under the microscope
under the microscope
By Adeyemi A . Olanrewaju
Exploring Exosomes as a Diagnostics Platform for Infectious Disease
Finding novel platforms to diagnose infectious disease has been in the forefront of research over the past decades . It has been particularly challenging because groups of organisms can share the same characteristics . This has always led to some cross-reactivities and has led to issues in the diagnosis of various infectious diseases .
Exosomes are very small biological molecules that are part of a larger family of extracellular vesicles ( EVs ). EVs are vesicles surrounded by membranes . They are cell-derived , carrying several types of molecular cargos such as DNA , RNA , lipids , proteins , and metabolites . EVs were first known to be derived from platelet as a procoagulant particles in normal plasma , which was reported by Chargaff and West in 1967 . This was later referred to as platelet dust in 1967 by Wolf . In the early 1980s , extensive ultrastructural studies showed that EVs are released by the fusion of multi vesicular bodies ( MVBs ) to the cell membrane . These EVs are known to mediate intracellular communication .
Following up on these studies , it was discovered that EVs can be isolated from all cell types and biological fluids such as plasma , breast milk , seminal fluid , amniotic fluid , saliva , urine , aspirations and nasal fluids . EVs have different subtypes which have varying sizes that overlap , and these create difficulty in separating them based on sizes .
It is important to have a robust method to separate these vehicles which will allow scientists to narrow down the exact type of vesicle responsible for a particular biological function . Exosomes will aid in the early detection of infections which will prevent the spread of disease to different parts of the body . It will also increase the specificity in the diagnosis of infectious disease and allow for more targeted use of antimicrobial therapy rather than using a broad-spectrum antibiotics .
The specific content of exosomes is influenced by environmental factors and the types of cells that produce them . Also , the physiological and pathological states of the cell affect their content . Based on this , exosome-associated proteins or nucleic acids can serve as indicators of disease . The work from several laboratories has demonstrated that specific components of pathogens get packaged within the host exosomes during infection . The mechanisms by which pathogen components are packaged into exosomes have been investigated although there is still much to learn . Generally , regarding infectious disease , the content of exosome will include some proteins or nucleic acid from the pathogen . For instance , cells from patients that are infected with Rift Valley Fever Virus ( RVFV ) releases exosomes that contain the nucleic acids and proteins from the virus . This platform represents a targeted approach to determine infection with an organism , with reduced potential for cross-reactivity . Because of the specificity in the potential use of exosome as a diagnostics marker , there will be an improved approach in the use of antibiotics , which will in turn reduce antimicrobial resistance .
The size of exosomes ranges from 30 nm to 150 nm in diameter . They originate from the invagination of the late endosome , which later forms an enclosed substance called the intraluminal vesicles ( ILVs ) containing cytoplasmic constituents . Later , these ILVs give rise to multivesicular bodies ( MVBs ), which
are then transported via the microtubule and cytoskeletal networks to the plasma membrane of the cells . From here , they are released to the extracellular space . Several methods are currently being used to purify exosomes from biological fluids . There are several methods utilized to purify exosomes and other extracellular vesicles . The most used method is the differential ultracentrifugation . This method uses ultracentrifuge at a very high speed ( 120,000-160,000 × g ) to separate exosomes from other extracellular vesicles . Other methods include size exclusion chromatography , magnet-based isolation , affinity chromatography , charged-based isolation , and microfluidic based isolation . Ultracentrifugation is laborious and it requires expensive equipment . Kit isolation of exosomes can therefore be used , which will be less expensive and requires less time .
Exosomes for diagnosis of infectious disease
Exosomes are found in all biological fluids of patients with different disease conditions such as infectious disease , cancer and inflammatory disease . The composition of these exosomes largely depends on the disease state . Therefore , we can say that exosome composition of healthy individuals is different from that of diseased patients . Because of the specific content of exosomes , they can be used as biomarkers .
Examples for the use of exosomes as a biomarker for diagnosis has been suggested for the following infectious diseases :
● Mycobacterium tuberculosis : More than 20 mycobacterial proteins that are involved in pathogenesis ( survival ) have been identified in exosomes purified from the serum of M . tuberculosis infected patients . Also , it has been observed that mRNA content of exosomes between patients with active tuberculosis , patients with latent tuberculosis and healthy subjects differs .
● HIV : Several HIV proteins such as gp120 , Nef , p24 and Pr55 have been observed to be present in exosomes that are purified from HIV virus infected cells . These proteins will be absent in healthy subjects , and this can be used as a good diagnostic tool for HIV .
● Rift Valley Fever Virus ( RVFV ): In vitro studies have demonstrated the presence of RVFV proteins such as the L , NSs , N and glycoprotein Gn and Gc in exosomes purified from RVFV infected cells and these proteins have been shown to be absent in healthy individuals .
Isolation of exosome is non-invasive and because of the specificity of its content , it serves as a good platform for diagnosis of infectious disease . The use of this in diagnosis is still in its infancy stage . Several basic research are ongoing , and it is projected to be fully in use within the next decade .
Adeyemi Olanrewaju , PhD , is an assistant professor in the medical laboratory science program in the college of health professionals at Texas State University . The main focus of his research is understanding the pathogenesis of pathogenic viruses and utilizing chemical compounds as a novel therapeutic countermeasure against these viruses . He obtained his bachelor of science degree in medical laboratory science ) from Ladoke Akintola University of Technology in Nigeria , and his master ’ s degree in microbiology from Western Illinois University . He received his PhD in infectious disease and microbiology from George Mason University .
8 • www . healthcarehygienemagazine . com • december 2024