TRANSGENIC ANIMALS AND DISEASE RESISTANCE

BY: Reddy Sailaja M (MSIWM030)

TRANSGENIC ANIMALS

‘Transgenesis’ is a molecular method of introducing a foreign gene (of interest) into the genome of an organism to express the desired trait or characteristic and further pass the trait to the progeny successfully. The gene that is being introduced is called a ‘transgene’.

A transgenic animal or genetically modified animal is the one that is being introduced with a desired foreign gene into its genetic material through recombinant DNA technology, a molecular biology technique.

Transgenesis has been widely applied in most of the domestic animals, aquaculture and agriculture that aids in human welfare and development.

Ralph Brinster and Richard Palmiter were the pioneers in creating first transgenic animal – “Super mouse” in 1982 by introducing human growth hormone in the mouse genome. The offspring produced were larger in size than the parent.

Figure 1: Transgenic super mouse (right) produced by recombinant DNA technology

Pig, goat, sheep, fish, cattle and insects like Drosophila melanogaster (fruit fly) are the most common transgenic animals that are being used in basic and applied research for human welfare.

PRODUCTION OF TRANSGENIC ANIMALS

Two methods are principally followed to generate transgenic animals

  1. Embryonic stem cell method
  2. Pronucleus method by microinjection
  1. Embryonic stem cell method for Transgenesis:
  2. Inner cell mass of mammalian blastocysts contain embryonic stem cells (ESCs). ESCs have the ability to produce all kinds of organisms’ cells, including gametes.
  3. Desired gene is selected from the donor organism.
  4. Vector DNA is chosen that carries the desired DNA to the host cell.
  5. Vector contains promoter and other regulatory sequences that are crucial for transgene transfer, selection and expression in the host organism.
  6. ESCs were cultured along with the vector containing desired DNA
  7. Successfully transformed cells will be selected based on the selection methods like antibiotic resistance.
  8. The transformed cells are injected into inner cell mass of embryonic blastocysts of the mouse for further propagation.
  9. A pseudo pregnant mouse (stimulus of mating results in making mouse uterus receptive for the blastocysts due to hormonal changes) was prepared and the transformed blastocyst stage embryo was introduced into the uterus.
  10. Blastocyst would implant successfully and the mouse gives birth to pups. 10-20% pups will be having the transgene and is heterozygous in nature (only one copy of the gene was transformed and the other was wild).
  11. Heterozygous mice are allowed to mate to get homozygous offspring (1 in 4, Mendelian ratio) was selected and propagated further to generate transgenic trait.

Figure 2: Embryonic stem cell method for transgenic animal generation

  • Microinjection method:
  • Manipulation of the pronucleus is the most common method to create a transgenic animal and is first described by Gordon et al.
  • Superovulating female is induced with specific hormones and the eggs are harvested.
  • The male and female pronuclei are visible under microscope several hours after the sperm is allowed to enter into the oocyte. As male pronucleus is larger in size, the transgene is microinjected easily into it.
  • Pronucleus stage is advantageous as it allows early incorporation of the transgene into the host DNA and the entire host cells could express it.
  • Once the transgene is introduced, male and female pronuclei are allowed to fuse to form a fertilized egg.
  • Once the blastocyst stage is reached, it was implanted into the pseudo pregnant mother and the progeny was checked for the transgene expression as in the ESCs method. 

Figure 3: Generation of transgenic animal by microinjection method

Other techniques followed to generate transgenic animals are listed in table 1.

TRANSGENIC TECHNIQUESINTERPRETATION
Cre-lox techniqueIdeal technique with more control over resulting phenotype; time-consuming
Viral vectorsdifficult; largely restricted to avian species
Cytoplasmic injectionLess efficient than direct pronuclear microinjection
Primordial germ cellsChimeric animals result
Nuclear transferLarge potential for genetically modifying livestock
Spermatogonial manipulationTransplantation into recipient testes

Table 1: Other techniques used to generate transgenic animals

APPLICATION OF TRANSGENESIS

  1. Disease resistant transgenic animals:

Selection and cross breeding of animals is a natural way of producing superior quality livestock animals with respect to disease resistance, more milk production, larger size etc. However, maintaining these qualities to be passed to the generations is unpredictable.

  1. Neurodegenerative disease resistant animals: Spongiform encephalopathy (Scrapie disease) in sheep, bovine spongiform encephalopathy (BSE) (Mad cow disease) in cattle, Creutzfeldt Jacob disease in humans are some of the major neurodegenerative diseases. These diseases occur because of the expression and misfolding of “prion” protein. ‘Gene knock out’ of ‘prion protein through rDNA technology helps in generating prion protein free livestock and resistant to neurodegenerative disorders. RNA interference (RNAi) is the new rDNA technique that helps in knocking down of the desired gene by forming a double stranded DNA construct and suppressing its expression.

RNAi method has extensive applicability, one of which is to generate knock down transgenic animals that can survive RNA based viral infections like foot and mouth disease, classic swine fever and the most resent SARS-CoV-2 disease.

Figure 4: Prion free sheep (Denning et al 2001)

  • Bacterial disease resistant cattle: Mastitis is a bacterial infection of the mammary gland in the cows that affects quality and quantity of the milk being produced. Scientists have developed transgenic cattle that express lysostaphin protein, which kills mastitis causing bacteria by cleaving their cell wall.

Similarly, lysozyme producing transgenic goats are also generated that prevents mastitis causing bacterial lysis and healthier mammary glands.

  • Disease resistance in fishes: Catfish often prone to microbial infection and death. Cecropin B is a small protein expressed in Hyalophora cecropia moth that has anti-microbial protperties. Scientists have generated Cecropin gene expressing transgenic catfish that confers resistance against microbial infections.
  • Disease resistant cattle against Brucellosis: Brucellosis is a deadly zoonotic disease, that can spread across animals without limit and even to humans. A large number of animals in American Bison area have been affected badly and the grazing cattle used to acquire the infection that lead to abortions, low fertility rates, reduced milk production etc. In humans, the disease is called undulant fever and its effects are severe. Recently, it was discovered that bovineNRAMP1 gene is efficient in providing resistance to brucellosis. Transgenic cattle with this gene offer protection against the disease.

Mastitis resistant transgenic cow (Agricultural research service, US)

Identification and integration of genes through transgenesis is the need of the hour that provides disease resistance and improved immune response in livestock and poultry. Scientists are focusing on genetic engineering based disease resistant animal models that would help livestock show resistance to diseases as shown in the table 2.

Table 2: Diverse applications of disease-resistant genetically engineered animals.

  • Medical applications:
  • Disease models: Understanding the disease and its effects are crucial for effective drug development and vaccine creation. Transgenic method is widely applied to generate disease models to understand causes and effects of human diseases. For example, mouse with various cancers or cystic fibrosis were produced through rDNA technology. These models give insights into the disease and further effective drug development and treatment.
  • Understanding gene functions: Mouse/rat genetic composition is closely related to humans. Hence, mice or rat models are chosen to produce genetically modified organism with alteration of gene like, gene knock-out (removal of a particular gene) or gene knock-in (insertion of a gene) or damaging the gene.

This category of transgenic models helps to understand the crucial functions of the gene and its role in human development and disease. This method is widely used to produce transgenic animals with superior quality. For example, transgenic cow – with disease resistance and improved milk production.

  • Production of therapeutic proteins and antibodies: Animals like horse, goat and cows were genetically modified to develop and secrete useful chemical substances like antibodies, and therapeutic proteins that help treat the human diseases efficiently. For example, transgenic cow that secretes egg proteins into its milk. These transgenic animals with therapeutic reagents production are also called ‘walking pharmacies’.
  •  Production of xenotransplants: Scientists have developed transgenic farm animals by ‘knocking out’ the gene that is responsible for eliciting immune response and rejection of an organ when introduced into the human body. For example, knock out transgenic pig organs can be now used for organ transplantation in humans. This method solves the issue of organ donor shortage and saves many critical lives.
  • Transgenic fishes: Transgenic fishes are produced by introducing genes responsible for disease resistant/temperature tolerant/ better growth etc. For example, a company called Aqua Bounty Farms has requested United States Food and Drug Administration (USFDA) to approve its genetically modified salmon that has the ability to grow three times bigger size than normal within a year of its growth.

DISADVANTAGES OF TRANSGENIC ANIMALS

  • Genetically modified animals (like disease laboratory study models) will show negative impact on ecosystem if they escape and released into the environment.
  • May act as human disease reservoirs for critical pathogens like virus, prions etc.
  • May cause severe allergies in humans as it is not a natural product.
  • Genetically modified animals may show alterations in its behavior if the foreign gene undergoes any changes like mutations, leading to any unexpected harm to the mankind.

Even though, the chance of adverse effects is minimal, one can’t rule out completely.

ETHICAL CONCERNS OF TRANSGENIC ANIMALS

It is considered as unethical to produce transgenic animals because it is kind of violating animal rights and disrespect to animals.

Unless there is a balance maintained between need and the production of transgenic animals and effective application in human welfare like medical purpose, agriculture and scientific understanding and application.

CONCLUSION

From its origin, transgenic method has been creating revolutionary output for human well being by producing therapeutic proteins, superior quality breeds of animals and plants and xenografts. A transgenic animal has full potential to play a significant role in biomedical field. However, it is important to maintain ethical standards in effective usage of transgenic method for human welfare. As there is an equal chance of enormous harm that may cause to humans and the environment with the misuse of the technique.

Histoplasmosis

BY- K. Sai Manogna (MSIWM014)

Histoplasma capsulatum is a dimorphic fungus that, at ambient temperatures, stays in a mycelial shape and develops as yeast in mammals at body temperature. Histoplasmosis is caused by infection. While the fungus that causes histoplasmosis can be found worldwide in temperate climates, it is native to the U.S. valleys of the Ohio, Missouri, and Mississippi Rivers. Internationally, in North and Central America, eastern and southern Europe, and parts of Africa, East Asia, and Australia, the fungus is mainly found in river valleys. 

The soil in areas endemic to histoplasmosis provides a high organic acid damp environment that is good for mycelial development. Near regions populated by bats and birds, such as caves and chicken coops, highly infectious soil is found. Decaying trees and river banks also provide vital incubation habitats. Birds cannot be infected by fungi and do not spread the disease; bird excretions, however, contaminate the soil, enriching the mycelium growth medium. In comparison, bats may become infected, and by droppings, they spread histoplasmosis. For years, infected soil can be potentially contagious. Building and construction practices that disturb infected soil have been associated with histoplasmosis outbreaks. Moreover, as airborne spores can fly hundreds of miles, travelers to endemic areas are at risk of histoplasmosis. 

Pathophysiology: 

Life cycle:

1. H.capsulatum develops in a mycelial form in the saprobic state. 

2. On the hyphae of mycelium; macroconidia, and microconidia are formed. They are converted under temperature-controlled regulation to the yeast form. 

3. Conidia and mycelial fragments from polluted soil are aerosolized, resulting in alveolar deposition through inhalation. 

4. The host defense involves neutrophils and macrophages’ fungistatic properties. 

5. In restricting the degree of infection, T lymphocytes are essential. With weakened cellular host defenses, vulnerability to dissemination is markedly increased. 

6. Intracellularly, transfer from the mycelial to the pathogenic yeast type occurs. 

7. After macrophage phagocytosis, the yeast replicates within approximately 15-18 hours. Multiplication occurs throughout the phagosomes despite fusion with lysosomes. 

8. Proposed theories suggest that yeasts may produce proteins that inhibit the activity of lysosomal proteases. 

9. The growth of yeast stops within 1-2 weeks after exposure as the host immune response grows. 

10. Systemically, cytokines activate the fungistatic activity against intracellular yeasts in macrophages. 

11. Delayed-type hypersensitivity to histoplasmal antigens occurs with the cell-mediated response (3-6 weeks following exposure). 

12. Approximately 85-90 percent of immunocompetent people develop a positive response to the Histoplasma species skin antigen examination. 

13. The inflammatory response develops, over weeks to months, calcified fibrinous granulomas with areas of caseous necrosis. 

14. With continued exposure to large inoculums, clinical symptoms of histoplasmosis occur. The initial pulmonary infection, with hematogenous propagation, can spread systemically and produce extrapulmonary manifestations. 

15. There may be hematogenous spread to regional lymph nodes through the lymphatic or liver and spleen.

In adult hosts, which are immunocompetent, progressive disseminated histoplasmosis is rare. In patients with compromised cellular immunity, systemic spread usually occurs and generally includes the CNS, liver, spleen, and rheumatologic, ocular, and hematologic systems. 

Epidemiology: 

Sex: 

The rates of positive skin test results for exposure to H capsulatum antigens are identical in males and females. In women, rheumatologic manifestations appear to occur primarily. 

Age: 

While histoplasmosis may affect individuals of any age due to immature or degraded immune defenses, those in severe age ranges are more vulnerable to developing an infection. 

Prognosis:

A positive result is associated with acute pulmonary histoplasmosis. There is a long-term, long path of recurrent, progressive disseminated histoplasmosis, lasting for years, with long asymptomatic periods. Subacute progressive disseminated histoplasmosis progresses to death within 2-24 months if untreated. 

A 50 percent relapse rate is associated with, if treated, acute progressive disseminated histoplasmosis. With life-long antifungal maintenance, the incidence decreases to 10-20 percent. Without care, death is inevitable. 

Morbidity/mortality:

Morbidity and mortality are linked to systemic infection length and magnitude. 

Around 90 percent of acute pulmonary histoplasmosis patients are asymptomatic. In as many as 5% of symptomatic patients, acute pericarditis can happen. Generally, the pericardial fluid is exudative. In 40-60 percent of pericarditis patients, pleural effusions form. In patients with underlying lung disease, chronic pulmonary histoplasmosis occurs. Patients form cavities that can expand and result in necrosis. Progressive pulmonary fibrosis that results in cardiac failure, respiratory, and recurrent infections may result from untreated histoplasmosis. 

In infected infants, older people, and immunosuppressed people, progressive disseminated histoplasmosis occurs. In the subacute form, in untreated situations, death occurs within 2-24 months. The acute form results in death within weeks if untreated. 

Symptoms: 

Most individuals who are exposed to the Histoplasma fungus never have symptoms. Some individuals can have flu-like symptoms that typically go away by themselves. 

Histoplasmosis signs include fever, chills over, coughing, headache, fatigue, chest pain, and body pains.

In Severe Histoplasmosis: 

Histoplasmosis can evolve into a long-term lung infection in certain persons, usually those with compromised immune systems, or it can spread from the lungs to other areas of the body, such as the CNS.

Transmission: 

Usually, histoplasmosis is acquired by inhalation of airborne microconidia, often after polluted material disturbance (e.g., practices such as spelunking, chicken coop washing, or construction). It is exceedingly rare to have primary cutaneous histoplasmosis and solid organ donor-derived histoplasmosis. 

Risk and Prevention for Histoplasmosis: 

If they’ve been to a place where Histoplasma resides in the atmosphere, everyone can get histoplasmosis. Activities that disturb the soil, particularly soil containing bird or bat droppings, are often associated with histoplasmosis. Some classes of individuals are at greater risk of developing extreme types of histoplasmosis: 

For example, people who have weakened immune systems, individuals who: 

a. Have HIV/AIDS

b. Who took corticosteroids or TNF inhibitors

c. Children and adults of age 55 years above

d. Who had an organ transplant.

Histoplasmosis is unable to propagate between individuals or between people and animals from the lungs. However, the infection can be passed by an organ transplant of an infected organ in rare cases. 

Diagnosis: 

The most commonly used and most sensitive tool for diagnosing disseminated histoplasmosis and acute pulmonary histoplasmosis following exposure to a large inoculum is Histoplasma antigen detection in urine and serum. Antibody studies, culture, and microscopy are other methods.

Detection of antigens: Enzyme immunoassay (EIA) is typically performed on serum and urine, but also be used for bronchoalveolar lavage in cerebrospinal fluid.

Antibody testing: Antibody tests are not as effective as antigen detection tests for the diagnosis of acute histoplasmosis or for immunosuppressed individuals that may not have an excellent immune response, as it may take two to six weeks to develop antibodies to Histoplasma.

Immunodiffusion (ID): checks for the presence of precipitin bands of H (indicating chronic or severe acute infection) and M (developing within weeks of critical illness and can linger for months to years after the disease has ended); sensitivity of approximately 80%. 

Complement Fixation (CF): After infection, complement-fixing antibodies can take up to 6 weeks to appear. CF is more sensitive than immunodiffusion but less precise. 

Culture: May be done on tissue, blood, and other body fluids. They may take up to 6 weeks to become positive; most useful in diagnosing severe histoplasmosis types. You may use a commercially available DNA probe to confirm this. 

Microscopy: Low sensitivity in tissue or body fluids for the detection of budding yeast, but can provide a quick, proven diagnosis if positive.

Polymerase Chain Reaction (PCR): PCR is still experimental but promising for the identification of Histoplasma directly from clinical specimens. 

Histoplasmosis Treatment: 

Mild histoplasmosis cases confined to the lungs will be cured in about a month without special treatment. Treatment with antifungal medications is needed for severe infections or disseminated cases of histoplasmosis. Antifungal drugs that treat histoplasmosis are itraconazole, fluconazole, and amphotericin B. For several months, a person might need to continue treatment.

THE PROCESS OF THE INFLAMMATION

BY : K. Sai Manogna (MSIWM014)

Inflammation is a systemic reaction for several causes, such as tissue damage and infections. An acute inflammatory response typically has a fast onset and lasts for a short time. In general, acute inflammation is followed by a systemic reaction known as an acute-phase response, marked by a rapid shift in the concentrations of many plasma proteins. Persistent immune activation in certain diseases can lead to chronic inflammation, which often has pathological implications.

An essential role of neutrophils in inflammation:

1. The predominant cell type infiltrating the tissue is the neutrophil at the early stages of an inflammatory response.

2. Within the first six hours of an inflammatory response, neutrophil penetration into the tissue peaks, with development of neutrophils in the bone marrow growing to meet this need.

3. An average adult produces more than 1010 neutrophils per day, but during a time of acute inflammation, neutrophil production can increase by as much as tenfold.

4. The bone marrow is left by the neutrophils and circulates inside the blood.

5. Vascular endothelial cells increase their expression of E- and P-selectin in response to the mediators of acute inflammation.

6. Increased P-selectin expression is caused by thrombin and histamine; cytokines like IL-1 or TNF-induce increased E-selectin expression. The circulating neutrophils express mucins such as PSGL-1 or the tetrasaccharides Lewisa sialyl and Lewisx sialyl bind to E- and P-selectin.

7. This binding mediates the attachment or tethering of neutrophils to the vascular endothelium, enabling the cells to roll in the direction of blood flow.

8. Chemokines such as IL-8 or other chemoattractants function on the neutrophils during this time, causing an activating signal mediated by G-protein that leads to a conformational shift in the molecules of integrin adhesion, resulting in neutrophil adhesion and subsequent transendothelial migration.

9. When in tissues, activated neutrophils also express elevated levels of chemoattractant receptors and thus show chemotaxis, migrating up the chemoattractant gradient.

10. Several chemokines, complement split products (C3a, C5a, and C5b67), fibrinopeptides, prostaglandins, and leukotrienes are among the inflammatory mediators that are chemotactic to neutrophils.

11. Furthermore, microorganism-released compounds, such as formyl methionyl peptides, are also chemotactic to neutrophils.

12. Increased levels of Fc antibody receptors and complement receptors are expressed by activated neutrophils, allowing these cells to bind more efficiently to antibody- or complement-coated pathogens, thereby increasing phagocytosis.

13. The triggering signal also activates the metabolic pathways into a respiratory burst, creating intermediates of reactive oxygen and intermediates of reactive nitrogen.

14. In the killing of different pathogens, the release of some of these reactive intermediates and the release of mediators from neutrophil primary and secondary granules (proteases, phospholipases, elastases and collagenases) play a significant role.

15. The tissue damage that can result from an inflammatory reaction also leads to these substances. The aggregation, along with accumulated fluid and different proteins, of dead cells and microorganisms, makes up what is known as pus.

Inflammatory Responses:

A complex cascade of non-specific events, known as an inflammatory response, is caused by infection or tissue injury, which provides early protection by minimising tissue damage to the site of the infection or tissue injury. Both localised and systemic responses are involved in the acute inflammatory response.

LOCALISED INFLAMMATORY RESPONSE:

Redness, swelling, pain, heat, and loss of function are the hallmarks of a localised acute inflammatory response first identified almost 2000 years ago. There is an increase in vasodilation within minutes of tissue injury, resulting in an increase in the area’s blood volume and a decrease in blood flow. The increased volume of blood heats the tissue and causes it to turn red. Vascular permeability also increases, leading to fluid leakage, especially in postcapillary venules, from the blood vessels. This results in the fluid deposition in the tissue and, in some cases, leukocyte extravasation, which leads to the area’s swelling and redness. The kinin, clotting, and fibrinolytic processes are triggered when fluid exudes from the bloodstream. The direct effects of plasma enzyme mediators like bradykinin and fibrinopeptides, which induce vasodilation and increased vascular permeability, are responsible for many of the vascular changes that occur early in the local response. Some of these vascular changes are due to the indirect effects of histamine-released complement anaphylatoxins (C3a, C4a, and C5a) that induce local mast-cell degranulation.

1. Histamine is a potent inflammatory mediator, inducing vasodilation and contraction of smooth muscle.

2. Prostaglandins may also contribute to the acute inflammatory response associated with vasodilation and increased vascular permeability.

3. Neutrophils bind to the endothelial cells within a few hours of the initiation of these vascular changes and move from the blood into the tissue areas.

4. These phagocytose neutrophils invade pathogens and release mediators that lead to the inflammatory reaction.

5. The macrophage inflammatory proteins (MIP-1 and MIP-1), chemokines which attract macrophages to the inflammation site, are among the mediators. Around 5-6 hours after an inflammatory response starts, macrophages arrive.

6. These macrophages are activated cells that show increased phagocytosis and increased release of mediators and cytokines that contribute to the inflammatory response.

7. Three cytokines (IL-1, IL-6, and TNF-𝛼) that induce activated tissue macrophages secrete many of the localised and systemic changes, which observed in the acute inflammatory response.

8. All three cytokines function locally, causing coagulation and vascular permeability to increase.

9. Both TNF-𝛼 and IL-1 induce increased expression of adhesion molecules on vascular endothelial cells. For example, TNF-𝛼 stimulates the expression of E-selectin, a molecule of endothelial adhesion that binds adhesion molecules to neutrophils selectively. IL-1 induces increased ICAM-1 and VCAM-1 expression, which binds to lymphocyte and monocyte integrins.

10. Neutrophils, monocytes, and lymphocytes circulating identify these adhesion molecules on the walls of the blood vessels, bind to them and then pass into the tissue spaces via the vessel wall.

11. IL-1 and TNF-𝛼 also act on macrophages and endothelial cells to induce the development of chemokines that, by increasing their adhesion to vascular endothelial cells and by acting as potent chemotactic factors, contribute to the influx of neutrophils.

12. Besides, macrophages and neutrophils are activated by IFN-𝞬 and TNF-𝛼, facilitating increased phagocytic activity and increased release of lytic enzymes into tissue areas.

13. Without the overt intervention of the immune system, a local acute inflammatory response may occur.

14. Cytokines released at the inflammation site also promote both the adherence of immune system cells to vascular endothelial cells and their migration into tissue spaces through the vessel wall.

15. This results in an influx of lymphocytes, neutrophils, monocytes, eosinophils, basophils, and mast cells to the tissue damage site, where these cells are involved in antigen clearance and tissue healing.

To monitor tissue damage and promote the tissue repair processes that are important for healing, the length and strength of the local acute inflammatory response must be carefully controlled. TGF-β has been shown to play an essential role in limiting the response to inflammation. It also encourages fibroblast aggregation and proliferation and the deposition of an extracellular matrix necessary for proper tissue repair. Clearly, in the inflammatory response, the leukocyte adhesion processes are of great importance. As exemplified by leukocyte-adhesion deficiency, a failure of proper leukocyte adhesion may result in disease.

SYSTEMIC ACUTE-PHASE RESPONSE:

The systemic response is known as the acute-phase response accompanies the local inflammatory response. This response is characterised by fever induction, increased hormone synthesis such as ACTH and hydrocortisone, increased white blood cell development (leukocytosis), and the production of a large number of liver acute-phase proteins.

1. The rise in body temperature prevents a variety of pathogens from rising and tends to strengthen the immune response to the pathogen.

2. A prototype acute-phase protein whose serum level increases 1000-fold during an acute-phase response is a C-reactive protein.

3. It is made up of five similar polypeptides by noncovalent interactions kept together.

4. The C-reactive protein binds and activates complements to a wide range of microorganisms, resulting in the accumulation of opsonin C3b on the surface of microorganisms.

5. The C3b-coated microorganisms can then readily phagocytose phagocytic cells, which express C3b receptors.

6. The combined activity of IL-1, TNF-𝛼 and IL-6 is linked to several systemic acute-phase effects. To cause a fever response, each of these cytokines works on the hypothalamus.

7. Increased levels of IL-1, TNF- and IL-6 (as well as leukaemia inhibitory factor (LIF) and oncostatin M (OSM)) induce hepatocyte development of acute-phase proteins within 12–24 h of the onset of acute-phase inflammatory response.

8. To induce colony-stimulating factors (M-CSF, G-CSF, and GM-CSF) secretion, TNF-𝛼 also acts on vascular endothelial cells and macrophages.

9. These CSFs induce hematopoiesis, causing the number of white blood cells required to combat the infection to increase temporarily.

10. Redundancy in the capacity of at least five cytokines (TNF-𝛼, IL-1, IL-6, LIF, and OSM) to induce liver acute-phase protein development results from the induction of NF-IL6, a common transcription factor, after the receptor interacts with each of these cytokines.

11. Amino-acid sequencing of cloned NF-IL6 showed that it has a high degree of sequence identity with C / EBP, a liver-specific transcription factor.

12. NF-IL6 and C / EBP both contain a leucine-zipper domain and a simple DNA-binding domain, and in the promoter or enhancer of the genes encoding different liver proteins, both proteins bind to the same nucleotide sequence.

13. C / EBP, which stimulates albumin and transthyretin production, is hepatocyte-constitutively expressed.

14. Expression of NF-IL6 increases and that of C / EBP decreases as an inflammatory response arises, and the cytokines interact with their respective receptors on liver hepatocytes.

15. The inverse relationship between these two transcription factors reflects the observation that serum protein levels such as albumin and transthyretin decrease during an inflammatory response while those of acute-phase proteins increase.

CHEMOKINES

BY: K Sai Manogna (MSIWM014)

1. Chemokines are the superfamily of small polypeptides, most of which contain residues of 90-130 amino acids are chemokines. 

2. They regulate the adhesion, chemotaxis, and activation of several kinds of leukocyte populations and sub-populations selectively, and sometimes explicitly. 

3. Consequently, they are significant leukocyte traffic regulators. Some chemokines are predominantly involved in inflammatory processes; others are expressed constitutively and play essential roles in the homeostatic or developmental activity. 

4. Housekeeping chemokines are manufactured in lymphoid organs and tissues or in non-lymphoid sites such as the skin where normal lymphocyte trafficking is directed, such as deciding the correct placement of newly-generated hematopoiesis leukocytes arriving from the bone marrow. 

5. Chemokines are constitutively expressed by the thymus, and normal B cell lymphopoiesis often depends on suitable chemokine expression. 

6. Effects mediated by chemokines are not confined to the immune system. 

7. Mice missing either the CXCL12 chemokine (also referred to as SDF-1) or its receptor display significant defects in brain and heart production. 

8. In the formation of blood vessels and wound healing, members of the chemokine family have also been shown to play regulatory roles. 

9. In response to infection, inflammatory chemokines are usually induced. 

10. The expression of inflammatory cytokines as inflammation sites is regulated by interaction with pathogens or the action of pro-inflammatory cytokines, such as TNF-𝛼. 

11. By inducing, adherence of these cells to the vascular endothelium, chemokines induce leukocytes to migrate into different tissue locations. 

12. Leukocytes are drawn to high localised concentrations of chemokines after migration into tissues, resulting in the selective recruitment of phagocytes and lymphocyte effector populations to inflammatory sites. 

13. The assembly of leukocytes at infection sites, coordinated by chemokines, is an integral part of mounting an infection response that is correctly oriented. 

Of more than 50 chemokines and at least 15 chemokine receptors have been identified. The chemokines have four residues of conserved cysteine and almost all fall into one or the other of two distinctive subgroups based on the position of two of the four invariant cysteine residues: 

■ Chemokines of the C-C subgroup, in which conserved cysteines are contiguous; 

■ Chemokines of the C-X-C subgroup, in which several other amino acids (X) distinguish the conserved cysteines. 

Receptors whose polypeptide chain traverses the membrane seven times mediate chemokine action. CC receptors (CCRs), which recognise CC chemokines, and CXC receptors (CXCRs), which recognise CXC chemokines, are two subgroups of receptors. 

As with cytokines, there is a high affinity (Ka > 109) and high specificity for the interaction between chemokines and their receptors. For instance, at least six different chemokines are recognised by CXCR2, and several chemokines can bind to more than one receptor. 

It activates heterotrimeric large G proteins when a receptor binds an appropriate chemokine, activating a signal-transduction method that generates such potent second messengers as cAMP, IP3, Ca2 +, and activated small G proteins. Chemokine-initiated activation of these signal transduction pathways carries out drastic changes. The addition of adequate chemokine to leukocytes induces sudden and widespread changes in shape within seconds, the promotion of more outstanding adhesion to endothelial walls by activating leukocyte integrins, and the development of phagocyte microbicidal oxygen radicals. These signal-transduction pathways facilitate other changes, such as granular material release, neutrophil and macrophage proteases, basophil histamine, and eosinophil cytotoxic proteins.  

Chemokine-Receptor Profiles Mediate Activity of leukocytes: 

1. Neutrophils express CXCR1, -2, and -4 among the central populations of human leukocytes; eosinophils have both CCR1 and CCR3. 

2. Some activated T cells have CCR1, -2, -3, and -5, CXCR3 and -4, and possibly others, whereas resting naive T cells show a few types of chemokine receptors. 

3. Consequently, variations in the expression of chemokine receptors by leukocytes coupled with the development by destination tissues and sites of distinctive chemokine profiles provide rich opportunities for the differential control of the activities of the various populations of leukocytes. 

4. Indeed, variations in chemokine-receptor expression patterns occur both within and between different populations of leukocytes. 

Fig : Patterns of expressions on some principle chemokine receptors on the human leukocytes.

Note: Their various cytokine output patterns can distinguish the TH1 and TH2 subsets of TH cells. Different profiles of chemokine receptors also show these subsets. CCR3 and -4 are expressed by TH2 cells, and a variety of other receptors are not expressed by TH1 cells. TH1 cells, on the other hand, express CCR1, -3, and -5, but most TH2 cells do not. 

The Other Inflammatory Mediators: 

Several other mediators released by cells of the innate and acquired immune systems activate or improve particular aspects of the inflammatory response in addition to chemokines. Tissue mast cells, blood platelets, and several leukocytes, including neutrophils, monocytes/macrophages, eosinophils, basophils, and lymphocytes, release them. 

The plasma comprises four interconnected mediator-producing processes in addition to these sources: the kinin system, clotting system, fibrinolytic system, and the complement system. The first three systems share the Hageman factor, a common intermediate. These four systems are triggered when tissue damage occurs, to form a network of interacting systems that produce several inflammation mediators. 

The Tissue Injury Stimulates the Kinin System: 

1. The kinin mechanism is an enzymatic cascade that starts with tissue injury, a plasma clotting factor, called Hageman factor, is activated. 

2. In order to form kallikrein, the activated Hageman factor then activates prekallikrein, which cleaves kininogen to generate bradykinin. 

3. This inflammatory mediator is a potent fundamental peptide that enhances vascular permeability, causes vasodilation, pain, and induces smooth muscle contraction. 

4. By cleaving C5 into C5a and C5b, kallikrein also works directly on the complement mechanism. 

5. An anaphylatoxin that induces mast-cell degranulation, resulting in the release of several inflammatory mediators from the mast cell, is the C5a complement portion. 

The Clotting System Yields Inflammation Mediators Produced by Fibrin: 

1. Another enzymatic cascade caused by blood vessel disruption yields significant amounts of thrombin. 

2. To generate insoluble strands of fibrin and fibrinopeptides, thrombin works on soluble fibrinogen in tissue fluid or plasma. 

3. Clot formation acts as a barrier to the spread of infection, for which the insoluble fibrin strands cross each other. 

4. After tissue damage, the clotting mechanism is activated very quickly to avoid bleeding and limit the spread into the bloodstream of invading pathogens. 

5. As inflammatory mediators, the fibrinopeptides act, inducing increased vascular permeability and neutrophil chemotaxis. 

The Fibrinolytic System Yields Inflammation Mediators produced by Plasmin: 

1. The fibrinolytic method completes the elimination of the fibrin clot from the damaged tissue. 

2. The end product of this pathway is the plasmin enzyme, which is generated by plasminogen conversion. 

3. Plasmin breaks down fibrin clots into degradation products that are chemotactic for neutrophils, a potent proteolytic enzyme via activating the classical complement pathway.

4. Plasmin also contributes to the inflammatory response. 

Anaphylatoxins Formed by the Complement System: 

1. Activation by both classical and alternative pathways of the complement system results in the development of several complement split products that serve as essential inflammation mediators. 

2. Binding of anaphylatoxins such as C3a, C4a, and C5a to receptors on the membrane of tissue mast cells induces degranulation with histamine release and other pharmacologically active mediators.

3. Such mediators cause contraction of smooth muscles and increase vascular permeability. 

4. C3a, C5a, and C5b67 function together to induce the adherence of monocytes and neutrophils to vascular endothelial cells, extravasate through the capillary endothelial lining, and migrate to the tissue site of complement activation. 

5. Thus, activation of the complement system results in fluid inflows carrying antibody and phagocytic cells to the entry site of the antigen. 

Lipids as Inflammatory Mediators: 

1. Phospholipids in the membrane of many cell types (e.g., macrophages, monocytes, neutrophils, and mast cells) are degraded into arachidonic acid and lyso-platelet-activating factor following membrane disturbances. 

2. Subsequently, the latter is transformed into a platelet-activating factor (PAF) that induces platelet activation and has several inflammatory consequences, including eosinophil chemotaxis, neutrophil and eosinophil activation and degranulation. 

Arachidonic acid metabolism:

a. Arachidonic acid metabolism produces prostaglandins and thromboxanes through the cyclooxygenase pathway. 

b. Various cells produce various prostaglandins: 

i. monocytes and macrophages produce large quantities of PGE2 and PGF2; 

ii. neutrophils produce moderate amounts of PGE2, which is released by mast cells. 

There are various physiological effects of prostaglandins, including increased vascular permeability, increased vascular dilation, and neutrophil chemotaxis induction. Thromboxanes cause platelet aggregation and blood vessel constriction. 

The lipoxygenase pathway also metabolises arachidonic acid to yield four leukotrienes: LTB4, LTC4, LTD4, and LTE4. Three of these (LTC4, LTD4, and LTE4) together make up what was formerly referred to as a slow-reacting anaphylaxis material (SRS-A); these mediators cause contraction of smooth muscle. LTB4 is a potent neutrophil chemoattractant. Several cells, including monocytes, macrophages, and mast cells, make leukotrienes.

Some cytokines are essential mediators of inflammation:

1. In the formation of an acute or chronic inflammatory response, several cytokines play a significant role. 

2. There are redundant and pleiotropic effects of IL-1, IL-6, TNF, IL-12, and several chemokines that together lead to the inflammatory response. 

3. Besides, IFN-contributes to the inflammatory response, functioning later in the acute response and by attracting and stimulating macrophages, leading in a significant way to chronic inflammation. 

4. The differentiation of the pro-inflammatory TH1 subset is caused by IL-12.

COMPONENTS OF BLOOD

BY: Reddy Sailaja M (MSIWM030)

BLOOD

Blood is a specialized body fluid, comprises of plasma and the cells that circulate throughout the human body. It supplies oxygen, glucose, antibodies, vitamins, electrolytes, heat, hormones, and immune cells across the body for life survival. It removes carbon dioxide and other waste generated from the cells in the body.

BLOOD COMPONENTS

Blood is made up of three main components: plasma, blood cells (red and white) and platelets.

Plasma: Plasma occupies 55% of blood fluid in human beings. Plasma comprises of 92% water and the remaining 8% contains carbon dioxide, glucose, hormones, proteins etc.

 Blood cells: Blood cells comprise of 45% of total blood fluid. These are produced in bone marrow by the process called ‘hematopoiesis’ from a common precursor cell (hematopoietic stem cells). Then these blood cells mature into red blood cells (RBCs), white blood cells (WBCs) and platelets. The organs like lymph nodes, liver and spleen regulate the generation, destruction and regulation of blood cells.

Figure 1: Blood cells production by hematopoiesis

Red blood cells: RBCs are also called erythrocytes. They are double concave shaped structures without nucleus. Approximately, 4.5 – 6.2 million/microliter are present in the blood. Their main function is to carry oxygen from lungs to all parts of the body. RBCs contain a special protein called ‘Hemoglobin’ that aids in oxygen transport. RBCs have a life span of 120 days.

White blood cells: WBCs are otherwise known as leucocytes. WBCs are vital in fighting against invading pathogens and infections. Approximately 3700 – 10500/microliter are present in the blood. Apart from fighting infection, WBCs also help heal wounds by ingesting dead cells and debris, protects against foreign entity that enter blood stream and fights against cancerous cells.

The following are the types of WBCs that are produced in response to the kind of infection (bacterial vs fungal vs viral vs parasitic).  Life span varies from hours to days to years depending upon the type of WBCs. WBCs are majorly divided into two subtypes: Granulocytes and agranulocytes. Granulocytes contains protein containing granules in their cytoplasm. Eosinophils, basophils and neutrophils constitute granulocytes. Monocytes and lymphocytes constitutes agranulocytes. The following table explains the types of WBCs’, their nature and function.

Type of white blood cellPercentage of abundance in WBCS (%)Function
Basophils0.5 – 1Basophils produce in response to parasite infections, allergy and bone marrow damage. It secretes histamine – involve in allergic reactions and heparin – an anticoagulant that aids blood clotting at the site of injury and subsequent wound healing.
Eosinophils2 – 4Eosinophils defend against bacterial and parasite infections by releasing toxic substances and results in immflamatory reaction.
Neutrophils60 – 70First line of defense against invading pathogens. They attack the pathogens, engulf and digest them by phagocytosis process and maintain normal health.
Monocytes3 – 8Maintains tidiness of the blood and other tissues by clearing the dead pathogen particles and damaged cells and their debris.
Lymphocytes20 – 25B – cells produce antibodies against bacterial, viral and fungal infection. T – cells are two types, cytotoxic T-cells kills the antigens and helper T-cells aid antibody production from B-cells. Natural killer cells attack any foreign object that comes in contact with the body.

Figure 2: Blood and its components

Blood platelets: These are also called thrombocytes. Approximately 1,50,000 – 4,00,000 platelets/microliter are present in the blood. Platelets help clot the blood to stop bleeding during injury by protects the wound against further infection.

In brief, blood functions include:

  • Oxygen supply to cells and tissues
  • Supply essential nutrients – glucose, aminoacids, fatty acids,
  • Removal of waste material – carbon dioxide, urea, lactic acid
  • Fighting against infections
  • Regulating body temperature and pH balance
  • Transport hormones and transmits neuromessages

Blood disorders:

Blood disorders often cause life threatening situations as the infection spreads out throughout the body by blood circulation. General blood disorders are as follows:

RBCs disorder – Anemia: Low number of RBCs in blood cause anemic situation. This results in low oxygen supply in the body, fatigue and pale skin.

WBCs disorder – Cancer: Lymphoma, myeloma and leukemia are the major blood related cancers.

Platelets disorder – Internal blood clots: these clots block blood supply and can be dislodged and spread through various organs like lungs, heart, brain etc, which can be fatal to the body.

CYTOKINES

BY: Ria Fazulbhoy (MSIWM031)

Cytokines are an important group of proteins or glycoproteins which play a major role in cell-to-cell communication between cells like lymphoid cells, inflammatory cells and hematopoietic cells. They are secreted by white blood cells (WBCs) and other cells of the body. They respond to stimuli and assist in the regulation of development of immune effector cells and sometimes have a direct effect of their own as well. The cytokine binds to the target cell by the presence of specific membrane receptors present on the target cell. (very high affinity-cytokines work at picomolar concentrations)

Mode of action

  1. Autocrine

The cytokine is released from a cell and binds to the membrane receptor present on the same cell.

  • Paracrine

Cytokine is released from the producer cell and binds to the target cell which is in close proximity

  • Endocrine

Cytokine binds to the target cell which is in a distant part of the body.

Four major groups of cytokines are Hematopoietic family (interleukins-ILs), Interferons (IFs) family, Tumor necrosis factors (TNF) family and chemokine family.

A variety of cells secrete cytokines, but the major principal producer cells are Th cells and macrophages. Cytokines secreted from these cells activate an entire network of interacting cells.

   Macrophage and Th cells are major producers of cytokines in the body.

Some biological functions of cytokines include:

  • Cellular and humoral immunity development
  • Inflammatory response induction
  • Control of cellular proliferation and differentiation
  • Healing of wounds
  • Development of innate/acquired immunity
  • Hematopoiesis

NOTE: Cytokines have a non-antigen specific mode of action and have very short half-lives.

Functions of some cytokines

Cytokine secretion by Tн cell subsets: Tн1 and Tн2

Difference in the pattern of cytokine secretion amongst Tн cell subsets determines the immune biological response made to a particular antigenic challenge. These two subsets are Tн1 and Tн2, which secrete different cytokines and mediate in different ways. Both these subsets secrete IL-3 and GM-CSF.

Tн1 and Tн2 have the following functional differences:

1) Tн1 subset:

  • It is responsible for mainly cell-mediated immune responses like activation of Tc cells and delayed hypersensitivity reactions.
  • Helps in promotion of excessive inflammation and tissue injury
  • Helps in production of opsonization-promoting IgG antibodies.
  • Effective in viral infections and intracellular pathogens.
  • IFN-४, IL-12 and IL-18 are responsible for the development of Tн1 cell response.
  • E.g.: IFN-४ and TNF-ß mediates inflammation and delayed hypersensitivity.
  • E.g.: IL-2 and IFN-४ promote differentiation of cytotoxic cells Tc from CD8 precursors.

2) Tн2 subset:

  • Responsible for secretion of antibodies for immune response.
  • Stimulates eosinophil activation and differentiation
  • Helps B cells
  • Promotes production of large amount of IgM and IgG
  • Supports allergic reactions
  • IL-4 is essential for the development of Tн2 response.
  • E.g.: IL-4 and IL-5 induce production of IgE and helps eosinophil attack on helminth or roundworm infections.

Tн2 development is favoured over Tн1. The cytokines produced by the two subsets are cross regulated. The cytokines produced by a subset (Tн1 or Tн2) promote the growth of their subset and simultaneously inhibit the activity and development of the opposite substrate (cross regulation). Two transcription factors known as T- Bet and GATA-3 are important in determining the cross regulation of the two subsets

  • T-Bet drives cells to differentiate towards Tн1 cells.
  • GATA-3 drives cells to differentiate along Tн2 cells.

EXTRAVASATION OF LYMPHOCYTES

BY: K. Sai Manogna (MSIWM014)

At inflammatory sites and secondary lymphoid glands, different subsets of lymphocytes exhibit directed extravasation. Therefore, lymphocyte recirculation is closely monitored to ensure that sufficient populations of B and T cells are recruited into various tissues. Extravasation of lymphocytes involves interactions between a variety of cell-adhesion molecules, as with neutrophils. The overall process is similar to what occurs during the extravasation of neutrophils and involves the same four stages of touch and rolling, activation, arrest and adhesion and, eventually, transendothelial migration.

Sites of Lymphocyte Extravasation:

1. Some regions of vascular endothelium consist of specialised cells with a plump, cuboidal (‘high’) form in the postcapillary venules of different lymphoid organs; such regions are referred to as high-endothelial venules or HEVs.

2. In appearance, their cells contrast strongly with the flattened endothelial cells that line the rest of the capillaries. Each of the secondary lymphoid organs comprises HEVs, except the spleen.

3. There are about 1.4 × 104 lymphocytes extravasate into a single lymph node every second through HEVs.

4. Cytokines developed in response to antigen capture influence the production and maintenance of HEVs in lymphoid organs.

5. In order to prevent the antigen from entering the node, the role of lymphocyte antigenic activation in the preservation of HEVs has been demonstrated by surgical blocking of afferent lymphocyte vasculature of the node.

6. The HEVs demonstrate impaired function within a short period and gradually return to a more flattened morphology.

7. High-endothelial venules express several cell-adhesion molecules. HEVs, like other vascular endothelial cells, express CAMs from the selectin (E- and P-selectin) family, the mucin-like (GlyCAM-1 and CD34) family, and the superfamily of immunoglobulins (ICAM-1, ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1).

8. In a tissue-specific way, some of these adhesion molecules are distributed. These tissue-specific adhesion molecules have been named vascular addressins (VAs) because they help to guide the extravasation to specific lymphoid organs of various populations of recirculating lymphocytes.

Receptor Profiles and Signals Guided by Lymphocyte Homing:

1. Related to neutrophil extravasation, the general lymphocyte extravasation mechanism is similar.

2. The fact that different subsets of lymphocytes migrate differently into different tissues is a significant aspect that separates the two processes. This method is known as trafficking or homing.

3. The numerous lymphocyte subset trafficking patterns are regulated by unique combinations of adhesion molecules and chemokines; homing receptors are called receptors that guide the circulation of different lymphocyte populations to specific lymphoid and inflammatory tissues.

Researchers have established several lymphocytes and endothelial cell adhesion molecules that are involved in lymphocyte interactions with HEVs and endothelium at tertiary sites or sites of inflammation.

Recirculating Naive Lymphocytes into Secondary Lymphoid Tissue:

Until it has been triggered to become an effector cell, a naive lymphocyte is not able to mount an immune response.

1. In specialised microenvironments within secondary lymphoid tissue (e.g., peripheral lymph nodes, Peyer patches, tonsils, and spleen), activation of a naive cell occurs.

2. Dendritic cells catch antigen inside these microenvironments and present it to the naive lymphocyte, resulting in its activation.

3. Naive cells do not display a preference for a specific form of secondary lymphoid tissue but instead circulate indiscriminately across the body to secondary lymphoid tissue through recognising HEV adhesion molecules.

4. The initial attachment to HEVs of naive lymphocytes is usually mediated by the binding of the L-selectin homing receptor to HEV adhesion molecules such as GlyCAM-1 and CD34.

5. The naive cell trafficking pattern is designed to keep these cells continuously recirculating across secondary lymphoid tissue, the primary purpose of which is to trap antigen transmitted by blood or tissue.

6. They are activated and enlarged into lymphoblasts until naive lymphocytes encounter antigen trapped in secondary lymphoid tissue. Activation takes approximately 48 h, and the blast cells are retained in the paracortical area of secondary lymphoid tissue during this time.

7. The antigen-specific lymphocytes cannot be identified in the circulation during this process, called the shutdown phase.

8. During the shutdown point, rapid proliferation and differentiation of naive cells occur. Then the effector and memory cells that this process produces leave the lymphoid tissue and begin to recirculate.

Lymphocytes of Effector and Memory follow distinct patterns of trafficking:

1. Effector and memory lymphocyte trafficking patterns vary from those of naive lymphocytes.

2. By recognising inflamed vascular endothelium and chemoattractant molecules produced during the inflammatory response, effector cells appear to be home to regions of infection.

3. On the other hand, memory lymphocytes selectively house the type of tissue in which antigen was first encountered.

4. This presumably ensures that a specific memory cell returns to the tissue where the antigen it recognises is most likely to re-encounter a subsequent threat.

5. Memory cells and effector cells express increased levels of specific molecules of cell adhesion, such as LFA-1, which interact with ligands present in additional tertiary lymphoid tissue (such as skin and mucosal epithelial) and at inflammation sites, allowing these sites to be accessed by effector and memory cells.

6. Naive cells lack the corresponding molecules of cell-adhesion and do not house these sites.

7. A variety of adhesion molecules, including E- and P-selectin and the Ig-superfamily molecules VCAM-1 and ICAM-1, are expressed in inflamed endothelium and bind to receptors expressed at high levels in the memory and effector cells.

8. Subsets of the memory and effector populations display tissue-selective homing activity, unlike naive lymphocytes.

9. Such tissue specificity is imparted by multiple combinations of adhesion molecules rather than by a single adhesion receptor.

10. A mucosal homing subset of memory/effector cells, for example, has high levels of LPAM-1 (4 7) and LFA-1 (Lb2) integrins that bind to MAdCAM and various ICAMs on venules of intestinal lamina propria.

11. However, since they have low levels of L-selectin that would promote their entry into secondary lymphoid tissue, these cells prevent direction to secondary lymphoid tissues.

12. Preferential homing to the skin is shown by the second group of memory/effector cells. Low levels of L-selectin are also expressed in this subset, however high levels of cutaneous lymphocyte antigen (CLA) and LFA-1, which bind to E-selectin and ICAMs on skin dermal venules, are seen.

13. While effector cells and memory cells that express decreased L-selectin levels do not appear to reach peripheral lymph nodes via HEVs, they may enter peripheral lymph nodes via afferent lymph vessels.

Adhesion-Molecule Interactions Play Extravasation Vital Roles:

1. A multi-stage mechanism involving a cascade of adhesion-molecule interactions is the extravasation of lymphocytes into secondary lymphoid tissue or regions of inflammation, similar to those involved in bloodstream neutrophil emigration.

2. This shows the usual interactions in the extravasation of naive T cells into lymph nodes through HEVs.

Mechanism:

1. In the first stage, a selectin-carbohydrate interaction similar to that seen with neutrophil adhesion.

2. L-selectin, which acts as a homing receptor which directs the lymphocytes to specific tissues expressing a corresponding mucin-like vascular addressin such as CD34 or GlyCAM-1, initially binds naive lymphocytes to HEVs.

3. The rolling of lymphocytes is less pronounced than neutrophil rolling.

4. Although the initial interaction of selectin-carbohydrate is minimal, the slow rate of blood flow in postcapillary venules, especially in regions of HEVs, reduces the possibility that the tethered lymphocyte can dislodge the sheer force of the flowing blood.

5. In the second stage, chemokines that are either localised on the endothelial surface or secreted locally mediate an integrin-activating stimulus.

6. To maintain these soluble chemoattractant variables on the HEVs, the thick glycocalyx covering of the HEVs can work.

7. If, as some have indicated, HEVs secrete lymphocyte-specific chemoattractants, it will clarify why, while they express L-selectin, neutrophils do not extravasate into lymph nodes at the HEVs.

8. As happens in neutrophil extravasation, chemokine binding to G-protein – coupled receptors on the lymphocyte contributes to the activation of integrin molecules on the membrane.

9. The integrin molecules interact with the adhesion molecules of the Ig superfamily (e.g., ICAM-1) once activated, so that the lymphocyte adheres tightly to the endothelium.

10. In the final stage, molecular mechanisms involved in the transendothelial migration are poorly understood.

HUMAN INSULIN PRODUCTION

BY: Reddy Sailaja M (MSIWM030)

INSULIN

Insulin is a peptide hormone that plays a critical role in human metabolism. It is synthesized and secreted by beta cells of Islets of Langerhans in the pancreas. It is the first peptide hormone to be discovered (by Frederick Banting and Charles Herbert Best, 1921). It is the first protein to be sequenced in 1951 by Frederick Sanger. Dorothy Hodgkin has determined the crystal structure of insulin in 1969. Nevertheless, it is the first hormone to be synthesized by recombinant DNA technology.

Figure 1: Structure of insulin

STRUCTURE AND FUNCTION

 Human insulin is made up two polypeptide chains of 51 amino acids (A-chain- 21 amino acids and B-chain, 30 amino acids) with a molecular mass of 5808 Daltons.  Insulin is an anabolic hormone that plays a crucial role in the metabolism of carbohydrates and fats by converting the free glucose available in the blood into glycogen that can be stored in the muscles.

Figure 2: Functions of insulin

SYNTHESIS

 Insulin is synthesized as a single polypeptide called ‘preproinsulin’ along with a 24 residue signal peptide in the pancreatic beta cells. The signal peptide guides preproinsulin to endoplasmic reticulum (ER), where the signal peptide gets separated, resulting in ‘proinsulin’ formation. In the ER, the proinsulin is further processed and folded with the formation of three disulphide bonds and gets transported to golgi complex. In golgi, the folded proinsulin is converted to ‘active insulin’ by cellular endopeptidases, namely prohormone convertases 1 & 2 and exoprotease carboxypeptidase E. These endonucleases cleave at two positions in the proinsulin, resulting in the separation of a fragment called C-peptide. The active and mature insulin now consists of two chains: A-chain (21 amino acids) and B-chain (30 amino acids), both liked to each other by two disulphide bonds.

Figure 3:  Synthesis of active insulin from precursor

INSULIN MALFUNCTION AND THE ASSOCIATED DISEASES

Insulin helps maintain blood sugar level normal at all the times. When the blood sugar level is high, insulin directs liver to store glucose in the form of glycogen. In need, insulin directs the liver and muscles to release the stored glycogen in the form of glucose to boost energy to the body.

When the insulin production is less or uncontrollable, malfunction of the hormone results in the development of a condition called as diabetes mellitus (DM), where the body is unable to maintain balance between normal blood sugar levels and sysnthesis or breakdown of glycogen. Malfunction of Insulin hormone leads to two major types of diabetes milletus: Type 1 and Type 2.

Type 1 DM: It is an autoimmune disease, where one’s own immune system attacks the pancreatic cells and results in low or no insulin production. Environmental factors, genes and certain viruses trigger the immune system to damage the pancreatic cells.

Type 2 DM: The condition develops either by low insulin production by pancreatic cells or inability of the body to utilize the released insulin for glycogen synthesis. Insulin resistance is the condition developed when the major organs like muscles, body fat and liver starts ignoring the signals form insulin and fail in converting free glucose into glycogen. As more insulin is being produced, pancreatic cells get damaged and the free glucose (that was not being stored) affects the body with surge of energy. This, type 2 DM is a lifestyle disease that results majorly of  over body weight, lack of exercise, smoking, lower belly fat etc.

HUMAN INSULIN PRODUCTION BY RECOMBINANT DNA TECHNOLOGY

 Recombinant DNA technology is a revolutionary technique, where DNA molecules from two different organisms are joined together and inserted into a host organism in order to generate new genetic combination that adds value to varied fields like science, health care, agriculture, poultry and industry.

Human insulin is being produced by recombinant DNA technology using E.coli or Saccharomyces cerevisiae as host organisms in many ways. The popular one is the production of insulin A-chain and B-chain separately in two E.coli strains and then joined together by disulphide bonds to produce active insulin.

The mRNA sequence of A-chain (basically, mRNA is a blue print of functional protein after modifications) is fused with ß-galactosidase gene (lac Z) present in the pBR322 plasmid (now called recombinant plasmid) and inserted into E.coli by transformation process. The recombinant bacteria is allowed to grow in the presence of an antibiotic, so that only transformed E.coli with A-chain will be selected. The whole lacZ gene and the fused A-chain will synthesize ß-galactosidase enzyme and A-chain. Similarly the B-chain was also synthesized separately.

Both the chains are purified from bacteria, combined, oxidized and reduced to form disulphide bridges to produce active insulin.

Figure 4: Human insulin production using recombinant DNA technology

Recombinant human insulin was first approved in 1982 for human administration. Humulin, is the first human insulin that was released into market in 1986.

Major insulin manufacturers include: Novo Nordisk A/S (Denmark), Sanofi S.A. (France), Eli Lilly and Company (U.S.), Bioton S.A. (Poland), Wockhardt Ltd. (India) and Julphar (UAE).

ANTIGEN – ANTIBODY INTERACTIONS

BY: RIA FAZULBHOY (MSIWM 031)

Antigen- antibody interactions are used to detect a number of immune diseases, check for humoral immunity and identify biological molecules. There is noncovalent interaction between the epitopes/antigenic determinants of antigens and the variable region (Vh & Vl) domain of antibodies. Noncovalent bonds include ionic bonds, hydrophobic bonds, hydrogen bonds and van der Waals forces.

Noncovalent bonds between antigen and antibody

Different Antigen – Antibody interactions:

  1. PRECIPITATION REACTIONS:

Precipitation reactions occur between antibodies (Ab) and soluble antigens (Ag) present in aqueous solution. They bind by noncovalent bonds to give Ag-Ab complexes known as lattices, which are in turn seen as precipitate. The term precipitins is given to antibodies which aggregate with soluble antigens.

The formation of Ag-Ab lettuces depends on the valencies of both antigens and antibodies:

  • Antibodies must be bivalent.
  • Antigens must be bivalent or polyvalent.

Precipitation reactions in fluids form a precipitation curve:

  1. Constant amount of antibodies is taken in a series of test tubes.
  2. Soluble antigens are added in an increasing amount to each test tube.
  3. Precipitates are formed in each test tube, and this precipitate is then centrifuged in order to form a pellet. Amount of precipitate is measured by the pellet.
  4. By plotting the graph of the amount of precipitate against increasing antigen concentration, we get a precipitin curve.
  5. Maximum precipitation occurs in the zone of equivalence where ration of antigen : antibody is optimum.
  6. If there is an excess of antigens or antibodies, such extensive lattices are not formed and precipitation is not seen.

                                                                       Excess Antibodies   Equivalence   Excess Antigens

    2) AGGLUTINATION REACTIONS

Definition of agglutination states that it is the interaction between antibodies and particulate antigens which results in visible clumping known as agglutination. Antibodies participating in such reactions are called agglutinins. Agglutination reactions gave a principle similar to precipitation reactions (based on cross-linking of polyvalent antigens). Excess of antibodies inhibits agglutination and this effect is known as the prozone effect.

There are 2 types of agglutination reactions:

  1.  Active (natural) agglutination
  2. Epitopes of the antigen are naturally found on the test particle.
  3. Eg. Antigens found on RBCs, bacteria, and fungal cells
  • Passive (chemically fixed) agglutination
  • Epitopes and soluble antigens do not occur naturally on the surface of the cells or particles
  • They need to be chemically fixed onto either RBCs (with the help of tannins/ chromium chloride) or synthetic materials like latex beads and polystyrene.
  • The synthetic materials offer more stability, uniformity and consistency.
  • Eg: soluble antigens, viral diseases.

            Examples of agglutination reactions

  1. Hemagglutination in blood typing

This is done to detect the blood group of patients and carry out proper blood transfusion. In typing for ABO antigens, red blood cells are mixed on a slide with antisera to the A or B blood group antigens. If antigen is present on the cells, they visibly clump due to agglutination taking place.

  1. Agglutination inhibition

Agglutination inhibition is used to detect use of illicit drugs and also used in pregnancy tests. It is also used to detect viral infections in patients.

3) RADIOIMMUNOASSAY (RIA)

Radioimmunoassay is a technique used to detect the binding of antigen and antibodies in the given sample. It is based on the principle that there is a competition for binding between radio-labelled antigens and unlabelled antigens when they are in the same vicinity as high affinity antibodies. The antibody does not distinguish between labelled and unlabelled antigens, thus there is competitive binding between the two.

>The radio-labelled antigen is generally labelled with:

  • Gamma emitting isotope like I125
  • Beta emitting isotope like 3H (tritium)

>The test sample which contains unlabelled antigens is a complex mixture like serum or other body fluids.

How does Radioimmunoassay take place?

  1. First the radio-labelled antigen (Ag*) is mixed with the antibodies at a concentration such that the antigens saturate the antigen binding site of the antibody. This concentration of antibodies which should bind to labelled antigens should be anywhere between 50-70%.
  2. An increasing amount of unknown test sample of non labelled antigens is added to the mixture.
  3. As the amount of non labelled antigens increase and bind to the antibody, the number of radio labelled antigens which bind to the antibody decreases. They compete to bind to the samples.
  4. To determine the amount of labelled and non labelled antigens bound to the antibody, the Ag-Ab complex is precipitated to separate from unbound, free antigen.
  5. Unbound antigens are separated by various methods like use of formalin killed S.aureus, other antibodies which react with free antigens, use of solid-phase RIAs, etc.
  6. The precipitated Ag-Ab complex’s radioactivity is measured with the help of a radiation counter.
  7. A standard curve can be obtained in order to plot and determine the amount of antigen present in the test sample.

Disease Associated With Viruses

BY- REDDY SAILAJA M (MSIWM030)

Viral disease:

A Virus is a genetic entity that comprises of either DNA or RNA, surrounded by a protein coat and require a host to survive. Viral disease is a condition, when a pathogenic virus attacks host, weakens its immune system and replicate inside host cells to further spread the infection.

Origin of virus:

Human infectious viruses have emerged from non-human reservoirs like – poultry, farm mammals, wild animals and rarely arthropods. About 75% of emerging human infectious viruses are due to zoonosis, which means spread of virus from animals or insects to humans. Viruses generally reside in nose, throat, upper or lower respiratory system and also attacks gastrointestinal, nervous and reproductive systems of the host. Common viral reservoirs include: deer, rat, pigs, bats, boar, mangoose, camel, goat, ferret, rabbit etc.

Figure 1: Animal to human viral transfer

Spread of virus:

  • Unprocessed foods
  •  Uncooked meat – zoonotic viral reservoirs
  • Infectious air droplets that spread through air
  • Poor personal sanitization and hospital environment
  • Unsterilized hospital equipment
  • Insect/animal bite
  • Sexual transmission
Figure 2: Common viral diseases symptoms.

Major types of viral diseases were stated in the following table with all the information like – symptoms, mode of transmission, treatment, prevention along with the examples.

Type of viral diseaseSymptomsMode of transmissionTreatmentPreventionExamples
Gastrointestinal viral disease: Virus attacks digestive system of the host and lead to inflammation of stomach and small intestine called gastroenteritis. .-Abdominal cramps -Diarrhea -Vomiting-Food/water contaminated with virus containing feces. -sharing objects infected with the virus-Intake of lots of water to prevent dehydration because of vomiting/diarrhea-Proper sterilization of cooking utensils. -Disinfection of surroundings -Proper washing of hands before food intake and after toilet usage-Rotavirus -Norvovirus – Astrovirus
Respiratory viral disease: Virus that invades nose, throat, upper air ways and deep lungs-Cough -Cold -Runny nose -Fever Body pains-Droplets of cough and sneeze. -Contaminated objects with contagious droplets like door knobs etc-Over the counter medication like pain relievers, cough suppressants, decongestants, antiviral drugs  -Personal hygiene -Social distance -Covering mouth face during cough and sneeze-Influenza virus -Common cold -Respiratory syncytial virus -Severe acute respiratory syndrome (SARS) – SARS-CoV-2  
Hepatic viral disease: Virus that infects liver and cause inflammation -Bodily fluids – Viral infected objects -Contaminated food/water-Anti viral drugs -Intake of fluids-Vaccination – No sharing of blades/razors – Safe sex-Hepatitis A – Hepatitis B -Hepatitis C
Neurologic viral disease: Virus that infects brain and associated systems-Fever -Drowsiness -Seizures -Confusion-Infected animal/insect -Contaminated objects-Rest -Intake of fluids -Anti inflammatory drugs-Proper rest -Intake of fluids – Anti-inflammatory drugs-Polio -Rabies -Viral Encephalitis -Viral meningitis
Exanthematous viral disease: Virus that causes skin rashes  -Fever -Body pains-Droplets of cough and sneeze. -Contact with infectious skin lesions -Mosquito bite (Chickun gunya virus)-Fever reducing medication -Pain relievers-Vaccination – Protecting from viral vectors (mosquitoes)-Measles – Rubella -Chicken pox -Chikun gunya

Treatment to viral diseases:

Most viral symptoms are mild and go away in a few days in most of the people. Only a few, with weak immune system suffers. Treatment against viral diseases comprise mainly of over-the-counter medications to relieve symptoms, subside cold or cough irritations. Drinking plenty of fluids – to keep hydrated, self isolation – to prevent spread of the virus, act as effective as a drug. Not a single antiviral medication is effective against viruses. Anti viral medicines act on viruses by preventing – viral entry into host cells, viral DNA/RNA replication, viral machinery assembly and spread. However, anti viral drugs are effective when taken during the early onset of infection or during outbreak of virus in a particular season.

Viral vaccines:

Vaccines against viruses stimulate host’s immune system to be defensive against the invading virus. Viral vaccines include: measles/mumps/rubella, polio, hepatitis A, hepatitis B, chicken pox, human papillomavirus, rotavirus, yellow fever and other common viruses.

Over all, prevention is better than cure. Maintaining personal hygiene, minimal social contact, intake of healthy food, fully cooked meet are the best way to prevent the spread of viruses, both existing and the emerging ones.