omega-3 fatty acids in suppressing atrial fibrillation
Paper instructions:
1) Appropriately focuses topic on a specific area. Describes one or more aspects of molecular pathogenesis of disease in more detail.
2) Prepares clear and well-organized outline, using appropriate format, correct spelling, etc.
3) Outlines recent advances in the field/subject/area: for example, new approaches, models, drug trials, mechanistic insights. Describes one or more research studies or clinical trials from literature.
4) Add five new annotated references from good quality sources.
—Annotation of the references (#4 above) should address the following questions:
• What is the purpose or hypothesis of the study and why was it important to test?
• What specific techniques / reagents / strategies are used to test the hypothesis?
• What are the major conclusions of the study?
• What is the significance?
2014-2015 Syllabus
Appendix D : PRE – PRESENTATION OUTLINE PART B WITH ANNOTATED REFERENCES
An example of a Pre-Presentation Outline Part B with Annotated References is given below .
Note that your outline should be more polished with additional details and more in-depth
descriptions of the underlying basis of disease than that presented in the Pre -Presentation
Outline Part A. At least five references should be included. To annotate each reference,
provide the following information under each reference:
x What is the purpose or hypothesis of the study and why was it important to test?
x What specific techniques / reagents / strategies are used to test the hypothesis?
x What are the major conclusions of the study?
x What is the significance?
The outline should be in the “bullet” format as show n below in the example.
PRE – PRESENTATION OUTLINE AND ANNOTATED BIBLIOGRAPHY EXAMPLE
Title: Stevens-Johnson Syndrome
Presenter: Ben Chen
Etiology-Molecular Understanding:
x Stevens-Johnson Syndrome, or SJS (and the more serious form of the disease, toxic epidermal
necrosis, or TEN), are considered type IV hypersensitivities (delayed T- cell mediated
hypersensitivities).
x SJS’s patho -mechanism involves HLA (human leukocyte antigens) – restricted presentation of a
drug or its metabolites for T-cell activation, specifically the activation of cytotoxic T lymphocyte
(CD 8+) responses as well as natural kil ler (NK) cells; this mechanism is strongly supported by
genetic associations between certain HLA alleles and specific drug -induced forms of SJS.
x Because the number of infiltrating inflammatory cells is too few to explain the widespread
epidermal necrosis in SJS, other key mediators have been demonstrated in the disease, primarily
Fas-FasL, perforin/granzyme B, and primarily, granulysin.
Genetic Markers and Predisposition to SJS:
x The highly variable HLA polymorphism provides diverse interaction between different kinds of
drug antigens.
x The strong association between HLA alleles and SJS (and subsequent ethnic specificity) was first
found in a study of Han Chinese in 2004, with 100% of carbamazepine (CBZ) -induced SJS patients
carrying the HLA -B *1502 allele; this allele is relatively absent from northeast Asians and
Caucasians and seems uniquely limited to Han Chinese ancestral Asians.
x Other studies of southeast Asian populations have shown high prevalence of this seemingly
ethnic -specific allele in inciden ces of CBZ- induced SJS (another 100% found in a study of Thais).
x For allopurinol -induced SJS, HLA -B *1501 is a genetic marker that has shown strong association
in studies of Taiwanese and Thais. Once more, an ethnic component has been implicated, with
the allele showing low frequency in Caucasians.
x For abacavir -induced SJS, HLA -B *5701 has been shown to be an excellent predictor of sensitivity
Date Created/Revised: __ 7/24/14__ By: _______ Page 31
2014-2015 Syllabus
amongst Caucasians (with those of Asian ancestry being rare carriers)
Key Initiators and Mediators of SJS
x Three ma jor initiators have been implicated in inducing the immune reactions in SJS: drug
antigen/metabolite, HLA genetic predisposition, and TCR (T -cell receptor) repertoire.
x Two hypotheses explaining how the drug is presented to the PCR have been proposed:
a.) Hapten/prohapten hypothesis proposed that the drug covalently bonds to a peptide carrier and this
complex is then presented by HLA to the TCR; the main problem with this hypothesis is its failure to
explain how some causative drugs induce SJS without cellu lar processing.
b.) P -i (direct pharmacological interaction) hypothesis suggests the drug metabolite or peptide-loaded
HLA directly interacts with the TCR. This proposal is now widely accepted.
x The basic effectors inducing extensive keratinocyte apoptosis in SJS are Fas -FasL,
perforin/granzyme B, and granulysin.
a.) Fas -FasL has been the most extensively studied. FasL has been shown to be presented on
keratinocytes in SJS patients. Furthermore, sFasL is in high levels in the serum of SJS patients. A pop tosis
of cultured keratinocytes was induced by adding high levels of sFasL containing serum isolated from a
SJS/TEN patient, and blocked by addition of anti-FasL monoclonal antibody.
b.) Granzyme B is in high concentrations in SJS/TEN blister fluid. It is proposed that perforin produced by
activated CTL and NK cells lead to the eventual apoptosis of keratinocytes by granzyme B and the
activated caspase cascade. Studies have also shown cytotoxicity from the mononuclear cells can be
blocked by inhibitors of perforin/granzyme B.
c.) Granulysin, as an effector released by CTL, has been recently been implicated as the primary
mediating culprit in keratinocyte apoptosis in SJS.
Current Research
Granulysin demonstrated as the major factor of keratinocyte apoptosis in SJS
ႛ In SJS/TEN blister cells, RNA expression of granulysin is markedly increased and
importantly, the level of granulysin proteins was also much higher (by the order of 2 to 4
times) than the protein levels of perforin, granzyme B and sFasL.
ႛ A purified 15 kDa granulysin showed significant toxicity in vitro, whereas sFasL, perforin,
and granzyme B concentrations in the SJS/TEN blister fluid had minimum cytotoxicity in
in vitro studies.
ႛ An injection of granulysin into mouse skin resulted in a SJS -like ski n necrosis.
Mouse model
x Researchers have developed a novel human -oriented SJS/TEN mouse to promote
Date Created/Revised: __ 7/24/14__ By: _______ Page 32
2014-2015 Syllabus
diagnostic and therapeutic approaches to human treatment.
x Peripheral blood mononuclear cells (PBMC) from SJS patients were injected into
immunocompromised m ice followed by oral administration of the causative drug. Other
immunocompromised mice had skin grafts of patients who had recovered from SJS and
were then given the patient derived PBMC and the causative drug.
Novel/Controversial Therapies
Systemic Corticosteroids
x Despite lack of strong evidence that would support their use, systemic corticosteroids
have been used for years for the purpose of reducing inflammation in SJS patients.
Suggested mechanisms include stabilization of lysosomal membranes, the suppression
of prostaglandin synthesis, the inhibition of the transcriptions of pro -inflammatory
cytokines , and reducing circulating CD 4+cells.
x Of studies published the last 15 years, none have shown increased mortality when
treated with systemic co rticosteroids.
x One study evaluating the efficacy of corticosteroids on those patients who had the more
severe toxic epidermal necrosis (TEN) had the troubling result of doubled mortality
(66%) to (33%) compared to TEN patients receiving supportive treatment alone; 15
patients were involved in each test group.
x In another study, visual acuity and ocular involvement significantly improved after
instituting systemic corticosteroids, especially when administered within 5 days of acute
symptom onset.
Cyclosporin/Cyclophosphamide
x Cyclosporin has been suggested as a treatment for SJS by inhibiting CD8 + T cells and
subsequent epidermal destruction.
x Studies have shown profound, favorable results from using
cyclosporin/cyclophosphamide based on re -epithelialization time, disease progression,
organ failure, and death.
Intravenous Immunoglobulin (IVIG)
x IVIG has been used to treat SJS under the notion that the antibodies would saturate the
Fas (CD95) binding site and prevent keratinocyte apoptosis by the Fa s- FasL interaction.
x No definitive studies have been performed to address outcomes of SJS from IVIG
treatment, although small trials have shown promise.
Annotated References
Chung WH, Hung SI, Yang JY, et al. Granulysin is a key mediator for disseminated keratinocyte death
in Stevens-Johnson syndrome and toxic epidermal necrolysis. Nat Med. 2008;14(12):1343 -50.
Date Created/Revised: __ 7/24/14__ By: _______ Page 33
2014-2015 Syllabus
x Purpose or hypothesis: Examining the role of granulysin as a mediator of keratinocyte apoptosis
in SJS and contrasting its role and importance with other noted mediators of SJS apoptosis,
including Fas-FasL and perforin/granzyme B. The article also attempts to reason why these
latter two mediators are inadequate factors in explaining the widespread keratinocyte apoptosis
found in SJS.
x Specific t echniques / reagents / strategies used to test the hypothesis : Quantitative PCR and
immunohistochemistry staining to measure granulysin RNA levels in SJS blister cells;
concentration measurements of certain proteins in SJS blister fluid by ELISA; direct injection of
granulysin purified from SJS blister fluid into mouse skin.
x Major conclusions: Granulysin is the most highly expressed cytotoxic molecule in blister cells
from SJS skin lesions; it also has the highest concentration (specifically a 15 kDa protein) in
blister fluid (up to 2 to 4 times) than other cytotoxic molecules. Injection of purified granulysin
into mouse skin yields an SJS -mimicking skin lesion.
x Significance : Although Fas -FasL and perforin/granzyme B levels are shown to be linked to the
severity of SJS, granulysin has been identified as the major mediator of keratinocyte apoptosis in
SJS. Future therapeutic approaches can target this mediator in patients with SJS.
Chung WH, Hung SI. Genetic markers and danger signals in stevens-johnson syndrome and toxic
epidermal necrolysis. Allergol Int. 2010;59(4):325 -32.
x Purpose or hypothesis: Provide an overview of the genomic and immunologic perspectives of
SJS, particularly in understanding immune mechanisms and bio -markers for disease prevention
and early diagnosis.
x Specific techniques / reagents / strategies used to test the hypothesis : Review past studies of
HLA association with certain drug-induced SJS and different effectors of keratinocyte apoptosis
in SJS, particular Fas-FasL, perforin/granzyme B, and granulysin.
x Major conclusions: The article provides a concise overview of the genetic susceptibility to SJS via
certain HLA alleles and sums the patho -mechanisms of SJS well, especially in incriminating
granulysin as the main mediator of keratinocyte apoptosis.
x Significance : A better understanding of the immune mechanisms of SJS allows innovations to be
made in future therapy and diagnosis. For example, a screening for a HLA -allele associated with
a particular drug -induced form of SJS could lead to earlier diagnosis or disease prevention by
avoiding that drug.
Kim KH, Park SW, Kim MK, Wee WR. Effect of age and early intervention with a systemic steroid,
intravenous immunoglobulin or amniotic membrane transplantation on the ocular outcomes of
patients with stevens -johnson syndrome. Korean J Ophthalmol. 2013;27(5):331-40.
x Purp ose or hypothesis: Compare the efficacy of early treatment with systemic corticosteroid or
intravenous immunoglobulin (IVIG) on ocular outcomes for SJS
x Specific techniques / reagents / strategies used to test the hypothesis: Retrospective case study
of 51 patients divided by age (above or below 18) with best corrected visual acuity (BCVA) and
ocular involvement scores (OIS) as outcome assessments.
x Major conclusions: Early intervention for the adult group showed marked improvement of mean
BCVA and OIS but not in the pediatric group. The odds ratio for improvement was 1.2 to 1.8 for
the corticosteroid group compared to supportive treatment alone, and a much better 1.2 to 2.4
Date Created/Revised: __ 7/24/14__ By: _______ Page 34
2014-2015 Syllabus
for the IVIG group.
x Significance : Although the results are not statistically profound and the group size small, it is
nonetheless noted that treatments of SJS shows promise for ocular outcomes, specifically in
adults, and more for IVIG over corticosteroids. This could lead way to future trials using these
treatment options.
Saito N, Yoshioka N, Abe R, et al. Stevens-Johnson syndrome/toxic epidermal necrolysis mouse
model generated by using PBMCs and the skin of patients. J Allergy Clin Immunol. 2013;131(2):434 -41.e1-9.
x Purpose or hypothesis: To produce a novel mouse model for SJS by u sing peripheral blood
mononuclear cells (PBMC) and skin from patients who had recovered from SJS. Hopefully, this
model could be used to promote future therapies and diagnoses.
x Specific techniques / reagents / strategie s used to test the hypothesis : PBMCs from SJS patients
were injected into immunocompromised mice followed by oral administration of the causative
drug. Other immunocompromised mice had skin grafts of patients who had recovered from SJS
and were then given the patient-derived PBMC and the causative drug.
x Major conclusions: Mice injected with the PBMCs and given the causative drug showed
conjuctival congestion and death of conjuctival epithelium. Mice injected with PBMCs from
patients with ordinary drug skin interactions (not SJS) showed no symptoms. Also CD8+ T -lymphocyte depleted PBMCs injected into mice also elicited no symptoms.
x Significance : CD8+ T -lymphocytes were proven to be important in SJS pathogenesis. The mouse
model shows promise in future approaches in diagnosis and therapy of SJS.
Singh GK, Chatterjee M, Verma R. Cyclosporine in Stevens Johnson syndrome and toxic epidermal
necrolysis and retrospective comparison with systemic corticosteroid. Indian J Dermatol Venereol
Leprol. 2013;79(5):686-92.
x Purpose or hypothesis: Compare the efficacy of cyclosporine and corticosteroid treatments of
SJS.
x Specific techniques / reagents / strategies used to test the hypothesis: 11 patients were
enrolled, all having SJS, SJS/TEN overlap, or TEN. They were given 3mg/kg cyclosporine for 7
days, then tapered for 7 days. The results were compared retrospectively to a group of 6
similarly aged patients who were treated with corticosteroids. Duration of hospital stay and
time for re-epithelialization were used to assess the results.
x Major conclusions: Hospital stay between the cyclosporine and corticosteroid groups were 18
and 26 days, respectively, and 14 and 23 days for re -epithelialization time.
x Significance : Taking into account the small size, there is a promising role of cyclosporine in th e
treatment of SJS in reducing morbidity. In several isolated patients, the turnaround was
remarkable with cyclosporine.
Wei CY, Chung WH, Huang HW, Chen YT, Hung SI. Direct interaction between HLA -B and
carbamazepine (CBZ) activates T cells in patients with Stevens -Johnson syndrome. J Allergy Clin
Immunol. 2012;129(6):1562 -9.e5.
Date Created/Revised: __ 7/24/14__ By: _______ Page 35
2014-2015 Syllabus
x Purpose or hypothesis: To determine the underlying molecular mechanism of HLA pathology in
delayed type hypersensitivities.
x Specific techniques / reagents / strategies u sed to test the hypothesis : Cytotoxic T lymphocytes
(CTL) were expanded in -vitro from patients with CBZ- induced SJS and the interactions were
monitored between the specific HLA-B*1502 analog, CBZ, and TCR by measuring CTL response
and using a peptide -binding assay, computer modeling, surface plasmon resonance, and site-directed mutagenesis.
x Major conclusions: The endogenous peptide -loaded HLA -B *1502 presented CBZ to the CTL
without involvement of drug metabolism or antigen processing
x Significance : The study demonstrated a direct interaction between the HLA and the drug and
confirmed the p -i hypothesis of the interaction between HLA and the TCR, showing the hapten -prophapten hypothesis having little or no role.
Other References
Ko TM, Chen YT. T-cell receptor and carbamazepine -induced Stevens -Johnson syndrome and toxic
epidermal necrolysis: understanding a hypersensitivity reaction. Expert Rev Clin Immunol.
2012;8(5):467 -77.
Worswick S, Cotliar J. Stevens -Johnson syndrome and toxic epidermal necrolysis: a review of
treatment options. Dermatol Ther. 2011;24(2):207 -18.
PLACE THIS ORDER OR A SIMILAR ORDER WITH US TODAY AND GET AN AMAZING DISCOUNT 🙂