Red Cabbage: The Disease-Fighting, Gut-Healing Superfood

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Fish Collagen: The Anti-Aging Protein with the Best Bioavailability
Would Ahiflower be absorbed similar to Flax and hemp? Refer to the Chiropractic and Stress Page for more information. For example, erythrocytes , macrophages and plasma cells are produced in the anterior kidney or pronephros and some areas of the gut where granulocytes mature. Birds , including intensively farmed poultry, appear to have a higher infection rate and carriage of Campylobacter spp, especially C jejuni jejuni , than other animals. Campylobacter jejuni , gram-stained smear of intestine.

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Why fish stomps flax as a source of omega-3

Clinical and patho-anatomic effects are also described. For more detailed information standard reference works should be consulted. Active chlorine 2 can be discharged into water courses, lakes and ponds in effluents from textile and paper plants.

Chlorine and compounds that release active chlorine into water are used as disinfectants in both public health and veterinary medicine. Thus, chlorine can be discharged in water from public swimming pools and from sterilizing procedures for equipment in dairy farms.

Chlorinated lime is used for a total disinfection of pond bottoms application rate of kg per ha , fish storage ponds and other facilities for fish culture and transport. If fish suffer from a gill disease, a recommended remedial proceedure is to spread chlorinated lime on the surface of the pond at a rate of 10—15 kg per ha if the average depth of the pond is 1 m.

However, overdosage or improper handling of chlorine or chlorine-releasing compounds can damage or kill fish. Marketed fish may also be harmed by chlorine if retailers keep them in tanks supplied with chlorinated tapwater which contains 0. Higher, rapidly lethal, concentrations can occur if the water supply works abstracts water containing a high content of organic matter; excessive chlorine then has to be used to disinfect the water.

If chlorinated water from a public supply has to be used, it should be passed through an activated charcoal filter to remove the chlorine; for small-scale use, small amounts c. Low concentrations of chlorine can be naturally absorbed by organic matter in the water and in sediments. Active chlorine is very toxic to fish. Its toxicity largely depends on water temperature: In general, a prolonged exposure to active chlorine concentrations of 0.

Active chlorine may affect specific parts of the fish e. The systemic effect manifests itself mainly as nervous disorders. The clinical symptoms of chlorine intoxication include a considerable restlessness, leaping out of the water, muscle tetanus, lying on one side, and spasmic movement of the mouth, fins and tail. The buccal spasms hinder respiration, so that the fish suffocate, and ultimately die. The skin and gills of the poisoned fish are covered with a thick layer of mucus and if the concentration of active chlorine is very high the gills become congested and can haemorrhage.

The body surface of such fish becomes pale and the margins of the gill filaments and fins are covered with a grey-white coating. Histopathologically, there is a marked dystrophy and necrobiosis leading to necrosis, with desquammation of the gill respiratory epithelium and of the epidermis of the skin.

Cyanides do not occur naturally in waters; they can be discharged in various industrial effluents, particularly from metal plating works and from the thermal processing of coal e. Cyanides may be present in water either as simple compounds nondissociated HCN, simple CN ions or as complex compounds e.

Simple cyanides are very toxic or extremely toxic to fish species; lethal concentrations for the majority of species are in the range of 0. Cyanide toxicity is affected by the pH of the water; if the pH is low the proportion of nondissociated HCN increases and so does the toxicity Table 2.

Cyanide toxicity is also markedly enhanced by an increase in water temperature and a decrease in the concentration of dissolved oxygen in the water. With complex cyanides, the toxicity varies according to their ability to dissociate into metal and HCN.

For example, the complex iron cyanides which do not dissociate are of low to very low toxicity to fish but the complex cyanides of zinc, cadmium, copper and mercury which do are highly toxic. The concentrations of different cyanide compounds proposed as maximum admissible levels for fish culture are in the range of 0. The mechanism of the toxic action of cyanides is based on their inhibition of respiratory enzymes i.

This blocks the transfer of oxygen from the blood to the tissues, reduces tissue respiration and leads to tissue asphyxia. The clinical symptoms of the cyanide poisoning of fish include increased depth of respiration, nervous disorders, and loss of equilibrium. If the fish are transferred to clean water while they are in the early stages of overturning, they will recover in 1 to 2 hours. The characteristic features of the patho-anatomic examination in cases of cyanide poisoning include a cherry-red colour of the gills and sometimes also leakage of tissue fluid mixed with blood into the body cavity.

Trace quantities of metals present in waters may be of natural origin. If waters are polluted with metals at greater concentrations, the source may be traced back to ore mining and processing, to smelting plants, rolling mills plants for the surface treatment of metals, film, textile and leather industries and other sources.

Atmospheric precipitation can wash out metals in dust and aerosols generated by the burning of fossil fuels, by the exhaust gases of motor vehicles, and from other sources. The mechanism of the toxic action of metals on fish is varied. Most of the metals have a great affinity for amino acids and the SH groups of proteins: The toxicity of metals to fish is significantly affected by the form in which they occur in water.

The ionic forms of metals or simple inorganic compounds are more toxic than complex inorganic or organic compounds. The toxic action of metals is particularly pronounced in the early stages of development of the fish. Another potentially harmful property of many metals is their ability to accumulate in the sediments and in the aquatic flora and fauna bioaccumulation.

Hence, the concentration of these metals in water does not provide a true indication of the total pollution of the aquatic medium; it is better to use the content of metals in the sediments, and especially also in the bodies of predatory fish which are the final link in the food chain, as an indicator. The metals found to be of highest importance to fisheries in practice include aluminium, chromium, iron, nickel, copper, zinc, arsenic, cadmium, mercury and lead. The toxicity of aluminium to fish depends to a large extent on the physico-chemical properties of the water and particularly on its pH.

Aluminium is soluble at pH values below 6. At higher pH values, aluminium is precipitated as the hydroxide, which can flocculate in the water.

It is possible that freshly precipated aluminium as a colloid may be toxic; the fully flocculated hydroxide has a low toxicity similar to that of suspended solids in general.

In toxicity tests, rainbow trout fry were exposed to different aluminium concentrations at a pH of 7. A concentration as low as 0. When an even lower concentration, 0. A mass kill of maraena and peled fry, reared in a public supply water clarified by flocculated aluminium sulphate, is a practical example. The aluminium concentration of the water was up to 0. All the fry of maraena and peled died within 10—14 days of hatching. It is not known whether this was due to ionic aluminium or to micro flocs affecting fish respiration.

Of these two forms, chromium III is poorly soluble and is readily adsorbed onto surfaces, so that the much more soluble chromium VI is the most common form in fresh water. For this reason, maximum admissible concentrations for chromium are generally based on toxicity data for the hexavalent form. Chromium compounds in the trivalent state III are more toxic to fish and other aquatic organisms than are those in the hexavalent state VI.

From the LC50 data obtained for different fish species, chromium III compounds are among those substances with a high toxicity to fish LC50s of 2. The toxicity of chromium compounds to fish is also considerably affected by the physico-chemical properties of water, especially the pH value and the concentrations of calcium and magnesium. At a high pH and a high concentration of calcium, the toxicity of chromium to aquatic organisms is reduced, compared to that in soft acid water.

With acute poisoning by chromium compounds, the body surface of the fish is covered with mucus, the respiratory epithelium of the gills is damaged and the fish die with symptoms of suffocation.

Fish suffering from chronic chromium intoxication accumulate an orange yellow liquid in their body cavity. In surface waters, iron occurs in ferrous state II soluble compounds or ferric state III mostly insoluble compounds.

The ratio of these two forms of iron depends on the oxygen concentration in the water, the pH and on other chemical properties of the water. Fish may be harmed by iron compounds in poorly oxygenated waters with a low pH where the iron is present mainly in the form of soluble compounds. Because the gill surface of the fish tends to be alkaline, soluble ferrous iron can be oxidized to insoluble ferric compounds which then cover the gill lamellae and inhibit respiration.

At a low water temperature and in the presence of iron, iron-depositing bacteria will multiply rapidly on the gills and further contribute to the oxidation of ferrous iron compounds.

Their filamentous colonies cover the gills; at first they are colourless but later the precipitated iron gives them a brown colour. The precipitated iron compounds and tufts of the iron bacteria reduce the gill area available for respiration, damage the respiratory epithelium and may thus suffocate the fish. In a similar toxic action, iron compounds can precipitate on the surface of fish eggs which then die due to a lack of oxygen.

The lethal concentration of iron for fish is not easy to measure because it depends to a large extent on the physico-chemical properties of the water. In cyprinid culture, it is generally accepted that the concentration of the soluble ionized forms of iron should not exceed 0. Nickel can be discharged into surface waters in effluents from metal plating plants. Nickel compounds are of medium toxicity to fish. With short periods of exposure, the lethal concentration is between 30 and 75 mg per litre.

As with the toxicity of other metals, the toxicity of nickel compounds to aquatic organisms is markedly influenced by the physico-chemical properties of water. For example, in soft waters with low calcium concentrations, the lethal concentrations of nickel compounds for the stickleback were less than 10 mg per litre.

In such cases nickel can be regarded as highly toxic to fish. After toxic exposure to nickel compounds, the gill chambers of the fish are filled with mucus and the lamellae are dark red in colour. Although copper is highly toxic to fish, its compounds are used in fish culture and fisheries as algicides and in the prevention and therapy of some fish diseases.

The physical and chemical properties of the water exert a strong influence on the toxicity of copper to fish. In water containing high concentrations of organic substances copper can become bound into soluble and insoluble complexes. Compounds that are slow to dissolve or are insoluble are unlikely to be taken up to any extent into the fish body, so their toxicity to fish is low. A good example of this effect of solubility is a comparison between the different LC50s for carp recorded during 48 hours exposure to CuSO 4.

The maximum admissible copper concentration in water for the protection of fish is in the range of 0. The characteristic clinical symptoms of fish poisoned by copper ions and copper compounds include laboured breathing and, in cyprinids, gasping for air at the water surface. The typical patho-anatomic appearance includes a large amount of mucus on body surface, under the gill covers and in the gills. Acute copper intoxication can be diagnosed on the basis of a chemical analysis of the gills in which the concentration of copper is much greater than in other parts of the body of the fish.

The gills of fish caught in waters free of copper contamination contain up to 10 mg of copper per 1 kg of dry matter. Zinc poisoning of fish is most frequently encountered in trout culture.

Rainbow trout and brown trout, and especially their fry, are extremely sensitive to zinc and its compounds. The lethal concentrations are around 0. Resistance to zinc compounds increases with age. The toxicity of zinc to fish is influenced by the chemical characteristics of water; in particular, increasing calcium concentrations reduce the toxicity of zinc.

The clinical symptoms and patho-anatomic picture of zinc poisoning in fish are similar to those found for copper. The best remedy to avoid frequent occurrences of zinc toxicity in trout culture is to avoid using galvanized pipes for the supply of water and to avoid using galvanized containers and equipment, especially in areas where the water is soft and acid. As a rule arsenic occurs in water in the oxidation state V but some of it may also be present in non-stable forms, i.

As with mercury see later biological particularly bacterial activity may lead to the formation of organic methyl derivatives of arsenic. The main sources of arsenic pollution in surface waters include industrial effluents e. Arsenic is able to accumulate in large quantities in the sediments on the bed of water courses and reservoirs, and in aquatic organisms. Arsenic compounds in the third oxidation state arsenites are absorbed fairly rapidly into fish and are more toxic than arsenic compounds in the oxidation state V arsenates.

From concentrations found to be lethal to different species of fish during 48 hours of exposure, diarsenic trioxide, for example, can be included among those substances which have a medium to high toxicity to fish; lethal concentrations are between 3 and 30 mg per litre. Cadmium in surface waters is usually found together with zinc but at much lower concentrations. The cadmium present in surface waters may be either dissolved or insoluble. Of the dissolved forms, those which may be poisonous to fish include the simple ion and various inorganic and organic complex ions.

Apart from an acute toxic action which is similar to that of other toxic metals damage to the central nervous system and parenchymatous organs , very small concentrations of cadmium may produce specific effects after a long exposure period.

Chief among these specific effects are those exerted on the reproductive organs. An adverse influence of long exposure to cadmium upon the maturation, hatchability and development of larvae in rainbow trout was recorded at concentrations as low as 0.

The acute lethal concentration of cadmium for different species of fish is from 2 to 20 mg l 1. Cadmium toxicity is reduced with increasing levels of calcium and magnesium in the water. For salmonids, the maximum admissible cadmium concentration in water is 0. Mercury is transported to the aquatic environment mainly in discharges of industrial effluents and by atmospheric precipitation. Unpolluted waters will contain trace amounts of mercury which do not exceed 0.

Mercury concentrations found in surface waters are not a true measure of the actual total amount of mercury present and therefore do not represent the extent of the mercury pollution there; this is because mercury is transferred from water to the sediments on the bed of water courses, lakes and reservoirs where it accumulates mainly as the sulphide. Elementary mercury and its organic and inorganic compounds can undergo methylation a process induced by the activity of microorganisms in the sediments.

The toxic end-product of this methylation, methyl mercury, enters the food chains and bioconcentrates in increasing amounts in aquatic organisms up the food chain. Mercury can be taken up into fish from food via the alimentary tract; the other routes are through the gills and skin.

Absorption from the alimentary tract has proved to be of the greatest importance in methyl mercury accumulation; evidence for this has been provided by the results of investigation at sites in the drainage area of the Berounka River in Central Bohemia.

The total mercury content in the flesh of fish from these localities is about 10 times that recorded in their food. This coefficient of bioaccumulation can be compared with the food efficiency coeficient of fish living in open waters and feeding on the aquatic invertebrates.

Of the other aquatic organisms in the drainage area of the Berounka River, the greatest mercury levels were recorded in leeches and this can be ascribed to their exclusively predatory mode of feeding.

With their wide distribution in different types of waters, leeches e. Helobdella stagnalis may be considered as good indicators of mercury contamination of the aquatic medium. Carnivorous fish contain the highest amounts of mercury because they form the final link in the aquatic food chain. The problem of mercury in the aquatic medium is important not only for environmental and hygienic reasons but also from the viewpoint of fish culture.

It has been shown that mercury compounds can cause damage to some vital tissues and organs in fish and may also have a harmful effect on reproduction. At very low concentrations they reduce the viability of spermatozoa, reduce egg production and affect the survival rate of developing eggs and fry. Acute lethal concentrations of inorganic mercury compounds are in the range of 0. The acute lethal concentrations of commonly found organic mercury compounds are from 0.

For salmonids the maximum admissible concentration of inorganic forms is about 0. For fish in general, the maximum admissible concentration of mercury in organic compounds has been suggested to be as low as 0.

A significant source of airborne lead contamination, and therefore of surface waters, is the exhaust fumes of motor vehicles which contain the break-down product of tetraethyl lead. In surface waters, lead largely accumulates in bottom sediments at concentrations about four orders of magnitude greater than in the water. Lead toxicity to fish and other aquatic organisms is significantly influenced by the water quality and depends on the solubility of lead compounds and on the concentration of calcium and magnesium in water.

The water solubility of lead compounds is reduced with increasing alkalinity and pH value of the water. Also, the toxicity of lead is known to be reduced with increasing calcium and magnesium concentrations in water.

The acute toxic concentrations in different types of water are in the range of 1 to 10 mg per litre for salmonids and of 10 to mg per litre for cyprinids. Acute lead toxicity is characterized initially by damage to the gill epithelium; the affected fish are killed by suffocation. The characteristic symptoms of chronic lead toxicity include changes in the blood parameters with severe damage to the erythrocytes and leucocytes, and degenerative changes in the parenchymatous organs and damage to the nervous system.

In trout, a characteristic symptom is a blackening of the caudal peduncle; a biochemical effect is the inhibition of amino levulinic acid dehydrase ALA-D in fish blood. The maximum admissible lead concentration in water is 0. Phenols occur in surface waters from discharges of industrial effluents, especially from the thermal processing of coal, from petroleum refineries, and from the production of synthetic fabrics.

Phenols are either monobasic e. Phenols can give an unacceptable taint to water and fish, especially chlorophenols which are formed from the chlorination of phenols. The maximum concentrations admissible for fish culture are 0. Prolonged exposure to a concentration of 0. Based on the lethal concentrations for fish, the different phenol compounds can be ranked as follows: Phenols are anaesthetics which affect the central nervous system.

The clinical signs of intoxication are characterized by increased activity and irritability, leaping out of the water, loss of balance and muscular spasms.

The post-mortem appearance include a conspicuous whitening of the skin which is heavily coated with mucus; at high temperature this may be accompanied by haemorrhages on the under side of the body.

Long exposure to low concentrations leads to dystrophic to necrobiotic changes in the brain, parenchymatous organs, circulation system and gills. Polychlorinated biphenyls are recognized as very important environmental pollutants.

PCBs are among the most environmentally persistent of organic compounds; although their solubility in water is very low, they are readily soluble in nonpolar solvents and can accumulate in fats. Mixtures of a large number of PCBs isomers are used in the heavy electrical equipment industry e.

In response to a growing concern about rising levels of PCBs in the environment from diffuse sources, their accumulation in biota, and uncertainty about their toxic effects, the production of polychlorinated biphenyls was restricted in , and successive controls placed on its use and disposal.

The main concern is that, once in the natural environment, they cannot be recovered or removed. In surface waters, PCBs occur at concentrations from 1. Polychlorinated biphenyls have a high capacity for accumulation in the bottom sediments and in aquatic organisms for which the bioaccumulation coefficient is from 10 3 to 10 5 , depending on the fat content. PCBs present a very difficult ecotoxicological problem; there are individual PCBs, each one with different toxicological properties.

Toxicity tests are carried out on commercial formulations which are identified by the extent to which they are chlorinated, and not by the specific PCBs that they contain. This makes it difficult to assess their toxicity in the environment, because differential uptake of the individual compounds leads to a different ratio being found in organisms when compared to that in the tested formulations.

Therefore, any assessment of the toxicity of PCBs can be made only in general terms on the basis of tests with commonly used formulations. PCB formulations are very toxic to extremely toxic to fish, especially in their early developmental stages; their 48 hour LC50s are below 1 mg per litre. Of the various toxic actions of PCBs reported, they have been found to adversely affect the enzyme systems within the microsomal fraction of the liver. If fish are exposed for a long time to low sublethal PCBs levels, the compounds accumulate in the body and can cause, mainly in the fry, deformities in the skeleton, damage to the skin and fins the fins disintegrate , to the parenchymatous organs mainly in the liver where hypertrophy, local dystrophy, and necrobiotic to necrotic changes can occur , and to the gonads.

These effects can cause a subsequent mortality during hatching, high mortality of early fry and an increased occurrence of different deformities among the survivors.

The maximum admissible PCBs concentrations in water range from 1. Lower admissible concentrations are recommended during hatching and rearing of the early stages of the fry.

However, analytical measurement of these concentrations in solution may be difficult; also, a significant proportion of the uptake of PCBs will be from the food. Analysis of fish tissue will give an indication of the degree of exposure, but the concentrations found must be correlated with the tissue fat content. Where significant amounts occur, the analysis should identify and quantify a number of key individual PCBs for an expert evaluation of the potential hazard.

Surfactants are compounds which, by lowering the surface tension of water, can facilitate the formation of emulsions with otherwise immiscible liquids such as oils and fat. They are widely used domestically and in industry. In recent years, the traditional soaps have been replaced by detergents that contain synthetic surfactants and other ingredients; for domestic washing of garments, these may contain water softeners, optical brighteners, and perfumes.

Surfactants are either ionic liable to electrolytic dissociation or nonionic nodissociating in water. Ionic surfactants are subdivided into anionic dissociating to a surface active anion and an inactive cation , cationic dissociating to a surface active cation and an inactive anion , and ampholytic assuming either anionic or cationic properties, depending on ambient conditions.

The anionic surfactants are those most widely used in industry. Because of the large number of synthetic surfactants in production, it is not surprising that they span a wide range of chemical toxic actions for aquatic organisms. However, they do have a common physico-chemical effect in that they can damage the lipid components of cell membranes.

Because the surface tension of the ambient water is decreased, the lipids are less water repellant and this leads to hydration and enlargement of the cell volume. At low surfactant concentrations this enlargement is reversible. Higher concentration can cause a suppression of metabolic processes in the cells. Long-term exposure may damage the cells which then become necrotic in the later stages.

These changes result mainly in an impairment of the gill respiratory epithelium. In addition, the exposure of fish to some surfactants can cause changes in the activity of respiratory enzymes, especially cytochromoxidase.

Surfactants can also damage the protective layer of mucus on the skin; the layer loosens and the resistance of the fish to infection decreases. Sublethal surfactant concentrations can also damage eggs and sperm. The toxicity of surfactants to fish is influenced by a number of biotic and, especially, abiotic factors. The age of the fish is a particularly important biotic factor. During embryonic and larval development, the sensitivity of fish to surfactants is sometimes greater by an order of magnitude in comparison with the juvenile and adult stages.

Of the abiotic factors, the molecular structure of the surfactant and the physico-chemical properties of water exert the greatest influence on their toxicity. The results of investigations into the relationship between toxicity and molecular structure indicate, for example with linear alkylbenzene sulphonates, that the toxicity to fish is markedly increased with the length of the molecular chain.

A similar correlation between toxicity and chain length was observed with other surfactants. Among the physico-chemical properties of water, increasing calcium and magnesium concentrations have the greatest effect on reducing surfactant toxicity and some influence is also exerted by the pH. This may be important where surfactants are incorporated into a detergent containing water softening chemicals e.

Where both cationic and anionic surfactants are present in waste waters their toxicity is much reduced, due to the formation of insoluble complex. The acute toxicity of surfactants varies considerably with the species of fish. Nevertheless, these compounds and the detergents that contain them are highly toxic to fish in the majority of cases, the hour LC50 ranging between 1 and 10 mg per litre. A small proportion of surfactants can be classified as having a medium toxicity hour LC50 between 10 and mg per litre and a few have a very low toxicity hour LC50 to 10 mg per litre.

For the majority of surfactants, no significant differences in their toxicity to fish were recorded between the anionic, cationic and nonionic groups. As stated above, surfactants can cause damage to the gill respiration epithelium e.

Therefore, the clinical signs of poisoning include respiratory disorders increased respiration rate, and cyprinids gasp for air at the water surface and later by inactivity.

The characteristics in the patho-anatomic examination are an increased amount of mucus on the skin and in the gills, and congestion to oedematous swelling of the gill apparatus.

The mucus is easily removed from the body surface and gills. In recent years, the number of pesticides available and the quantity used has considerably increased. Pesticides are chemicals which have a specific toxic action to which the pest species is particularly sensitive. The chemical is then applied at a concentration which kills the pest but does not affect a wide range of non-target organisms.

The ideal pesticide is a chemical which is extremely pest-specific; for the pesticide user it should also be persistent in order to avoid the need for repeated applications. However, on environmental grounds, pesticides should be non-persistent to avoid concentrations building up in environmental compartments and causing unsuspected side-effects. For example, the insecticide DDT is very persistent and thus can build up in food chains to ultimately affect the egg-shell thickness of birds of prey.

Because pesticides are designed and used to kill living organisms, and because of the possibility of unsuspected side effects, it is tempting to implicate them in any incident of fish poisoning where there is no other obvious cause of the damage.

There are many cases, therefore, where pesticides have been assumed to be the cause of damage but where the real cause was some other factor.

Some cases of pesticide poisoning of fish are obvious; accidental discharges from road accidents, factory disasters, overspraying of water, or careless disposal of unwanted spray and pesticide containers, can be clearly identified as causes of mortality, especially if the concentrations measured or calculated in the water exceed the 96 hour LC50 by a significant margin.

Less easily identified are cases of long-term leaching of persistent pesticides from fields and forests. Besides these acute and chronic direct effects, an indirect action may be important. Inexpert application of aquatic herbicides or algicides to the water, or the accidental contamination of surface waters with these chemicals, may kill excessive quantities of aquatic plants and algae.

The rapid decomposition of this organic matter forms a considerable dissolved oxygen demand on the water. This will lead to an oxygen deficit and the fish may die of suffocation. Another potentially serious indirect consequence of pesticide contamination of the aquatic biota is the reduction or complete destruction of the natural food supply of the fish. Many of the organisms on which the fish feed are much more sensitive, particularly to insecticides, than the fish themselves. Besides the active ingredient, pesticide formulations contain a number of other chemicals which may sometimes be much more toxic to fish than the active ingredient itself.

When a pesticide enters the aquatic environment, the active ingredient may undergo chemical and biological degradation.

In some cases the degradation products may be more toxic to fish than the original active ingredient. For example, parathion is biodegraded to form paraoxon, which is a more toxic compond; similarly, trichlorphon is degraded to form the more toxic compound dichlorvos. It follows that the absence of a specific active ingredient in water cannot guarantee that harmful degradation products are not present. Some herbicides are used in fish culture and water management to kill unwanted aquatic plants e.

Trichlorphon based organo-phosphorus insecticides, e. Soldep, Masoten, Neguvon, etc. Pesticides based on copper oxychloride may be used to control fish parasites, including the control of gastropod intermediate hosts, and to kill excessive growths of algae. However, in the majority of cases pesticides have the potential to cause damage to fish.

The most toxic pesticides are those based on chlorohydrocarbons e. DDT, dieldrin , organo-phosphorus compounds, carbamates and thiocarbamates, carboxylic acid derivatives, substituted urea, triazines and diazines, synthetic pyrethroids, and metallic compounds.

These pesticides act as nerve poisons. Because of their chemical structure and their persistance, their use is now strictly controlled or banned.

The clinical signs of fish poisoning by organochlorine pesticides on the basis of chlorohydrocarbons include increased activity, followed by a long stage of reduced activity. There is no specific patho-anatomic picture in these cases of intoxication; dystrophic alterations have been recorded in the liver and kidneys.

The mechanism of the toxic action of organo-phosphorus pesticides on fish follows the same pattern as their action on homoiothermic animals, in that some hydrolytic enzymes, particularly acetylcholine hydrolase, are inhibited. The degree of inhibition of cerebral acetylcholine hydrolase in fish varies with the specific organo-phosphorus compound causing the effect. The toxicity of these pesticide formulations to fish also varies; from the 48h LC50s obtained they are ranked among those substances of very high to medium toxicity to fish 0.

Also, salmonids are very sensitive to organo-phosphorus pesticides. The typical sign of fish poisoning with these pesticides is a darkening of the body surface at the onset of uncoordinated activity. The water flea Daphnia magna is very sensitive to organo-phosphorus pesticides; from the 48h LC50s obtained for these substances, they can be classified as extremely toxic.

It is interesting to note that the water flea was found to be sensitive to trichlorphon and dichlorvos at concentrations close to the level of detection by gas-liquid chromatography. Daphnia magna can be regarded, therefore as a sensitive indicator of organophosphorus pesticide pollution. Carbamate and thiocarbamate compounds also inhibit the activity of acetylcholine hydrolase. However, unlike the toxic action of organo-phosphorus compounds, the inhibition of enzyme activity is readily reversed after carbamate and thiocarbamate poisoning.

The toxicity levels of these substances to fish vary from very high to low toxicity 48h LC50s in the range of 1 to mg per litre. The clinical and patho-anatomic pictures of fish poisoning by these pesticides are not specific. A number of these pesticides are based on phenoxyacetic acid; the main representative of this group is 2-methylchlorphenoxyacetic acid MCPA.

Most of the MCPA-based products are of medium to low toxicity to fish 48h LC50s in the range of 10 to mg per litre. The clinical signs of poisoning are mostly characterized by increasing narcosis. There is no marked patho-anatomic picture in fish poisoned by these herbicides. Herbicides formulated from substituted ureas are of high to low toxicity to fish 48h LC50s are in the range of 1 to mg per litre.

The patho-anatomic picture is characterized by an increased amount of mucus on the darkened body surface, hyperaemia of the gills and the presence of a small amount of exuded fluid in the body cavity of the fish. Triazine-based pesticides are of high to medium toxicity to fish 48h LC50s range from 1 to mg per litre. The clinical signs of fish poisoning by these chemicals are largely characterized by progressive narcosis. The presence of exuded fluid into the body cavity and into the digestive tract is an especially characteristic patho-anatomic sign, particularly in rainbow trout.

The presence of exudates causes a marked swelling of the body cavity; in rainbow trout it has even led to a rupture of the body wall in some cases. Diazine-based herbicidal preparations are less toxic to fish than are triazine-based preparations. Most of the former are of low to very low toxicity to fish 48h LC50 ranging from to 10 mg per litre. The clinical course of intoxication is characterized by stages of immobility.

The patho-anatomic picture is not specific to these compounds. The 48h LC50s of these pesticides show that they rank among those substances of high toxicity up to 10 mg per litre to extreme toxicity less than 0. The clinical signs of poisoning are not specific and include respiratory disorders.

The most conspicuous patho-anatomic change is the presence of a small amount of exuded fluid in the body cavity. These include primarily the fungicides formulated from compounds containing copper, mercury and aluminium. In the majority of cases, their toxicity to fish and the clinical and patho-anatomic symptoms correspond to those found in fish poisoned by the respective metals.

Oils and refined products have been responsible for many of the recently recorded pollution incidents in surface and underground waters. Between to these substances were responsible for the majority of water pollution accidents recorded on a worldwide basis. These accidents were not associated with problems in sewage treatment plants; most of them were due to careless storage and handling of oil, transport accidents, and defective equipment, all of which can be ascribed either directly or indirectly to human error.

However, oils and refined products can also be discharged into the aquatic environment with industrial effluents. The petrochemical industry is mainly responsible for such effluents; other important sources of pollution include the engineering and metallurgical industry and car and truck repair and service stations. Most of these sources have discharged polluting effluents for many years. To some extent, the large number of reported oil-related pollution incidents is due to the very visible surface film that is formed; it therefore needs no chemical analysis for its detection.

For this reason, few discharges of oil go unnoticed. The harmful effects of such discharges depend on the physical effects of the surface film, and on the transfer of water soluble products into the water.

However, few of the constituent of oil and refined oil products will readily dissolve in water. The complement system can be activated by at least three separate pathways. The two evolutionary older pathways are the so-called "alternative" and the lectin pathways.

Both are activated on many bacterial surfaces, contributing to innate immunity. The third pathway, which is mainly antibody-activated and hence part of the adaptive immune system, developed much later, but was identified first.

Somewhat unfairly, it is therefore called the "classical pathway". The alternative pathway of complement activation starts with the spontaneous hydroysis of an internal thioester bond in the plasma complement component C3 to result in C3 H 2 O.

The changed conformation of C3 H 2 O enables binding of the plasma protein factor B which is in turn cleaved into fragments Ba and Bb by the plasma protease factor D. While BY diffuses away, the C3 H 2 O Bb complex is a soluble C3 convertase which proceeds to cleave a number of C3 molecules, resulting in small, soluble C3a and a larger fragment, C3b, which normally is rapidly inactivated.

In case C3b is generated near a bacterial or cellular surface, it binds covalently to this surface. The process just described now repeats on the membrane: The further development depends on the nature of the surface in question.

If C3b binds to the membrane of one of our own cells, the process of activation is inhibited by one of several different protective proteins, preventing damage to the cell. A bacterial surface lacks these inhibitors, allowing the complement cascade to proceed. Facilitated by the bacterial surface, factor P properdine stabilizes the membrane-bound C3bBb complex..

This complex, the C3 convertase of the alternative pathway, subsequently works as an amplifying tool, rapidly cleaving hundreds of additional C3 molecules. Soluble C3a diffuses into the surroundings, recruiting phagocytes to the site of infection by chemotaxis.

C3b fragments and their cleavage products C3d, C3dg and C3bi are deposited on the bacterial surface in increasing numbers and are recognized by specific complement receptors CR1-CR4 present on the membrane of phagocytes. This function, making the bacterium a "delicacy" for phagocytes, is called opsonization, from the Greek word for goody. The complement cascade does not stop at this point: The smaller cleavage products C3a, C4a, C5a, sometimes called "anaphylatoxins", have additional functions in their own right: The lectin pathway of complement activation exploits the fact that many bacterial surfaces contain mannose sugar molecules in a characteristic spacing.

These, by cleaving C4 and C2, generate a second type of C3 convertase consisting of C4b and C2b, with ensuing events identical to those of the alternative pathway. The classical pathway usually starts with antigen-bound antibodies recruiting the C1q component, followed by binding and sequential activation of C1r and C1s serine proteases. Yet, this pathway can also be activated in the absence of antibodies by the plasma protein CRP C-reactive protein , which binds to bacterial surfaces and is able to activate C1q.

This is desirable when unwanted complement activation causes hemolysis, as in paroxysmal nocturnal hemoglobinuria or in some forms of hemolytic uremic syndrome. For the lytic pathway's importance in fighting meningococcal infections, Eculizumab treatment increases the risk of these infections, which may be prevented by previous vaccination. Frequently, coagulation more about that in cardiovascular pathophysiology and kinin systems are activated simultaneously by a process called contact activation.

As its name implies, this process is initiated when a complex of three plasma proteins is formed by contact with certain negatively charged surfaces. Such surfaces may be collagen, basal membranes, or aggregated platelets in case of a laceration, or bacterial surfaces in case of an infection.

Factor XII is activated by contact with the negatively charged surface, starting the entire coagulation cascade. In addition, factor XII cleaves prekallikrein, releasing the active protease kallikrein that in turn releases the nonapeptide bradykinin from HMWK. Bradykinin enhances small vessel permeability, dilates small vessels indirectly via the endothelium but otherwise favors contraction of smooth muscle and is the strongest mediator of pain known.

Bradykinin and other kinins have a short half life, being inactivated by peptidases including angiotensin converting enzyme ACE. ACE inhibitors , frequently used to lower blood pressure, have the common side effect of inducing cough. This is believed to be due to an increase in bradykinin activity. The upshot of these plasma protein cascades is the start of an inflammatory reaction, and the blocking of small venules by coagulation, which is useful to prevent spreading of an infection via the blood.

Driven by blood pressure, plasma is filtrated out of the vessels showing enhanced permeability, forming tissue lymph. This is diverted to the regional lymph nodes, where phagocytes and other leukocytes are waiting to initiate further defense measures. Activation of the plasma protein cascades is in many regards a precondition for the next step, the activation of cellular systems at the infection site.

How are participating cells activated? Neutrophil granulocytes frequently designated PMN, for polymorphonuclear leukocytes are able to directly recognize and phagocytize many bacteria, but not the most crucial polysaccharide-capsulated pathogens. These agents are only recognized and phagocytized following opsonization with complement, via complement receptors on the neutrophil.

How do neutrophils find their way from the blood stream to their site of action? From the site of infection, a host of molecules diffuse in all directions, eventually reaching endothelial cells of neighboring vessels.

These molecules include LPS lipopolysaccharide derived from bacteria, C3a, C4a, C5a and signaling molecules from the first macrophages on the scene, e.

Endothelial cells quickly react to these signals with changes in their expression pattern, exposing new proteins such as ICAM-1 and -2 on their membranes which are then tightly bound by cell-cell contact proteins of neutrophils and other leukocytes rolling past. Neutrophils are normally rolling along the endothelium by dynamic contacts between their sialyl-Lewis-x-carbohydrates and selectin proteins on the endothelial plasma membrane. It squeezes through between two endothelial cells and, along the chemotactic gradient, approaches the focus of infection.

There, neutrophils phagocytize and kill bacteria. In the process, they quickly die, as the harsh conditions necessary to kill bacteria also lead to irreparable cell damage.

Their apoptotic bodies are picked up by macrophages. Mast cells are activated to degranulate and release histamine by a broad spectrum of stimuli: Later, following an adaptive immune response, mast cells may degranulate in response to cross linking of antibodies of the IgE type.

Endothelial cells and thrombocytes. To avoid too much redundancy, we will take a closer look at the activation of endothelial cells and platelets in cardiocascular pathophysiology. Activation of macrophages and dendritic cells via pattern recognition receptors. To sense the presence of pathogens, macrophages and dendritic cells express a much broader spectrum of receptors than neutrophils.

These pattern recognition receptors PRRs recognize pathogen-associated molecular patterns PAMPS , structures that are conserved in broad classes of pathogens for their functional importance. Many of these receptors reside at the plasma membrane: One group of receptors, C-type lectins , recognize certain sugar units that are typically located at the terminal position of carbohydrate chains on pathogen surfaces.

The "mannose receptor" recognizes terminal mannose, N-acetyglucosamin or fucose, in a parallel to mannan binding lectin. The polynucleotide-binding TLRs check the endosomal compartment. On activation, NLRs form a large cytoplasmic complex, the inflammasome.

The inflammasome contributes to cell activation and is instrumental in cleaving IL-1 and other cytokines from their inactive precursors. Therefore, some NLRs serve as unspecific receptors for "danger threatening cells". For long periods of time, they seem to have been a core tool in multicellular organisms' competition with bacteria.

The sea urchin genome, for example, contains more than receptors each for Toll-like receptors and NOD-like receptors. In addition to these direct pattern recognition receptors PRRs , complement receptors , e. Activation of these macrophage receptors leads to phagocytosis and in most cases killing and break-down of ingested bacteria. Via the bloodstream, these cytokines also reach the liver, where they launch another tool of non-specific defense, the production of acute phase proteins.

On activation, macrophages and dendritic cells also express certain membrane-associated proteins, e. B7-molecules CD80 and CD86 that are required to initiate an adaptive immune response.

What is difference between macrophages and dendritic cells? Macrophages are more on the non-adaptive side of defense. They are "heavy earth moving equipment", as their name implies, able to phagocytize large amounts of particulate matter.

Dendritic cells are mainly on the adaptive side of defense: They are able to phagocytize, but don't do the heavy lifting. Many antigens are taken up by macropinocytosis "drinking a whole lot" , a mechanism of taking up large gulps of surrounding fluids with all soluble antigens.

A third way for dendritic cells to take up antigens is by being infected with viruses, which, as we shall see later, is important to start an adaptive antiviral immune response. Many of our dendritic cells are quite long-lived, having originated during developmental stages before birth from hematopoietic cells in the wall of the yolk sac or the fetal liver.

Later, dendritic cells are also produced in the bone marrow. Dendritic cells have two stages of life: Where they go is determined by chemokine receptors, with which they follow the chemokine trail into peripheral tissues. When everything is quiet, they sit in their target tissues for years on end, but a "traumatic" infection with heavy TLR signaling can make them mature and rush to the lymph node in an instant, now following chemokines that are recognized by newly expressed chemokine receptor 7 CCR7.

Mature dendritic cells have lost the ability to take up antigen, but have everything needed for a productive relation with T cells, most prominently lots of MHC and B7 molecules.

By secreting chemokine CCL18, these battle-hardened, worldly-wise dendritic cells are especially attractive to young, naive T cells, the implications of which will only become clear later.

Our innate defence system contains cells that look just like B or T lymphocytes in the microscope, yet express neither B nor T cells receptors. We call them innate lymphoid cells. These cells may be activated by cytokines released by macrophages or dendritic cells and contribute to non-adaptive defence.

Our notion of these cell types is still incomplete. Of this group, we will only look at natural killer cells in more detail.

Histamine is released from mast cell granules, resulting in vascular dilatation and an increase in permeability. It is produced by decarboxylation of the amino acid histidine.

There are four types of histamine receptors, all of the G protein-coupled 7TM family. Proinflammatory functions of histamine are mediated by the H1 and H4 receptors. Drugs blocking these receptors are frequently used in the treatment of allergies, unwanted aspects of inflammation runny, stuffed nose and motion sickness.

H2 receptor blockers are used to decrease gastric acid production. Via H1 receptors, histamine increases small vessel diameter and permeability; via H4 receptors, it recruits eosinophils and other leukocytes. Serotonin is mainly released from activated, aggregating thrombocytes. It activates additional platelets and enhances their ability to bind clotting factors. Serotonin is synthesized from tryptophan. Proteases acid hydrolases, collagenase, cathepsins, etc.

However, a frequent unwanted side effect of these activities is tissue destruction, as proteases are also released from the cells. Many cell types synthesize prostaglandins and leukotrienes from arachidonic acid, a poly-unsaturated fatty acid component of phospholipids.

On demand, arachidonic acid is mobilized from the membranes by phospholipases and metabolized in either of two directions: Two cyclooxygenase isoenzymes are expressed and regulated differentially. COX1 is expressed constitutively in many tissues. It is instrumental, e. COX2 is induced whenever the natural immune system is activated.

Due to their very short half-life, prostaglandins primarily influence the immediate neighborhood of the producing cell. They have very different functions in different tissues; their pro-inflammatory functions are just a small part of their spectrum. For these reasons, it does not do prostaglandins justice to describe their functions in generalized terms: Looking at pro-inflammatory effects in isolation, prostaglandins PGE2 and PGD2 promote vasodilatation the "2" in prostaglandin designations indicates the number of double bonds in the molecule.

PGE2 triggers pain, not by itself, but by potentiating the effect of pain-causing stimuli such as bradykinin and elevated extracellular potassium. Two other prostaglandins have opposing effects on blood coagulation: In the hypothalamus,PGE2 is instrumental in triggering fever. The mechanism increases set temperature in the hypothalamus. Fever reduces proliferation rates of many pathogens, as their enzymes are optimized to function at normal body temperature.

At the same time, some steps required for an adaptive immune response antigen presentation are accelerated. From an evolutionary point of view, fever is an old trick in fighting infections: Therefore, it's not justified to lower fever as a matter of routine via pharmacologic means.

Leukotrienes C4, D4, E4 cause bronchial constriction and enhance vascular permeability, making them key players in bronchial asthma. Leukotriene B4 is chemotactic and activates PMN. Due to their broad spectrum of effects, prostaglandins and leukotrienes offer numerous opportunities to interfere pharmacologically, with, unsurprisingly, equal opportunities for unwanted side effects.

Cortisol and related glucocorticosteroids inhibit the phospholipase which releases arachidonic acid from phospholipids. As this curtails synthesis of both prostaglandins and leukotrienes, glucocorticoids have a strong anti-inflammatory effect.

However, as conventional COX inhibitors inhibit both of the two isoenzymes, they tend to cause typical side effects, including gastritis, intestinal bleeding and ulceration, as well as nephropathy in case of prolongued use. When it became clear that it would suffice to block one of the cyclo-oxygenase enzymes, COX2, for anti-inflammatory effects, COX2-specific drugs with the promise of reduced side effects were developed. In principle, this worked: Low doses of acetylsalicylic acid are being used to reduce the risk for thromboembolic events.

The main bifurcation in arachidonic acid metabolism may result in hyperactivity of one pathway in case the other is blocked. Leukotriene effects can be pharmacologically inhibited by leukotriene receptor blockers e. It has many pro-inflammatory effects, including platelet activation, increasing vascular permeability, bronchial constriction and neutrophil chemotaxis and activation. Following phagocytosis or stimulation by mediators like PAF, neutrophils and macrophages rapidly activate their NADPH oxidase enzyme complex, producing chemically extremely aggressive oxygen-derived reactants like peroxide radicals.

O 2 - , hydrogen peroxide H 2 O 2 , superoxide-anion O 2 2- , singlet oxygen 1 O 2 or hydroxyl radicals. This virtually explosive process is called respiratory or oxidative burst. In a further step, another enzyme, myeloperoxidase, produces hypochloric radicals. These reactive oxygen species ROS are extremely toxic, chemically modifying all kinds of bacterial macromolecules. This works very well to kill phagocytized pathogens, but also kills the phagocyte and frequently damages surrounding tissue.

Nitrogen oxide NO , produced by endothelial cells and macrophages, has two functions: Endothelial cells sensing mediators of inflammation activate their endothelial NO synthase eNOS , producing large amounts of NO to relax adjacent smooth muscle cells.

The term "cytokine" is somewhat fuzzy. It denotes a polypeptide signaling molecule produced primarily, but not exclusively, by cells of the immune system with the aim of coordinating the defense functions of many different cell types. There are many different cytokines, with vastly different spectra of functions and target cells.

Unfortunately, their names are not at all intuitive. A fairly large subgroup of cytokines mediate chemotaxis. Depending on the relative positions of the cysteines which determine tertiary structure, they are classified into four subfamilies: To improve on the bewildering chaos of traditional designations, a unified nomenclature was introduced.

Chemokines are named for their subfamily, with an "L" for ligand and a number: Receptors get an "R" instead, e. Receptors, too, have a common structure: The guiding system of chemokine-gradient fields and chemokine receptors enables all cells of the immune system to arrive in the right place at the right time.

Let's take a look at the cytokine cocktail released by macrophages in response to their activation via pattern recognition receptors. IL activates NK and ILC1 natural killer and innate lymphoid-1 cells and helps to direct differentiation and maturation of a specific T cell subset these cell types are explained later on in sections 1.

They have local as well as remote effects. Several cytokines are produced as recombinant proteins and used as drugs, for example, G-CSF e. Counteracting some of these cytokines can be helpful in inhibiting unwanted immune responses. Cortisol and other glucocorticoids at higher than physiologic concentrations are highly immunosuppressive.

This is for a large part due to a suppressive effect on the expression of many cytokines, e. Recombinant proteins counteracting specific cytokines can be used to inhibit limited aspects of an immune reaction without exposing the patient to the danger of generalized immune suppression. Receptor activation results in expression of genes, the products of which contribute to defending the organism against infection.

Purpose of the molecule: Coordination of a non-adaptive defense reaction on a local and a systemic level. We will first consider abstract strategy, then practical mechanisms. In case an epithelial barrier is breached, it is essential to confine the ensuing bacterial infection to this area. The most dangerous development possible would be the distribution of these pathogens via the blood over the entire organism, a life-threatening complication termed sepsis.

This can be prevented by enhancing permeability of the small blood vessels and closing the draining venules by clotting. The lymph node with its many phagocytes acts as a filter, preventing further spreading. At the same time, leukocytes are recruited from the blood to the primary infection area and endothelial cells are instructed to help them pass. Occasionally, they come too late, and the bacteria have already spread. Everywhere in the body, macrophages are activated by the distributed bacteria.

Everywhere in the body, the coagulation cascade is kicked off, together with the fibrinolytic cascade, consuming all available clotting factors disseminated intravascular coagulation and causing profuse bleeding. Once these processes are under way, they are extremely difficult to stop. Most patients in this condition are lost. This causes fever, the sensation of feeling sick with conservation of energy, but mobilization of energy to produce more defense equipment: All these effects increase the chances of successfully fighting back the infection.

The induction of proteases in inflammatory cells may lead to considerable tissue destruction, as seen in rheumatoid arthrits and in fistulating Crohn's disease. Viruses seem to be less readily detected by non-adaptive mechanisms than bacteria, fungi or parasites. This is probably due to the fact that they are produced in human cells, making their appearance "less unfamiliar" than that of other pathogens. We are therefore equipped with special innate systems to deal with viruses: Interferons IFNs were named for their ability to interfere with virus replication.

Three types of interferons were originally described, depending on the cell type used for purification: They have therefore been subsumed under the heading "type I-interferons".

Type-I-interferons are signaling molecules secreted by virus-infected cells with the aim of slowing or inhibiting virus replication in neighboring cells. Again, this buys time to mount a more efficient, adaptive immune response.

Most viruses, when replicating in human cells, give rise to intermediates consisting of long double-stranded RNA. This type of RNA normally does not exist in human cells, which only contain RNA-molecules with very short double-stranded parts between loops.

Consequently, the appearance of long stretches of double-stranded RNA is a pathogen-associated molecular pattern for potential viral infection, stimulating expression and secretion of type I-interferons.

In contrast to some other PRRs, these are expressed by virtually all cell types. One of the induced proteins is P1-kinase. By phosphorylating eukaryotic translation initiation factor eIF2, it inhibits ribosomal mRNA translation.

This severely restricts replication opportunities for any virus infecting these cells, as it relies on the host cell machinery to produce virus proteins.

Of course, this harsh measure negatively affects host cell functioning as well. A second anti-viral mechanism is activated by induction of the oligoadenylate synthase enzyme. This enzyme oligomerizes ATP by catalyzing unusual 2'-5' bonds normally, nucleotide connections are 3'-5'. Additional proteins induced by type I-interferons facilitate the initiation of an adaptive immune response to eventually eliminate the virus.

These include MHC class I molecules see section 2. R ecombinant type I interferons are injected as therapeutics. Viral infections would seem like logical indications, but interferons are both expensive and have considerable adverse effects, e.

Their application is therefore limited to life-threatening viral diseases, e. Additional applications are unrelated to viral infections, but are a logical consequence of interferons' effects. Natural killer NK cells are similar in appearance and function to cytotoxic T lymphocytes, but lack the receptor T cells are using to identify virus-infected cells the T cell receptor: So how do they recognize cells that should be killed?

One of the cellular properties activating NK cells may be characterized by the catch phrase missing or altered self. NK cells are important in the early phases of defense against certain viruses, but also against other infectious agents, as well as for the elimination of rogue cells to prevent tumor formation. They express two types of receptors: The inhibiting receptors KIR- once acronym for killer inhibiting receptors , now more neutrally killer cell immunoglobulin-like receptors sense the presence of normal MHC-I molecules on cells probed by the NK cell.

A cell with normal MHC-I will be left alone. Many viruses, especially herpes viruses, inhibit MHC-I expression in infected cells. Viruses using this trick have a selective advantage later on, as these cells cannot be identified as infected by cytotoxic T cells explained in sections 2.

Yet, with this strategy they make themselves vulnerable to attack by NK cells. In addition, NK cells may be activated by alternative mechanisms. In some cells, this happens as the result of oncogenic transformation. The importance of this mechanism has been shown in the early defense against the protozoon Leishmania , which is spread by sand flies. Although NK cells are part of the non-adaptive immune system, they can also be directed to target structures by antibodies, in a mechanism termed antibody-dependent cellular cytotoxicity ADCC.

One big problem in defending against pathogens is that they reside in different compartments: To be able to fight pathogens in all these various circumstances, a broad spectrum of tools had to be developed.

Especially useful tools to combat extracellular pathogens are antibodies. IgM always consists of five joined immunoglobulin units, IgA sometimes of two. A few technical terms used in immunology: Functionally, an antibody has a variable and a constant region. While the constant region is encoded in the genome, and as such determinate like any other protein, the variable region is generated by a most unusual process referred to as rearrangement, involving cutting and pasting DNA.

The immunoglobulin's variable region binds antigen. An antigen is everything that is able to elicit an adaptive immune response. Its chemical composition is of minor importance. Antigens include, but are not limited to, polypeptides, carbohydrates, fats, nucleic acids and less frequently than commonly perceived synthetic materials.

A certain minimum size is required. Very small molecules only function as antigens, so-called haptens, when coupled to larger carriers. Antibodies recognize fairly large, three-dimensional surface structures.

Any non-covalent binding force can be used to establish this contact: Antigen binding is therefore reversible. In most cases, a biological macromolecule contains several independent structures able to elicit an antibody response, so-called antigenic determinants or epitopes.

Conversely, two very different macromolecules which by chance share a certain three-dimensional structure may be bound by the same antibody, a phenomenon known as cross-reaction. All these statements refer to antigens bound by antibodies.

Antigens recognized by T-lymphocytes are more narrowly restricted: If a certain protease is used to digest the Y-formed antibody, three fragments result: In early experiments, this fraction was successfully crystallized, giving the fragment the name Fc fraction crystallizable. As this is the "back" end of an antibody, many cells of the immune system have receptors binding to it: The affinity of most of these receptors is too low to bind single, free antibodies for longer periods of time.

Only after antigen-binding, resulting in larger immune complexes, cooperative binding between several Fc ends and their receptors leads to rapid internalization by phagocytosis, providing a mechanism for rapid antigen clearance.

Bacteria, viruses and parasites in general are antigenic. After a lag phase of at least five days, which we must survive with the help of innate immunity, B-lymphocyte-derived plasma cells will produce specific antibodies. These antibodies then bind to the pathogens.

How does this help us? Depending on pathogen, antibodies can help by at least five different mechanisms: For example, these may be virus-infected cells exposing viral envelope proteins in their cell membrane. Neutralizing viruses or toxins means studding them from all directions with antibodies, so that they are no longer able to make contact with their receptors.

To enter a cell, each virus makes contact with one specific protein, which we call its receptor. Of course, the protein was not intended to be a virus receptor; it has some physiological function that is quite different.

For example, HIV human immunodeficiency virus misuses the lymphocyte transmembrane protein CD4 as its receptor. CD4 is important for lymphocyte functioning, which we will look at in section 2.

For some viruses unfortunately not for HIV , it is possible to induce neutralizing antibodies, either by the infection itself or by vaccination. For example, vaccination against hepatitis B virus HBV is very effective. The vaccine contains recombinant envelope protein, HBs-antigen, and induces neutralizing antibodies. If HBV later enters the body, it is immediately studded with antibodies. Unable to enter the liver cell, it remains completely harmless and is soon phagocytized and degraded.

Some bacterial diseases, like tetanus or diphtheria, are not so much caused by the bacteria themselves, but rather by toxins they produce. These bacterial toxins also work by binding and misusing cellular proteins, directing the cells to do something that is in the interest of the bacteria. Vaccinating babies with inactivated versions of these toxins produces neutralizing anti-toxin antibodies. If a child later is infected, it will not even notice, as the disease-causing toxins cannot bind to their receptors: Complement-activation via the classical pathway: IgM and two of the four subclasses of IgG activate complement.

The Fc portion of these antibodies binds complement component C1q, with further steps unfolding as described in section 1. Free soluble antibodies are not able to activate complement. How is this important, as complement is also activated via the alternative and lectin pathways?

Antibodies make the process much more efficient: More complement pores are formed, with a better chance of bacterial lysis. In addition, immunoglobulins are opsonizing in their own right, via Fc-receptors on phagocytes. Complement receptors are also important for immune complex-waste management. CR1 is not only present on leukocytes, but also on red blood cells, binding to C3b that has been deposited on immune complexes.

With that, erythrocytes become the garbage truck for immune complexes, transporting them to spleen and liver, where phagocytes will take them off their backs. If this transport system is overwhelmed, soluble immune complexes will deposit at sites of filtration, e. IgM is a pentamer consisting of five Y-formed units arranged in a circle.

It is always the first immunoglobulin coming up in response to an infection, gradually declining afterwards. The ability of IgM to activate complement is so strong that a single bound IgM-"crab" functions as a landing platform for C1q. This is different from IgG, where at least two IgG molecules have to bound at a distance allowing C1q to go in between. By its size, IgM is mainly confined to blood plasma; it is simply too big to squeeze through between endothelial cells.

IgG is the standard model antibody, appearing later during an immune response than IgM. IgG is the only class of antibodies transported across the placenta, equipping a newborn child for months with antibodies against pathogens "seen" by its mother. Half-life of IgG in blood is approximately 21 days, about double that of IgM.

IgG reach high molar concentrations in plasma, a prerequisite for effective neutralization of viruses or toxins. IgA , of which two subclasses exist IgA1 and IgA2 , can be found as a monomer in the blood, but its main function is to protect "outer" epithelial surfaces.

To get there, it has to be produced in the submucosa as a dimer joined by a J-chain. An epithelial cell, e. There, it is released by cleavage of the receptor. SC protects sIgA from proteolytic digestion in the intestinal tract. Its strong glycosylation localizes and concentrates sIgA in the thin mucus layer lining the epithelium.

There, sIgA prevents viruses, bacteria and toxins to make contact with their respective receptors by keeping them near the surface of the mucus lining, a mechanism termed immune exclusion.

IgE developed as a tool to fight parasites worms and protozoa. If a worm penetrates the epithelial barrier, it binds to and crosslinks specific IgE, resulting in mast cell degranulation. Additional IgE will bind to the parasite.

Mast cells release histamine and other molecules attracting eosinophils. An inflammatory reaction, induced via H1 receptors, facilitates the movement of eosinophils, which are guided in their chemotaxis by H4 receptors. In developed countries, parasite infections today are less common.

A problem arises when the immune system confuses innocuous entities such as inhaled tree or grass pollen with dangerous parasites. Normally useful IgE then becomes a liability, inducing hay fever or bronchial asthma. IgD is found together with IgM on the cell membrane of newly produced B lymphocytes, and in negligible amounts in plasma. Soluble IgD is not currently thought to have a function in defense.

In patients, it is possible to measure concentrations of either an entire immunoglobulin class e. In the past, antigen-specific antibody concentrations were routinely expressed as a "titer". One typical example for such a vintage test would be the complement binding reaction, where upon the addition of a serum dilution and complement, test erythrocytes either lyse or don't lyse. A patient's serum was diluted 1: If lysis was seen at dilutions 1: Frequently, it was expressed reciprocally: We will look at three of the numerous test systems to determine antibody concentrations: For all three, monoclonal antibodies are required.

Originally, simple antisera were used to detect specific biomolecules, including human antibodies. A laboratory animal such as a rabbit was immunized with the purified molecule in question example: Yet, such an antiserum, in lab jargon called "polyclonal antibody" is far from a precision tool. It contains a smorgasbord of antibodies against all antigens the lab animal has been in contact with. These side specificities can completely distort the test results. A monoclonal antibody obviates the specificity problem, as it constitutes amplified replicas of a single antibody produced by a single B cell.

However, generating a monoclonal antibody is a time-consuming and tedious procedure. In the usual procedure, a mouse is repeatedly immunized with the antigen of interest, in our example human IgM. After several weeks of injections with human IgM, the mouse will produce antibodies against human IgM.

Many of the B cells producing these antibodies will reside in the mouse's spleen, which is removed to get hold of these cells. At this point, it would seem straightforward to take these cells into culture and simply harvest the desired antibody, yet the cells would stop proliferating and die very soon. To endow them with unlimited survival and proliferation potential, they are fused to a mouse tumor cell line that has exactly these properties.

In addition, the tumor cells have a biochemical Achilles' heel that is later used to get rid of unwanted cells. Fusion of cells can be performed by a simple lab procedure using polyethylene glycol. It's the goal of the next step to have only the desired fusion cells survive. Unfused or fused B cells are no problem- they die automatically after a few days.

Unfused or fused tumor cells are a problem: To kill them, a trick is used. The tumor cell line is deficient in an enzyme important to recycle purine nucleotides, hypoxanthine-guanine phosphoribosyltransferase HGPRT. To survive, the tumor cells constantly synthesize new purine bases, for which they need tetrahydrofolic acid. The trick is to block the regeneration of tetrahydrofolic acid by adding its antagonist aminopterin to the culture.

Following fusion, the bulk of cells is cultivated in HAT -media, named for containing h ypoxanthine the recycling starting point , a minopterin and t hymidine which also could not be produced without tetrahydrofolic acid.

Tumor cells die, as they are now completely unable to produce purine nucleotides. B cells die anyway. After some time in culture, only these cells remain, which we refer to as hybridoma cells, implying a fusion cell that grows like a lymphoma. These represent all varieties of B cells originally present in the mouse spleen. Many will not produce any antibody at all, many will produce antibodies unrelated to our antigen, and only few will produce high-affinity antibodies to human IgM.

How to find them and get rid of the others? The next step is limiting dilution: The volume is chosen in a way that statistically, there is only one single hybridoma cell in every other well. Whatever grows up will thus be monoclonal, meaning stemming from one single cell. Hybridoma cells secrete their antibody into the medium, or culture supernatant.

The last remaining challenge is to find the two, three or five cell clones producing antibody against our antigen among the hundreds or thousands of clones producing something else or nothing at all. For that, an immunological assay usually ELISA, see below is used with our antigen, human IgM, as a bait to test all culture supernatants for the presence of antibody binding it.

Once found, the hybridoma cell clone can be expanded and cultured virtually indefinitely, and monoclonal antibody can be purified from its culture medium in large quantities. Today, monoclonal antibodies against most diagnostically important macromolecules are commercially available. In addition, monoclonal antibodies are increasingly being used as drugs, e. However, as they mostly originate from the mouse, they would elicit an immune response in humans HAMA: Therefore, "humanized" monoclonals are used, where all parts of the mouse antibody not directly required for antigen binding are replaced by their human counterparts.

Antibody concentrations in patients' sera can be measured by many methods; the most common one is ELISA e nzyme- l inked i mmuno s orbent a ssay.

To ascertain a recent infection with a specific virus, a test for IgM against that virus could be performed as follows. First, the wells of a microtiter plate are coated with virus or virus protein. Then, the wells are incubated with diluted patient serum: After washing thoroughly, monoclonal mouse antibody against human IgM is added.

This is the same antibody we produced in the previous section, but now has been linked to an enzyme such as horse radish peroxidase. If there was anti-virus IgM in the patient's serum, the enzyme-linked antibody will bind, too. If the serum contained no anti-virus IgM, the enzyme-linked antibody will be subsequently washed away.

Finally, a colorless substrate molecule is added, which is metabolized to a bright color pigment by horse radish peroxidase. The amount of color, proportionate to the amount of anti-virus IgM in the patient serum, is photometrically quantified.

Color means the patient has IgM against the virus; no color means no anti-virus IgM is present. An analogous parallel test could be run using another monoclonal antibody against human IgG, to check whether the patient had been infected with the same virus a longer time ago.

Western blots are used, for instance, as a confirmation test to diagnose HIV infection. HIV proteins are denatured and solubilized using the detergent SDS, separated via a polyacrylamide gel and transferred to a paper-like membrane. This blot with bound virus proteins is then subjected to basically the same steps as described above for the virus-coated plastic well in the ELISA. The membrane is first treated with diluted patient serum, then with an enzyme-linked monoclonal antibody against human antibody, finally with substrate, with washing steps in between.

If the patient has antibodies against HIV, this will show in the form of colored bands on the membrane. Sometimes, for instance in autoimmune disease, it is important to test whether a patient has antibodies against certain tissue structures, without knowing the exact molecule the antibody might recognize.

To assay whether a patient has anti-nuclear antibodies, cells or a tissue section are applied to a glass slide and incubated with a droplet of diluted patient serum. If antibodies are present that bind to some nuclear structure, they can again be detected using a mouse monoclonal against human antibody, in this case coupled to fluorescent dye.

If the patient has antinuclear autoantibodies, the nuclei will be brightly visible in the fluorescence microscope; in the absence of ANA, they will remain dark.

For an overview whether normal amounts of IgM, IgG and IgA are present in human serum, immunoelectrophoresis is informative. First, serum proteins are separated electrophoretically in a gel.

Then, rabbit anti-human serum is applied to a groove running in parallel to the axis of separation. The rabbit antiserum diffuses through the gel towards the separated human proteins.

Precipitation arcs form where serum proteins and antibody meet, allowing to identify three separate arcs for IgM, IgG and IgA. In case of IgA deficiency, that specific arc would be missing. How is it possible that we are able to form antibodies against virtually any antigen on the globe?

Antibodies are made of polypeptide chains, and polypeptides are genetically encoded, yet the human genome only consists of approximately 25, genes. Even if the majority of them encoded antibodies, that wouldn't do the trick by far. The answer to this conundrum has been found: The variable region of an immunoglobulin is formed by portions of both the heavy and the light chain.

The variable portion of the heavy chain is not linearly encoded in the genome, bat rather in separated gene segments of three types, V, D and J v ariable, d iversity and j oining. Importantly, each of these segments is present in multiple, slightly different variations: A complete heavy chain variable region exon is randomly cobbled together by juxtaposing one V, one D and one J segment by a cut and paste process at the DNA level. Then, normal DNA repair proteins directly rejoin the segments.

In all, there are 65x27x6 ways to recombine the segments, resulting in 10, different heavy chain possibilities just by rearranging the building blocks. But that is not all. The rejoining process is somewhat messy: This mechanism is called junctional diversity or imprecise joining. Light chain genes are individually manufactured along the same lines, with the difference that they do not have D segments, just V and J segments.

Combining randomly generated heavy with randomly generated light chains adds another level of variability. Somatic recombination is performed in immature B cell precursors in the bone marrow. Maintenance of a productive reading frame is monitored by specific quality control mechanisms. Successful assembly of a heavy chain, for example, is signaled via a specific kinase, BTK Bruton's tyrosine kinase.

In the absence of a BTK signal, implying frame shifts in both heavy chain genes, the now useless maturing B cell enters apoptosis. Once an entire antibody has successfully been assembled, it is expressed as a transmembrane protein in the form of a B cell receptor.

The difference between B cell receptor and secreted antibody is in a transmembrane domain, encoded by a separate exon, that can be added or omitted by alternative splicing. In the course of an adaptive immune response, especially if the antigen cannot be eliminated quickly, an additional mechanism adding to overall variability and allowing development of high-affinity antibodies comes into play: In B cells rapidly proliferating in germinal centers of lymphoid follicles, those regions within the rearranged VDJ heavy chain or VJ light chain exons that encode the protein loops making direct contact with the antigen undergo somatic mutation at a rate that is approximately thousandfold of normal.

These complementarity determining regions are therefore also called hypervariable regions. What is the mechanism behind this mutation rate? In all cells, one of the most frequent forms of DNA damage is spontaneous hydrolytic deamination of cytosine, resulting in uracil.

AID is only active in genomic regions that are intensely transcribed, as the two DNA strands have to be slightly separated for the enzyme to work. Deamination is equivalent to a point mutation: Some of these mutations will increase antibody affinity, and the respective B cells will be able to hold on to antigen for longer and consequently receive a stronger stimulus to proliferate.

Somatic hypermutation over time thus favors a shift to antibodies of higher affinity. In summary, four different mechanisms contribute to the generation of antibody diversity: Antibody diversity is thus caused by a DNA-based random generator. That seems kind of an oxymoron: How is it possible that a random generator develops in this rigid system?

Comparing different species, we find that all vertebrates, from fish to man, use some form of RAG-based random generator to enhance defense against infections.

Interestingly, a few primeval jawless fish species like lamprey and hagfish do not. If we take a look at our genome, we do not find a sleekly designed, minimalistic high tech machine. Rather, it resembles a confusing accumulation of ancient sediments. Between and overlapping active genes, it contains many copies of "molecular nonsense machines" like retroviruses and transposons, most of them inactivated by mutations.

What do I mean by "molecular nonsense machines"? Imagine a contraption with the sole ability to produce copies of itself. Given sufficient resources, that would soon result in an avalanche of these machines. Viruses, in principle, are nothing else. Another type of nonsense machine is a unit of DNA containing the information required to produce enzymes with the ability to excise the unit from surrounding DNA and implanting it elsewhere.

This is what we call a transposon. In the Silurian, to million years ago, the following genetic accident happened in a fish: Yet, it could still be healed if the transposon re-excised itself. This structure was the nucleus of our antibody- and T cell receptor-loci, which evolved by numerous locus doublings followed by mutational drift.

B and T cell receptors correspond to the original transmembrane protein, the RAG proteins to the transposon's nucleases. Usually, all that remained from the original transposon were its left and right demarcations for excision, short base sequences we now call recombination signal sequneces RSS.

Of all transposon copies, only one, on chromosme 11, maintains active nucleases: Once a variable region has been successfully generated by rearrangement, it can be handed down from one isotype to another. These cells now produce IgG, having undergone class switch. Note that the variable region has remained exactly the same.

The antibody binds the same antigen with the same affinity, only it's now of the IgG isotype. Probability and type of class switch are influenced by cytokines released by T-lymphocytes and other cells. Class switch occurs spatially and temporally parallel to somatic hypermutation, in the germinal centers of secondary follicles. Both processes are initiated by the same enzyme, AID. Gene segments for heavy chain constant regions have switch regions that easily form single chain DNA loops.

In these temporary loops, AID deaminates cytosine, leading to uracil. This is in fact a targeted and accelerated version of a process occurring regularly in our cells, spontaneous deamination by hydrolysis.

Uracil in DNA constitutes a "wrong" base that is quickly eliminated by a dedicated repair system. If the same happens at the opposite strand a few nucleotides further down, a double strand break occurs. In case of class switch recombination, this form of DNA cleavage occurs simultaneously at two distant locations. Isn't it dangerous to have antibodies generated randomly? One would expect some useful antibodies, depending on the type of infections encountered. But more antibodies are likely to be useless and some might be even dangerous, causing autoimmune disease if they by chance bind to structures of our own body.

B cell clones having rearranged antibodies recognizing ubiquitous self-antigens undergo apoptosis at an early stage clonal deletion or change into a "frozen" state from which they cannot be reactivated clonal anergy.

However, these protective mechanisms do not work perfectly, sometimes allowing autoantibodies to be produced. The distinction between useful and useless antibodies is made by infecting pathogens.

New antibodies are rearranged all the time in newly developing B cells in the bone marrow. Once it is clear that they don't recognize frequent self-antigens, they migrate to peripheral lymphatic tissues and wait. Most wait in vain, and eventually die. In case of an infection, an invading pathogen will encounter a broad array of antibodies, sitting as "B cell receptors" on resting B cells in lymph nodes or other lymphoid tissue.

If one out of a million of B cell receptors fits an antigen of the pathogen, this specific B cell is induced to proliferate, while all other B cells don't react. This is called "clonal selection": The difference between B cell receptor and secreted antibody is a transmembrane domain at their terminus of the heavy chain that is included or excluded by alternative splicing. As our immune system is constantly engaged fighting subliminal infections, there are a lot of "useful" proliferating B cells at any point in time.

Thus, the proportion of useful B cells among the total is actually higher than expected from the randomness of antibody generation. Antibodies are sharp-edged tools, always involving the risk of autoimmune damage. It would be extremely dangerous if a single contact between B cell receptor and antigen were sufficient to unleash large-scale antibody production.

Therefore, in analogy to a gun, the release of a "safety catch" is required as a safeguard before a B cell can be activated.

This is accomplished by a complex process summarily designated "T cell help". An exception to this rule are so-called T cell independent antigens. In many cases, these are linear antigens with repetitve epitopes which are able to crosslink multiple B cell receptors or additional pattern recognition receptors.

This activation merely leads to production of IgM, usually of modest affinity. Neither class switch nor affinity maturation is possible in the absence of T cell help. To understand how T cells function and interact with other cells, some information on lymphoid tissues and organs, T cell receptor and MHC is required.

In the bone marrow, hematopoietic stem cells give rise to lymphoid progenitor cells. From these, B cells differentiate in the bone marrow, although the name B cell is derived from a gut-associated organ in birds, the b ursa Fabricii , that doesn't exist in humans.

Lymphoid progenitors also migrate to the t hymus located on top of the heart , where they undergo complex quality assurance procedures that allow only a small fraction of these thymocytes to leave the thymus as mature naive T cells explained in section 2. Lymphocytes travel mainly via the bloodstream. APC leave the bloodstream to widely roam tissues. Eventually, all types of cells meet again at the peripheral lymphatic organs: LYMPH NODES seem static in the microscope, but should better be compared to the transit area of a big international airport, with oodles of cells arriving and leaving all the time.

Lymph nodes have several inlets and an outlet. Afferent lymphatic vessels reaching the most peripheral lymph nodes transport the interstitial fluid filtrated from blood capillaries.

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