Exploring Vasculitis: Insights into Diagnosis, Treatment, and Genetic Modulation

By Aryana Adur | Jericho Senior High School, Jericho, New York, United States

I. Introduction

Angiitis, or more commonly known as Vasculitis, is an autoimmune disease that leads to the inflammation of blood vessels. (1) Vasculitis can be caused by various types of infectious agents that can be separated into two categories, direct and indirect agents. (2) Direct infectious agents, such as fungi and protozoa (3) directly destroy the vascular wall, instigating an inflammatory response/ reaction. Furthermore, a handful of microbial agents, or indirect infectious agents, (e.g. Mycobacterium tuberculosis & Mycobacterium leprae), stimulate an autoimmune response resulting in the swelling/inflammation of the blood vessel wall tissue. Although there is no cure available for Vasculitis, reducing blood vessel inflammation can be an effective method of managing the underlying cause of Vasculitis. This can be achieved by taking anti-inflammatory medications such as glucocorticoids (e.g. prednisone or methylprednisolone) (4), though the type of medication received will correlate with the severity of the disease. In rare cases, Vasculitis can cause various types of aneurysms to form, resulting in the need to surgically remove the affected blood vessel wall to reduce the risk of it rupturing and causing permanent damage.

Keywords: Vasculitis; Diagnosis; Treatment, Genetic modulation; Autoimmune disease; ELISA  

II. Methods and Techniques

The research article, “ELISA is the Superior Method for Detecting Antineutrophil Cytoplasmic Antibodies in the Diagnosis of Systemic Necrotising Vasculitis” encompasses the idea of using ELISA (enzyme-linked immunosorbent assay) to detect or antineutrophil cytoplasmic antibodies, or “ANCA'', as a biomarker for Vasculitis. ANCA is a type of autoantibody that attack the white blood cells and neutrophils. The binding of ANCA onto certain neutrophil receptors results in 1. the release of toxic materials from the neutrophil cell, inflicting damage on neighboring blood vessel walls, 2. the displacement of neutrophils through walls of certain blood vessels, causing harm to nearby tissues, and 3. the spread of signaling factors that attract a higher amount of neutrophils that cause inflammation and the loss of small blood vessels. (5) In the past, the most utilized method of determining ANCA concentration was direct immunofluorescence, yet it was suggested that ELISA may be a more reliable, accurate, and efficient alternative method. Being able to more effectively detect ANCA antibodies present in the bloodstream could help increase the chances of early detection of Vasculitis. Early diagnosis and treatment can help minimize symptoms and decrease the development and progression of the disease throughout the bloodstream. (6) ELISA, the enzyme-linked immunosorbent assay, is used to evaluate certain antibodies, antigens, or glycoproteins present in a biological sample. There are three types of ELISA assays: direct ELISA, indirect ELISA, and Sandwich ELISA. To perform the procedure for the most complex type of ELISA, the Sandwich ELISA, first coat the ELISA plate with a capture antibody (used to detect the antigen of interest), and any residual, unbound substance should be washed from the plate. Next, the biological sample tested is added. Any antigens present in the sample would bind to the capture antibodies on the wells. Samples are added in duplicate or triplicate with varying concentrations to ensure that it falls within the detection levels of the assay, and any excess samples should be washed from the plate. The detection antibody (usually labeled with an enzyme such as horseradish peroxidase or alkaline phosphatase) is added, which binds to a target antigen bound to the plate. Last, the substrate is added, which is then converted to a colored product, as ELISA assays are usually chromogenic. The antigen concentration can then be determined by using a standard curve and calculating the optical density. (7) Research suggests that ELISA is a better alternative to using direct immunofluorescence (another method to determine ANCA concentration) to test for ANCA concentration within the bloodstream. Direct immunofluorescence has a higher sensitivity (appx. 65%) compared to ELISA (appx. 60%). In contrast, it was also discovered that ELISA had a higher positive predictive value when compared to direct immunofluorescence. It has a higher rate of accuracy, unlike direct immunofluorescence, which falsely determined the presence of ANCA in various subjects with no history of Vasculitis. Despite these results, it was concluded that both methods were not precise enough to conclude the subtype of Vasculitis present in a subject. ANCA concentration can be approximately determined with both types of methods, though both types of data cannot provide the proper level of precision to accurately determine the subtype of Vasculitis present. Furthermore, the research article, “MicroRNA-223 Regulates the Development of Cardiovascular Lesions in LCWE-Induced Murine Kawasaki Disease Vasculitis by Repressing the NLRP3 Inflammasome”, focuses on a subtype of Vasculitis known as “Kawasaki Disease”, or “KD”. Kawasaki Disease is a form of Vasculitis, with inflamed blood vessels centrally located near the heart. Scientists discovered that the molecule microRNA-223 (miR-223) can aid in controlling and minimizing the autoimmune response caused by Kawasaki Disease. The occurrence of this autoimmune response is mainly triggered by the binding of a signaling molecule, interleukin-1 (IL-1), and the molecule, NLRP3 Inflammasome. In this experiment, mice were genetically engineered to lack the miR223 molecule, of which was later compared to the control group of mice (that contained the molecule) on the basis of reactions to KD. The miR223 molecule can help greatly reduce the activity of NLRP3 Inflammasome, in addition to serving as a biomarker for KD and helping take a step closer to finding a treatment for the disease. Genetic engineering, or genetic modification, is the application of different laboratory technologies to modify the sequences of nucleotide bases in an organism. One example of genetic modification technology is CRISPR- Cas9, discovered in 2012 by Jennifer Doudna, Feng Zhang, and Emmanuel Charpentier. (8) CRISPR- Cas9 works by first, selecting a certain genetic location to cut a segment of DNA. Insertion or deletion of nucleotide bases occurs, which can affect levels of gene expression. The first step in performing genetic modification is extracting the DNA from the specimen. This DNA will contain the nitrogenous base sequence template for the genetic insertion/deletion. Next, gene cloning separates the desired gene from the rest of the DNA. Once a gene has been cloned, it has to be designed to function in another organism. Restriction enzymes are now able to cut the gene apart and replace certain regions. (9) The gene is then placed in a specimen, in this case, the gene producing miR223 is deleted in the experimental group of mice. (10) The results show that mice that lacked the gene to produce miR223 had higher levels of NLRP3 Inflammasome activity. This suggests that organisms with the miR223 gene can help lower the severity of symptoms, whereas organisms lacking this molecule will not be able to efficiently combat this disease. It was concluded that miR223 can serve as a premature biomarker for KD Vasculitis, and could possibly be stabilized for pharmaceutical purposes in the future. (11)

III. Conclusion

Both conclusions acquired from the articles seemed to properly correlate with the results from each experiment. Given the results, the conclusions make sense, though parts of the second article, “MicroRNA-223 Regulates the Development of Cardiovascular Lesions in LCWE-Induced Murine Kawasaki Disease Vasculitis by Repressing the NLRP3 Inflammasome”, could be tweaked slightly to accurately mimic the role of miR223 in a human. For example, another test animal could have been used (e.g. Capra aegagrus hircus or Equus caballus) to accurately replicate the body systems of humans to closely see the results of the molecule’s role in a human body. (12) The article, “ELISA is the Superior Method for Detecting Antineutrophil Cytoplasmic Antibodies in the Diagnosis of Systemic Necrotising Vasculitis”, concluded that neither direct immunofluorescence nor ELISA could accurately determine the subtype of Vasculitis present, yet perhaps longer studies of ANCA concentration in humans with the disease could signify a correlation with autoantibody concentration and either disease subtype or disease progression. (13) “MicroRNA-223 Regulates the Development of Cardiovascular Lesions in LCWE-Induced Murine Kawasaki Disease Vasculitis by Repressing the NLRP3 Inflammasome” also shows that miR223 has a large role in controlling the expression and progression of KD. Other forms of miRNA could also play a role in controlling this disease, which can give a higher possibility of finding a therapeutic treatment for KD.

IV. References

1. Brigham and Women's Hospital. "Vasculitis." Brigham and Women's Hospital. Accessed July 7, 2023.https://www.brighamandwomens.org/medicine/rheumatology-inflammation-immunity/services/vasculitis.

2. "Enzyme-linked immunosorbent assay (ELISA)." British Society for Immunology. Accessed July 7, 2023. https://www.immunology.org/public-information/bitesized-immunology/experimental-techniques/enzyme-linked-immunosorbent-assay.

3. "GENETIC ENGINEERING." National Human Genome Research Institute. Last modified July 1, 2023. Accessed July 7, 2023.https://www.genome.gov/genetics-glossary/Genetic-Engineering

4. Harris, Anne, Grace Chang, Matthew Vadas, and David Gillis. "ELISA is the superior method for detecting antineutrophil cytoplasmic antibodies in the diagnosis of systemic necrotising vasculitis." National Library of Medicine. Last modified 1999. Accessed July 7, 2023. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC501542/pdf/jclinpath00282-0038.pdf

5. "Making a Genetically Engineered Crop." University of Nebraska- Lincoln. Accessed July 7, 2023. https://cropwatch.unl.edu/biotechnology/makinggmo.

6. Maruyama, Daisuke, Begüm Kocatürk, Youngho Lee, Debbie Moreira, Shuang Chen, Michael C. Fishbein, Rebecca A. Porritt, Magali Noval Rivas, and Moshe Arditi. "MicroRNA-223 Regulates the Development of Cardiovascular Lesions in LCWE-Induced Murine Kawasaki Disease Vasculitis by Repressing the NLRP3 Inflammasome." National Library of Medicine. Last modified May 2021. Accessed July 7, 2023. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8138581/

7. Mayo Foundation for Medical Education and Research (MFMER). "Vasculitis." Mayo Clinic. Accessed July 7, 2023. https://www.mayoclinic.org/diseases-conditions/vasculitis/symptoms-causes/syc-203634

8. McClure, Mark E., and Rachel B. Jones. "Anti-neutrophil cytoplasm antibodies (ANCA)." Vasculitis UK. Accessed July 7, 2023. https://www.vasculitis.org.uk/about-vasculitis/what-is-anca

9. Mukherjee, P., S. Roy, D. Ghosh, and S. K. Nandi. "Role of animal models in biomedical research: a review." National Library of Medicine. Accessed July 7, 2023. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9247923/.

10. Regents of the University of Michigan. "Vasculitis Treatments." University of Michigan Health. Accessed July 7, 2023. https://www.uofmhealth.org/conditions-treatments/rheumatology/vasculitis-treatments

11. Satta, R., and G. Biondi. Vasculitis and infectious diseases. N.p.: PubMed, 2015. https://pubmed.ncbi.nlm.nih.gov/25876145/

12. Suza, Walter, Donald Lee, Marjorie Hanneman, and Patricia Hain. "Genetic Engineering." Iowa State University. Accessed July 7, 2023. https://iastate.pressbooks.pub/genagbiotech/chapter/genetic-engineering/.

13. Vlachopanos, Georgios. "Overview of Infections as an Etiologic Factor and Complication in Patients with Vasculitides." Review by Panagiotis Theofilis, Aikaterini Vordoni, Maria Koukoulaki, and Rigas G. Kalaitzidis. Rheumatology International 42, no. 5 (February 14, 2022): 759-70. https://doi.org/10.1007/s00296-022-05100-9.

The Scientific Review Article published, “Exploring Vasculitis: Insights into Diagnosis, Treatment, and Genetic Modulation,” was received on July 10, 2023, and was reviewed and accepted on July 15, 2023. To contact editors and reviewers please click here.