Revision for my CAD exam:
Recognise it? :fed001: I hope to finish the rest of it at some point
Holy shet is that a manta ray?! 
maybe…
Is this what u trying to make?
You guys are mean That took me forever to make!
millan OP…
That’s all I can do:
It’s the matrix! 
Tillo OP, this is all I can do…
A. Abstract
Systemic lupus erythmatosus is an autoimmune B cell disease where critical system organs are attacked by autoreactive germinal center B cells. No cure exists, but there are several mechanisms of action that are proposed that attempt to explain the cause of autoreactive B cells. One such mechanism is damage to the CD84/Ly108 genes, where the loss of tolerance stems from this damage. Another such mechanism is the expression of the Sle1b gene in mice, where SLE symptoms manifest after the expression of this gene.
We propose a study in where we combine both viral gene therapy of CD84/Ly108 and the viral knockdown/interference of the Sle1b gene as a potential treatment method or cure of SLE. We will investigate the effects of concurrently reintroducing functional CD84/Ly108 and interfering with the function of Sle1b in lpr mice, and quantify the manifestation of tolerance and SLE within these mice.
B. Historical Review
The discovery of lupus can be divided into three stages: the classical, neoclassical, and modern periods (1). The classical and neoclassical periods saw gradual development from a mere clinical description of cutaneous lesions by the thirteenth century doctor Rogerius to the deduction that these lesions along with other symptoms were indicative of a systemic problem by the nineteenth century doctor Kaposi (2). Finally, the modern period arrived after advances in the cellular and microscopic observation of lupus, such as immunofluorescent tests for antinuclear antibodies (ANA) (3). It is known that lupus is a autoimmune disease, where autoreactive B cells are activated/fail to undergo negative selection. These autoreactive B cells synthesize reactive autoantibodies and cause inflammation, pain, and damage on a systemic level (4).
One of the most defining characteristics of lupus is the highly elevated antinuclear antibody (ANA) levels of a patient. While other symptoms like rashes, lesions, and systemic damage can indicate an autoimmunity disorder, elevated ANA titers in combination with these visible symptoms is a classic diagnosis of lupus (4). There are three different forms of lupus: systemic lupus erythematosus (SLE), drug-induced lupus erythematosus (DILE), and cutaneous lupus erythematosus (CLE) (5). DILE is caused, unsurprisingly, by certain prescription drugs. Symptoms of DILE are very similar to those of SLE, but they rarely affect major systemic organs. Drugs most commonly associated with the development of DILE are hydralazine, procainamide, and isoniazid (5). DILE is an acute disorder, where symptoms will typically disappear within six months. CLE is a readily visible chronic form of lupus, where symptoms only manifest on the skin. Symptoms of CLE include the lesions and rashes indicated by classical and neoclassical doctors. A small percentage - approximately 10% - of people who suffer from CLE will develop SLE in their lifetimes (5). SLE is a chronic disease, where symptoms can range from mild to severe - encompassing pulmonary hypertension, kidney inflammation, atherosclerosis, and nervous system inflammation (5). Systemic lupus is by far the most common form of lupus, and is potentially the most problematic, as it may affect critical organs within patients. SLE is the most widely studied form of lupus, with mouse models emulating human lupus.
There is currently no cure for lupus. Even though the disease was first observed in the thirteenth century, the exact mechanism of action for lupus is largely unknown. On a cellular level, Arce et al. (2001) determined that there was an abnormality with B cell counts in children suffering from SLE. They found that naive and memory B cells are ~90% reduced, and that plasma cell precursors are expanded 3-fold. Interestingly, they also determined that the predominant blood B cell subset within these SLE children are pre-germinal center B cells (6). Murakami et al. (1995) also have determined that autoimmune symptoms within mice were capable of being prevented by the elimination of peritoneal B1 cells. They subjected the B1 cells within neonatal autoimmune-prone mice to hypotonic shock, eliminating them, and found that by selectively eliminating these peritoneal B1 cells, the symptoms of autoimmunity were suppressed. Obviously speaking, this is by no means a cure since B1 cells self-renew in the peritoneal cavity, but this study was central in determining the possible role of B1 cells in B cell autoimmunity (7). As a secondary result, Murakami et al. also noted that this hypotonic therapy significantly decreased ANA titer levels in the sera of these mice, and also reduced the pathological changes in the mices’ kidneys. Other studies like Grammer et al.'s (2003) have determined that spontaneous germinal center response is another potential cause of lupus manifestation in human patients (8).
Biochemically speaking, several mechanisms of action have been proposed for the failure of tolerance within these cells. Ma et al. (2015) have proposed that B-cell intrinsic toll-like receptors (TLRs) play a central role during lupus pathogenesis in mouse models and human patients. They proposed that TLR2, 4, 7, 8, and 9 are the main receptors that have malfunctioned, and are binding to self-antigen and are erroneously activating these B cells to react against the self (9). Other studies found that CD40 is key receptor in the erroneous activation of germinal center lupus-prone B cells. Grammer et al.'s (2003) same study also proposed this role of CD40 interactions. The found that by deliberately blocking CD40 interactions on peripheral B cells in vitro, the cells spontaneously proliferated and secreted antibodies that were self-reactive (8). Similar findings were indicated by Wang et al.'s (2002) study, where they found that anti-CD40 ligand coupled with CTLA4 antibody signifcantly suppressed the onset of SLE in lpr mice. However, this study further examined the role of CD40 by determining the mechanism of effect. They found that their therapy partially suppressed B cell class switching and also decreased the frequency of mutations in the VHBW-16 gene, which is expressed by pathogenic ANAs (9).
The causes of SLE on a genetic level are still very elusive. Beginning in 1971, Grumet et al. and Waters et al. determined that HLA histocompatibility factors could provide a genetic predisposition to SLE (10,11). They determined concurrently that mutations in the MHC gene within B cells significantly raised the chances of developing SLE in mice. However, the exact cause of mutation and subsequent onset of SLE is still unclear, since there is extensive linkage disequlibrium within the MHC gene (12). Other genetic predispositions raise the chances of developing SLE, including classical complement components (13, 14), Fc gamma receptor mutations (15), and the STAT4 gene (16). Some genes have also been identified within mouse models that, when activated, develop SLE symptoms. One such gene is Sle1b (17), where upregulation of this gene was demonstrated to cause the manifestation of SLE within lpr mice (18).
C. Hypothesis and Specific Aims
Previous research has attempted to find a way to develop a cure based on the suppression and upregulation of these genes involved in SLE. Wong et al. (2015) manipulated the CD84/Ly108 genes and forced lpr mice to overexpress those genes. Remarkably, they found that the overexpression of these genes led to a significant reduction in the spontaneous germinal center response and autoantibody production observed by Grammer et al. (8, 19). Additionally, another study conducted by Wong et al. (2012) found that an altered Sle1b gene within female mice that is upregulated will spontaneously provoke a germinal center response, where the response is specifically to generate ANA specific antibody forming B cells (20). To further reinforce the idea that CD84 plays a central role in the generation of an appropriate germinal center B cell response, Cannons et al. (2010) found that CD84 is necessary for prolonged T cell:B cell interactions, and is needed for optimal germinal center formation in vivo. Moreover, they found that Ly108 is another T cell:B cell adhesion protein that has synergistic activity with CD84 (21).
From all of these recently found interactions in germinal center B cells, it is unknown whether a coordinated effect of both reintroducing CD84/Ly108 functionality and disruption of Sle1b activity would provide an effective treatment for SLE. One of the major milestones that I hope to achieve in this proposed research would be the development of a viable genetic therapy system for SLE, where modified vectors could deliver functional and healthy SLE-susceptible genes to germinal center B cells. The central goal of this gene delivery method would be to ensure the restoration of germinal center B cell tolerance. In observing the restorative effects of reintroducing functional CD84 and Ly108 to B cells, and the suppressive effects of interfering with Sle1b’s activity on the onset of SLE, I hypothesize that the reintroduction of functional CD84 and Ly108 genes into autoreactive germinal center B cells within lpr mice via viral payload coupled with the therapeutic suppress of Sle1b via RNAi should restore germinal center B cell tolerance and mitigate the symptoms of SLE.
Specific Aim 1: Preparation of lpr mice via knockout of CD-84 and Ly108 in mature mice GC B cells. Since lpr mice already have the Sle1b gene encoded, no splice-in of Sle1b genes are necessary. However, we must knock out the CD84 and Ly108 genes within the mice to verify that the germinal centers of the mice will be spontaneously active. By knocking out these genes, we can confirm the spontaneous activation of germinal centers that Grammer et al. (2010) observed. The knockout of these genes is necessary to provide a “blank slate” for the introduction of functional CD84/Ly108 genes.
Specific Aim 2: Develop and deliver modified adenovirus with tetracycline-inducible CD-84/Ly108 plasmid payload and Sle1b RNAi. A viral delivery system will ensure the specificity of our Tet-inducible payload. Since the adenovirus has been a long-favored vector of delivery (CITATION NEEDED), we will use a transformed version of that to deliver our functional payload. The tetracycline inducibility is necessary to manipulate CD84/Ly108 expression. In short, we will be able to manually activate these genes to ensure that the gene needs to be overexpressed in order to restore tolerance. Obviously, we must also verify the success of the adenovirus payload. This will be elaborated on later.
RNAi of Sle1b will need to be inserted manually. Upon encounter with the transcription of Sle1b, our engineered RNAi will interfere with the effects of this gene and will stop the effects of this gene from occurring. Verification steps will be taken to ensure that the product of Sle1b is being inhibited by the RNAi.
Specific Aim 3: Measure effects of CD84/Ly108 activation and Sle1b interference. The final aim of our study is to measure the effects of the combined therapy. The key to this specific aim is to concurrently activate the CD84/Ly108 and simultaneously introduce the RNAi into the model. After some time, we will measure the effects of this activation by comparing ANA titer levels before activation to after activation. In addition, verification of levels of pre germinal center B cells within the blood will be compared and cross referenced with the findings of Arce et al. (2001) (6). Immunofluorescent flowcytometry will also be used to quantify autoreactive B cell levels within the mice after treatment.
D. Research Design and Methodology:
The major, central aim of this research project is to verify the success of aggressive therapeutic methods in treating - and possibly even curing - SLE. Similar to aggressive AIDS treatments (CITATION NEEDED), this research will combine two known possible methods for suppressing autoreactive B cells and permanently restoring tolerance to these cells. In order to do so, we propose to develop an effective delivery system for functional restorative SLE-susceptible genes, and pair it with aggressive RNAi knockdown of Sle1b.
Specific Aim 1: Preparation of lpr mice via knockout of CD-84 and Ly108 in mature mice GC B cells.
Rationale: Logically speaking, lpr mice will have deliberately mutated CD84 and Ly108, and will contain the Sle1b sublocus. Knockout of CD84 and Ly108 will ensure that the germinal center will not appropriately form as Cannons et al. (2010) states, and that the knockout will ensure the manifestation of lpr. Nothing will need to be done to the Sle1b sublocus in this specific aim, as it will be targeted for interference later on.
The lpr mice will need to be fully developed, as knockout of these genes in embryonic stages will lead to the improper development of the fetus (CITATION NEEDED). However, knockout of these genes in fully developed mice will not lead to problems (CITATION NEEDED).
Experimental design: Fully mature, unmodified custom B6.Cg-Sle1NZM2410/Aeg Yaa/DcrJ mice will be purchased from The Jackson Laboratory and will be bred in house (22). These mice will have the loxP promoter inserted into the CD84/Ly108 regions per our custom order. We will then purchase Cre recombinase from New England Biolabs, and excise the targeted regions within the mice (23).
To verify the success of this, we will utilize an αCD84 and αLy108 monoclonal marker antibody and expose biopsied lpr mice lymph node tissue to it in vitro. These monoclonal antibodies will be custom ordered from Neo Scientific (24). One biopsy/exposure will occur before the Cre knockout, and a second biopsy/exposure will occur after the Cre knockout. The levels of fluorescence will be quantified using a flowcytometer, and will determine the success of the knockout process.
Specific Aim 2: Develop and deliver modified adenovirus with tetracycline-inducible CD-84/Ly108 plasmid payload and Sle1b RNAi.
Rationale: In order to specifically reintroduce functional and manually activatable CD84/Ly108 into only B cells, a specific vector must be used. Classically speaking, the best specific vector to use would be a modified, non-reproducing adenovirus transformed to bind to B cell receptors. This custom adenovirus will then be able to splice in functional CD84 and Ly108 into the mices’ B cells. The reason for a tetracycline promoter is so that we can manually control the activation of these genes manually. By manipulating the activation of these genes, we can ascertain the restoration of tolerance within germinal center B cells as Wong et al. (2015) so suggest (19).
The second part of this specific aim is to develop a second adenovirus type that carries the gene necessary for synthesis of RNAi molecules for Sle1b. Injection of this second adenovirus type will ensure the knockdown of the Sle1b gene.
Experimental design: Adenoviruses containing Tet-inducible and functional CD84/Ly108 genes will be custom-ordered from Vector BioLabs (25). These viruses will be injected into the experimental lpr mice group. Verification of success of reintroduction will be done with biopsies and immunofluorescent flowcytometry, similar to the verification process in Specific Aim 1. The control group of lpr mice will not be injected with any adenoviruses whatsoever.
To introduce RNAi into the mice, we will also order adenoviruses containing the DNA sequence for synthesis of Sle1b RNAi from Vector BioLabs. Very similarly, we will inject these adenoviruses into the experimental group of lpr mice and confirm success of introduction via immunofluorescent flowcytometry. The control group of lpr mice will also not have any of these adenoviruses injected into them. To keep from cross-contamination, we will separate the control and experimental groups from each other in clinical conditions. Each group will be kept in quarantine under sterile conditions.
Specific Aim 3: Measure effects of CD84/Ly108 activation and Sle1b interference.
Rationale: We must then turn on the CD84/Ly108 genes and the Sle1b RNAi interference gene. We must verify the success of this activation by quantifying the ANA levels of the mice before activation and after activation. A comparison between the ANA levels of the mice between the experimental and control groups is also necessary for us to be able to statistically determine the effectiveness of our therapy.
Experimental Design: We will purchase tetracycline from Sigma-Aldritch (26) and dissolve within saline solution. We will then inject both groups of mice with this tetracycline solution to activate our functional genes. After a period of time (SPECIFIC DELTA T NEEDED), we will then extract blood serum from both groups and perform a serial dilution to verify ANA titer levels. In addition, we will purify B cells from the blood serum and statistically examine the types using immunofluorescent flowcytometry. These results will be compared to those of Arce et al. (6).
E. Interpretation and Implications:
The range of results within this study will depend on the results of the Specific Aim experiments before it. Should the knockout of the CD84/Ly108 work, then the fluorescence levels of knockout GC B cells should be significantly lower than those of GC B cells from control group mice. Similarly, if the reintroduction of functional restorative CD84/Ly108 work, then the fluorescence levels of “repaired” GC B cells should be significantly higher than those of the control group. Very similarly, the fluorescence levels of cells with Sle1b RNAi gene should also be significantly higher than those of the control group.
Finally, to quantify the success of our therapy, we can expect for significantly lower ANA titer levels in “repaired” B cells in the experimental group than in the control group. Even though the control group was also exposed to the tetracycline, it should not have any effect on the ANA titer levels. Similarly, we can expect to see a significantly lower amount of pre-germinal center B cells in our analysis of B cell composition in experimental group blood samples.
These results, if they are obtained, would support our hypothesis of the effectiveness of our gene therapy. In the context of the original problem, our supported hypothesis indicates that this is a possible avenue of aggressive therapy for SLE. Similar to AIDS therapy, this new SLE therapy would potentially help treat and possibly even cure SLE, assuming results are supportive.
Other experiments that can be conducted based on the results of this study is to develop a viral gene therapy system for MHC - another SLE-susceptible gene. This follow-up study would not be able to use adenoviruses, however, seeing as the maximum capacity for a gene within an adenovirus will not be able to fit the large MHC gene. Thusly, an alternative delivery method will be required to make this gene therapy possible.
References
1. Lupus.org. What is the history of lupus? Lupus Foundation of America 2013. Web. 10 Nov 2015.
http://www.lupus.org/answers/entry/what-is-the-history-of-lupus
2. Kaposi M. 1872. Neue Beitrage zur Keantiss des lupus erythematosus. Arch Dermatol Syphilol. 4: 36.
3. Moore J.E., Lutz W.B. 1955. The natural history of systemic lupus erythematosus: An approach to its study through chronic biological false positive reactions. J Chron Dis. 2: 297.
4. lupusny.org. What IS Lupus and How Does It Affect the Body. SLE Lupus Foundation 2013. Web. 10 Nov 2015.
http://lupusny.org/news/foundation-news/2013/01/18/what-lupus-and-how-does-it-affect-body
5. Lupus.org. Are there various forms of lupus? Lupus Foundation of America 2013. Web. 10 Nov 2015
http://www.lupus.org/answers/entry/forms-of-lupus
6. Arce E., Jackson D.G., Gill M.A., Bennett L.B., Banchereau J., Pascual V. 2001. Increased Frequency of Pre-germinal Center B Cells and Plasma Cell Precursors in the Blood of Children with Systemic Lupus Erythematosus. J Immunol 167.4: 2361 - 2369.
7. Murakami M., Yoshioka H., Shirai T., Tsubata T., Honjo T. 1995. Prevention of autoimmune symptoms in autoimmune-prone mice by elimination of B-1 cells. Int Immunol 7.5: 877-882.
8. Grammer A.C., Slota R., Fischer R., Gur H., Girschick H., Yarboro C., Illey G.G., Lipsky P.E. 2003. Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J Clin Invest 112.10: 1506-1520.
9. Ma K., Li J., Fang Y., Lu L. 2015. Roles of B Cell-Intrinsic TLR Signals in Systemic Lupus Erythematosus. Int J Mol Sci 16.6: 13084-13105.
10. Grumet FC, Coukell A, Bodmer JG, Bodmer WF, McDevitt HO. 1971. Histocompatibility (HL-A) antigens associated with systemic lupus erythematosus. A possible genetic predisposition to disease. N Engl J Med 285:193–196.
11. Waters H, Konrad P, Walford RL. 1971. The distribution of HL-A histocompatibility factors and genes in patients with systemic lupus erythematosus. Tissue Antigens 1:68–73.
12. Ramos P.S., Brown E.E., Kimberly R.P., Langefeld C.D. 2010. Genetic Factors Predisposing to Systemic Lupus Erythematosus and Lupus Nephritis. Semin Nephrol 30.2: 164-176.
13. Agnello V, De Bracco MM, Kunkel HG. 1972. Hereditary C2 deficiency with some manifestations of systemic lupus erythematosus. J Immunol 108: 837–840.
14. Hauptmann G, Grosshans E, Heid E. 1974. Lupus erythematosus syndrome and complete deficiency of the fourth component of complement. Boll Ist Sieroter Milan 53
15. Brown EE, Edberg JC, Kimberly RP. 2007. Fc receptor genes and the systemic lupus erythematosus diathesis. Autoimmunity 40: 567–581.
16. Remmers EF, Plenge RM, Lee AT, Graham RR, Hom G, Behrens TW, et al. 2007. STAT4 and the risk of rheumatoid arthritis and systemic lupus erythematosus. N Engl J Med 357: 977–986.
17. Jax.org. Sle1bNZM2410/J. Mouse Genome Index 2015. Web. 11 Nov 2015.
http://www.informatics.jax.org/allele/MGI:2154451
18. Wong E.B., Khan T.N., Mohan C., Rahman Z.S.M. 2012. The Lupus-Prone NZM2410/NZW Strain–DerivedSle1bSublocus Alters the Germinal Center Checkpoint in Female Mice in a B Cell–Intrinsic Manner. J Immunol 189.12: 5667-5681.
19. Wong E.B., Soni C., Chan A.Y., Domeier P.P. et al. 2015. B Cell–Intrinsic CD84 and Ly108 Maintain Germinaln Center B Cell Tolerance. J Immunol 194.4: 4130-4143.
20. Wong E.B., Khan T.N., Mohan C., Rahman Z.S.M. 2012. The Lupus-Prone NZM2410/NZW Strain–DerivedSle1bSublocus Alters the Germinal Center Checkpoint in Female Mice in a B Cell–Intrinsic Manner. J Immunol 189.12: 5667-5681.
21. Cannons J.L., Qi H., Lu K.T., Dutta M., et al. 2010. Optimal Germinal Center Responses Require a Multistage T Cell:B Cell Adhesion Process Involving Integrins, SLAM-Associated Protein, and CD84. J Immuni 10: 253-265
22. Jax.org. B6.Cg-Sle1NZM2410/Aeg Yaa/DcrJ Mice. The Jackson Laboratory 2015. Web. 11 Nov 2015
https://www.jax.org/strain/021569
23. Neb.com. Cre Recombinase. New England BioLabs Inc. 2015. Web. 12 Nov 2015
https://www.neb.com/products/m0298-cre-recombinase
24. Neobiolab.com. Monoclonal Antibody Services. Neo Scientific 2015. Web. 12 Nov 2015.
http://neobiolab.com/monoclonal-antibody
25. vectorbiolabs.com. Ad-HQ Adenovirus Construction Services. Vector BioLabs 2015. Web. 12 Nov 2015
Tillo OP, this is all I can do…
I told you… Sports are not worth it!
Test drugs on pplz!
Figure out and create LSD v2 or something.
*lest keep throwin some BS thingies*
I can also do this… kinda
Ahh work, don’t we all love it 
pshhhh, please…
classified
Seems similar to the shape of Jaguar model, frontal part.
pshhhh, please…
classified
there is a button that shows you full BBcode, dont have to open spoilers.
I told you… Sports are not worth it!
Test drugs on pplz!
Figure out and create LSD v2 or something.
*lest keep throwin some BS thingies*
I can also do this… kinda
Tillo pls you know I can’t do that. And plus, I want free football tickets!
And here, have some more vaguely science-y stuff - this time, a BS paper review I did during the hour before it was due:
A soluble form of B cell maturation antigen, a receptor for the tumor necrosis factor family member APRIL, inhibits tumor cell growth. - A review
In this study, Rennert et al. demonstrated that they were capable of interfering with proliferation-inducing ligands (APRILs) within cancerous B cells. To achieve this, they produced B cell maturation antigen (BCMA) which was artificially introduced to female SCID mice with B cell tumors. They discovered that BCMA receptors naturally bound to APRILs. Upon binding to APRILs, the cancerous B cells would proliferate very quickly; they suggest is one of the autocrine factors in rapid cancerous cell proliferation is this APRIL binding to BCMA receptors. Consequently, they discovered that upon introduction of tagged APRIL to these cells, the cells displayed a 30-40% increase in proliferation rates. In addition, injection of a strong competitive inhibitor for BCMA (termed BCMA-Fc) into SCID mice that had these cancer cells xxxx tumor growth signficantly, where the growth slowed to about two thirds volume.
The most important line of evidence that Rennert et al. discovered was the highly significant reduction in tumor growth rates upon the introduction of BCMA-Fc. By measuring tumor size and tagging the inhibitor, they quickly found that the BCMA receptor had a much higher affinity for the synthetic inhibitor than APRIL. Thusly, BCMA-Fc was found to interfere with autocrine APRIL growth signals. Another line of evidence that supports this claim is when they analyzed supernatant from cells that expressed tagged APRIL and BCMA-Fc. Interestingly, the supernatant contained a majority of APRIL, which supports their claim of BCMA-Fc having a higher affinity than APRIL.
Interesting secondary evidence that Rennert et al. saw were the binding tendencies of BCMA-Fc in the presence of membrane-bound APRIL and a costimulator BAFF. BCMA binds more strongly to APRIL than to BAFF, which suggests that while BCMA are both receptors for BAFF and APRIL, specificity of interaction is probably at work, where BCMA prefers APRIL.
The most important implication of these findings is that APRIL’s growth-stimulatory capability in transformed cancerous B cells suggests that APRIL plays a significant and potentially critical role in tumorigenesis. This is supported by the apparent interference of tumor growth rates with the injection of synthetic inhibitor BCMA-Fc. Future research should definitely include manipulation of this signaling pathway with inhibitory ligands similar to BCMA-Fc. This research should also include the tracking of tagged APRIL within tumors to discover potential modes of attacking cancerous B cells.
there is a button that shows you full BBcode, dont have to open spoilers.
I know, but some people might fall for it At least for a couple of clicks… I know I did when I first saw this!!
I know, but some people might fall for it
i just did it to see the wonderfull piramid effect looks awesome (it even looks like it has shadows)
Yeah, well, can any of you do this?
Find all differentiable functions 
such that
For all real numbers x and all positive integers n.
You guys are all OP, this is all I could do:
A B C…X Y Z
1+1 = 2
2+2 = 4
You guys are all OP, this is all I could do:
Should we nerf them?