Cell Regeneration PDF

5. CELL REGENERATION (De Jonge) In order to restore organ func on, both func onal cell mass and ssue architecture must be restored. The (r)evolu on of ideas began with:  Lower animals spontaneously generate from ma er (Aristotle)  Harvey proposed “epigenesis” with the animal growing from u...

5. CELL REGENERATION (De Jonge) In order to restore organ func on, both func onal cell mass and ssue architecture must be restored. The (r)evolu on of ideas began with:  Lower animals spontaneously generate from ma er (Aristotle)  Harvey proposed “epigenesis” with the animal growing from undifferen ated mass (Ex ovo omnia, 1651)  Spallanzani published his historical book “Prodromo” in 1768 describing several types of regenera on in many animals such as frog, earth worm, snail and salamander. Spontaneous regenera on became one of the most studied theories of the me. “In Spallanzani's absence, his rivals conspired against him. Under the instruc ons of Giovanni Scopoli, Spallanzani's assistant in the museum, visited his family home in Emilia, and asking to see his private museum. He then accused Spallanzani of stealing objects from the Pavia Museum and pu ng them in his own collec on. The news spread throughout Europe and reached Spallanzani, by now in Vienna on his way home. Bizzozero began to inves gate the histology of bone marrow. At that me, there were two main views on the func ons of this ssue, which dated back to the ancient me of Hippocrates and Aristotle. According to these views, the bone marrow either cons tuted an ‘excrement’ of the bone, or, on the contrary, it represented its nutri onal ‘matrix’. Bizzozero discovered a par cular kind of nucleated red cell in this ssue, and he considered these cells to be the precursors of the mature red cells of the circula ng blood. It began in the seventeenth century, when Hooke, van Leeuwenhoek and others, discovered the cellula as a building block of many organisms. Then, in the first half of the nineteenth century, Schleiden and Schwann established the ‘cell theory’, according to which all organisms are composed of ny units, the cells. Schleiden and Schwann assumed that cells are formed de novo from an intercellular substance in some kind of crystalliza on (‘free cell forma on’) - an assump on that misled many scien sts and inhibited research into cell division for almost three decades. Through his ar cles, Bizzozero became a pre-eminent proponent of social and poli cal measures against the spread of infec ous diseases, and an ac ve promoter of public understanding of the benefits of science. Bizzozero wrote on the advantages of vaccina on, and on the prac cal applica on of the principles of hygiene for the improvement of public health. He even realized that cancer and smoking are related and, in a popular ar cle, he suggested giving up smoking to reduce the spread of this disease. Hence, there’s a switching of beliefs, from mys cism and religion to preforma on (growth from a smaller organism) un l epigenesis and regenera on. Many of the conclusions were defined in the Interna onal Medical Congress in Rome (1984). Bizzozero’s cell renewal classifica on was:  Labile cells – regenerate con nuously; e.g., epithelial cells, spleen cells, bone marrow  Stable cells – divide at a very slow rate normally; e.g., example: hepatocytes, renal tubular cells, bone, car lage, and connec ve ssue  Permanent cells – no regenera ve capacity; e.g., nerve cells, myocardium, skeletal muscle cells Skin regenera on can be used as an example for regenera on because skin is an important barrier o en under a ack. The process involved in wound healing and regenera on is firstly hemostasis, but also inflamma on (not pain or loss of blood) and regenera on. There are two types of skin wound healing processes:  By first inten on, with a surgical cut with apposed margins and minimal loss of ssue;  By second inten on, with a larger wound, characterized by a large open area, loss of ssue and is o en asep c. 5.1 - HEMOSTASIS The first priority of the body is hemostasis. Upon damage of the ssue, there’s vascular constric on that stops the blood flow, which is done via mechanical contrac on (vascular spasm, which lasts 30 minutes) of the vessel structure, leading to an immediate contrac on of the smooth muscle cells, reducing blood flow and trapping blood cells, but also immediately ac va ng cells present in the blood and ssue. This leads to forma on of a platelet plug and the coagula on of the blood. Bizzozero had previously described coagula on: blood platelets, swept along by the blood stream, are held up at the damaged spot as soon as they arrive at it. At first one sees only 2–4–6 (platelets); very soon the number climbs to hundreds. Usually, some white blood cells are held up amongst them. Li le by li le the volume increases and soon the thrombus fills the lumen of the blood vessels, and impedes the blood stream more and more. In a first step towards clot forma on, platelets are recruited to the site of vessel injury by now-exposed molecules of the vessel wall, such as collagen and von Willebrand factor; this factor mediates the linking of platelets to collagen via a specific receptor in the platelet membrane. The resul ng change of shape of the platelet from its res ng state into the dendri c form indicates ac va on; the ac vated platelet, in turn, releases prothrombo c molecules, such as adenosine diphosphate (ADP). By binding to its receptors, ADP induces aggrega on and it recruits further platelets to the site; thromboxane is another important mediator of platelet ac va on, and, in aggrega on under its influence, the platelets cross-link with each other. These inter-locking mechanisms cause platelet ac va on. The clot grows rapidly; ac vated platelets also trigger the coagula on cascade and thus the forma on of thrombin. Thrombin, in turn, s mulates platelet ac va on even further, in a con nuous feedback loop. Addi onally, thrombin induces the forma on of fibrin for the mesh, stabilizing the clot. The self-reinforcing process of platelet ac va on, crucial in the forma on of blood clots, is an obvious therapeu c target in condi ons caused by an appropriately triggered blood coagula on. Coagula on is hence a complex but well-regulated cascade: FACTOR NAME TYPE OF MOLECULE SOURCE PATHWAYS I Fibrinogen Plasma protein Liver Common; converted into fibrin II Prothrombin Plasma protein Liver* Common; converted into thrombin III Tissue thromboplas n or ssue factor Lipoprotein mixture Damaged cells and platelets Extrinsic IV Calcium ions Inorganic ions in plasma Diet, platelets, bone matrix En re process V Proaccelerin Plasma protein Liver, platelets Extrinsic and intrinsic VI Not used Not used Not used Not used VII Proconver n Plasma protein Liver* Extrinsic VIII An hemophilic factor A Plasma protein factor Platelets and endothelial cells Intrinsic; deficiency results in haemophilia A IX An hemophilic factor B (plasma thromboplas n component) Plasma protein Liver* Intrinsic; deficiency results in haemophilia B X Stuart-Power factor (thrombokinase) Protein Liver* Extrinsic and intrinsic XI Plasma thromboplas n antecedent Plasma protein Liver Intrinsic; deficiency results in haemophilia C XII Hageman factor Plasma protein Liver Intrinsic; ini ates clo ng in vitro also ac vates plasmin XIII Fibrin-stabilizing factor Plasma protein Liver, platelets Stabilizes fibrin; slows fibrinolysis A er hemostasis, the consequent process is an inflamma on reac on, seen in all ssues, even in the absence of infec on. Mast cells ac va on (i.e., degranula on) is a fundamental passage for inflamma on; it occurs via:  Cytokines/chemokines (IL-1b, IL-4, IL-6, IL-8, IL10, MCP-1, TNF-α)  Vasoac ve amines (Histamine and Serotonin)  Growth Factors (FGF-2, PDGF, TGF-β1, VEGF)  Proteases (Chymase and Tryptase)  Proteoglycans (Heparin)  Lipid mediators (LTB4, LTC4, PAF, PGD2, PGE2) Important roles for mast cells are:  Monocyte recruitment in the inflammatory stage  Neutrophil recruitment in the inflammatory stage  Mediators in (or of) the inflammatory stage  Affect kera nocytes in the prolifera ve stage  Affect fibroblasts in the prolifera ve stage  Promo ng angiogenesis at the site of injury The final stage of inflamma on is to clear the site from pathogens and cell debris. Then the proper rebuilding (regenera on) occurs: fibrin joins both sides (“cement”) with collagen as “glue”; capillaries easily bridge the small gap and epidermal cells close it; there is hence minimal remodeling needed. However, most wounds are not surgical, but second inten on; hence, the rebuilding involves: immediate filling of the gap, removal of pathogens and cell debris, infiltra on with new cells and ECM and cellular and structural regenera on. A second inten on wound healing is characterized by a large open area with wound edges far apart, loss of ssue and o en asep c (natural wounds). In second inten on wound healing: In the phase of granula on ssue and capillary forma on, a scaffold of fibrin is built a er the wound area is cleared of debris and pathogens; there’s a consequent fibroblast invasion and collagen deposi on (crea ng a scab); the fibroblasts then differen ate into myofibroblasts and the outgrowth of capillary loops (VGEF) occurs. Specifically, TGF- β1 induces differen a on with ECM. The pulling forces of wound contrac on are hence: cells within the fibrin and collagen network (myofibroblasts and platelets) and drying (evapora on). Re-epithelializa on by migra on and prolifera on is a method used in lab known as the “Wound or Scratch assay”, in which HaCaT cells are spontaneously immortalized, human kera nocyte line. Complete epidermal reprogramming is needed: epidermal cell migra on is driven by mesenchymal (fibroblasts-secreted) growth factors (HGF/SF, KGF/FGF7) and macrophage, platelet, and mast cell GFs, guided by extracellular matrix components (collagen). This results in change of integrin expression and secre on of matrix metalloproteases (MMPs). This is not an epithelial-to-mesenchymal transi on (EMT). Ac va on of prolifera on is also fundamental: it’s composed of epidermal cell prolifera on (growth factors HGF/SF, EGF, KGF, FGF-10, TGF-a, IGF-1) and endothelial cell prolifera on (growth factors HGF/SF,...), resul ng in gene expression driving mitosis. 5.2 - SKIN WOUND HEALING Skin consists of two basic layers: the epidermis, a superficial thin layer composed of epithelial ssue, and the dermis, a deeper thick layer composed primarily of connec ve ssue. When the skin is damaged, the type of healing process that occurs depends on the depth of the injury:  Epidermal wound, such as a minor abrasion or minor burn, does not penetrate the dermis, hence here healing is a simple process in which surrounding basal epidermal cells mul ply and migrate across the wound un l it is covered;  Dermal wound, however, penetrates the dermis and involves mul ple ssue layers; the healing process is different and more complex than epidermal healing and occurs in three phases: 1. The first phase involves hemostasis and inflamma on: fibrin and blood platelets form a loose blood clot to prevent further blood loss. The damaged ssue causes release of histamine, which triggers vasodila on and increases the permeability of the blood vessels. This in turn increases the delivery of white blood cells which help remove microbes and foreign par cles via phagocytosis. 2. Two or three days later, the prolifera on and migratory phase begin: in the ini al por on of this phase, the clot exterior dries forming a scab; fibroblasts infiltrate the wound and secrete collagen to strengthen the clot. Fibroblasts also trigger the endothelial cells surrounding the wound to proliferate and injured blood vessels to start regrowing. These cells form a delicate mesh known as granula on ssue. 3. The third phase is the matura on and remodeling phase which may last between 3 weeks to 6 months. The scab sloughs off collagen fibers; it becomes more organized and fewer fibroblasts are present. The blood vessels are restored to normal. Finally, scar ssue forms by a process called fibrosis; the scar ssue differs from normal skin in that it has a denser arrangement of collagen fibers and a reduced elas city. The histologies of normal and scarred skin are quite similar, because the result is o en nearly perfect: hair follicles are plen ful in normal skin but absent in scarred skin. The scarred dermis is a rela vely acellular expanse of collagen. All ssues in the body respond to injury:  GENERAL WOUND-HEALING RESPONSE  FULL TISSUE REGENERATION DEPENDENT ON - Hemostasis - Presence of labile cells (epidermal, epithelial cells) - Inflamma on with immune cell - Presence of stable cells (hepatocytes, fibroblasts, endothelial cells, progenitor or - infiltra on (ac va on by IL-1, IL-6, TNF-α, prostaglandins, ROS) stem cells) - Damaged cell removal by proteases - Presence of permanent cells (neurons and cardiac muscle cells) - An -inflammatory molecules (TGFb, histamine, L-4, IL-10) - Forma on of extracellular matrix (ECM) The cells at the damaged site respond to:  Local release of signaling molecules from damaged cells: DAMPs and alarmins (signaling through Toll-like receptors on immune cells)  Exposure to normally “hidden” ECM components: collagen, laminin, fibronec n, hyaluronic acid  Granular content released from mast cells, neutrophils, and macrophages (IL-4, IL-6, TGF-b, ROS, etc.)  Receptors involved in signaling; each cell has surface receptors for specific molecules (interleukin receptors, histamine receptor, etc.). Cells also have receptors for ECM components; e.g., integrins. These adhesion receptors also sense tensile strength and roughness. However, not all organs can regenerate sufficiently (skin, liver, intes ne, muscle vs heart, kidney and brain) and not all organs are affected the same way by scar ssue (skin, liver, intes ne, muscle vs heart, kidney and brain). It’s important to select the right model system: e.g., in studies of the protec ve effect of Met receptor agonist 1K1 in infarcted heart, 3 animals with 8 square-shaped full-thickness wounds were used. Each animal carries own nega ve control; 4 treatments were tested: K1K1 TL 1 nM in vehicle; K1K1 TL 50 nM in vehicle; vehicle alone; physiological serum. 400 μl of treatments were administered topically twice a week. Analysis me was on day 7, 24 and 35. Skin seeding (mul -faceted enhancement) is an accelerated re-epitheliza on and reduced contrac on in wounds treated with MSTCs. Wound margins were ta ooed with black ink. Micro skin ssue columns (MSTCs) can be seen in the wound bed as short, white, rod-like structures. Another interes ng ongoing research is how decellularized liver scaffolds promote liver regenera on a er par al hepatectomy; in rela on to this, 3D bioprin ng scaffolds can be used for wound healing. This is not only interes ng because of the biology but also due to its revenue, as it has important applica ons: chronic wounds, diabe c foot ulcers, pressure ulcers, venous leg ulcers, other chronic wounds, acute wounds, surgical wounds, trauma c wounds and burns. 6. DISEASE AND GENETICS According the WHO, health is a state of complete physical, mental and social well-being and not merely the absence of disease or infirmity. The enjoyment of the highest a ainable standard of health is one of the fundamental rights of every human being without dis nc on of race, religion, poli cal belief, economic or social condi on. The health of all people is fundamental to the a ainment of peace and security and is dependent on the fullest co-opera on of individuals and States. Governments have a responsibility for the health of their people which can be fulfilled only by provision of adequate health and social measures. A disease:  It has a cause: e ology  It has a mechanism: pathogenesis  It has structural and func onal features: manifesta on  It can have secondary consequences: complica ons and sequelae  It can have a predicted development (cure or progression): prognosis  Epidemiologists might know the incidence (new cases) and prevalence 6.1 - ETIOLOGY Some e ological (causa ve) agents include: gene c abnormali es (although it’s hard to define normal alleles), infec ve agents (bacteria, viruses, fungi, parasites), chemicals, radia on, mechanical trauma (from cars or sharks), combina ons of the above. There’s also a rela onship between the amount and the probability of disease: a. [A] Physical agents. For example, the risk of trauma c injury to pedestrian increases in propor on to the kine c energy of the motor vehicle. When spending more me in traffic the chance of an accident increases. b. [B] Infec ous agents. Many infec ous diseases result only if sufficient numbers of the microorganism (e.g. bacterium, virus) are transmi ed; smaller numbers are capable of being eliminated by the non-immune and immune defenses. Low-dose exposure can increase resistance. c. [C] Allergens. In sensi zed (i.e. allergic) individuals, minute amounts of an allergen will provoke a severe anaphylac c reac on. Allergen-specific immunotherapy can help in “desensi zing” pa ents. d. [D] J-shaped curve. “Best” exemplified by alcohol*, of which small doses (c. 1–2 units per day) reduce the risk of premature death from ischemic heart disease, but larger doses progressively increase the risk of cirrhosis. Protec ve effects have been caused by low exposure to pathogens. However, it has been suggested that an increase in IgG4 levels could have a protec ng role by preven ng immune over-ac va on, similar to that occurring during successful allergen-specific immunotherapy by inhibi ng IgE-induced effects. However, emerging evidence suggests that the reported increase in IgG4 levels detected a er repeated vaccina on with the mRNA vaccines may not be a protec ve mechanism; rather, it cons tutes an immune tolerance mechanism to the spike protein that could promote unopposed SARS-CoV2 infec on and replica on by suppressing natural an viral responses. Allergen immunotherapy is a form of therapeu c vaccina on for established IgE-mediated hypersensi vity to common allergen sources such as pollens, house dust mites and the venom of s nging insects. The classical protocol, introduced in 1911, involves repeated subcutaneous injec on of increasing amounts of allergen extract, followed by maintenance injec ons over a period of 3 years, achieving a form of allergen-specific tolerance that provides clinical benefit for years a er its discon nua on. There are two types of causa on:  Gene c  Environmental  Mul factorial (both gene c and environmental); it is in fact o en a complex interplay between both. What about epigene cs (modifica on of DNA)? It can’t be classified exactly in the gene c or environmental categories. In fact, there’s o en a complex interplay between both. It is, however, important to know that risk factors are not causes, unless they are. The causes of accidents are studied in the belief that by finding causes, accidents can be prevented by removing or controlling their causes. It follows that the risk factors that have tradi onally been regarded as contribu ng to accidents can only be regarded as causes if it is possible to alter them by means of one or more road safety measures. Risk factors are causes if their rela onship to accidents can be changed by implemen ng one or more road safety measures influencing the risk factors. 6.1.1 - GENETIC ABNORMALITIES Causality has been studied especially by Hill and Doll. The Egyp ans had very interbred families; the Habsburg also presented this phenomenon, and all of them eventually suffered from a gene c disease called “Habsburg-Unterlippe” (difficul es in chewing food, difficul es in ar cula ng well); later genera ons suffered mental illness, hunchback, and drooling. Queen Victoria of Great Britain (1819–1901) was believed to be a carrier of hemophilia. Her descendants extensively married royal families in neighboring countries, eventually spreading hemophilia to other royal families in Europe. The human muta on rate per genera on (es mated from trio sequencing) has revealed an almost linear rela onship with the age of the parents. Fathers contribute to about three mes as many muta ons per year as mothers. The muta on rate es mates around 0.43 × 10−9 bases per year; hence, 1 base change in 2,325,581,395 bases per year leading to 60 to 100 new muta ons in each newborn genome. Female eggs undergo some damage over life, while sperm are con nuously produced. Useful heterogeneity is a phenomenon usually described by the peppered moth: with the industrial revolu on, the smoke and debris coming from factories turned the trees into a dark black color, thus rendering the white peppered moths an easy target; over me, Haldane believed that a new species of black moth had been discovered, when in reality it was simply the same species who had undergone selec on, only allowing the darker one (gene c modifica on) to survive. He described the importance of useful heterogeneity in the Peppered moth, thanks to polygenic inheritance, muta on rate and disease, as well as heterozygote persistence. Haldane introduced the important idea that immunity to infec ous diseases played an important part in human evolu on and that the individuals heterozygous for thalassemia (and sickle cell polymorphism) may possess greater resistance to Plasmodium falciparum malarial infec on. “What shape are your cells? At the microscopic level, small changes can have huge consequences. And while some adapta ons change these shapes for the be er, others can spark a cascade of debilita ng complica ons. This is the story of sickle-cell disease. Sickle-cell disease affects the red blood cells, which transport oxygen from the lungs to all the ssues in the body. To perform this vital task, red blood cells are filled with hemoglobin proteins to carry oxygen molecules. These proteins float independently inside the red blood cell’s pliable, doughnut-like shape, keeping the cells flexible enough to accommodate even the niest of blood vessels. But in sickle cell disease, a single gene c muta on alters the structure of hemoglobin. A er releasing oxygen to ssues, these mutated proteins lock together into rigid rows. Rods of hemoglobin cause the cell to deform into a long, pointed sickle. These red blood cells are harder and s ckier, and no longer flow smoothly through blood vessels. Sickled cells snag and pile up– some mes blocking the vessel completely. This keeps oxygen from reaching a variety of cells, causing the wide range of symptoms experienced by people with sickle-cell disease. Star ng when they’re less than a year old, pa ents suffer from repeated episodes of stabbing pain in oxygen-starved ssues. The loca on of the clogged vessel determines the specific symptoms experienced. A blockage in the spleen, part of the immune system, puts pa ents at risk for dangerous infec ons. A pileup in the lungs can produce fevers and difficulty breathing. A clog near the eye can cause vision problems and re nal detachment. And if the obstructed vessels supply the brain the pa ent could even suffer a stroke. Worse s ll, sickled red blood cells also don’t survive very long— just 10-20 days, versus a healthy cell’s 4 months. This short lifespan means that pa ents live with a constantly depleted supply of red blood cells; a condi on called sickle-cell anemia. Perhaps what’s most surprising about this malignant muta on is that it originally evolved as a beneficial adapta on. Researchers have been able to trace the origins of the sickle cell muta on to regions historically ravaged by a tropical disease called malaria. Spread by a parasite found in local mosquitoes, malaria uses red blood cells as incubators to spread quickly and lethally through the bloodstream. However, the same structural changes that turn red blood cells into roadblocks also make them more resistant to malaria. And if a child inherits a copy of the muta on from only one parent, there will be just enough abnormal hemoglobin to make life difficult for the malaria parasite, while most of their red blood cells retain their normal shape and func on. In regions rife with this parasite, sickle cell muta on offered a serious evolu onary advantage. But as the adapta on flourished, it became clear that inheri ng the muta on from both parents resulted in sickle-cell anemia. Today, most people with sickle-cell disease can trace their ancestry to a country where malaria is endemic. And this muta on s ll plays a key role in Africa, where more than 90% of malaria infec ons occur worldwide. Fortunately, as this “adapta on” thrives, our treatment for sickle cell con nues to improve. For years, hydroxyurea was the only medica on available to reduce the amount of sickling, blun ng symptoms and increasing life expectancy. Bone marrow transplanta ons offer a cura ve measure, but these procedures are complicated and o en inaccessible. But promising new medica ons are intervening in novel ways, like keeping oxygen bonded to hemoglobin to prevent sickling, or reducing the s ckiness of sickled cells. And the ability to edit DNA has raised the possibility of enabling stem cells to produce normal Hb”. The Hardy-Weinberg law states that allele and genotype frequencies in a popula on will remain constant from genera on to genera on in the absence of other evolu onary influences. It’s relevant in the following cases: random ma ng, no selec on, no muta on and no migra on. In an ini al popula on of A1A1 (20), A1A2 (40), and A2A2 (140), the frequencies are: Frequency A1A1 = 20/200 = 0.1; Frequency A1A2 = 40/200 = 0.2; Frequency A2A2 = 140/200 = 0.7. Allele frequencies are therefore: Frequency A1 = 0.2; Frequency A2 = 0.8, because: A1 is in all 20 of the first and half of the second: 20 + 20 = 40 (over 200); A2 is in all 140 of the second and half of the first: 140 + 20 = 160 (over 200). Offspring (random ma ng): - Frequency A1A1= 0.22 = 0.04; - Frequency A1A2= 2(0.2)*(0.8) = 0.32 - Frequency A2A2= 0.82 = 0.6 Why do we have gene c diseases? If natural selec on is supposedly removing bad genes, why do these genes s ll exist? These are very interes ng ques ons, which leads to one of the major reasons why the study of human evolu on is important. There are several reasons why we s ll have gene c diseases and gene c disease risks. One reason why gene c diseases persist relates to the way in which alleles for the disease are expressed. For example, does it require you to have two copies of the allele, one from both parents, for it to affect you? Or is one copy from one parent enough? When an autosomal trait only requires one allele, we refer to it as dominant. If the autosomal trait requires an allele from both parents, we call it recessive. Most major gene c diseases are recessive, which allows there to be many carriers of the disease who are unaffected because they have only one copy. A carrier is typically not under any pressure from natural selec on. Inbreeding This is also why too much inbreeding for a popula on can be a problem. With high inbreeding there's a greater chance that unfavorable recessive traits will come together, resul ng in a less healthy popula on. If a child is born from parents that are gene cally dissimilar, there is much less chance of these recessive diseases will come about and will be observed. Now allele expression and the effects of inbreeding are very complex and important subjects. There are gene c traits that may or may not be expressed regardless of whether they are in one copy or two. Also, there are mes when limited inbreeding can be helpful for a popula on. Purifying is another reason why gene c diseases persist is that natural selec on does not operate in a simple cut and dry way. Natural selec on certainly purifies the genome of traits that would be immediately deadly. Purifying selec on is especially relevant if the trait prevents the offspring from ever being born. In fact, up to 20% of known pregnancies end up in a spontaneous, natural abor on. Many miscarriages during pregnancy are caused by these natural abor ons. The actual number of spontaneous abor ons is likely much, much higher, but they go undetected because they don't always result in obvious symptoms. Of course, these miscarriages are terrible moments for families, but it's important and maybe a li le reassuring to know that many miscarriages are normal biological accidents, and they do not mean that the families did anything wrong. Most families can have a normal reproduc ve success even if they've had a miscarriage in their past. Purifying selec on is very strong when the trait has a drama c effect on the offspring's survival, but o en gene c diseases are less deadly. If the trait s ll allows some level of reproduc ve success, then natural selec on enters compe on with the expression of the trait and the other forces of evolu on. Also, if the disease has no major impact on reproduc on, such as those diseases that happen in our twilight years, then they would have li le impact on natural selec on. 6.1.2 - INFECTIVE AGENTS Recent decades have seen repeated pathogen emergence from wild or domes c animal reservoirs into human popula ons, from HIV-1 and HIV-2, to the 1918 influenza virus, to Middle East respiratory syndrome coronavirus, to SARS- CoV-2. For a novel pathogen to become a threat to human popula ons, first, contact between humans and the animal reservoir must occur; the pathogen must either have or evolve the capacity for human- to- human transmission; and finally, this human-to-human transmission must enable expansion of the pathogen’s geographical range beyond the zone of spillover. Recent global changes have affected each of these steps. History defined a massive increase in global movement:  Post-Columbus contact and European coloniza on: smallpox, measles and other diseases (15th-18th century);  Classical an quity trade and war: Antonine plague (second century); Plague of Cyprian (third century); Jus nian plague (sixth century);  Transatlan c slave trade and European coloniza on: P. falciparum malaria (16th-19th century)  Interna onal air travel: SARS epidemic (2002-2004) Urbaniza on also had a strong impact on infec ous diseases: urban expansion and land-use change lead to novel human-wildlife interac ons, air pollu on increases disease suscep bility, improved health-care access, more persistent outbreaks of respiratory disease in urban se ngs, poor sanita on and high popula on density increase spread of enteric infec ons (e.g., Aedes aegyp and albopictus adapted to urban se ngs, and breed in standing water; the rapid spread of Aedes albopictus across the world may be due to the interna onal commercializa on of used res and its strong ecological plas city which allows it to adapt to different environments 5), global transit networks increase risk of impor ng and expor ng pathogens (e.g., Anopheles spp. Less adapted to urban areas, leading to possible declines in malaria). Girolamo Fracastoro was an Italian physician, poet, and scholar in mathema cs, geography and astronomy, the first to theorize the “small and invisible”. In his major clinical work (“On Contagion, Contagious Diseases and Their Cure”), Fracastoro dis nguished three forms of contagion:  Diseases, such as syphilis and gonorrhea were only transmi ed by direct contact;  Other diseases were transmi ed by fomites as clothing, that had been in contact with the sick;  Diseases such as tuberculosis and smallpox were capable of infec ng persons at a distance from the sick and were transmi ed by air. He speculated that these infec ons were caused by transferable seed-like beings, seminaria or germs; he believed they were vola le chemicals. Pasteur is the “father of microbiology”, and he established the germ theory of diseases: he proved that all life, even microbes, arose only from their like and not de novo. Pasteur had not only resolved the controversy of origin of microorganisms but also had shown how to keep solu ons sterile with this one single experiment. Germ theory of diseases describes that diseases are caused by ny germs; this is an accepted scien fic theory for many diseases. these ny germs invade human beings and animals: their reproduc on within the host caused diseases. Many scien sts had proposed the germ theory even before Pasteur’s experiment, but all those proposals remained unrecognized. Fracastoro suggested that diseases are caused by invisible living organisms transmi ed from one person to another; this was further supported by Marcus von Plancies, who not only stated that living germs are the causa ve agents of diseases, but also said that different germs are responsible for different diseases. However, these views were dominated by Galen’s theory. The concept of germs to diseases had become quite general during the 18th century; in 1842, Holmes believed that puerperal fever, a disease of childbirth, was caused by a germ carried from one mother to another by midwives and physicians. At the same me, Semmelweis started using an sep cs for hand disinfec on during obstetrical opera ons and also explained the importance of hand disinfec on to other physicians. This has gradually reduced the rate of deaths caused due to infec ons associated with child births; Semmelweis published the concept and prophylaxis of childbed fever in 1861. The concept of an sepsis was brought into life by the experiments of Lister in England; later, the importance of an sepsis was completely understood. On the other side, Pasteur had successfully worked on the fermenta on issues and Pebrine disease, a silkworm disease that ruined the silk industry. Pasteur was then challenged to work on anthrax, a disease of ca le, sheep and rarely human beings; he isolated the microbes from the blood of infected animals. Simultaneously, Koch was also working on anthrax in Germany because of his interest in bacteriology; he con nued to work on anthrax and discovered that the germs that cause anthrax are rod-shaped. He also isolated these germs from the blood of animals that died of anthrax; in his experiments, he inoculated the isolated germs into a healthy animal; then, he observed that these animals had developed similar kind of symptoms of anthrax. He finally provided the first evidence that bacteria are the causa ve agents of diseases in animals, which led him to establish Koch’s postulates, a series of four principles to support germ-to-disease concept: 1. The organism must always be present in every case of the disease; 2. The organism must be isolated from a host containing the disease and grown in pure culture; 3. Samples of the organism taken from pure culture must cause the same disease when inoculated into a healthy animal in the laboratory. 4. The organism must be isolated from the inoculated animal and must be iden fied as the same original organism that was first isolated from the originally diseased host Although Koch’s postulates provided the guidelines to iden fy the cause of disease, there are some inherent limita ons that could not be answered in those mes; some of these limita ons are: a. The postulates explain some infec ous diseases, but not viruses as techniques were not developed to isolate viruses during 1800s; therefore, it was believed that Koch’s postulates are applicable to only a few infec ous diseases, as per the third postulate. b. The experimental animal should develop the disease, but it did not happen in every case because of asymptoma c carriers, immunity and gene c resistance. Koch’s postulates fail to explain prion diseases and other agents that cannot be grown in cultures; for our understanding, prion is a type of protein that can trigger normal proteins in the brain to fold abnormally; prion diseases can affect humans and animals. Hill wanted to expand a postula on beyond infec on and developed the “Bradford Hill criteria” or “Hill's criteria” for causa on, which pioneered the modern randomized clinical trial and established a convincing link between smoking and lung cancer. b.1.3 - CHEMICALS Bizzozero had realized that cancer and smoking are related and, in a popular ar cle, he suggested giving up smoking to reduce the spread of this disease. These associa ons, however, could be confounding (e.g., associa on between reduced pancrea c cancer risk and high caffeine consump on). The use of the Bradford-Hill criteria was modernized by introducing nine aspects of associa on: Strength of associa on (lots of sta s cal tools) Temporality (the cause must precede the outcome) Coherence (rela onship is consistent with knowledge) Consistency (consistent outcome in mul ple studies) Biological gradient (dose-response) Experiment (randomly assigned treatment vs. outcome) Specificity (single cause for a single effect) Plausibility (a molecular/biological explana on) Analogy (a similar agent should cause a similar effect) Individual exposures can cause epigene c modifica ons to parental DNA that lead to observed effect in future offspring, even though there’s no direct exposure. Epidemiologists are interested in understanding factors related to health and disease the smokers tend to die younger than non-smokers: it certainly looks that way, but disentangling cause-and-effect can be difficult. Smokers are different on average from non-smokers: they're more likely to drink heavily and have less healthy diets, but even if we measure these confounding factors, we may not measure them perfectly or there may be others we haven't measured; also, as people become ill, they may give up smoking, which could wrongly suggest the reduce smoking. We could randomly assign 50,000 people to smoking, 50,000 people to not smoke and follow them up to monitor their health; this removes the possibility that any other factor could be responsible for any differences we see in their long-term health, but this is neither ethical nor prac cal. Fortunately, at the point of concep on, our genes, which have passed on randomly from genera on to genera on, influence how much we eat drink, smoke and more. These gene c influences are not affected by anything else: you may or may not choose to do in your life, they're not related to confounding factors: we can use this knowledge to learn about cause and effect, grouping people according to their gene c code: this method is called Mendelian randomiza on (e.g., smokers carrying one version of a gene called CHRNA tend to smoke less heavily than those who carry a different version). Hence, Mendelian randomiza on consists in using people's varia on in gene cs to approximate an exposure a dietary exposure. When grouping people according to which version of this gene they have, we find that the people with the version of the gene associated with heavier smoking do die younger, but is the gene influencing how long we live in some other way? We don't think so: when we look at the same gene in non- smokers, there's no effect on life expectancy, so if that must be driven by smoking. Using this method shows that smoking causes lung cancer, heart and respiratory disease, and many other diseases, but doesn’t influence depression or anxiety. Mendelian randomiza on has already begun to tell us about factors that influence our risk of disease; now we're using the same approach in other ways to look at several risk factors together and to look at what influences disease progression which may help us to develop new treatments. 5. How does a mosquito bite regulate the inflammatory response in the skin? How does Plasmodium sporozoite trafficking to the mosquito salivary glands impact the secre on of certain salivary proteins, which in turn influence sporozoite infec vity? What host receptor interacts with salivary proteins? Which salivary proteins might serve as puta ve biomarkers for epidemiological analysis of malarial transmission? What role does the skin microbiota plays in mosquito bites and Plasmodium infec on? Could a cocktail of Anopheles salivary an gens – stand-alone or in combina on with Plasmodium an gens – be used as an effec ve candidate an -malarial vaccine?