Surfactant and Alveolar Proteinosis

                                        Surfactant and Alveolar Proteinosis

                                             PKGhatak, MD

A Phopholipoprotein compound present in the lung alveoli (air sacs) acts as a Surface Tension Lowering Agent. Alveoli are tiny air sacs, lined by just one layer of pneumocyte type1 cell - inside the sac outside air enters and leaves and outside the alveoli the blood circulates and the exchange of gases takes place. If the surfactant is removed, the wall of the alveoli will come in apposition and will not be separated by the force generated during breathing in the air (inspiration).

If you take two glass slides and put a drop of water on one slide, then place the other slide over it; then try to separate those glass slides by pulling them apart, no matter how strong your hands are, you will not be able to separate the slides. Now repeat the same experiment with one drop of soap water. The slides will come apart easily. Soap is a surface tension lowering agent. That is the reason the doctors are asking you to wash your hands with soap water as soon as possible to minimize the COVID infection if you were out.

A surfactant molecule has two ends, one end is a Hydrophobic end the other is a Hydrophilic end. The hydrophobic end is turned toward the air, and the other end towards the blood. Thereby keeping air and blood separate.

The Alveolar type 2 cells secrete surfactant. The surfactant molecules inside the cells are called lamellar bodies and the extracellular surfactant is chemically the same. Chemically the surfactant is Dipalmitoylphosphatidylcholine. Surfactant is also present in the intestine and inner ear.

There is a balance between surfactant production and removal from the alveoli. Alveolar type 2 cells play a major role in both the formation and removal of the surfactant. In addition, several other factors are also involved in the removal of the surfactant from the lung. The alveolar macrophages, airway epithelial cells and ciliary escalator effect and reabsorption in blood in airways, breakdown of surfactant to its constituents by enzymes, lipid and protein components are all thought to be involved in this process. Granulocyte-Macrophage stimulating factor plays a crucial role by increasing the production of Macrophages, which engulf surfactants.

Group B streptococcus can degrade surfactants, pulmonary infections, pulmonary edema fluid and serum protein, which also destroy surfactants.

Chromosome 10 carries two genes that code the four proteins that are parts of the surfactant molecule. The deficiency of these genes is inherited as autosomal dominant and also as recessive modes. Congenital absence of surfactant is not compatible with life. In the fetal lung, surfactants are present in small amounts. Maturation of Alveolar type 2 cells correlated with the capacity to generate an adequate amount of surfactant. This becomes a crucial point in the survival of preterm infants with Acute Respiratory Syndrome.

Alveolar Proteinosis:

 

 Alveolar proteinosis:

When the balance between production and removal of surfactant is disrupted and removal grossly lags behind the accumulated surfactant in the alveoli completely fills up the alveolar space; the exchange of gases is markedly impaired and hypoxemia develops. The patients become short of breath, complain of tightness of the chest, and develop fever and marked hypoxemia.

Pulmonary alveolar proteinosis is a rare disease. The incidence is 1:000,000 population, higher in males and high in the age group 30 to 60 years.

The causes of alveolar proteinosis are three. Congenital, Autoimmune and Secondary.

Congenital alveolar proteinosis.

The granulocyte-macrophage colony stimulating factor (GM-CSF) deficiency impairs the clearing of surfactant from alveoli. The disease is inherited as an Autosomal recessive mode. The GM-MSF gene is located in the 5q 3.1 location on chromosome 5.

Autoimmune alveolar proteinosis.

Autoimmune proteinosis accounts for 90 % of all cases. Antibodies to GM-CSF are demonstrated in some cases but not all instances. The response to the removal of antibodies by plasmapheresis and IV therapy, and also Rituximab, a monoclonal antibody to phospholipids and also decreases the lymphocyte B 20 activities, results in the improved outcome, supporting this theory.

Secondary alveolar proteinosis.

The clinical picture develops the following conditions - cancers like Chronic Myeloid Leukemia, post exposure to inhaled toxic gases, exposure to a toxic level of Nickel.

Laboratory findings and diagnosis.

Hypoxemia is generally severe - often below 87%, with a low PaO2 when on oxygen at 100 %. Clubbing of fingers is present; evidence of weight loss is common. Chest X-ray shows lower and middle lobe dense consolidation, more centrally located giving an appearance of "Bat Wings". Bronchoscopy shows frothy, profuse secretion filling the airways and lung parenchyma. Fluid obtained from bronchoalveolar lavage fluid shows Periodic Acid Schiff (PAS) positive proteinous materials is diagnostic.

Treatment. Maintaining a near normal PaO2 by any means is essential, including intubation and mechanical ventilation.

GM -CSF delivered by inhalation is most commonly employed, it may also be given IV. Plasmapheresis can also be undertaken. Bronchoalveolar lavage was a standard mode of treatment until GM-CSF therapy became available.

In resistant cases, Lung Transplantation is the only option that remains.

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IgA and IgA associated diseases

 

                               Immunoglobulin A (IgA) and disease associated with IgA

                                               PKGhatak, MD

Immunoglobulin A is the dominant Immunoglobulin secreted by the submucosal lymphoid tissues of the gastrointestinal tract, salivary glands, ducts of secretory glands, respiratory and genitourinary tracts; in addition, IgA is also present in tear and breast milk. The combined mucosal lymphoid tissue is the largest lymphatic system and much more than the combined lymphoid tissues of the spleen, lymph nodes and liver.

IgA plays a prominent role in keeping infectious agents from entering or invading the body through these tracts.

IgA is present in two subtypes. IgA1 or serum IgA and IgA2 or secretory IgA. The secretory IgA is 2/3 of the total IgA.

The amount of IgA secreted in a day is much greater than IgM and IgG combined. It is estimated that 5 Gm of IgA is produced daily.

Regulation of IgA production.

Polysaccharide moiety of a compound antigenic for IgA, instead of a protein that acts as the antigen for other immunoglobulin synthesis. The antigen is recognized by the dendritic cells and T-cells are located in the lymphoid tissue of the submucous layer of different tracts, and subsequent communication with B cells, memory cells, and plasma cells work in the same way as the antibody production of other sites.

During fetal development T cells migrate from the thymus to GI, Respiratory, GU tracts, breast tissue and other sites.

The initial production of secretory IgA by the mucosal B-cells is a polymer IgA (pIgA). A specific pIgA receptor (sIgA R) binds with pIgA and carries pIgA molecules across the epithelial cells into the lumen. In the lumen, pIgA is broken down as a secretory IgA (sIgA) monomer. sIgA binds with the mucous layer of epithelium and prevents the attachment of invading pathogens to the surface epithelium. The secretary IgA acts also as an anti-inflammatory agent. But the secretory IgA does not bind with complement, so the pathogenic organisms are not phagocytized by the macrophages but eliminated by peristalsis. Epithelial membrane continuity is essential for mucosal defense. If the mucous layer is disrupted, as happens in Ulcerative colitis and Crohn's disease, the bacterial invasion of tissue must be met with other standard methods of immunity and inflammation.

The differentiation of T cells to secretory immunocytes and memory cells is due to the action of cytokines; of the cytokines, the T-cells derived growth factor (TDGF) and IL5 are important.  The serum IgA is produced in the bone marrow, spleen and lymph nodes.  The liver is the main source of the breakdown of IgA.

Pathogens that evade secretory IgA:

Neisseria gonorrhoeae, Streptococcus pneumoniae, Hemophilus influenza type B, and certain spores of the fungus are capable of destroying secretory IgA. The drug Vancomycin can also degrade secretory IgA.

Congenital deficiency of IgA:

Some patients may have no or very low IgA from birth, in others, a low IgA is due to the presence of autoantibodies. If such an individual is transfused with blood or plasma products, severe allergic and anaphylactic reactions may occur due to the presence of IgA in the transfused products. There is a high association between autoimmune diseases with IgA deficiency.

IgA associated diseases:

IgA Nephropathy (IgA N).  It used to be known as Berger's disease.

IgA Nephropathy is the most common type of Nephropathy in Asia and also in the world.

It is postulated that the respiratory tract bacteria produce Degalactosylation of IgA molecule. This degalactosyzed IgA (dIgA) becomes an antigen and antibodies are produced. Then antibodies and dIgA combine and this Antigen + Antibody complex is deposited in the extracellular matrix of the Glomeruli of the kidney leading to IgA Nephropathy. The previously held theory was that aberrant glycosylation of IgA leads to polymerization of IgA and because of the complex's large size, these complexes are deposited in the mesangium of glomeruli. Inflammation starts as a result, and glomerulonephritis progresses to membranous glomerulonephritis with crescent formation.

IgA N may run in families, but the abnormal gene/genes are not identified, and the mode of inheritance is unknown.

Clinically two groups are identified.

Benign and Aggressive types.

Benign IgA N:

Gross hematuria develops in a day or two following a URI (upper respiratory infection). Hematuria stops after a few days but may persist for a period as microscopic hematuria. Some of these patients go on to develop proteinuria. Over a period of 10 to 20 years, the protein loss in the urine becomes very significant, and they develop edema of the hands, feet and face. Renal functions gradually deteriorate and eventually end in chronic renal failure.

Aggressive IgA N.

At the onset, the symptom is gross hematuria following a URI but soon or simultaneously developing Henoch Schönlein purpura, Dermatitis herpetiformis, Bulbous epidermolysis bullosa, vasculitis of skin, liver and heart.

The deterioration of renal function is rapid and if treatment is delayed, then renal failure develops.

Diagnosis:

IgA levels are higher than normal, but the disease activities do not correlate with blood levels of IgA. A kidney biopsy is required for diagnosis. Immune-florescence staining shows deposition of IgA and complement C in the glomerular mesangium.

Henoch Schönlein purpura.

Purpuric eruptions develop following a URI. The purpura is mostly seen in the legs, buttocks and sometimes in the hands, face and trunk. The purpura is more abundant along with the pressure points like the sock line and the waistband. Purpura is palpable and does not fade away upon applying pressure. Children between 2 and 6 are mostly affected. Male children and white and Asian children are more vulnerable to HS purpura. The underlying pathology is a vasculitis of small vessels of the skin, mucous membrane and joints. Lesions in the mouth and arthritis may develop. GI symptoms are common and some also develop IgA nephropathy.

Skin lesions.

Dermatitis herpetiformis.

Clusters of raised red, intensely itchy lesions develop on elbows, knees, buttocks,  occasionally on arms and on the scalp. The lesions are intensely itchy. Often blisters develop and scars form when opened up from scratches.

People of the age group 30 to 40 of European heritage develop recurrent lesions. Oral and GI lesions also occur. Aphthous ulcers in the mouth, inner side of cheeks, gum, tongue and plate. Pits and fissures on the enamel of teeth may appear. The GI manifestations are bloating, cramps and diarrhea.

Cause.

IgA antibodies develop against the skin epidermal transglutaminase. It is the skin manifestation of Gluten enteropathy from gluten sensitivity.

Diagnosis.

Demonstration of IgA deposition in the upper layers of the dermis by immunofluorescence.

Dysmorphic Epidermolysis Bullosa.

IgA deposition at the junction of the basal layer of the epidermis and papillary cells of dermal held by collagen fibrils are damaged by the process. The epidermis is easily lifted off from the dermis with minor trauma. Blister formation and secondary infections are common. Epidermolysis Bullosa is an inherited disease. It is inherited by autosomal recessive and autosomal dominant modes and multiple gene mutations are present.

There are several clinical types based on clinical presentation and the associated involvement of other systems. GI, GU, Respiratory, and ENT may variously be involved.  It may be present at birth or at anytime later in life. It is a serious disease. There are serious complications if patients initially respond to treatment. Esophageal stricture and cancer are usual.  Diagnosis is made by immunofluorescence staining of the biopsy tissues. The prognosis is variable but not good in general.

IgA is the principal immunoglobulin in keeping the GI tract and other mucous membranes of hollow organs free of pathogens. Because of constant contact with bacteria, some of the bacterial degradation products/secretory enzymes become antigenic and antibodies are produced. Antibodies can react with normal tissues like nerve cells, GI cells, etc. This hypothesis has generated a new look into the pathogenesis of Multiple sclerosis, Parkinson's disease, etc. In the future, more and more laboratory confirmations will be available

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Hemoglobin and Beta Thalassemia Trait

 

                                        Hemoglobin and Beta Thalassemia Trait

                                                PKGhatak, MD

Hemoglobin is a metalloprotein. Four molecules of Ferrous iron are attached to four individual porphyrin rings known as protoporphyrin in a tetrahedron configuration. This compound is called Heme. The porphyrin rings are interlinked and attached to the Polypeptide chains (protein) called Globins. Of the total four globins,  two identical globins are Alpha globins and the other two are Beta globins. The alpha and beta globins differ from each other by the numbers and sequence of amino acids composition.

In the developing human fetus, globin chains are different. Two alpha globins are the same but the beta globins are replaced by Gamma globulins and the hemoglobin is called fetal hemoglobin or hemoglobin F. In a newborn, the fetal hemoglobin is rapidly replaced by adult hemoglobin (A).

The variations in globin amino acids sequence, substitution or deletion produce many abnormal hemoglobins, most of them are not important for humans but a significant one is hemoglobin S of Sickle cell disease and the other is the hemoglobin of Thalassemia.

The genes responsible for coding goblin chains are inherited by the recessive mode. When both parents are carriers of abnormal globin genes, the progeny will be homozygous and the resultant disease is called Thalassemia. If one parent is the carrier then out of four pregnancies, two children will be carriers and the condition is known as the Thalassemia trait.

The defective coding gene of the Beta globin results in deficiency of the beta chain synthesis, it is located on chromosome #11p15.5 location. Due to the deficiency of the beta chain, the metalloporphyrin joins with two globin chains called the Delta chain. In normal circumstances, the delta chain is present in a minuscule amount. But in Beta Thalassemia trait the delta chain becomes significant. The resultant hemoglobin (designated alfa2 delta2) or A2. The A2 hemoglobin does not function like normal hemoglobin. The red cells (RBC) containing A2 hemoglobin have a shorter life span and are broken down by the spleen. The blood count of Beta Thalassemia trait patients shows a picture almost like iron deficiency anemia. The red cells are smaller, a bit paler in color and some of the RBC show bluish spots called basophilic stippling and the presence of target cells and occasional nucleated RBC. The total number of red cells is either normal or higher, the MCV is high normal or just above normal, the reticulocyte count is high. The serum iron, ferritin and ferritin saturation are high normal, or just above normal levels. The rest of the hemogram appears like iron deficiency anemia. Another major difference in beta thalassemia is the red cell mean width distribution called RWD. The RWD remains normal in beta thalassemia trait, whereas, in iron deficiency, the RWD is over 15 %.  The confirmation of the Beta Thalassemia Trait requires Hemoglobin electrophoresis.

Beta Thalassemia Trait:

Beta Thalassemia trait is prevalent in India, Mediterranean counties and the North African Arab population. Most of the patients remain symptoms free. Beta thalassemia trait patients are often mistakenly diagnosed with Iron deficiency anemia and are prescribed iron therapy. Iron is not only useless in beta thalassemia but iron overload in men and post-menopausal women may produce liver disease, heart disease and gastrointestinal diseases.

Blood picture of minor and moderate beta thalassemia.

Blood test	minor	moderate
Hb	Low	Lower
Hct	28 -40 %	15 -10 %
MCV	50 -75 fL	40 -60 %
MCH	Low	Lower
Reticulocytes +Target cells + poikilocytes	+	++
Basophil stippling	+	++
Nucleated RBC	0	+
Hb Electrophoresis. Hb A.-alfa2beta2 HbA2.-alfa2 delta2 Hb F.-alfa2gamma2	85 % + 4%+ 2%+	Less than 80% More than 5 % More than 5 %

Beta thalassemia clinically presents as mild or moderate. The degree of deficiency of Beta globin determines the clinical groups.

In mild disease, the patients are asymptomatic almost their entire lives. The hemoglobin level is slightly low. On electrophoresis, the pattern is listed above. These patients need only genetic counseling.

In the moderate beta Thalassemia trait, the anemia is moderate and patients in their adult life show signs of easy fatigue and low capacity of strenuous exercise. The spleen may be enlarged and bone marrow may be hypercellular. There is a much higher rate of RBC turnover, as a result, the bilirubin in serum may be above normal levels. In many cases, periodic blood transfusion is required for anemia. Some patients may require splenectomy.

Association of beta thalassemia and autoimmune disease:

Patients with beta Thalassemia have shown an increased incidence of autoimmune diseases like Diabetes mellitus, fibromyalgia, asthma and arthritis. It is postulated that the location of an abnormal gene in beta thalassemia (11p15.5) is in proximity to immune-regulating eight genes. This association may be a spillover effect.

Another alternative explanation is that when hemoglobin is broken down to hemoporphyrins and hematoporphyrin bind with opioid receptors and act as anti-inflammatory agents. In beta thalassemia, the concentration of hemoporphyrins is low, thereby increasing the chance of autoimmune inflammation.

The increasing numbers of Vietnamese immigrant population found to have Thalassemia E disease. Thalassemia E is due to a different variation of amino acids in the beta globin. Patients are generally asymptomatic but mildly anemic. The hemogram shows a picture similar to the beta thalassemia trait. Genetic counseling is advocated to limit the thalassemia E in the population.

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Iron and Hemochromatosis

 

                                              Iron and Hemochromatosis

                                                 P.K. Ghatak, MD

Iron is an essential element for humans. Iron acts as an electron receptor and donor (redox). The iron is present in the enzyme system in which reduction and oxidation take place. Iron when reacts with hydrogen peroxide (H2O2) releases an oxygen radical that is a poison, and unless H2O2 is neutralized the cells will die. During iron transport and storage, the iron molecule is combined with proteins which makes iron less reactive to cells.

 Most of the iron is present in the hemoglobin. Hemoglobin transports oxygen(O2) from the lungs to every cell of the body.  The mitochondria of the cell utilize O2 for oxidative phosphorylation and the released energy is utilized for cell functions.

Knowledge in Iron absorption, transport, utilization, and storage mechanisms has greatly evolved in the last 10 years, a brief summary will be presented here.

Iron absorption is tightly regulated by a hormone Hepcidin produced primarily by Hepatocytes of the liver, and also by small amounts in the brain. muscles .and other tissues and use locally. 

The amount of iron absorbed varies according to the iron storage status. When the store is full, the absorption may be only 5 %, whereas, in iron deficiency, 35 % of iron is absorbed. Hepcidin is released in response to increased iron of the body. Hepcidin prevents further absorption of iron in the small intestine and in the distal renal tubules.

Iron in the body.

The adult male has 4.5 gm of iron, and an adult female has 3.5 gm of iron in the body. Iron is lost on a regular basis in the female on account of menstrual blood loss and transfer of iron to the developing child during pregnancy.

In premenopausal women, iron storage may be as low as 500 mg. Iron is stored in bone marrow, liver, spleen and in muscles as myoglobin, in tissues in the megakaryocytes. Organic iron ferritin in mitochondria and cytochrome enzyme system acts as a coenzyme. In the blood, in addition to hemoglobin, a small amount of about 4 mg is present as transferrin. Muscles retain iron as myoglobin.

The daily requirement of iron.

Adult female of reproductive age requires 18 mg of iron daily. Whereas, adult males need only 8 mg and children need 15 mg daily.

An individual with normal dietary habits gets about 15 mg of iron in the food. Plant based food containing a good amount of iron is mulberries, fruit, lentils, tofu, potatoes, and spinach. Of the animal source, a high amount of iron is present in red meat, liver, giblets, and egg yolk.

Gastric acid is needed for releasing iron from food. Vitamin C helps iron absorption. Alcohol increases iron abortion. High calcium in the food, zinc, manganese, and excessive amount of tea and coffee decrease iron absorption.

Daily loss of iron from the body is about 1 mg /day in adult males, and about 2 mg a day in females in the reproductive age. Loss of iron is due to the shedding of cells in the GI tract and from the skin.

Hemochromatosis.

Hemochromatosis is due to the overload of the body with iron. The liver is commonly affected. Other organs damaged are the pancreas, heart, skin, gonads, adrenals, pituitary and joints.

There are two distinct groups of hemochromatosis.  One is hereditary and the other is acquired causes.

Hereditary Hemochromatosis.

The gene encodes Hepcidin, the HFE gene. It is inherited by an Autosomal recessive pattern. Two important HFE gene mutations are C282Y (C stands for amino acid cysteine 282 is band location is replaced by amino acid tyrosine Y), and H63D (histidine is replaced by amino acid aspartate)

Hereditary hemochromatosis is clinically classified as type I, type II, type III and type IV. Types I, II, III are inherited as autosomal recessive mode, and type IV is the autosomal dominant mode.

Type Ia Hereditary hemochromatosis.

The mutations are on chromosome 6 and in the C282Y gene known as the HFE gene. Iron accumulation begins in the 20s in male and become symptomatic when reaching 40 -50 yrs. of age. In females, the onset of symptoms is after menopause. The mode of inheritance is autosomal recessive.

Type Ib. The patients have one copy of a chromosome containing the mutated gene C282Y from one parent and another copy of the gene H63D from the other parent. This group constitutes about 2 % of all hemochromatosis patients. Diabetes and fatty liver are common presentations.

Type II. This disorder results in the early development of cardiac symptoms due to iron deposit results in cardiac fibrosis and heart failure.

Type III is a disorder of the Transferrin receptor protein due to a mutation of the TFR2 gene. The severity of symptoms is between type II and type I.

Type IV. In this type, one copy of the mutated SLC40A1 gene that encodes Ferroprotein produces hemochromatosis.

The clinical picture is common in all types of hemochromatosis.

Initial symptoms are nonspecific like fatigue and pain in joints. Later, dark discoloration of the skin, abdominal discomfort, hepatomegaly, and the onset of diabetes mellitus develop. If left untreated, congestive heart failure, cardiac arrhythmias, cardiomegaly and various symptoms due to hypogonadism manifest in young men and in women after menopause. Patients are susceptible to infection by iron-loving bacteria like Vibrio, Listeria and Yersinia.

Diagnosis of hemochromatosis.

Serum ferritin levels over the normal limit of 200 nanogram/ml in adult males and over 300 nanogram/ml in postmenopausal women and ferritin saturation over   45 % in patients with clinical suspicion of hemochromatosis should be confirmed by finding gene mutation by chromosome study. MRI of the liver is not essential for diagnosis but MRI R2/T2 images show the degree of the iron store. The liver biopsy tissue, when stained with Prussian-blue, iron loaded hepatocytes and bile ducts become evident.

 

 Secondary Hemochromatosis.

Secondary hemochromatosis develops when repeated blood transfusions are required for a prolonged period of time. Each unit of blood has 200 to 250 mg of iron. In children, 10 units of blood transfusion can overload the body with iron and in adults, 20 units will do the same. In iron overload conditions, the blood levels of ferritin reach over 1000 micrograms/ L.

Type of anemia where repeated transfusion is necessary.

Thalassemia, Myelodysplasia, Chronic hemolytic anemia, Acquired bone marrow aplasia, Multiple myeloma, Acute leukemia, Lymphoma, and Chemotherapy induced bone marrow depression.

To prevent iron overload, a Chelator Agents is used. At present three agents are approved for use in secondary hemochromatosis.

Deferoxamine. Deferoxamine is given IV, and the dose is 25 -50 mg/ Kg body weight. It used to be commonly used but now it is not. IV infusion site pain and infection are the main reason the compliance is poor.

Deferiprone. Deferiprone is given by mouth, the dose is 75 mg/ Kg body weight, and the drug must be given in 3 divided doses. The main adverse effect is agranulocytosis. The patient's compliance is good with deferiprone.

Deferasirox. Deferasirox is also an oral drug, given only once a day, the dose is 10-30 mg /Kg body weight. GI symptoms are a major side effect, and compliance is also good. At present, it is frequently prescribed chelating drug.

The goal of Chelating agent therapy is to bring down Ferritin levels to the normal range.

Complications:

In hereditary hemochromatosis: enlarged liver, enlarged spleen, cirrhosis of the liver. hepatocellular carcinoma, arthritis, impotency in males, early menopause in females, enlarged heart, cardiac arrhythmias, congestive heart failure, hypothyroidism and dark skin.

Prevention of Hereditary Hemochromatosis.

Hereditary hemochromatosis: Genetic counseling.

Diet.

 The patients should avoid oysters and clams in order to avoid infection by iron loving bacteria.

Treatment of Hereditary Hemochromatosis.

Phlebotomy. Patients are required to undergo phlebotomy once a week until the blood ferritin reaches near normal levels. Maintenance phlebotomy is generally required, on average, 2 to 3 times a year to keep the ferritin levels in the normal range.

In hereditary hemochromatosis, chelating therapy usually is not required.

Prognosis.

In early detected cases the life expectancy should be normal. In late detected cases the prognosis varies according to the degree of damage to organs and the organs involved.

written in memoriam of classmate Rama Mukherjee. 

edited: August 2025.

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