continuously monitored with
an infrared eye tracking system (240 Hz, ETL-400; ISCAN). Experimental control was handled by NIMH Cortex software (http://www.cortex.salk.edu/). Trials were aborted if eye position deviated more that 1° from fixation. At the beginning of each recording session, neuronal RFs were mapped to determine the approximate spatial extent over which stimuli elicited selleck chemicals a visual response. Monkeys fixated a central point during which each neuron’s RF was mapped using subspace reverse correlation in which Gabor (eight orientations,
80% luminance contrast, spatial frequency 1.2 cpd, Gaussian half-width 2°) or ring stimuli (80% luminance contrast) appeared at 60 Hz. Each stimulus appeared at a random location selected from a 19 × 15 grid with 1° spacing in the inferior right visual field. The orientation Nintedanib in vitro of the Gabor stimuli and the color of all stimuli (one of six colors or achromatic) were randomly selected. This resulted in an estimate of the spatial RF, orientation, and color preference below of each neuron. Recordings were often made from multiple electrodes, and the preferences of units on separate channels did not always match. The stimuli for the main experiment were centered on the estimated
spatial RF of the best-isolated units. The monkey began each trial by fixating a central point for 200 ms and then maintained fixation through the trial. Each trial lasted 3 s, during which neuronal responses to a fast-reverse correlation sequence (16 ms stimulus duration, exponential distributed delay between stimuli with mean delay of 16 ms, i.e., 0 ms delay p = 1/2, 16 ms delay p = 1/4, 32 ms delay p = 1/8, and so on) were recorded. The stimuli were composed of oriented bars (eight orientations) or bar composites (16 orientations × 5 conjunction angles, total of 72 unique stimuli, Figure 1A). These latter stimuli were constructed from the conjunction of three bars at conjunction angles of 0°, 22.5°, 45°, 67.5°, and 90° between the end elements and the center. The five conjunction levels created five categories of shapes. These were enumerated as 0 (zero curvature/straight), 1 (low curvature), 2 (medium curvature), 3 (high curvature), and 4 (C).
These ongoing and planned trials should provide an answer to the questions raised above as to whether targeting Aβ in symptomatic AD patients will GSK J4 molecular weight have any efficacy at all. However, available phase 2 data would suggest if these compounds are going to have disease-modifying effects and improve the course of cognitive decline in this patient population, the effect is going to be quite modest. Although most therapeutic activity
in AD with respect to potentially disease-modifying therapy has focused on anti-Aβ therapies designed to decrease Aβ production or aggregate formation or remove preexisting aggregates, both tau-targeted and more general neuroprotective agents among others are also being developed (Golde et al., 2010 and Salloway et al., 2008). Development of anti-tau therapies has been hindered by a lack of clear insight into what is the optimal desired effect on tau (e.g., decreasing phosphorylation, blocking proteolysis, or preventing aggregation). Though animal modeling studies do provide evidence that Aβ aggregates promote www.selleck.co.jp/products/BIBF1120.html some aspects of tau pathology (Götz et al., 2001 and Lewis et al.,
2001), the precise mechanistic links between Aβ and tau pathologies have not been established, thus hindering not only our ability to appreciate the biological connection but also to develop better animal models and identify druggable therapeutic targets. Neuroprotective strategies are rational approaches but have generally not gained much traction with little progress in terms of new investigational drugs
for AD (Salloway et al., 2008). The reasons for this may stem from (1) a lack of understanding regarding the mechanisms of neural injury, (2) uncertainty regarding the best targets for neuroprotection in AD, (3) the inadequacy of current animal models as exemplified by the relative paucity of neurodegeneration in transgenic mice that are primarily models of amyloid deposition and do not exhibit the full spectrum of AD pathologies, or (4) the poor track record of successful translation of neuroprotective drugs from first the preclinical to clinical phase in other neurological disease such as stroke or any neurodegenerative condition. In any case, if one assumes the temporal sequencing in the Aβ aggregate cascade is correct, then one would predict that anti-tau therapy will be most effective in the very early pathological phases of the disease and not after the stage when robust Aβ deposition, synaptic loss, and neurofibrillary changes have begun. In contrast, general or focused neuroprotective strategies might be efficacious even in such later stages, as there is evidence for continued neuronal demise as the clinical dementia worsens.
Dasatinib purchase (32) and (33). Any treatment suppressing CCNA2 expression would be beneficial in inhibiting tumor growth. In our study, CCNA2 was decreased in HCT-116 cells when treated with PPD in both microarray screening and inhibitors real-time PCR arrays. CCNE2 (cyclin E2), a significant overexpression gene in tumor-derived cells, was downregulated by PPD. Cyclin E2 is reported to specifically interact with CIP/KIP family of CDK inhibitors, and plays a role in cell cycle G1/S transition. The expression of cyclin E2 peaks at the G1-S phase and exhibits a pattern of tissue specificity distinct from that of cyclin E1 (34) and (35). PI3K activation In addition,
although not involved in top 20 upregulated gene list, CDKN1A (p21) was significantly upregulated by the treatment of PPD, which is consistent with previous reports that PPD analogs increased p21 expression in protein level (36) and (37). The p21 binds to all G1/S cyclin-cdk complexes, in preventing the G1-S transition, leading to G1 arrest and inhibiting cell proliferation (38). Our cell cycle and gene expression assays suggested that the PPD-induced G1 cell cycle checkpoint blockage might result from the regulation of a number of gene clusters such as CDKN1A, CCNE2 and CCNA2. An important issue was pathway activation or suppression. In our gene expression analysis, apoptosis regulation, NF-κB, and m-TOR pathways, were transcriptionally activated when treated with PPD. A number of studies have investigated Cell press that these pathways are the crucial and essential in tumor initiation and progression (39), (40) and (41).
General housekeeping, Safety and Maintenance as well other projects/assignments will be assigned by Supervisor/Manager during machine downtime or production shutdown.
5-7 years in Internet related system development for a public-facing internet web site. (Candidate must have prior experience with public-facing/internet web site development)
Experienced Java Developer with knowledge in web framework based development and internet technologies
Strong knowledge of concepts and technology such as Responsive Design, HTML5, CSS3, and mobile web site development is a major plus
Strong oral and written communications skills with ability to create software design and engineering documents
Ability to write complex programs, analyze code changes and make suggestions to improve code complexity for maintainability and scalability.
Ability to reason logically and creatively, identifying problems, drawing valid conclusions from the data available, and developing effective solutions while applying creative thinking in the design and development of high performing web applications
We are looking for a senior consultant that could provide guidance, help build the vision, the business model and the road-map to achieve our ambitious goal. This consultant will bring its expertise and experience to accelerate the learning curve of the team, the identification of meaningful business problems where analytics could have an impact and the development of the platform.
The consultant will work closely with the head of solution business operations leading the initiative.
The Solutions Architect's role is to strategically design and implements information systems and networked software architectures that support core organizational functions, and assure their high availability.
This individual will gain organizational commitment for all technology and software plans, as well as evaluate and select all technologies required to complete those plans.
In addition, the Solutions Architect provides technical leadership across the organization, from strategic decision making down to the project planning level.
Troubleshooting MagicINFO server related issues when requested from the project. Resolve the issue or capture the problem with detail information to escalate it to HQ.
Design and implement MagicINFO solutions for the project. Set-up and configure MagicINFO server for customer’s IT environment.
To support pre-sales, gather customer requirement, identify risks and respond to customer regarding technical questions.
Experience in server side development with Java. Actual coding is not mandatory for this position, but development experience is critical for troubleshooting and support customer technically.
Technical Literacy- Solution architects possess a high level of technical literacy, which helps them determine how a software solution fits into an organization's current structure and assists in the development of specifications and requirements.
Analytical Assessment- This role requires a high level of analysis since solution architects need to examine a client's current systems and determine overall project needs and scope
Communication - solution architects are responsible for communicating with team members, clients, and external vendors, so they exhibit excellent written and verbal communication skills
Problem-Solving- Solution architects need to be excellent problem-solvers, able to quickly change direction based on updated client specifications or system limitations
Database skills such as PostSQL, MS-SQL for troubleshooting or identify the problem. DB setup, clustering, management skill is not required.
Team Leadership- Because solution architects oversee and direct development teams throughout the project development life-cycle, experience with team leadership and motivation is essential
Schedule Management- Solution architects need extensive time management skills to determine development schedules and milestones and ensure that deliverables are completed on time
5+ years of work experience in a relevant role Willingness to work independently an d as part of a team
Note: This job description summarizes the main aspects of the job, but does not cover all the duties that the jobholder may be required to perform. This document is intended to enhance the understanding between the Manager and the employee and may be changed/amended as the job/business needs require.
Professional Salon Concepts is a leading wholesale distributor of professional hair care products servicing licensed hairdressers and salons in Illinois and Indiana. Our vision is supported through exceptional customer service, cutting edge educational programs, a luxury boutique Space location, and a team that is committed to supporting every aspect of the salon business.
Salon Consultant covering the surrounding areas of North Western Indiana to develop and support current and prospective salons. We are looking for a candidate to build longevity with the company. Almost every salon you see is an opportunity for PSC Salon Consultants. We capture the emotional power of a Salon to create an experience for our clients that their customers will remember.
Be able to assist the salon in increasing a salons service dollars and retail dollars through a consultative sales approach.
Be able to work some evenings and weekends as requested by PSC and/or Directors of Sales/Business Development.
Ability to learn technical applications as it relates to Salon, digital signage, televisions, and other audio equipment.
Working closely with the Directors of Sales/Business Development, Sales Support, and Field Leadership on a daily basis targeting prospects and scheduling appointments to sell all PSC products and services.
Additionally, performance will be measured based on New Recurring Revenue generated from appointments set.
The Salon Consultant will be responsible for making an average of at least 10 outbound calls each day.
All quarterly and annual quota objectives must be met or exceeded by setting qualified appointments, making the required outbound sales calls and generating revenue from the appointments set.
The Salon Consultant is responsible for setting “Qualified” appointments. A qualified appointment consists of a scheduled meeting with a decision-maker for a face-to-face discussion about how PSC can help their business. If there is a pattern of no-shows or unqualified appointments, this will be addressed as a performance issue. A minimum of 90% of all scheduled appointments must be qualified and completed.
We are seeking an ambitious, self-motivated sales professional in your area who will sell with integrity and is driven to succeed based upon the PSC culture and systems.
The Registered Nurse –On Call will provide direct patient care services to hospice patients and supportive care to the patient’s family after normal business hours on an on-call basis; manage an interdisciplinary group consisting of Licensed Practical/Vocational Nurses, Certified Nursing Assistants and support staff to enhance the quality of hospice care; and, case manages all assigned patient clinical records.
The Social Worker (MSW) is that member of the interdisciplinary team whose primary function is to provide psychosocial support to the patient/family unit based on an assessment that identifies the needs, goals, interventions and services indicated accordance with the established plan of care and to utilize professional training and personal judgment in monitoring the psychosocial assessment process
Performs psychosocial assessments, case management, education and ongoing evaluation of patients and families.
Completes comprehensive psychosocial assessments within 5 days. Maintains an appropriate clinical record, completing documentation by the next business day.
Develops a written plan of care to provide information and direction to other interdisciplinary team members regarding the provision of social work services.
Documents all findings, needs, problems, measurable goals, plans, interventions, and patient responses in the clinical record.
Communicates plans and changes to the physician and other members of the interdisciplinary team through the care plan, coordination notes, and participation in IDG meetings.
Monitors safety of patient environment, including following reporting policy regarding possible abuse/neglect and suicide, ability of patient and family to adjust to the care of the patient in the home; and the financial impact of patient’s illness upon the family.
Shares areas of expertise and initiates interdisciplinary referrals including community outreach regarding end of life care issues.
Educates team members and patient’s families about community resources and how to access them appropriately.
Participates in training and in-service education and leads training modules relating to SW role in hospice process. This includes training/mentoring of newly hired social workers to include orientation, policy standards and process review.
Assists in the development, implementation, and evaluation of the ongoing quality improvement process.
Acts as the field placement supervisor for social work students including the day-to-day supervision, evaluation, project management and communication/coordination with the university’s field placement coordinator.
Education: Master’s Degree in Social Work from a CSWE School. A degree in Human Services/Social Services may be considered.
Experience: One year experience as a Social Worker in a healthcare and/or hospice setting is required; three to five years preferred.
Equipment/Tools/Work-Aids: Must be able to operate computer, facsimile equipment, copier and cell phone/beeper. Must have and maintain transportation to be used for work.
Knowledge of terminally ill patients and their families along with understanding of hospice and the psychosocial dynamics of illness, loss, and death; ability to apply knowledge of the special needs of hospice patient and families; knowledge of roles of all disciplines providing hospice services; knowledge of community resources; good oral and written communication skills; documentation management.
Personal Traits, Qualities and Aptitudes: Sensitivity to the impact of life and death issues face by individuals with terminal illness; tact in dealing with these patients and their families; ability to support others at a time of crisis; ability to cope with the stress of repeated loss. Must be able to demonstrate flexibility by performing a variety of tasks, often changing from one to another of a different nature without loss of efficiency or composure. Communicate effectively with people of all socioeconomic backgrounds. Responsible functioning in the work place, maturity, cooperation, flexibility, tact in dealing with co-workers and all member of the hospice team.
Specific vision abilities include close vision, distance vision, depth perception, and ability to adjust focus
The Gordmans Sales Associate position is responsible for providing our Guest with a positive shopping experience by delivering elevated Guest Service. The purpose of this position is to assist guest, recovery sales floor, and support the management team in the day to day operations of the store. Associates may be directed to work in several departments including Point of sale.
Acknowledge and greet all guests in a pleasant and courteous manner and ensure all guests receive prompt service in all areas of the store.
Contribute to the overall effectiveness of the stockroom operations by unloading trailers, preparing merchandise for sales floor.
Understand basic guest inquiries regarding merchandise location and price including sale items, payment methods and related inquiries.
Must be knowledgeable and follow all Point of Sale register procedures including communicating the benefits of our company incentive programs; i.e. Credit, Synaps and g-Rewards.
Ensuring high levels of Guest satisfaction by being knowledgeable on all merchandise. And working in a team environment to provide excellent sales and service
Ability to stand and continuously perform essential job functions for up to an eight and one half hour shift.
Basic Mathematical skills, including addition and subtraction, necessary to verify payments and unit counts.
SPEAKING: Must be able to communicate effectively with the corporate office, subordinates, job applicants and customers.
Job Description Summary The Chemical Operator- Floater role requires the incumbent be flexible as s/he will be reassigned to different shifts as required to meet customer demand. This position is located in Valparaiso, IN. Monitor and review information from materials, events, or the environment, to detect or assess problems.
Patrol work areas to detect leaks or equipment malfunctions or to monitor operating conditions. Monitor gauges, recording instruments, flowmeters, or products to ensure specified conditions are maintained. Use either control mechanisms or direct physical activity to operate machines or processes.
Adjust controls to regulate temperature, pressure, feed, or flow of liquids or gases and times of prescribed reactions, according to knowledge of equipment and processes. Observe safety precautions and display leadership by taking responsibility for his/her safety and that of others. Analyze information and evaluate results to choose the best solution and solve problems.
Use knowledge of working area to make accurate and timely decisions. Agile and supportive of a culture of process improvement and problem solving. Demonstrate an openness to trying new approaches.
Observe, receive, and obtain information from all relevant sources. Record operational data, such as temperatures, pressures, ingredients used, processing times, or test results. Test product samples for specific gravity, chemical characteristics, pH levels, concentrations, or viscosities or send them to laboratories for testing.
Inspect equipment, structures, or materials to identify the cause of errors or other problems or defects. Drive results by demonstrating persistence and motivation to overcome obstacles and challenges. Provide information to supervisors and coworkers by telephone, in written form, e-mail, or in person.
Maintain customer focus and understands what internal and/or external customers need. Collaborate and generously share information and knowledge with others. Perform other duties as assigned.
Required Qualifications/Skills High School Diploma or equivalent Preferred: Prior experience in an Industrial Operations Environment or Military Engineering Knowledge of raw materials, production processes, quality control, costs, and other techniques for maximizing the effective manufacture and distribution of goods. Controlling operations of equipment or systems.
Demonstrates persistence and motivation to overcome obstacles and challenges. Consistently delivers goals and objectives. Plans, prioritizes and organizes work to deliver on time within given work time.
Takes responsibility for personal choices, decisions and behaviors. Contributes to a positive work environment; acts as a reliable and dependable member of the team. Willing to take on new challenges; takes steps to adapt thinking and behavior to support change.
Safety and Quality Minded. High Integrity and Detailed Oriented. Dependable, Cooperative, Team Player.
We offer a competitive salary and benefits package including 401(k), medical, dental, vision, prescription drug, tuition reimbursement, and a vacation package. At Lonza, we believe our employees are our greatest resource. Lonza is an equal opportunity employer.
All qualified applicants will receive consideration for employment without regard to race, religion, color, national origin, sex, sexual orientation, gender identity, age, status as a qualified individual with disability, protected veteran status, or any other characteristic protected by law. Lonza. The place to Go, Stay and Grow.