Nancy Uythoven, MD
Stephan Kohlhoff, MD
Robert Karp, MD
SUNY-Downstate Medical Center
Pre Test
Q1. The provitamin of vitamin A is
A. retinol
B. beta-carotene
C. retinoic acid
D. riboflavin
Q2. Humoral Immunity involves
A. Antibody production by B-cells
B. Killing of tumor cells by NK cells
C. Granuloma formation by macrophages
D. All of the above
Q3. True or False: Protein-energy Malnutrition (Pe-M) describes both kwashiorkor and marasmus.
Q4. The human body stores excess iron in association with what protein?
A. Transferrin
B. Albumin
C. Ferritin
D. None of the above
Objectives:
On completion of this module, residents and physicians will be able to:
1. Appreciate the importance of protein-energy malnutrition (Pe-M) in the development of the immune system and describe:
o Differing effects on cell-mediated and humoral immunity of P-e M
o Changes in thymic and other lymphoid pathology and histology
2. Recognize the role of vitamin A in measles/respiratory disease
o Effects on mortality
o Effects on incidence
o Use of supplementation
3. Recognize the potential protective effect of malnutrition for infectious illness
Facilitator's Preparation:
Butler JC, Havens PL, Sowell AL,, et al. Measles severity and serum retinol (vitamin A) concentration among children in the United States. Pediatrics. 1993;91(6):1176-81.
Glasziou, PP and Mackerras, DEM. Vitamin A Supplementation in Infectious Diseases: A Meta-analysis. BMJ 1993; 306: 366-70
Murray MJ, Murray NJ, Murray AB, Murray MB. Refeeding-malaria and Hyperferraemia. Lancet 1975; pp 653-654
Smythe PM, Brereton-Stiles GG, Grace HJ et. al. Thymolymphatic Deficiency and Depression of Cell-mediated Immunity in Protein-calorie Malnutrition. Lancet 1971; pp 939-43
Sommer A, Djunaedi E, Loeden AA, et. al. Impact of Vitamin A Supplementation on Childhood Mortality. Lancet 1986; pp 1169-1173
Woodward BD, Woods JW, Crouch DA. Direct Evidence that Primary Acquired Cell-Mediated Immunity is Less Resistant than is Primary Thymus-dependent Humoral lmmunity to Depressive Influence of Wasting Protein-energy Malnutrition in Weanling Mice. American Journal of Clinical Nutrition 1992; 555: 1180-5
Additional references are provided below
Introduction:
A child's nutritional status influences the development of their immune system and the effectiveness of their immune response. This can leave the child open to new infection and impair their ability to clear established ones. This relationship has been described in generalized undernutrition and specific micronutrient deficiencies.
Cell-mediated immunity (CMI) uses macrophages, natural killer (NK) cells, antigen specific cytotoxic T-cells, cytokines and other cell-mediators to respond to antigens. It includes the Th-1 response: when exposed to antigen, antigen-presenting cells (APC: macrophages, dendritic cells, and B-lymphocytes) as shown below.
APCs take up the antigen ' digest it ' present the epitopes (digested protein) on their surface, and ' then present the epitopes to T-helper cells.
T-helper cells activate the APCs ' increase their efficacy 'Activated APC cells acquire the ability to kill their intracellular pathogens. (Parham 2nd ed., 2005)
The mechanisms will be described in the body of the module.
Antibodies secreted by B-lymphocytes mediate humoral immunity. By binding to surface antigen on invading microbes, antibodies mark them for destruction by the complement system or macrophages.
Naive B-cell ' phagocytizess foreign antigen ' processes it and presents it on its surface ' presents these epitopes to T-helper cells ' T-cells then activates the B-cells ' these secretes antibodies specific to that antigen. (Parham 2nd ed., 2005)
Again, the mechanisms will be described in the body of the module.
An important concern, however, is that malnutrition may at times protect from infectious illness in that micronutrient deficiencies may prevent to organism or parasites from multiplying freely. There are many example of this phenomenon. There is, for an example, a hypothesis that the common C677TT mutation in Methyl Tetrahydro Folate Reductase (MTHFR) (Sabine has reference) may have provided protection against salmonella. Lactoferrin may bind E. e-coli so that there is less gastrointestinal disease with iron deficiency (see Murray, et al), a theoretical advantage to iron deficiency that must be ignored in clinical practice. There were claims in historical fiction by Somerset Maughan that iron deficiency protected against TB. The case we discuss is recrudescence of malaria with refeeding and the importance of including anti microbial/anti parasite with refeeding. (Murray, et al)
Case 1:
You are sent to West Africa to investigate the usefulness of tuberculin-skin testing as a screening tool for infection in the area. The first patient you meet is Geneva S. She is a 3-year-old girl who appears very thin, with prominent facial bones, wasting of her extremities and a distended abdomen.
Upon talking with her mother you find that she was diagnosed with measles 3 months ago, and despite treatment never fully recovered. You're told that the mother feels Geneva has been losing weight and coughing for the past 5 months. On exam you find her to be 11.5 kgs, which is the 5%ile for her age and gender. She has cervical, axillary, epitrochlear, and inguinal lymphadenopathy. You also discover she is severely anemic and has very low albumin levels. You diagnose her with Kwashiorkor and suspect she is infected with TB. You place a PPD and collect sputum to culture for AFB. When evaluated 3 days later the PPD is negative, but the culture for AFB comes back positive.
Q1. Why doesn't Geneva respond to the PPD with a positive sputum sample?
A1. Due to her impaired cellular immunity reflected in decreased Type IV hypersensitivity reaction, Geneva could not mount a proper Th-1 response to the antigen. Thus, she is unable to activate her macrophages to respond to the antigen and does not demonstrate the typical erythema and induration seen in someone who has been exposed to TB.
Woodward et. al used protein-restricted mice as an animal model of P-e M and found that these mice had a decreased delayed (Type IV) hypersensitivity reaction to sheep RBCs compared to controls. In contrast, they found no difference in anti-sheep RBC titers between the two groups, suggesting that CMI is more susceptible to P-e M than humoral immunity. (Woodward et. al 1992). In a similar study, Smythe et al. found that children with P-e M 70% did not respond to the antigen DNCB?, were anergic, while there was no child in the control group who did not respond (Smythe et. al 1971).
Q2. What are the specific changes that occur with P-e M and Cell Mediated Immunity?
A2. In P-e M the host loses its ability to turn out functional T-cells. Activated T-helper cells normally secrete IFN-?, and IL-2. IL-2 stimulates growth of T-helper and cytotoxic T-cells. IFN? inhibits the TH-2 response, or the humoral response. Without the function of the T-helper cells they are unable to activate their effector cells in order to propagate the immune response.
Once activated, cytotoxic T-cells are normally able to induce apoptosis in somatic cells presenting foreign epitopes on MHC I: tumor cells or virally infected cells. Without T-helper activation these cytotoxic T-cells cannot induce apoptosis and leave these infected cells to actively turn out new virus particles and propagate the infection.
Activated macrophages acquire the ability to kill their intracellular pathogens from activation by helper T-cells and are then also able to secrete cytokines such as IL-1, IL-6 and TNF- . . IL-1 and IL-6 are both pyrogenic cytokines, and induce fever to fight the infection systemically. TNF-? attracts and activates neutrophils, and NK cells, and increases the synthesis of IL-2 receptors. These cytokines are pro-inflammatory and stimulate the improved efficiency of the effector cells to clear the antigen. The now activated NK cells can then release their granules and induce apoptosis in virally infected cells.
If the T-helper cells are not functioning properly, not only will the pathogens continue to live inside the macrophages, but no cytokines will be released, neutrophils and NK cells will not be properly mobilized, and the immune response will be attenuated.
In humoral immunity, helper T-cells activate the B-cell and through cytokines (IL-4, IL-5) induce differentiation of the B-cell into a plasma cell, which then secretes antibodies specific to that antigen. IL-4 enhances class switching to IgG and IgE; IL-5 class switches to IgA. These secreted antibodies are free in the serum, flag antigens for destruction (by complement or macrophages) and can be transferred with the serum to passively protect another individual, unlike CMI. In absence of T-helper activation memory B-cells are still able to respond to previously encountered antigen and the ability to mount a response is unaffected. As Woodward writes,
"The differential sensitivity of cell- and antibody-mediated immunity to weanling PEM is generally attributed to the relatively greater involution of the T-cell system than of the B-cell system in PEM" (Woodward et. al 1992)
Tuberculosis is a classic example of an infection, whose incidence and severity is increased in malnutrition with its inhibitory effects on T-cell mediated immunity.
The Case continues:
Geneva's mother brings her back to you 3 weeks later over concerns about her daughter's "sleepiness". You notice she is less responsive and alert than at her previous visit. She is very lethargic, her mother says she sleeps all the time and has been vomiting more than 3 times a day. When awoken she is very irritable and confused as to where she is and who you are. She complains of headache and sensitivity to light. You fear she is now suffering from TB meningitis and start therapy immediately. Unfortunately, the meningitis was too far advanced and Geneva dies, despite your best efforts.
Q3. What are the histological effects of P-e M on the lymphoid organs that could be seen at autopsy?
A3. Once a child becomes protein deficient few T-cells are able to mature in the thymus and trigger an immune response. Humoral immunity remains relatively intact because early on the thymus is competent to turn out mature T-cells, which can in turn activate B-cells to put out antibody. These mature B-cells no longer need T-cell stimulation, and can put out antibody to previously encountered pathogens without help. The difficulty begins when the thymus has stopped putting out mature T-cells and there are none left to activate macrophages and NK cells to respond to new infections. (Smythe et. al 1971) With insufficient CMI a child is at increased risk of spread and severity of tuberculosis.
It was found that 76% of African children with P-e M had no visible tonsils, and tonsillar area was found to be about half that of well-nourished children. At autopsy, a greater number of thymuses in children with P-e M were found to be chronically atrophied. The weight of the thymus in children with kwashiorkor was found to be 30% of that which was expected based on the child's length. The mean thickness of Peyer's patches and lymphoid tissue in the appendix was also approximately half that in the controls.
Case concluded
A historical note: Prior to the age of anti mycobacterium therapy, the only treatment was rest, rehabilitation and diet. As Holt wrote in his 6th edition of Pediatrics (1911)
"The diet [in TB] is a matter of the utmost importance. Tuberculosis patients must be fed like most other sick children, care being taken not to disturb the digestion by the unnecessary use of drugs. For a staple article of diet, milk is the best, and when this is not well borne some of its substitutes-buttermilk, kumyss, matzoon, etc.-may be tried. Cream is almost as useful as codliver oil and should be given in one form or another whenever the child's digestion can tolerate it." (p. 1051)
Further readings in the history of TB can be found in Thomas Mann's The Magic Mountain, Somerset Maughan's Sanatorium, and the work of Edward Trudeau, (see appendix)
Case 2
You're working in "Doctors Without Borders" in Bangladesh. Golam M. is a 12-month-old boy who presents with a macular rash over his face and his chest. His mother gives a history of fever, cough, eye irritation and nasal congestion 4-5 days before the onset of the rash. She says that she first noticed the rash only on his face and since then has spread to his chest. On physical exam you notice he has injected sclera, inflamed nasal turbinates, and some 1 to 3 mm grayish elevations with an erythematous base, on the buccal mucosa opposite his molar teeth, that seem to be resolving. The rash is erythematous and macular and blanches when pressed on. You notice that the rash has now spread to his abdomen.
Q3. What is Golam's diagnosis?
A3. Measles. Measles classically presents with the prodrome of fever and the 3 "C's" of cough, coryza and conjunctivitis. The rash is macular and typically begins on the face spreading down to the neck, chest, abdomen, and extremities (centrifugal spread). Also, Koplik spots are pathognomonic of measles. They can be whitish, grayish or bluish. They are raised and on an erythematous base and usually on the buccal mucosa opposite the molars but can spread to involve all of the buccal and labial mucosa.
Q4. What specific nutrient deficiency will affect the mortality of this child's illness?
A4. A meta-analysis of vitamin A supplementation has shown that vitamin A lowers "all cause mortality rates in children by around one-third". A "stronger effect was seen in children hospitalized with measles, with a reduction of 66%." This effect is only in those children who are even marginally deficient in vitamin A. (Glasziou and Mackerras, 1993) In the united States, it was found that children entering emergency care for measles who were then admitted to hospital had substantially diminished Vitamin A levels when compared to children with measles who were sent home.
Q5. What effect does vitamin A have on the incidence of respiratory disease?
A5. Bloem et. al. found a dose dependent relationship between vitamin A and the incidence of respiratory disease. Children deficient in serum retinol were found to be 3.6 times more likely to develop respiratory disease in the 3 mos. of follow-up. Those with marginal serum retinol levels were found to be 2.4 times more likely to develop respiratory to disease. (Bloem et. al 1990). Sommer et. al found that respiratory disease was 2-4 times more likely in those children who showed physical signs of severe vitamin A deficiency (xerophthalmia) than in those without. (Sommer et. al 1986)
Q6. What role does Vitamin A supplementation play in measles vaccination?
A6. Benn et. al found that infants, in West Africa, receiving only one dose of the measles vaccine at 9 months of age and vitamin A supplementation had significantly higher seroconversion rates than those not receiving supplements and the effect of increased plasma antibody concentration was greater in boys than girls. Those receiving two doses of measles vaccine (6 mos and 9 mos.) showed no difference in seroconversion rates or antibody titers between the supplemented group and the placebo. (Benn et. al, 1997)
TB and measles at the turn of the 20th century.
Holt's 1911 Pediatrics provides a clear description of the consequence of overlapping TB and measles without an ability to intervene with antibiotics or other pharmaco-therapeutic agents. The figure below is the fever curve for a child with quiescent TB who contracts measles. Holt's description follows - see Appendix 2.
Case 3
You are in Central Africa treating children for malnutrition. Tupac S. is a 16 year old boy who is severely malnourished, with a BMI of 15, but reports no illnesses in the past 6 mos. You initiate refeeding with a supplemented diet including skim milk, sorghum and vitamins without iron. Over the first couple days of refeeding he appears much improved, but on the fifth day after initiation he suddenly deteriorates. He is now suffering from fever, chills, severe headaches, nausea and vomiting. He also reports muscle aches and passing bloody stools. On physical exam you notice the patient is febrile, jaundiced and has hepato-splenomegaly. You decide to take blood smears, stain it with Giemsa and find RBCs with intracellular gametocytes.
Q7. What is the etiology of Tupac's illness?
A7. Plasmodium falciparum is one of the intracellular parasites responsible for malaria. It is transmitted by the female Anopheles mosquito and is the leading cause of death in children younger than 5 years of age in sub-saharan Africa. P. Ffalciparum stimulates production of high levels of TNF- , IFN-? and IL-1, which leads to suppression of RBC production and high fever. P. falciparum infection initially causes splenomegaly, due to congestion of parasitized RBCs to be phagocytized. As the disease progresses the liver enlarges through deposition of parasites, malarial pigment and cellular debris. If not treated it will progress to cerebral malaria, where the cerebral blood vessels become plugged with parasitzed RBCs. These lead to multiple ischemic events which cause mental status changes, coma and death. (Robbins 2005 p401-403)
Q8. What is the relationship between refeeding and Tupac's deterioration on day 5?
A8. When refeeding the malnourished population of Central Africa, Murray et. al found an increase in malarial attacks in patients and their relatives who were switched from famine conditions to a hospital diet in those with a quiescent infection. Parasitemia was found to be increasing by 48 hrs and to be at maximum by day 5. The percentage of parasitized RBCs on arrival was 2% and peaked at 15% on day 5. Frequency of attacks also peaked at day 5 after refeeding was begun. This implies that the famine conditions in which these people live is a protective factor, preventing the multiplication of their parasitic infection, and keeping them from getting flare-ups of their malaria. Once refed the patients suddenly had an excess of nutrients which could now be used by the parasites for their own propagation. (Murray et. al, 1975)
Q9. What is iron's role in the pathogenesis of Tupac's illness?
A9. Murray also observed that shortly after resumption of a normal diet there was a dramatic increase in serum iron. It is postulated that during famine conditions the body sequesters iron, keeping it from being utilized by bacteria and other pathogens. Once refeeding has begun the mobilization of iron from body stores exceeds its rate of consumption, creating a hyperferremia. This excess serum iron can then be used by plasmodiummalaria (and other pathogens) for their own multiplication and precipitate an attack. Iron is only one factor implicated in the growth of plasmodia and the general nutrition status of the patient may also affect the precipitation of an attack. (Murray et. al 1975)
Q10. After a month of therapeutic food administration and significant weight gain - is Tupac out of the woods?
A10. Not quite, it is important to recognize that while therapeutic food uptake may quickly lead to disappearance of physical signs of malnutrition full reconstitution of the immune system takes a longer time so that seemingly improved children are still at increased risk for infection. (Chevalier et al, 1996)
Summary
Have residents review learning objectives in light of the information presented in the cases.
1. Appreciate the importance of protein-energy malnutrition (Pe-M) in the development of the immune system:
Pe-M has been shown to affect the development of the lymphoid system. Children with Pe-M are found to have impaired delayed type hypersensitivity (CMI) but normal antibody titers to sheep RBCs, which points to a defect in cell-mediated immunity. Autopsy studies of these children showed diffuse lymphoid atrophy, especially the thymus. This implies a defect in T-cell maturation. The lack of mature T-cells prevents activation of the effector cells crippling the cell-mediated arm of the immune system. The humoral arm of the immune system is not as T-cell dependent as CMI and is relatively spared.
2. Recognize the role of vitamin A in measles/respiratory disease:
Studies have shown that vitamin A lowers all cause mortality in deficient populations. This effect was shown to be stronger in those with respiratory disease, such as measles. Vitamin A status has also been found to affect the incidence of measles; those deficient were found to be almost four times more likely to contract a respiratory disease than those who were vitamin A replete. Even those who were only marginally deficient were at a higher risk for respiratory disease. (Butler, et al). Supplementation of vitamin A proved useful when the child has received only one dose of the measles vaccine. When the child received 2 doses the seroconversion rates and antibody titers were comparable between vitamin A supplemented and placebo groups.
3. Recognize the potential protective effect of malnutrition for malaria.
Living under famine conditions forces the body to conserve nutrients storing excess. This leads to limited resource allocation and makes it difficult for pathogens to use those resources for their own metabolism. One example is iron, which is sequestered by the body during famine conditions, making it unavailable for use by other organisms. Once begaun on a more nutritious diet, iron was released from stores at a greater rate than could be utilized. This left enough free iron to be used by pathogens, in this case P. falciparum, for their own metabolism.
Post-Test
Q1. Delayed hypersensitivity is part of
A Humoral immunity
B. Cell-mediated immunity
C. Both humoral and cell-mediated immunity
D. Neither humoral or cell-mediated immunity
Q2. A negative PPD in an African child with kwashiorkor is most likely due to
A. Lack of exposure to TB
B. Immunity to TB
C. Anergy
D. None of the above
Q3. Without proper maturation of T-cells
A. NK cells cannot reach their maximum effectiveness
B. Macrophages will not kill their intracellular pathogens
C. Cytotoxic T-cells cannot induce apoptosis
D. All of the above
Q4. Vitamin A deficiency is shown to increase the incidence of
A. Malaria
B. Tuberculosis
C. Measles
D. Mumps
Q5. Vitamin A supplementation is beneficial in
A. A 1-dose measles vaccination schedule at 9 mos. old
B. A 2-dose measles vaccination schedule at 6 and 9 mos. old
C. A 3 dose measles vaccination schedule at 3, 6 and 9 mos old
D. None of the above
Q6. True or False: Being malnourished always exacerbates a current infection.
Q7. True or False: When treating malnourished populations it is sufficient to just provide a nutritious and accessible diet.
Post Test Answers: Q1 B, Q2 C, Q3 D, Q4 C, Q5 A, Q6 False, Q7 False
References:
Benn CS, Aaby P, Bale C, et. al. Randomized Trial of Effect of Vitamin A Supplementation on Antibody Response to Measles Vaccine in Guinea-Bissau, West Africa. Lancet 1997; 350: 101-05
Bloem MW, Wedel M, Egger R, et. al. Mild Vitamin A Deficiency and Risk of Respiratory Tract Diseases and Diarrhea in Preschool and School Children in Northeastern Thailand. American Journal of Epidemiology 1990; 131: 332-39
Butler JC, Havens PL, Sowell AL,, et al. Measles severity and serum retinol (vitamin A) concentration among children in the United States. Pediatrics. 1993;91(6):1176-81.
Chevalier P, Sevilla R, Zalles L, Sejas E, Belmonte G, Parent G, Jambon B. Immuno-nutritional recovery of children with severe malnutrition. Sante 1996; 6: 201-8.
Glasziou, PP and Mackerras, DEM. Vitamin A Supplementation in Infectious Diseases: A Meta-analysis. BMJ 1993; 306: 366-70
Holt, L. E. (1913). Pediatrics (6th ed). New York: Appleton-Century Croftsb
Kumar V, Abbas A, Fausto N. Robbins and Cotran: Pathologic Basis of Disease. 7th ed. Elsevier Inc. 2005.
Murray MJ, Murray NJ, Murray AB, Murray MB. Refeeding-malaria and Hyperferraemia. Lancet 1975; pp 653-654
Parham Peter. The Immune System. 2nd ed. Garland Science Publishing. 2005.
Smythe PM, Brereton-Stiles GG, Grace HJ et. al. Thymolymphatic Deficiency and Depression of Cell-mediated Immunity in Protein-calorie Malnutrition. Lancet 1971; pp 939-43
Sommer A, Djunaedi E, Loeden AA, et. al. Impact of Vitamin A Supplementation on Childhood Mortality. Lancet 1986; pp 1169-1173
Woodward BD, Woods JW, Crouch DA. Direct Evidence that Primary Acquired Cell-Mediated Immunity is Less Resistant than is Primary Thymus-dependent Humeral Immunity to Depressive Influence of Wasting Protein-energy Malnutrition in Weanling Mice. American Journal of Clinical Nutrition 1992; 555: 1180-5
Appendix 1.
"Faced with what he believed to be a sure and speedy death, Trudeau left his medical practice in New York City and set off for his favorite resort in the Adirondacks to die. There, instead of wasting away, he steadily regained his strength, due entirely, he believed, to healthy diet and outdoor exercise. Experiments on tubercular rabbits in his lab at the cottage seemed to verify his belief. In February of 1885, Trudeau welcomed the first group of hopeful patients to his sanatorium in the woods." [see: http://www. healthsystem.virginia.edu/internet/library/historical/medical_history/alav/tuberculosis.cf
Appendix 2.
Annotated Answers
A1. The answer is B. Vitamin A is a fat-soluble vitamin, also known as the retinoids (retinol, retinal and retinoic acid) and are of animal origin. The carotenoids, such as beta-carotene, are provitamins to vitamin A and are derived from plants. A provitamin is a substance that can be turned into a vitamin by animal tissues. Riboflavin is vitamin B2 and a central component of the cofactor FAD.
A2. The answer is A. Humoral immunity is the segment of the immune system that utilizes antibodies produced by B-lymphocytes to fight infection. Lysing tumor cells and forming granulomas are functions of NK cells and macrophages, respectively, but they are part of the cell-mediated immunity, not humoral.
A3. The answer is True. The term protein-energy malnutrition (Pe-M) describes a spectrum of disorders that include marasmus, kwashiorkor, and intermediate states of marasmus-kwashiorkor. Marasmus is a result of insufficient intake of protein and calories and results in emaciation. Tradiutionally, kwashiorkor has been seen as an insufficient intake of protein with adequate caloric intake. In fact, caloric deprivation is the underlying condition for both. The hypoalbuminemia and edema of kwashiorkor isare a result of either the rapidity of the deprivation of protein and energy or an underlying infection triggereing the edematous state. These two states are not mutually exclusive, they exist on a spectrum and a child may have elements of both.
A4. The answer is C. Excess iron is mostly stored in the liver bound to ferritin. Transferrin is a plasma protein used for iron delivery. It interacts with receptors on the surface of cells, is endocytosed. The endosome is then acidified releasing the iron from the transferrin, to be utilized by the cell. Albumin is a serum protein which maintains the osmotic pressure of the intravascular compartment. It also binds bilirubin, free fatty acids, calcium and some drugs.
