Exosomes are cell-derived vesicles that are present in many and perhaps all biological fluids, including blood, urine, and cultured medium of cell cultures. The reported diameter of exosomes is between 30 and 100 nm, which is larger than LDL, but much smaller than for example, red blood cells.
We asked 10 prominent scientists to share their thoughts on science and in particular the field of exosomes research. The video-series tell the story and history of this exciting new area of research, and its impact on other research fields such as cancer and immunology. The video-series also discusses the potential future therapeutic and diagnostic applications that may come from exosome research.
Contributors: – Xandra Breakefield, Ph.D……….. Professor, Massachusetts General Hospital – Jan Lötvall, MD., Ph.D ………….. Professor, University of Gothenburg, President of ISEV – Suresh Mohla, Ph.D……………… Chief (TBMB) and Division Associate Director, NIH – Esther Nolte-‘t Hoen, Ph.D……… Senior Scientist, Utrecht University – Michiel Pegtel, Ph.D…………….. Assistant Professor, VUmc, Amsterdam – Graça Raposo-Benedetti, Ph.D.. Director of Research, CNRS, Institut Curie – Phillip A. Sharp, Ph.D…………… Nobel Laureate, Professor, MIT – Johan Skog, Ph.D……………….. CSO, Exosomes Diagnostics – Dima Ter-Ovanesyan……………..Ph.D Student, Harvard University – Clotilde Thery, Ph.D…………….. Director of Research, INSERM, Institut Curie, Secretary General of ISEV
Acknowledgements: Members of the research groups of Michiel Pegtel (Cancer Center Amsterdam, VUmc), Graça Raposo-Benedetti (Structure and Membrane Compartments, CNRS, Institut Curie) and Clotilde Théry (Immunity and Cancer, INSERM, Institut Curie)
is an international quality standard that is provided by ICH, an international body that defines standards, which governments can transpose into regulations for clinical trials involving human subjects. A similar guideline for clinical trials of medical devices is the international standard ISO 14155, that is valid in the European Union as a harmonized standard. These standards for clinical trials are sometimes referred to as ICH-GCP or ISO-GCP to differentiate between the two and the lowest grade of recommendation in clinical guidelines
GCP follows the International Conference on Harmonisation (ICH) of GCP guidelines. GCP enforces tight guidelines on ethical aspects of a clinical study. High standards are required in terms of comprehensive documentation for the clinical protocol, record keeping, training, and facilities, including computers and software. Quality assurance and inspections ensure that these standards are achieved. GCP aims to ensure that the studies are scientifically authentic and that the clinical properties of the investigational product are properly documented. Ongoing research shows that whether conducting research involving a new drug, a behavioral intervention, or an interview or survey, GCP provides investigators and their study teams with the tools to protect human subjects and collect quality data.
GCP guidelines include protection of human rights for the subjects and volunteers in a clinical trial. It also provides assurance of the safety and efficacy of the newly developed compounds.
GCP guidelines include standards on how clinical trials should be conducted, define the roles and responsibilities of clinical trial sponsors, clinical research investigators, and monitors. In the pharmaceutical industry monitors are often called Clinical Research Associates.
The Tuskegee syphilis experiment (/tʌsˈkiːɡiː/) was an infamous clinical study conducted between 1932 and 1972 by the U.S. Public Health Service to study the natural progression of untreated syphilis in rural African-American men in Alabama. They were told that they were receiving free health care from the U.S. government
The Public Health Service started working on this study in 1932 during the Great Depression, in collaboration with the Tuskegee Institute, a historically black college in Alabama. Investigators enrolled in the study a total of 600 impoverished sharecroppers from Macon County, Alabama. Of these men, 399 had previously contracted syphilis before the study began, and 201 did not have the disease. The men were given free medical care, meals, and free burial insurance for participating in the study. None of the men infected was ever told he had the disease, nor was any treated for it with penicillin after this antibiotic became proven for treatment. According to the Centers for Disease Control, the men were told they were being treated for “bad blood”, a local term for various illnesses that include syphilis, anemia, and fatigue.
The 40-year study was controversial for reasons related to ethical standards, primarily because researchers knowingly failed to treat patients appropriately after the 1940s validation of penicillin as an effective cure for the disease they were studying. Revelation in 1972 of study failures by a whistleblower led to major changes in U.S. law and regulation on the protection of participants in clinical studies. Now studies require informed consent communication of diagnosis, and accurate reporting of test results.
By 1947, penicillin had become the standard treatment for syphilis. Choices available to the doctors involved in the study might have included treating all syphilitic subjects and closing the study, or splitting off a control group for testing with penicillin. Instead, the Tuskegee scientists continued the study without treating any participants; they withheld penicillin and information about it from the patients. In addition, scientists prevented participants from accessing syphilis treatment programs available to other residents in the area. The study continued, under numerous US Public Health Service supervisors, until 1972, when a leak to the press resulted in its termination on November 16 of that year. The victims of the study included numerous men who died of syphilis, 40 wives who contracted the disease, and 19 children born with congenital syphilis.
The Tuskegee Syphilis Study, cited as “arguably the most infamous biomedical research study in U.S. history”,led to the 1979 Belmont Report and the establishment of the Office for Human Research Protections (OHRP) It also led to federal laws and regulations requiring Institutional Review Boards for the protection of human subjects in studies involving human subjects. The Office for Human Research Protections (OHRP) manages this responsibility within the USDepartment of Health and Human Services (HHS)
Dr. Nicholas Herten-Greaven lectures about the Tuskegee syphilis experiment as part of the Ethics in Clinical Research course at Oxford College
A randomized controlled trial (or randomized control trialRCT) is a type of scientific (often medical) experiment, where the people being studied are randomly allocated one or other of the different treatments under study. The RCT is often considered the gold standard for a clinical trial. RCTs are often used to test the efficacy or effectiveness of various types of medical intervention and may provide information about adverse effects, such as drug reactions. Random assignment of intervention is done after subjects have been assessed for eligibility and recruited, but before the intervention to be studied begins.
Presented by Dr. Brent Logan, PhD, Professor in the Division of Biostatistics, Medical College of Wisconsin. This lecture will provide an overview of study designs and statistical issues in all phases of clinical trials. We will start by describing dose-finding phase I designs, and then will cover phase II designs, including the framework for determining sample size and the use of two-stage designs. The remainder of the lecture will focus on major design issues in phase III clinical trials, including endpoint specification, eligibility, power and sample size calculation, blinding, randomization, stratification, and data monitoring
Database management systems (DBMS) are computer software applications that interact with the user, other applications, and the database itself to capture and analyze data. A general-purpose DBMS is designed to allow the definition, creation, querying, update, and administration of databases. Well-known DBMSs include MySQL, PostgreSQL, Microsoft SQL Server, Oracle, Sybase and IBM DB2. A database is not generally portable across different DBMSs, but different DBMS can interoperate by using standards such as SQL and ODBC or JDBC to allow a single application to work with more than one DBMS. Database management systems are often classified according to the database model that they support; the most popular database systems since the 1980s have all supported the relational modelas represented by the SQL language. Sometimes a DBMS is loosely referred to as a ‘database’.
Clinical trials are experiments done in clinical research. Such prospective biomedical or behavioral research studies on human participants are designed to answer specific questions about biomedical or behavioral interventions, including new treatments (such as novel vaccines, drugs, dietary choices, dietary supplements, andmedical devices) and known interventions that warrant further study and comparison. Clinical trials generate data on safety and efficacy.They are conducted only after they have received health authority/ethics committee approval in the country where approval of the therapy is sought. These authorities are responsible for vetting the risk/benefit ratio of the trial – their approval does not mean that the therapy is ‘safe’ or effective, only that the trial may be conducted.
Clinical Trials: Follow-up, Adherence to the Protocol and Post-Randomization Dennis Black, MA, PhD
Cancer is a class of diseases characterized by out-of-control cell growth. There are over 100 different types of cancer, and each is classified by the type of cell that is initially affected.
Cancer harms the body when damaged cells divide uncontrollably to form lumps or masses of tissue called tumors (except in the case of leukemia where cancer prohibits normal blood function by abnormal cell division in the blood stream). Tumors can grow and interfere with the digestive, nervous, and circulatory systems, and they can release hormones that alter body function. Tumors that stay in one spot and demonstrate limited growth are generally considered to be benign.
More dangerous, or malignant, tumors form when two things occur:
a cancerous cell manages to move throughout the body using the blood or lymph systems, destroying healthy tissue in a process called invasion
that cell manages to divide and grow, making new blood vessels to feed itself in a process called angiogenesis.
When a tumor successfully spreads to other parts of the body and grows, invading and destroying other healthy tissues, it is said to have metastasized. This process itself is called metastasis, and the result is a serious condition that is very difficult to treat.
How cancer spreads – scientists reported in Nature Communications(October 2012 issue) that they have discovered an important clue as to why cancer cells spread. It has something to do with their adhesion (stickiness) properties. Certain molecular interactions between cells and the scaffolding that holds them in place (extracellular matrix) cause them to become unstuck at the original tumor site, they become dislodged, move on and then reattach themselves at a new site.
The researchers say this discovery is important because cancer mortality is mainly due to metastatic tumors, those that grow from cells that have traveled from their original site to another part of the body. Only 10% of cancer deaths are caused by the primary tumors.
The scientists, from the Massachusetts Institute of Technology, say that finding a way to stop cancer cells from sticking to new sites could interfere with metastatic disease, and halt the growth of secondary tumors.
In 2007, cancer claimed the lives of about 7.6 million people in the world. Physicians and researchers who specialize in the study, diagnosis, treatment, and prevention of cancer are called oncologists.
Malignant cells are more agile than non-malignant ones – scientists from the Physical Sciences-Oncology Centers, USA, reported in the journal Scientific Reports (April 2013 issue) that malignant cells are much “nimbler” than non-malignant ones. Malignant cells can pass more easily through smaller gaps, as well as applying a much greater force on their environment compared to other cells.
Professor Robert Austin and team created a new catalogue of the physical and chemical features of cancerous cells with over 100 scientists from 20 different centers across the United States.
The authors believe their catalogue will help oncologists detect cancerous cells in patients early on, thus preventing the spread of the disease to other parts of the body.
Prof. Austin said “By bringing together different types of experimental expertise to systematically compare metastatic and non-metastatic cells, we have advanced our knowledge of how metastasis occurs.”
WHAT CAUSES CANCER?
Cancer is ultimately the result of cells that uncontrollably grow and do not die. Normal cells in the body follow an orderly path of growth, division, and death. Programmed cell death is called apoptosis, and when this process breaks down, cancer begins to form. Unlike regular cells, cancer cells do not experience programmatic death and instead continue to grow and divide. This leads to a mass of abnormal cells that grows out of control.
WHAT IS CANCER? – VIDEO
A short, 3D, animated introduction to cancer. This was originally created by BioDigital Systems and used in the Stand Up 2 Cancer telethon.
GENES – THE DNA TYPE
Cells can experience uncontrolled growth if there are damages or mutations to DNA, and therefore, damage to the genes involved in cell division. Four key types of gene are responsible for the cell division process: oncogenes tell cells when to divide, tumor suppressor genes tell cells when not to divide, suicide genes control apoptosis and tell the cell to kill itself if something goes wrong, and DNA-repair genes instruct a cell to repair damaged DNA.
Cancer occurs when a cell’s gene mutations make the cell unable to correct DNA damage and unable to commit suicide. Similarly, cancer is a result of mutations that inhibit oncogene and tumor suppressor gene function, leading to uncontrollable cell growth.
Carcinogens are a class of substances that are directly responsible for damaging DNA, promoting or aiding cancer. Tobacco, asbestos, arsenic, radiation such as gamma and x-rays, the sun, and compounds in car exhaust fumes are all examples of carcinogens. When our bodies are exposed to carcinogens, free radicals are formed that try to steal electrons from other molecules in the body. Theses free radicals damage cells and affect their ability to function normally.
GENES – THE FAMILY TYPE
Cancer can be the result of a genetic predisposition that is inherited from family members. It is possible to be born with certain genetic mutations or a fault in a gene that makes one statistically more likely to develop cancer later in life.
OTHER MEDICAL FACTORS
As we age, there is an increase in the number of possible cancer-causing mutations in our DNA. This makes age an important risk factor for cancer. Several viruses have also been linked to cancer such as: human papillomavirus (a cause of cervical cancer), hepatitis B and C (causes of liver cancer), and Epstein-Barr virus (a cause of some childhood cancers). Human immunodeficiency virus (HIV) – and anything else that suppresses or weakens the immune system – inhibits the body’s ability to fight infections and increases the chance of developing cancer.
WHAT ARE THE SYMPTOMS OF CANCER?
Cancer symptoms are quite varied and depend on where the cancer is located, where it has spread, and how big the tumor is. Some cancers can be felt or seen through the skin – a lump on the breast or testicle can be an indicator of cancer in those locations. Skin cancer (melanoma) is often noted by a change in a wart or mole on the skin. Some oral cancers present white patches inside the mouth or white spots on the tongue.
Other cancers have symptoms that are less physically apparent. Some brain tumors tend to present symptoms early in the disease as they affect important cognitive functions. Pancreas cancers are usually too small to cause symptoms until they cause pain by pushing against nearby nerves or interfere with liver function to cause a yellowing of the skin and eyes called jaundice. Symptoms also can be created as a tumor grows and pushes against organs and blood vessels. For example, colon cancers lead to symptoms such as constipation, diarrhea, and changes in stool size. Bladder or prostate cancers cause changes in bladder function such as more frequent or infrequent urination.
As cancer cells use the body’s energy and interfere with normal hormone function, it is possible to present symptoms such as fever, fatigue, excessive sweating, anemia, and unexplained weight loss. However, these symptoms are common in several other maladies as well. For example, coughing and hoarseness can point to lung or throat cancer as well as several other conditions.
When cancer spreads, or metastasizes, additional symptoms can present themselves in the newly affected area. Swollen or enlarged lymph nodes are common and likely to be present early. If cancer spreads to the brain, patients may experience vertigo, headaches, or seizures. Spreading to the lungs may cause coughing and shortness of breath. In addition, the liver may become enlarged and cause jaundice and bones can become painful, brittle, and break easily. Symptoms of metastasis ultimately depend on the location to which the cancer has spread.
HOW IS CANCER CLASSIFIED?
There are five broad groups that are used to classify cancer.
Carcinomas are characterized by cells that cover internal and external parts of the body such as lung, breast, and colon cancer.
Sarcomas are characterized by cells that are located in bone, cartilage, fat, connective tissue, muscle, and other supportive tissues.
Lymphomas are cancers that begin in the lymph nodes and immune system tissues.
Leukemias are cancers that begin in the bone marrow and often accumulate in the bloodstream.
Adenomas are cancers that arise in the thyroid, the pituitary gland, the adrenal gland, and other glandular tissues.
Cancers are often referred to by terms that contain a prefix related to the cell type in which the cancer originated and a suffix such as -sarcoma, -carcinoma, or just -oma. Common prefixes include:
Adeno- = gland
Chondro- = cartilage
Erythro- = red blood cell
Hemangio- = blood vessels
Hepato- = liver
Lipo- = fat
Lympho- = white blood cell
Melano- = pigment cell
Myelo- = bone marrow
Myo- = muscle
Osteo- = bone
Uro- = bladder
Retino- = eye
Neuro- = brain
HOW IS CANCER DIAGNOSED AND STAGED?
Early detection of cancer can greatly improve the odds of successful treatment and survival. Physicians use information from symptoms and several other procedures to diagnose cancer. Imaging techniques such as X-rays, CT scans, MRI scans, PET scans, and ultrasound scans are used regularly in order to detect where a tumor is located and what organs may be affected by it. Doctors may also conduct an endoscopy, which is a procedure that uses a thin tube with a camera and light at one end, to look for abnormalities inside the body.
Extracting cancer cells and looking at them under a microscope is the only absolute way to diagnose cancer. This procedure is called a biopsy. Other types of molecular diagnostic tests are frequently employed as well. Physicians will analyze your body’s sugars, fats, proteins, and DNA at the molecular level. For example, cancerous prostate cells release a higher level of a chemical called PSA (prostate-specific antigen) into the bloodstream that can be detected by a blood test. Molecular diagnostics, biopsies, and imaging techniques are all used together to diagnose cancer.
Cohort studies are observational analytical studies, the word ‘cohort’ is derived from the Latin word ‘cohors’, which means unit. For conducting cohort type of studies, the study population is chosen from general population both exposed to a certain agent suspected for disease development and unexposed to the cause. The population is followed for a longer period of time. The incidence in disease development in exposed group is compared with the non-exposed group. Therefore, the objective of a cohort study is to find out association between a suspected cause(s) and disease. If performed correctly, cohort studies can predict results comparable to the experimental analytical studies. The following measurements can be done in a cohort study design: absolute risk or incidence, relative risk (risk ratio or rate ratio), risk difference, and attributable proportion. Cohort studies are classified as prospective and retrospective studies based on the timing of enrollment of subjects and disease outcome.
In medical research, it is important for a researcher to know about different analytical studies. The objectives of different analytical studies are different, and each study aims to determine different aspects of a disease(s) such as prevalence, incidence, cause, prognosis, or effect of treatment. Therefore, it is essential to identify the appropriate analytical study associated with certain objectives.
Analytical studies are classified as experimental and observational studies. While in an experimental study, the investigator examines the effect of presence or absence of certain intervention(s), he does not need to intervene in a observational study, rather he observes and assesses the relation between exposure and disease variable. Interventional studies or clinical trials fall under the category of experimental study where investigator assigns the exposure status. Observational studies are of four types: cohort studies, case-control studies, cross-sectional studies, and longitudinal studies
While experimental studies are sometimes non indicative or not ethical to conduct or very expensive, observational studies probably are the next best approach to answer certain investigative questions. Well-designed observational studies may also produce similar results as controlled trials; therefore, probably, the observational studies may not be considered as second best options. In order to design an appropriate observational study, one should able to distinguish between four different observational studies and their appropriate application depending on the investigative questions. Following is a brief discussion on four different observational studies (each will be discussed in detail individually in my upcoming blogs):
Observational Analytical Study Designs
Cohort methodology is one of the main tools of analytical epidemiological research. The word “cohort” is derived from the Latin word “cohors” meaning unit. The word was adopted in epidemiology to refer a set of people monitored for a period of time. In modern epidemiology, the word is now defined as “group of people with defined characteristics who are followed up to determine incidence of, or mortality from, some specific disease, all causes of death, or some other outcome” (Morabia, 2004). In cohort studies, individuals are identified who initially do not have the outcome of interest and followed for a period of time. The group can be classified in sub sets on the basis of the exposure. For example, a group of people can be identified consisting of both smoker and non-smoker and followed them for the incidence of lung cancer. At the beginning of the study none of the individuals have lung-cancer and the individuals are grouped into two sub sets as smoker and non-smoker and then followed for a period of time for different characteristics of exposure such as smoking, BMI, eating habits, exercise habits, family history of lung cancer or cardiovascular diseases, etc. Over the time, some individuals develop the outcome of interest. From the data collected over time, it is convenient to evaluate the hypothesis whether smoking is related with the incidence of lung cancer. The following schematic shows the basic design of a cohort study. There are two types of cohort studies: prospective and retrospective. A prospective study is conducted at present but followed up to future i.e., waiting for the disease to develop. On the other hand, a retrospective study is carried out at present on the data collected in the past. This is also called as historic cohort study. In the next blog, I will discuss these in detail.
In terms of objective, case-control studies and cohort studies are same. Both are observational analytical studies, which aim to investigate the association between exposure and outcome. The difference lies in the sampling strategy. While cohort studies identify the subjects based on the exposure status, case-control studies identify the subjects based on the outcome status. Once the outcome status is identified the subjects are divided into two sets: case and control (who do not develop the outcome). For example, a study design which determines the relation between endrometrial cancer with use of conjugated estrogen. For this study, subjects are chosen based on the outcome status (endrometrium cancer) i.e., with disease present (case) and absent (control), and then these two subsets are compared with respect to the exposure (use of conjugated estrogen). Therefore, case-control study is retrospective in nature and cannot be used for calculating relative risk. However, odd ratio can be measured, which in turn, is approximate to relative risk. In cases of rare outcomes, case control study is probably the only feasible analytical study approach.
Cross-sectional study is a type of observational analytical study which is used primarily to determine the prevalence without manipulating the study environment. For example, a study can be designed to determine the cholesterol level in walker and non-walker without exerting any exercise regime or activity on non-walkers or modifying the activity of the walkers. Apart from cholesterol other characteristics of interest, such as age, gender, food habits, educational level, occupation, income, etc., can also be measured. The data collected at one time in present with no further follow up. In cross-sectional design, one can study a single population (only walkers) or more than one population (both walker and non-walker) at one point of time to see the association between cholesterol level and walking. However, the design of this study does not allow to examine the causal of a certain condition since the subjects are never been followed either in past or present.
Longitudinal studies, similar to cross-sectional studies, are also a type of observational analytical studies. However, the difference of this study design with the cross-sectional study is the following up the subjects for a longer time; hence, can contribute more to the association of causative to a condition. For example, the design that aims to determine the cholesterol level of a single population, say the walkers over a period of time along with some other characteristics of interest such as age, gender, food habits, educational level, occupation, income, etc. One may choose to examine the pattern of cholesterol level in men aged 35 years walking daily for 10 years. The cholesterol level is measured at the onset of the activity (here, walking) and followed up throughout the defined time period, which enables to detect any change or development in the characteristics of the population.
Following two tables summarize different observational analytical studies with regard to the objectives and time-frame.
 Morabia, A (2004). A History of Epidemiologic Methods and Concepts. Birkhaeuser Verlag; Basel: p. 1-405.
 Hulley, S.B., Cummings, S.R., Browner, W.S., et al (2001). Designing Clinical Research: An Epidemiologic Approach. 2nd Ed. Lippincott Williams & Wilkins; Philadelphia: p. 1-336.
 Merril, R.M., Timmreck, T.C (2006). Introduction to Epidemiology. 4th Ed. Jones and Bartlett Publishers; Mississauga, Ontario: p. 1-342.
 Lilienfeld, A.M., and Lilienfeld, D.E. (1980): Foundations of Epidemiology. Oxford University Press, London.
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