Overview of Acute Inflammation - in a FAQ style

This chapter will help you to:

• answer the frequently asked questions on acute inflammation
• prepare for the theory and viva exams in pathology
• get an easy refence tool for mediators of inflammation which might prove useful in pharmacology (autacoids) and microbiology (immunity) as well

General Considerations

1. Define inflammation.

Inflammation is defined as the protective response of a living vascularised tissue to a sub-lethal injury.

2. What are the different types of inflammation?

Inflammation may be of two types:
a) Acute inflammation
b) Chronic inflammation

3. State the cardinal signs of inflammation.

The cardinal signs of inflammation are:
Rubor (redness)
Tumor (swelling)
Calor (heat)
Dolor (pain)
Functio laesa (loss of function)

4. Compare acute & chronic inflammation.

Acute inflammation

It is of short duration
Characterized by tissue destruction followed by repair
Principal cells of inflammation are neutrophils (microphage)
Exudate formation is a hallmark


Chronic inflammation

It is of longer duration
Characterised by the simultaneous progress of tissue destruction and repair
Principal cells of inflammation are macrophages, lymphocytes and plasma cells.
Exudate formation is not so promine


5. What are the mechanisms underlying the genesis of the cardinal signs of inflammation?

Rubor – Due to increased vasodilation
Dolor – Due to local injury & release of pain producing mediators of inflammation
Tumor – Due to transudation & exudation of fluids into tissue spaces
Calor – Due to increased rate of oxygen utilization in the local tissue (respiratory burst) and vasodilatation.



ACUTE INFLAMMATION


6. Enumerate different events of acute inflammation.

The different events of acute inflammation are:

A. Vascular changes:
a) Changes in vascular flow & caliber
b) Increased vascular permeability

B. Leukocyte cellular events
a. Margination & rolling
b. Adhesion & transmigration
c. Chemotaxis & activation
d. Phagocytosis & degranulation
e. Leukocyte induced tissue injury


7. Write a short note on Phagocytosis.

Definition: It is a process of endocytosis by which a cell engulfs a semisolid material with subsequent formation of a phagocytic vacuole.

Mechanism:

A) FORMATION OF ENDOSOME

Phagocytosis can be basically of two types - Constitutive phagocytosis and receptor mediated phagocytosis. Costitutive phagocytosis is a continuous process that is not induced by any agent. Receptors meditative phagocytosis is produced in most cases by specialized clathrin-coated pits on the cell membrane. The latter is triggered by various ligands binding to their receptors on the cell surface. After the pit is full of ligands, the pit pinches off, forming a coated vesicle. This coated vesicle is now called early endosome. Now the endosome gets separated from the membrane and moves into the cytosol. This is now called a late endosome.

B) TRANSPORT OF ENDOSOME
The transport of vesicles is guided by different groups of targeting and docking proteins. On the surface of the endosome (vesicle) there is a specific V-snare protein that latches with its corresponding T-snare protein on the target.
Various small GTP-binding proteins of the Rab family are associated with acceleration of the transport, where as proteins of Sec 1 family slow down the transport process.

C) FATE OF THE ENDOSOME
The late endosome fuses with lysosomes and lysosomal enzymes digest their contents.

Importance of Phagocytosis:

• It helps to kill bacteria. Bacteria can be killed by ROS-mediated, non-ROS-mediated (enzymatic) & NO-mediated mechanisms
• Phagocytosis of dead tissue by scavenger cells (macrophages) helps in clearing tissue debris
• Receptors mediated endocytosis helps in internalization of LDL, insulin, epidermal growth factor, nerve growth factor, diphtheria toxin and a no. of viruses.

8. What are the morphological patterns, which an acute inflammatory process can present?

Morphological patterns of acute inflammation:

• Catarrhal – Superficial inflammation of mucous surfaces with hyper secretion of mucus by goblet cells.
  
  E.g. Common cold, Inflammation of bowel

• Serous – Formation of protein rich fluid exudates with minor cellular exudation
  
  E.g. Peritonitis, Synovitis
  
  
• Fibrinous – Here, exudates contains abundant fibrinogen which is precipitated as a thick fibrin coating
  
  E.g. Fibrinous pericarditis, lobar pneumonia
  
  
• Pseudo-membranous – Formation of a membrane made of fibrin, necrotic epithelium and inflammatory cells
  
  E.g. Pseudo-membrane of diphtheria
  
  
• Hemorrhagic – Associated with frank hemorrhage due to vascular damage by bacterial toxins
  
  E.g. Secondary bacterial infection in viral pneumonia
  
  
• Gangrenous – Inflammation associated with wide spread necrosis of the organ, probably resulting from superimposed thrombosis or vascular occlusion
  
  E.g. Strangulated hernia
  
  
• Purulent – Production of pus which is creamy yellow in color, high specific gravity, alkaline in reaction & semi fluid in consistency
  
  E.g. Abscess, Empyema
  
  
• Phlegmatous – Diffuse spread of exudates through loose and lax tissue spaces by virulent bacteria
  
  E.g. Erysipelas of face
  
  
• Allergic – Hyper sensitivity of the host immune system to an antigen which was previously introduced to the body
  
  E.g. Bronchial asthma

NOTE: Q. Why is diphtheric inflammation called Pseudo-membranous?

In diphtheria the membrane that is formed does not contain any basement membrane. It is composed entirely of fibrin, necrotic tissue &cellular exudates; hence it is called pseudo-membranous.


9. What are the fates of acute inflammation?

The fates of acute inflammation are:

• Resolution
  
• Progression to suppuration
  
• Progression to chronic phase with fibrosis

10. What are the cells taking part in host immunity? State their roles in the process of inflammation?

Natural cytolytic cells

a) Natural killer cells – Large granular lymphocytes, having Fc receptors for antibodies and kill anti body coated cells and virus infected tumor cells


Phagocytic cells

a) Neutrophils – Granulocytes with multilobed nucleus; help in phagocytosis and kill bacteria
b) Eosinophils – Granulocytes with bilobed nucleus and coarse brick-red cytoplasmic granules; take part in parasite defense and allergic response
c) Macrophages – Present in tissue, spleen, lymph nodes; bears Fc and C3 receptors on cell surface; takes part in inflammatory response anti-viral and anti-tumor activities


Antigen-presenting cells (APCs)

a) Monocytes – agranulocytes with Horse-shoe shaped nucleus that modulates immune response by releasing various cytokines
b) Macrophages – as above


Antigen -responsive cells

a) CD4 T cells – Mature in thymus, characterized by large nucleus, small cytoplasm; contains CD2, CD3, TCR, CD4 molecules on cell surface; Secretes IL-1 and other lymphokines and stimulate T and B cell growth
b) CD8 T killer cells – Involved in recognition of antigen; bears CD2, CD3, TCR, CD8 molecules on surface; Kill viral, tumor, transplant cells and secretes IL-2, IFN-gamma


Antibody producing cells

a) B cells – Mature in bursal equivalent; presents surface antibodies and class II MHC molecules and involved in Antibody production
b) Plasma cells – Activated B cells with eccentric small nucleus with Russel bodies in the cytoplasm that serve as antibody factories


Other cells

a) Basophils / mast cells – Granulocytes with coarse blue cytoplasmic granules; bears Fc-receptor of Ig E on surface; involved in Histamine release and allergic reactions



11. Enumerate the chemical mediators of acute inflammation.

The different chemical mediators of acute inflammation are classified into:

• Cell-derived mediators
• Plasma-derived mediators

The cell derived mediators include:

a) Preformed mediators, like Histamine, Serotonin & Lysosomal enzymes
b) Newly synthesized mediators, like Prostaglandins, Leukotrienes, PAF, ROS, NO, Cytokines, etc

The plasma derived mediators include:

Active by-products of the clotting system, fibrinolytic system, kinin system & complement system



12. Mention the functions of the mediators of inflammation with examples.

The most likely mediators involved in the following inflammatory changes are:

Vasodilatation:

a) Prostaglandins
b) Nitric oxide

Increased vascular permeability:

a) Vasoactive amines
b) C3a and C 5a
c) Bradykinin
d) LT C4, D4, E4
e) PAF

Chemotaxis and leukocyte activation:

a) C5a
b) LT B4
c) Bacterial products
d) Chemokines

Fever:

a) IL1, IL6s
b) TNF-alpha
c) Prostaglandins

Pain:

a) Prostaglandins
b) Bradykinin

Tissue damage:

a) Lysosomal enzymes
b) ROS
c) NO


A BRIEF DISCUSSION ON SOME IMPORTANT MEDIATORS OF INFLAMMATION:

Histamine: -

Derived from the amino acid histidine by decarboxylation
Present in mast cells
Released in response to physical injury, typeI hypersensitivity, C3a and C5a of complement, leukocyte-derived histamine-releasing proteins, neuropeptides and some cytokines.
Causes arteriolar dilatation and increased vascular permeability, venular endothelial contraction and widening of inter-endothelial cell junction.
Inactivated by histaminase



Serotonin: -

Derived from amino acid tryptophan by 5-hydroxylation and decarboxylation. Also called 5-hydroxytryptamine or 5HT
Found within platelet- dense body granules
Release is stimulated by platelet aggregation
Function is similar to histamine



Arachidonic acid metabolites (Eicosanoids): -

These are derivatives of eicosa-tetra-eneoic acid (20carbon unsaturated fatty acid) viz. arachidonic acid
Arachidonic acid is liberated from membrane-phospholipid during cell injury by the enzyme phospholipase A2. This enzyme is inhibited by glucocorticoids
From arachidonic acid, by cycloxygenase enzyme (COX) the prostaglandins (PG) & thromboxanes (TX) are produced. This enzyme is inhibited by NSAIDs
From arachidonic acid by lipoxygenase enzyme (LOX) the leukotrienes (LT) are produced. This enzymes is inhibited by zileuton


Functions of eicosanoids are:

1. PG I2 (prostacyclin)

Vasodilatation
Inhibits platelet aggregation


2. TX-A2 (thromboxane)

Vasoconstriction
Promotes platelet aggregation


3. PG-D2, PG-E2, PG-F2 alpha

Vasodilation
Edema


4. LT-B4

Chemotaxis

5. LT-C4, LT-D4, LT-E4

Vasoconstriction
Bronchospasm
Increased vascular permeability



Platelet-activating factor (PAF): -

It is a phospholipid- derived mediator. Chemically it is acetyl glycerol ether phosphocholine
Obtained from membrane phospholipids of leukocytes and other cells by the action of phospholipase-A2
It causes vasoconstriction in high doses and vasodilation at low doses .in addition it also causes broncho-constriction, increased leukocyte adhesion, chemotaxis, leukocyte degranulation and oxidative burst. It can also stimulate synthesis of eicosanoids



Nitric oxide (NO): -

It is a short acting soluble gaseous free radical
It was initially called EDRF (endothelium-derived relaxation factor)
It is synthesized from L-Arginine with the help of NO-synthase

L-Arginine + O2 + NADPH = Citrulline + NO + NADP

The reaction is catalysed by NO-synthase

The enzyme NO synthase has 3 isoforms:

I. Neuronal isoform, present in neurons, and constitutive in nature. It is also called nc NOS
II. An inducible isoform present in cardiac myocytes, hepatocytes and respiratory epithelium, called i NOS
III. Another constitutive isoform present in endothelium, called ec NOS

Of these, the function of the constitutive iso-enzymes (I and III) depends on the cytosolic Ca++ level.


Functions of NO include:

a. Smooth muscle relaxation by stimulating soluble Guanylyl cyclase enzyme
b. Formation of peroxynitrite radical by combining with super oxide radical. This radical breaks into toxic NO2 and OH- radical
c. It can combine with iron in presence of thio-compounds (R-SH) to form di-thio di-nitroso complex with iron. This depletes iron and leads to inhibition of several bacterial enzymes.



Complement system: -

It is a group of about 20 plasma proteins that help in non specific immune response
It comprises of 2 distinct pathways – the classical pathway and the alternate pathway
Classical pathway comprises of sequential activation of number of plasma proteases following activation by immune-complex
Alternate pathway is activated by the cell wall lipo-polysaccharide of bacteria
Both the systems converge into a final common pathway leading to the formation of a Membrane Attack Complex (MAC) that induces pore formation in the membrane of cells and leads to osmotic lysis

Role of complement components in inflammation are:

a) C 3a and C5a increases vascular permeability (anaphylatoxin)
b) C5a is chemotactic to leukocytes
c) C3b is an important opsonin
d) C5b- C7 complex is a chemo tactic agent
e) C5b-9 is the MAC



Kinin system: -

This system comprises of 2 vasoactive peptides- bradykinin and lysyl-bradykinin (kalidin)
Bradykinin is a Nona peptide whereas lysyl-bradykinin is a deca peptide having an extra lysine residue at the amino-terminal
They are derived from the High and the Low molecular weight kininogens (HMWK and LMWK)
They are inactivated by kininase I and kininase II.Kininase II is identical with ACE

Function of kinins are:

a) Contraction of smooth muscle
b) Dilatation of arterioles
c) Production of pain
d) Chemotaxis
e) Increased vascular permeability



Clotting system: -

The coagulation system plays important roles in the mediation of inflammation
Activation of the Hageman factor (factor XII) is the key process leading to activation of the plasma proteases
Fibrino-peptides formed due to conversion of fibrinogen to fibrin leads to increased vascular permeability
The fibrino-peptides are also chemotactic to neutrophils



Fibrinolytic system: -

Converts fibrin into fibrinopeptides which lead to:

a) Increased vascular permeability
b) Chemotaxis
c) Generation of C 3a
d) Activation of kinin system



Lysosomal products: -

Lysosomal granules of neutrophils and monocytes contain different inflammatory mediators
The proteolytic substances include acid proteases and neutral proteases
Acid proteases have acidic pH optima and active only in phagolysosomes
Neutral proteases include elastase, collagenase and cathepsin, which degrade extra-cellular matrix (ECM). It can generate C3a and C5a, and stimulate kinin system
To counteract the uncontrolled effects of proteases, a number of anti-proteases are present in serum and ECM




References:

• Pathologic Basis of Disease – 7th ed, Robbins & Cotran
• General Pathology – Walter Israel
• Review of Medical physiology – 19th ed, W F Ganong
• Harper’s Biochemistry – 25th ed, Murray et al
• Immunology – Roitt
• Medical microbiology – 22nd Jawetz, Melnick & Adelberg
• Pharmacology – 6th ed, Rang, Dale, Ritter

Pathology & Pharmacology: Pheochromocytoma Review

Pheochromocytomas are tumors of the adrenal medulla that produce, store, and secrete catecholamines. They are usually derived from the adrenal medulla but may develop from chromaffin cells in or about sympathetic ganglia (extraadrenal pheochromocytomas or paragangliomas).

The clinical features are mainly due to the release of catecholamines and, to a lesser extent, to the secretion of other substances. Hypertension is the most common sign, and hypertensive paroxysms or crises, often spectacular and alarming, occur in over half the cases.



PATHOLOGY

Location and Morphology

In adults, approximately 80% of pheochromocytomas are unilateral and sol Solitary lesions inexplicably favor the right side. Although pheochromocytomas may grow to large size (over 3 kg), most weigh less than 100 g and are less than 10 cm in diameter. The tumors are highly vascular, 10% are bilateral, and 10% are extraadrenal.

The tumors are made up of large, polyhedral, pleomorphic chromaffin cells. Less than 10% of these tumors are malignant. As with other endocrine tumors, malignancy cannot be determined from the histologic appearance; tumors that contain large numbers of aneuploid or tetraploid cells, as determined by flow cytometry, are more likely to recur. Local invasion of surrounding tissues or distant metastases indicate malignancy.

Catecholamine Synthesis, Storage, and Release

Pheochromocytomas synthesize and store catecholamines by processes resembling those of the normal adrenal medulla. These tumors are not innervated, and catecholamine release does not result from neural stimulation. Pheochromocytomas also store and secrete a variety of peptides, including endogenous opioids, adrenomedullin, endothelin, erythropoietin, parathyroid hormone-related protein, neuropeptide Y, and chromagranin A. These peptides contribute to the clinical manifestations in selected cases.

Most pheochromocytomas contain and secrete both norepinephrine and epinephrine, and the percentage of norepinephrine is usually greater than in the normal adrenal. Increased production of dopamine and homovanillic acid (HVA) is uncommon with benign lesions but may occur with malignant pheochromocytoma.



FAMILIAL PHEOCHROMOCYTOMA

In approximately 5% of cases, pheochromocytoma is inherited as an autosomal dominant trait either alone or in combination with other abnormalities such as MEN type 2a (Sipple's syndrome) or type 2b (mucosal neuroma syndrome), von Hippel-Lindau's retinal cerebellar hemangioblastomosis, or von Recklinghausen's neurofibromatosis.

A familial syndrome should be suspected in any patient with bilateral pheochromocytomas.



CLINICAL FEATURES

Pheochromocytoma occurs at all ages but is most common in young to midadult life. Most patients come to medical attention as a result of hypertensive crisis, paroxysmal symptoms suggestive of seizure disorder or anxiety attacks, or hypertension that responds poorly to conventional treatment. Most patients have hypertension in association with headaches, excessive sweating, and/or palpitations.

Hypertension Hypertension is the most common manifestation. In approximately 60% of cases the hypertension is sustained, although significant blood pressure lability is usually present, and half of patients with sustained hypertension have distinct crises or paroxysms. The other 40% have blood pressure elevations only during an attack. The hypertension is often severe, occasionally malignant, and may be resistant to treatment with standard antihypertensive drugs.

Paroxysms or Crises The paroxysm or crisis occurs in over half of patients. In an individual patient, the symptoms are often similar with each attack. The paroxysms may be frequent or sporadic, occurring at intervals as long as weeks or months. With time, the paroxysms usually increase in frequency, duration, and severity. The attack usually has a sudden onset. It may last from a few minutes to several hours or longer. Headache, profuse sweating, palpitations, and apprehension, often with a sense of impending doom, are common. Pain in the chest or abdomen may be associated with nausea and vomiting. Either pallor or flushing may occur during the attack. The blood pressure is elevated, often to alarming levels, and the elevation is usually accompanied by tachycardia. The paroxysm may be precipitated by any activity that displaces the abdominal contents. In some cases a particular stimulus may induce an attack in a characteristic fashion, but in others no clearly defined precipitating event can be found. Although anxiety may accompany the attacks, mental or psychological stress does not usually provoke a crisis.

Other Distinctive Clinical Features Symptoms and signs of an increased metabolic rate, such as profuse sweating and mild to moderate weight loss, are common. Orthostatic hypotension is a consequence of diminished plasma volume and blunted sympathetic reflexes. Both these factors predispose the patient with unsuspected pheochromocytoma to hypotension or shock during surgery or trauma. Secretion of the hypotensive peptide adrenomedullin may contribute to the hypotension in some patients.

Cardiac Manifestations Sinus tachycardia, sinus bradycardia, supraventricular arrhythmias, and ventricular premature contractions all have been noted. Angina and acute myocardial infarction may occur even in the absence of coronary artery disease. A catecholamine-induced increase in myocardial oxygen consumption and, perhaps, coronary spasm may play a role in these ischemic events. Electrocardiographic changes, including nonspecific ST-T wave changes, prominent U waves, left ventricular strain patterns, and right and left bundle branch blocks may be present in the absence of demonstrable ischemia or infarction. Cardiomyopathy, either congestive with myocarditis and myocardial fibrosis or hypertrophic with concentric or asymmetric hypertrophy, may be associated with heart failure and cardiac arrhythmias. Multiorgan system failure with noncardiogenic pulmonary edema may be the presenting manifestation. Elevated levels of amylase originating from damaged pulmonary endothelium and abdominal pain may suggest acute pancreatitis, although serum lipase levels are normal.

Carbohydrate Intolerance Over half of patients have impaired carbohydrate tolerance due to suppression of insulin and stimulation of hepatic glucose output. The impaired glucose tolerance rarely requires treatment with insulin and disappears after removal of the tumor.

Hematocrit The elevated hematocrit is secondary to diminished plasma volume. Rarely, production of erythropoietin by the tumor may cause a true erythrocytosis

Other Manifestations Hypercalcemia has been attributed to the ectopic secretion of parathyroid hormone-related protein. Fever and an elevated erythrocyte sedimentation rate have been reported in association with the production of interleukin 6. Elevated temperature more commonly reflects catecholamine-mediated increases in metabolic rate and diminished heat dissipation secondary to vasoconstriction. Polyuria is an occasional finding, and rhabdomyolysis with myoglobinuric renal failure may result from extreme vasoconstriction with muscle ischemia.

Pheochromocytoma of the Urinary Bladder Pheochromocytoma in the wall of the urinary bladder may result in typical paroxysms in relation to micturition. The location in the bladder wall is responsible for the occurrence of symptoms while the tumors are quite small, and, consequently, catecholamine excretion may be normal or minimally elevated. Hematuria is present in over half of patients, and the tumor can often be visualized at cystoscopy.



DIAGNOSIS

The diagnosis is established by the demonstration of increased excretion of catecholamines or catecholamine metabolites. The diagnosis can usually be made by the analysis of a single 24-h urine sample, provided the patient is hypertensive or symptomatic at the time of collection.


Biochemical Tests

The assays employed include those for vanillylmandelic acid (VMA), the metanephrines, and unconjugated or "free" catecholamines. The VMA assay is both less sensitive and less specific than assays of metanephrines or catecholamines. Accuracy of diagnosis is improved when two of three determinations are employed.

The following considerations apply to all the urinary tests:

(1) Despite claims for the adequacy of determinations made on random urine samples, analysis of a full 24-h urine sample is preferable. Creatinine should also be determined to assess the adequacy of collection.

(2) Where possible, the collection should be made when the patient is at rest, on no medication, and without recent exposure to radiographic contrast media. When it is not practical to discontinue all medications, drugs known specifically to interfere with these assays should be avoided.

(3) The urine should be acidified and refrigerated during and after collection.

(4) With high-quality assays, dietary restrictions are minimal and should be specified by the laboratory performing the analyses.

(5) Although most patients with pheochromocytoma excrete increased amounts of catecholamines and catecholamine metabolites at all times, the yield is increased in patients with paroxysmal hypertension if a 24-h urine collection is initiated during a crisis.


Free Catecholamines

The upper limit of normal for total urinary catecholamines is between 590 and 885 nmol (100 and 150 ug) per 24 h. In most patients with pheochromocytoma, values in excess of 1480 nmol (250 ug) per day are obtained. Measurement of epinephrine is often of value, since increased epinephrine excretion [over 275 nmol (50 ug) per 24 h] is usually due to an adrenal lesion and may be the only abnormality in cases associated with MEN. False-positive increases in catecholamine excretion result from exogenous catecholamines and related drugs such as methyldopa, levodopa, labetalol, and sympathomimetic amines, which may elevate catecholamine excretion for up to 2 weeks. Endogenous catecholamines from stimulation of the sympathoadrenal system also may increase urinary catecholamine excretion. Relevant clinical situations that cause such increases include hypoglycemia, strenuous exertion, central nervous system disease with increased intracranial pressure, severe hypoxia, and clonidine withdrawal.

Metanephrines and VMA

In most laboratories, the upper limit of normal is 7 umol (1.3 mg) of total metanephrines and 35 umol (7.0 mg) of VMA excretion per 24 h. In most patients with pheochromocytoma, the increase in these urinary metabolites is considerable, often to more than three times the normal range. Metanephrine excretion is increased by exogenous and endogenous catecholamines and by treatment with monoamine oxidase inhibitors; propranolol may cause a spurious increase in metanephrine excretion, since a propranolol metabolite interferes in the commonly used spectrophotometric assay. VMA is less affected by endogenous and exogenous catecholamines but is spuriously increased by a variety of drugs, including carbidopa. VMA excretion is decreased by monoamine oxidase inhibitors.

Plasma Catecholamines

Measurement of plasma catecholamines has a limited application. The care required in obtaining basal levels and the satisfactory results with urinary determinations make measurement of plasma catecholamines unnecessary in most cases. Plasma catecholamine levels are affected by the same drugs and physiologic perturbations that increase urinary catecholamine excretion.

When the clinical features suggest pheochromocytoma and the urinary assay results are borderline, measurement of plasma catecholamines may be worthwhile. Markedly elevated basal levels of total catecholamines support the diagnosis, although approximately one-third of patients with pheochromocytoma have normal or slightly elevated basal values. The usefulness of plasma catecholamine determinations may be increased by agents that suppress sympathetic nervous system activity. Clonidine and ganglionic blocking agents reduce plasma catecholamine levels in normal subjects and in patients with essential hypertension. These drugs have little effect on catecholamine levels in patients with pheochromocytoma. In patients with elevated or borderline basal catecholamine values, failure to suppress plasma or urinary levels with clonidine supports the diagnosis of pheochromocytoma.



DIFFERENTIAL DIAGNOSIS

Since the manifestations of pheochromocytoma can be protean; the diagnosis must be considered and excluded in many patients with suggestive clinical features.

• In patients with essential hypertension and "hyperadrenergic" features such as tachycardia, sweating, and increased cardiac output, and in patients with anxiety attacks associated with blood pressure elevations, analysis of a 24-h urine collection is usually decisive in excluding the diagnosis. Repeated determinations on urine collected during attacks may be necessary, however, before the diagnosis can be excluded with certainty. The clonidine suppression and glucagon stimulation tests may be helpful in excluding the diagnosis in difficult cases.
• Pressor crises associated with clonidine withdrawal and the use of cocaine or monoamine oxidase inhibitors may mimic the paroxysms of pheochromocytoma.
• Factitious crises may be produced by self-administration of sympathomimetic amines in psychiatrically disturbed patients.
• Intracranial lesions, particularly posterior fossa tumors or subarachnoid hemorrhage, may cause hypertension and increased excretion of catecholamines or catecholamine metabolites. While this is most common in patients with an obvious neurologic catastrophe, the possibility of subarachnoid or intracranial hemorrhage secondary to pheochromocytoma should be considered.
• Diencephalic or autonomic epilepsy may be associated with paroxysmal spells, hypertension, and increased plasma catecholamine levels. This rare entity may be difficult to distinguish from pheochromocytoma, but an aura, an abnormal electroencephalogram, and a beneficial response to anticonvulsant medications will often suggest the proper diagnosis.
• Related tumors that secrete catecholamines and produce similar clinical syndromes should be carefully ruled out. These include chemodectomas derived from the carotid body and ganglioneuromas derived from the postganglionic sympathetic neurons.
  
  
  
  
  TREATMENT
  
  
  Preoperative Management
  
  The induction of stable alpha-adrenergic blockade is the basis of preoperative management and provides the foundation for successful surgical treatment. Once the diagnosis is established, the patient should be placed on phenoxybenzamine to induce a long-lived, noncompetitive alpha-receptor blockade. The usual initial dose is 10 mg every 12 h with increments of 10 to 20 mg added every few days until the blood pressure is controlled and the paroxysms disappear. Because of the long duration of action, the therapeutic effects are cumulative, and the optimal dose must be achieved gradually with careful monitoring of supine and upright blood pressures. Most patients require between 40 and 80 mg phenoxybenzamine per day, although 200 mg or more may be necessary. Phenoxybenzamine should be administered for at least 10 to 14 days prior to surgery. Over this time, the combination of alpha-receptor blockade and a liberal salt intake will restore the contracted plasma volume to normal. Before adequate alpha-adrenergic blockade with phenoxybenzamine is achieved, paroxysms may be treated with oral prazosin or noncompetitive intravenous phentolamine. Selective alpha1 antagonists have been employed for preoperative preparation, but their role in preparative management should be limited to the treatment of individual paroxysms. They may be useful as antihypertensive agents in patients with suspected pheochromocytoma while workup is in progress, since they are usually better tolerated than phenoxybenzamine and will prevent serious pressor crises if pheochromocytoma is present.
  
  Nitroprusside, calcium channel blocking agents, and possibly angiotensin-converting enzyme inhibitors also reduce blood pressure in patients with pheochromocytoma. Nitroprusside may also be useful in the treatment of pressor crises.
  
  Beta-Adrenergic receptor blocking agents should be given only after alpha blockade has been induced, since administration of such agents by themselves may cause a paradoxic increase in blood pressure by antagonizing beta 2-mediated vasodilation in skeletal muscle. Beta blockade is usually initiated when tachycardia develops during the induction of alpha-adrenergic blockade. Low doses often suffice, and a reasonable starting dose is 10 mg propranolol three to four times per day, increased as needed to control the pulse rate. Beta blockade is effective for catecholamine-induced arrhythmias, particularly those potentiated by anesthetic agents.
  
  Preoperative Localization of the Tumor
  
  Surgical removal of pheochromocytoma is facilitated if the location of the tumor or tumors can be established preoperatively. Once pheochromocytoma is diagnosed, localization should be undertaken while the patient is being prepared for surgery.
  
  CT or magnetic resonance imaging (MRI) of the adrenals is usually successful in identifying intraadrenal lesions. Extraadrenal tumors within the chest can frequently be identified by conventional chest films or CT. MRI is useful in identifying extraadrenal tumors in the abdomen. If these studies are negative, abdominal aortography (once alpha-adrenergic blockade is complete) may identify extraadrenal pheochromocytomas in the abdomen, since these lesions are often supplied by a large aberrant artery.
  
  If aortography, CT, and MRI fail to localize the lesion, venous sampling at different levels of the inferior and superior vena cava may reveal catecholamine gradients in the region drained by the tumor; this area may then be restudied by selective angiography or scanning by CT or MRI.
  
  An additional localization technique involves a radionuclide scintiscan after administration of the radiopharmaceutical [I131] meta-iodo-benzyl-guanidine (MIBG). This agent is concentrated by the amine uptake process and produces an external scintigraphic image at the site of the tumor. This type of scanning may be useful in characterizing lesions discovered by CT when biochemical confirmation is indeterminate, as well as in localizing extraadrenal pheochromocytomas.
  
  Percutaneous fine-needle aspiration of chromaffin tumors is contraindicated; indeed, pheochromocytoma should be considered before any adrenal lesions are aspirated.
  
  Surgery
  
  Surgical treatment of pheochromocytoma is best performed in centers with experience in the preoperative, anesthetic, and intraoperative management of pheochromocytoma.
  
  
• Monitoring during the surgical procedure should include continuous recording of arterial pressure and central venous pressure as well as electrocardiography.
• Adequate fluid replacement is crucial.
• Intraoperative hypotension responds better to volume replacement than to vasoconstrictors.
• Hypertension and cardiac arrhythmias are most likely during induction of anesthesia, intubation, and manipulation of the tumor.
• Intravenous phentolamine is usually sufficient to control the blood pressure, but nitroprusside may be required.
• Propranolol may be given in the treatment of tachycardia or ventricular ectopy.
  
  
  
  Special situations
  
  Pheochromocytoma in Pregnancy Spontaneous labor and vaginal delivery in unprepared patients are usually disastrous for mother and fetus. In early pregnancy, the patient should be prepared with phenoxybenzamine, and the tumor should be removed as soon as the diagnosis is confirmed. The pregnancy need not be terminated, but the operative procedure itself may result in spontaneous abortion. In the third trimester, treatment with adrenergic blocking agents should be undertaken; when the fetus is of sufficient size, cesarean section may be followed by extirpation of the tumor. Although the safety of adrenergic blocking drugs in pregnancy is not established, these agents have been administered in several cases without obvious adverse effect. Antepartum diagnosis and treatment lowers the maternal death rate to that approaching nonpregnant pheochromocytoma patients; fetal death rate, however, remains elevated.
  
  Unresectable and Malignant Tumors In cases of metastatic or locally invasive tumor in patients with intercurrent illness that precludes surgery, long-term medical management is required. When the manifestations cannot be adequately controlled by adrenergic blocking agents, the concomitant administration of metyrosine may be required. This agent inhibits tyrosine hydroxylase, diminishes catecholamine production by the tumor, and often simplifies chronic management. Malignant pheochromocytoma frequently recurs in the retroperitoneum, and it metastasizes most commonly to bone and lung. Although these malignant tumors are resistant to radiotherapy, combination chemotherapy has had limited success in controlling them. Use of 131I-MIBG has had limited success in the treatment of malignant pheochromocytoma, due to poor uptake of the radioligand.
  
  
  
  PROGNOSIS AND FOLLOW-UP
  
  The 5-year survival rate after surgery is usually over 95%, the recurrence rate is 10%. After successful surgery, catecholamine excretion returns to normal in about 2 weeks and should be measured to ensure complete tumor removal. Catecholamine excretion should be assessed at the reappearance of suggestive symptoms or yearly if the patient remains asymptomatic. For malignant pheochromocytoma, the 5-year survival rate is 50%.
  
  Complete removal cures the hypertension in approximately three-fourths of patients. In the remainder, hypertension recurs but is usually well controlled by standard antihypertensive agents. In this group, either underlying essential hypertension or irreversible vascular damage induced by catecholamines may cause the persistence of the hypertension.
  
  
  
  REFERENCES
  
  Harrison’s Principles of Internal medicine – 15th and 16th ed
  http://www.harrisonsonline.com/

Microbiology: An overview of Bacterial Toxins

This chapter will help you to

• Define exotoxins and Endotoxins


• Differentiate between exotoxins and endotoxins


• Classify exotoxins according to mechanism of action


• Write short notes on: Pore forming toxins, Enzymatic toxins, Superantigens, Proteolytic toxins, Toxins acting on second messenger systems


• Write short notes on: Lipid A, Lipopolysaccharide (LPS), Lipooligosaccharide (LOS), O-antigen


• Discuss the pathogenesis of endotoxic shock
  

Ťoxigenesis, or the ability to produce toxins, is a mechanism by which many bacteria cause disease. There are two main types of bacterial toxins, lipopolysaccharides, which are associated with the outer membrane of the cell wall of Gram-negative bacteria, and proteins, which are released from bacterial cells. The cell-associated toxins are referred to as endotoxins and the extracellular diffusible toxins are referred to as exotoxins.
Endotoxins are cell-associated substances that are structural components of bacteria. Chemically, endotoxin refers to the lipopolysaccharide (LPS) or lipooligosaccharide (LOS) located in the outer membrane of Gram-negative bacteria.
Exotoxins are usually secreted by bacteria. However, in some cases, exotoxins are released by lysis of the bacterial cell. Exotoxins are usually proteins or polypeptides, that act enzymatically or through direct action on host cells and stimulate a variety of host responses.

Endotoxins and Exotoxins vary greatly in structure and function

Endotoxin is a molecular complex of lipid and polysaccharide; hence, the alternate name lipopolysaccharide. The complex is secured to the outer membrane by ionic and hydrophobic forces, and its strong negative charge is neutralized by Ca2+ and Mg2+ ions.

Members of the family Enterobacteriaceae exhibit O-specific chains of various lengths, whereas N gonorrhoeae, N meningitidis, and B pertussis contain only core polysaccharide and lipid A. Some investigators working on the latter forms of endotoxin prefer to call them lipooligosaccharides to emphasize the chemical difference from the endotoxin of the enteric bacilli. Nevertheless, the biologic activities of all endotoxin preparations are essentially the same, with some being more potent than others.

Exotoxins, unlike the lipopolysaccharide endotoxin, are protein toxins released from viable bacteria. They form a class of poisons that is among the most potent, per unit weight, of all toxic substances. Most of the higher molecular-sized exotoxin proteins are heat labile; however, numerous low molecular-sized exotoxins are heat-stable peptides. Unlike endotoxin, which is a structural component of all Gram-negative cells, exotoxins are produced by some members of both Gram-positive and Gram-negative genera. The functions of these exotoxins for the bacteria are usually unknown, and the genes for most can be deleted with no noticeable effect on bacterial growth. In contrast to the extensive systemic and immune-system effects of endotoxin on the host, the site of action of most exotoxins is more localized and is confined to particular cell types or cell receptors. Tetanus toxin, for example, affects only internuncial neurons. In general, exotoxins are excellent antigens that elicit specific antibodies called antitoxins. Not all antibodies to exotoxins are protective, but some react with important binding sites or enzymatic sites on the exotoxin, resulting in complete inhibition of the toxic activity (i.e., neutralization). Exotoxins can be made into toxoids, which can be used for active immunisation.

Bacterial Exotoxins

Bacterial exotoxins can be classified into 5 classes according to their mechanisms of action:

1. Toxins that damage membranes


2. Toxins that act as enzymes


3. Toxins that activate second messengers


4. Toxins that activate immune response


5. Toxins that act as protease

A) Toxins that damage membranes

These toxins get inserted into the membrane and form trans-membrane pores. This leads to influx of water by endosmosis, resulting in cellular swelling and lysis.

Examples:

Table 2

• Perfringiolysin O from Clostridium perfringens attach to cell membrane cholesterol and atke part in the pathogenesis of gas gangrene


• Alpha toxin from Staphylococcus aureus targets cell membrane and causes abscesses


• Pneumolysin from Streptococcus pneumoniae target cell membrane cholesterol and cause pneumonia


• Hemolysin from Escherichia coli damage renal tubular cell membrane and cause UTI


• Streptolysin O from Streptococcus pyogenes taget membrane cholesterol and is implicated in Sore throat
  
  B) Toxins that act as enzymes
  
  These toxins have an “A plus B Subunit” arrangement. Subunit A acts as the catalyst (active subunit) and subunit B helps in binding (binding subunit). Occasionally a translocation subunit (T subunit) may be present that helps “A subunit” to reach the target substrate in the cell. They ultimately catalyse some reactions in the cell, which inactivates the cellular protein synthetic apparatus.
  

Figure 1: Ribbon structure of Diphtheria toxin

Classical example of this type of toxin is the Diphtheria toxin. This toxin splits NAD into ADP-Ribose and Nicotinamide. The ADP-Ribose (ADPR) attaches to a regulatory protein called EF 2 that helps in protein synthesis, and covalently modifies it. This inactivates EF 2 and inhibits protein synthesis in the host cell, leading to cell death.

C) Toxins that activate second messengers

These toxins bind to cell surface receptors and act like hormones to activate one or more second messenger pathways in the cell. This subsequently causes changes in the cellular functions, leading to cell death.

Examples of toxins of this class are as follows:

Table 3

• E. coli secretes a heat-labile toxin that increases cellular ADP-ribosyl transferase and a heat-stable toxin that stimulates guanylate cyclase causing raised cyclic nucleotide levels in the intestinal cells. this leads to diarrhea.


• Bacillus anthracis, that causes Anthrax, secretes an edema factor which stimulates Adenylate cyclase


• Bordetella pertussis (causes whooping cough) liberates the pertussis toxin that acts via ADP-ribosyl transferase

D) Toxins that activate immune response

Several bacterial toxins can act directly on the T cells and antigen-presenting cells of the immune system, and stimulate them non-specifically. Impairment of the immunologic functions of these cells by toxin can lead to human disease. These antigens belong to a class of molecules called superantigens.


Figure 2: Schematic representation of a superantigen

Normally an antigen binds at the antigen-binding-groove (ABG) of the T cell receptor (TCR). Superantigens bind outside the ABG and causes widespread nonspecific stimulation of the T cells, leading to immunological dysfunction.

Examples of super-antigenic toxins are as follows:

Table 4

• Staph. aureus secretes enterotoxins that is implicated in Food poisoning, and exfoliative toxins that causes Staphylococcal Scalded Skin Syndrome


• toxic-shock syndrome toxin from Staphylococcus binds TCR and MHC II and causes Toxic Shock Syndrome


• Strep. pyogenes elaborates Pyrogenic exotoxins that cause Scarlet fever

E) Toxins that act as protease

These toxins cleave various regulatory proteins of the cell and impair cellular function. Examples of this group of toxins include:

Table 5

• B. anthracis secretes a Lethal factor which is a Metalloprotease


• C. botulinum secretes the Neurotoxins, A-G which are Zinc-metalloprotease in nature


• Clostridium tetani releases the tetanus toxin which act as Zinc-metalloprotease and cleaves neuronal regulatory proteins like Synaptobrevin.
  
  
  Summary of Exotoxins:
  


• Exotoxins are protein in nature


• They are actively secreted by the bacteria


• They can act by damaging membranes, Inhibiting protein synthesis, Activating second messengers, Activating immune response & as Proteases


• They can be made into toxoids
  
  
  
  Bacterial Endotoxins
  
  Bacterial endotoxins are characterized by the following features:


• Part of the outer membrane of the cell wall of Gram-negative bacteria


• Invariably associated with Gram-negative bacteria whether the organisms are pathogenic or not


• Although the term "endotoxin" is occasionally used to refer to any cell-associated bacterial toxin, in bacteriology it is reserved to refer to the LPS complex
  
  
  Chemical nature of Endotoxin
  
  
  Figure 1: Structure of endotoxin

LPS consists of three components or regions: Lipid A, an R polysaccharide and an O polysaccharide.

• Region I. Lipid A is the lipid component of LPS. It contains the hydrophobic, membrane-anchoring region of LPS. Lipid A consists of a phosphorylated N-acetylglucosamine (NAG) dimer with 6 or 7 fatty acids (FA) attached. Usually 6 FA are found. All FA in Lipid A are saturated. Some FA are attached directly to the NAG dimer and others are esterified to the 3-hydroxy fatty acids that are characteristically present. The structure of Lipid A is highly conserved among Gram-negative bacteria. Among Enterobacteriaceae Lipid A is virtually constant.

Figure 2: Structure of Lipid A

The primary structure of Lipid A has been elucidated and Lipid A has been chemically synthesized. Its biological activity appears to depend on a peculiar conformation that is determined by the glucosamine disaccharide, the PO4 groups, the acyl chains, and also the KDO-containing inner core.

Region II. Core (R) antigen or R polysaccharide is attached to the 6 position of one NAG. The R antigen consists of a short chain of sugars. For example: KDO - Hep - Hep - Glu - Gal - Glu - GluNAc -
Two unusual sugars, heptose and 2-keto-3-deoxyoctonoic acid (KDO), are usually present, in the core polysaccharide. KDO is unique and invariably present in LPS and so it has been used as an indicator in assays for LPS (endotoxin).
With minor variations, the core polysaccharide is common to all members of a bacterial genus (e.g. Salmonella), but it is structurally distinct in other genera of Gram-negative bacteria. Salmonella, Shigella and Escherichia have similar but not identical cores.

Region III. Somatic (O) antigen or O polysaccharide is attached to the core polysaccharide. It consists of repeating oligosaccharide subunits made up of 3 - 5 sugars. The individual chains vary in length ranging up to 40 repeat units. The O polysaccharide is much longer than the core polysaccharide, and it maintains the hydrophilic domain of the LPS molecule. A major antigenic determinant (antibody-combining site) of the Gram-negative cell wall resides in the O polysaccharide.
Great variation occurs in the composition of the sugars in the O side chain between species and even strains of Gram-negative bacteria. At least 20 different sugars are known to occur and many of these sugars are characteristically unique di-deoxy-hexoses, which occur in nature only in Gram-negative cell walls. Variations in sugar content of the O polysaccharide contribute to the wide variety of antigenic types of Salmonella and E. coli and presumably other strains of Gram-negative species. Particular sugars in the structure, especially the terminal ones, confer immunological specificity of the O antigen, in addition to "smoothness" (colony morphology) of the strain. Loss of the O specific region by mutation results in the strain becoming a "rough" (colony morphology) or R strain.

LPS and virulence of Gram-negative Bacteria

Both Lipid A (the toxic component of LPS) and the polysaccharide side chains (the nontoxic but immunogenic portion of LPS) act as determinants of virulence in Gram-negative bacteria.

The O polysaccharide and virulence
Virulence, and the property of "smoothness", is associated with an intact O polysaccharide. The involvement of the polysaccharide chain in virulence is shown by the fact that small changes in the sugar sequences in the side chains of LPS result in major changes in virulence. There are several postulated mechanisms of virulence:

1. O-specific antigens could allow organisms to adhere specifically to certain tissues, especially epithelial tissues.
2. Smooth antigens probably allow resistance to phagocytes, since rough mutants are more readily engulfed and destroyed by phagocytes.
3. The hydrophilic O polysaccharides could act as water-solubilizing carriers for toxic Lipid A. It is known that the exact structure of the polysaccharide can greatly influence water binding capacity at the cell surface.
4. The O antigens could provide protection from damaging reactions with antibody and complement. Rough strains of Gram-negative bacteria derived from virulent strains are generally non virulent. Smooth strains have polysaccharide "whiskers" which bear O antigens projecting from the cell surface. The O antigens are the key targets for the action of host antibody and complement, but when the reaction takes place at the tips of the polysaccharide chains, a significant distance external to the general bacterial cell surface, complement fails to have its normal lytic effect. Such bacteria are virulent because of this resistance to immune forces of the host. If the projecting polysaccharide chains are shortened or removed, antibody reacts with antigens on the general bacterial surface, or very close to it, and complement can lyse the bacteria. This contributes to the loss of virulence in "rough" colonial strains.
5. The O-polysaccharide or O antigen is the basis of antigenic variation among many important Gram-negative pathogens including E. coli, Salmonella and Vibrio cholerae. Antigenic variation guarantees the existence of multiple serotypes of the bacterium, so that it is afforded multiple opportunities to infect its host if it can bypass the immune response against a different serotype. Furthermore, even though the O polysaccharides are strong antigens, they seldom elicit immune responses which give full protection to the host against secondary challenge with specific endotoxin.

Lipid A and Endotoxic shock
The physiological activities of LPS are mediated mainly by the Lipid A component of LPS. Lipid A is a powerful biological response modifier that can stimulate the mammalian immune system.
Since Lipid A is embedded in the outer membrane of bacterial cells, it probably only exerts its toxic effects when released from multiplying cells in a soluble form, or when the bacteria are lysed as a result of autolysis, complement, phagocytosis, or antibiotic therapy.

The injection of living or killed Gram-negative cells or purified LPS into experimental animals causes a wide spectrum of nonspecific pathophysiological reactions, such as fever, changes in white blood cell counts, disseminated intravascular coagulation, hypotension, shock and death. The sequence of events follows a regular pattern:

(1) Latent period
(2) Physiological distress (diarrhea, prostration, shock)
(3) Death

How soon death occurs varies on the dose of the endotoxin, route of administration, and species of animal. Animals vary in their susceptibility to endotoxin.

The mechanism is complex. In humans, LPS binds to a lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane, which in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4). This triggers the signaling cascade for macrophage/endothelial cells to secrete pro-inflammatory cytokines and nitric oxide that lead to characteristic "endotoxic shock". CD14 and TLR4 are present on several cells of the immunological system cells, including macrophages and dendritic cells.

In monocytes and macrophages, three types of events are triggered during their interaction with LPS:

1. Production of cytokines, including IL-1, IL-6, IL-8, tumor necrosis factor (TNF) and platelet-activating factor. These, in turn, stimulate production of prostaglandins and leukotrienes. These are powerful mediators of inflammation and septic shock that accompanies endotoxin toxemia. LPS activates macrophages to enhanced phagocytosis and cytotoxicity. Macrophages are stimulated to produce and release lysosomal enzymes, IL-1 ("endogenous pyrogen"), and tumor necrosis factor (TNFalpha), as well as other cytokines and mediators.

2. Activation of the complement cascade. C3a and C5a cause histamine release (leading to vasodilation) and affect neutrophil chemotaxis and accumulation. The result is inflammation.

3. Activation of the coagulation cascade. Initial activation of Hageman factor (blood-clotting Factor XII) can activate several humoral systems resulting in:
a. Coagulation: a blood clotting cascade that leads to coagulation, thrombosis, acute disseminated intravascular coagulation, which depletes platelets and various clotting factors resulting in internal bleeding.
b. Activation of the complement alternative pathway (as above, which leads to inflammation)
c. Plasmin activation which leads to fibrinolysis and hemorrhaging.
d. Kinin activation releases bradykinins and other vasoactive peptides which causes hypotension.
The net effect is to induce inflammation, intravascular coagulation, hemorrhage and shock.

LPS also acts as a B cell mitogen, stimulating the polyclonal differentiation and multiplication of B-cells and the secretion of immunoglobulins, especially IgG and IgM.



Summary of Endotoxins:

• Endotoxins are LPS in nature
• They are integral part of the cell wall of gram negative bacteria
• O polysaccharide allows organisms to adhere specifically to certain tissues, especially epithelial tissues, allows resistance to phagocytes and act as water-solubilizing carriers for toxic Lipid A. They also provide protection from damaging reactions with antibody and complement and are the basis of antigenic variation among many important Gram-negative pathogens
• LPS binds to a lipid binding protein (LBP) in the serum which transfers it to CD14 on the cell membrane. This in turn transfers it to another non-anchored protein, MD2, which associates with Toll-like receptor-4 (TLR4). This triggers the signaling cascade for macrophage/endothelial cells to secrete pro-inflammatory cytokines and nitric oxide that lead to endotoxic shock
• Endotoxin causes shock by production of cytokines, activation of the complement cascade and activation of the coagulation cascade
• LPS also acts as a B cell mitogen
  
  
  
  References:


• Mechanisms of Bacterial pathogenesis – Toder’s online textbook of bacteriology: Toder K, University of Wisconsin-Madison Department of Bacteriology


• Bacterial Toxins: Friends or Foes? – Emerging Infectious Diseases: Vol 5, No 2, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA


• Pathogenesis of bacterial infection – Medical Microbiology: Jawetz, Melnick & Adelberg

For any Further Query, please email at: tirthankar82@yahoo.co.in