Innate Immunity, Gut Integrity, and Vitamin A in Gambian and Indian Infants

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1 S23 Innate Immunity, Gut Integrity, and Vitamin A in Gambian and Indian Infants D. I. Thurnham, 1 C. A. Northrop-Clewes, 1 F. S. W. McCullough, 1,a B. S. Das, 3 and P. G. Lunn 2,a 1 Northern Ireland Centre for Diet and Health, University of Ulster, Coleraine, and 2 MRC Dunn Nutrition Laboratories, Cambridge, United Kingdom; 3 Department of Biochemistry, Ispat General Hospital, Rourkela, India Gut integrity, which can be measured by the urinary lactulose:mannitol excretion test, deteriorates with the introduction of weaning foods. In The Gambia, gut integrity measured monthly over 15 months in 119 infants (aged 2 15 months) was least impaired from April to June. This coincides with the time of year of maximum vitamin A (VA) intake the mango season. Subsequently, two VA intervention studies were done in infants in India. Eighty infants attending a community health center received 16,700 IU weekly or placebo. In another study, 94 hospitalized infants were given 200,000 IU VA or placebo: 31 received VA on admission, while the rest (32 VA, 31 placebo) received treatment on discharge. All VA-treated groups had more rapid improvement in gut integrity than the placebo groups, but no group had gut integrity normalized by Western standards. The data suggest that VA status may influence gut integrity. Vitamin A (VA) has long been known for its anti-infective properties [1], but the mechanism by which it protects the body against infection is still unclear. We recently obtained evidence that one of the main targets for the immunomodulatory effects of VA may be the gut [2]. Most infective agents encountered by humans are prevented from entering the body by a variety of biochemical and physical barriers. The importance of the barrier is made abundantly clear when a person suffers serious burns and the barrier is broken. Most infective agents enter the body via the epithelial surface of the nasopharynx, gut, lungs, and genitourinary tract. These surfaces are protected by soluble factors (e.g., mucous, lysozyme, complement, acute-phase proteins) and phagocytes, and there is evidence that the integrity of the epithelial surface will influence the susceptibility of the organism to infection. In particular, where there is poor gut integrity, there is an increased risk of bacterial translocation across the gut wall with a consequent increased risk of infection within the body [3, 4]. Impaired gut integrity can be demonstrated by poorly me- The studies were approved by ethics committees of the Medical Research Council (The Gambia) and Ispat General Hospital (India). In all studies, informed consent was obtained from the parent or guardian of the child. Support: Hoffmann La Roche provided financial assistance and the vitamin A supplement used in the Indian studies. F.S.W.M. was supported by a Department of Northern Ireland distinction award while carrying out the studies in India. a Present affiliations: Manchester Metropolitan University, Hollings Faculty, Department of Food and Consumer Technology, Manchester, United Kingdom (F.S.W.M.); Department of Biological Anthropology, University of Cambridge, Cambridge, United Kingdom (P.G.L.). Reprints or correspondence: Prof. D. I. Thurnham, NICHE, School of Biomedical Sciences, University of Ulster, Coleraine BT 52 1SA, Northern Ireland (di.thurnham@ulst.ac.uk). The Journal of Infectious Diseases 2000;182(Suppl 1):S by the Infectious Diseases Society of America. All rights reserved /2000/18203S-0005$02.00 tabolized sugars that permeate the intestine by unmediated diffusion. The differential urinary excretion following ingestion of two sugars of different molecular weights can be used as an index of permeability for assessing mucosal damage. Lactulose and L-rhamnose [5] or lactulose and mannitol [6] are both useful probes for assessing small intestine integrity. Our interest in these techniques stemmed from observations that histologic data on villus atrophy obtained by endoscopy or biopsy of the small intestine of malnourished children correlated with the lactulose:mannitol (L:M) index of permeability [7]. Growth faltering is a feature of the weaning period in many Third World countries and remains a problem in Keneba, The Gambia, even after many years of medical, sanitary, and nutritional interventions by the Medical Research Council of the United Kingdom. Thus, it was of particular interest when the L:M permeability test showed that impaired gut integrity was strongly linked with growth faltering in infants [8]. The objective of our work was to show that gut integrity can be linked with factors that influence VA status. Initially, we observed that seasonal factors strongly correlated with measurements of gut permeability in Gambian infants in a reanalysis of the data originally reported by Lunn et al. [8]. We also report results from two placebo-controlled VA intervention studies in India. Methods Studies in The Gambia. The study was described in detail by Lunn et al. [8]. In brief, 119 breast-fed infants in three Gambian villages in the West Kiang region were recruited at ages of 2 15 months. Thereafter, other infants entered the study at age 2 months and left when 15 months old. Weight and length were recorded at regular monthly clinics when a finger-tip blood sample was taken to measure indices of immune function. Weaning foods were introduced at ages 3 4 months. Gut integrity was also measured

2 S24 Thurnham et al. JID 2000;182 (Suppl 1) Figure 1. Growth profile of Gambian infants. Data (from [13]) show that period when infants fail to gain satisfactory weight increments begins at onset of weaning and continues until about age months. monthly by the L:M dual-sugar permeability test (DSPT) [8]. Two sugars, lactulose and mannitol, were dissolved in a little water and given to the infant to drink. Urine was collected over the next 5 h in a urine bag (U-bag; Hollister Europe, Workingham, UK), and the urinary L:M ratio was measured as an index of gut permeability or damage. The two nonmetabolizable sugars cross the mucosa differentially. Lactulose crosses paracellularly and mannitol crosses transcellularly. Thus, damage from gut parasites tends to increase the passage of lactulose, but villus atrophy reduces the flow of mannitol. Both effects increase the urinary L:M ratio, which if [8] is considered abnormal. The acute-phase protein a-1- antichymotrypsin (ACT) was also measured monthly on plasma by an immunoturbidometric assay with antisera supplied by Dako (Copenhagen) and a centrifugal analyzer (Cobas-Bio; Roche Diagnostics, Welwyn, UK) [9]. The upper level of the reference range for ACT is 0.64 g/l [10]. Studies in India. Two studies were done in and around Rourkela, Orissa State. In the first, 144 hospitalized infants (mean age, 9 months) admitted for diarrheal or respiratory disease were randomly divided into 3 groups. Ninety-four infants completed the study [11]. Two groups received 200,000 IU of oral retinyl palmitate and 40 IU of a-tocopherol in 4 ml of ground nut oil either on day 1 of admission ( n p 31) or at discharge ( n p 32, up to day 5). The third group received 4 ml of oil plus vitamin E as placebo ( n p 31) at discharge. The DSPT was done on admission, at dis- charge, and 10 and 30 days after administration of VA or placebo. In a second study, 80 infants attending a community clinic for minor ailments were randomly allocated to receive 16,700 IU of VA weekly or placebo for 8 weeks [12]. The DSPT was done at baseline and at 4 and 8 weeks. foods, and the falling growth rates are closely linked to worsening gut integrity [13]. Growth faltering is a common feature in many Third World infants, beginning about the time that weaning foods are introduced, and often lasts for several months. The studies by Lunn et al. [8] suggested that 40% of growth faltering in infants aged 2 15 months was associated with abnormal L:M ratios. If lactose maldigestion (which is associated with villus atrophy) was included in the calculations, 60% of the variance in growth faltering was explained by damage to the mucosa of the small intestine [14]. Thus, it was of particular interest when a reanalysis of the permeability data showed a large seasonal influence on the permeability results [15]: The poorest values were in the rainy season and the best toward the end of the dry season (April to June; figure 2). Figure 2 shows a combined histogram in which changes in growth rate (mean monthly change in weight z score) and gut integrity (L:M ratio) are plotted in relation to month of the year. These data must be interpreted in terms of food availability and disease prevalence. The main crop harvest occurs in November, and food is generally plentiful until the end of May. The dry season occurs from December to May and is the health- Results Figure 1 illustrates how expected weight-for-age increases over the first 3 months of life but then falls over the next 9 months before stabilizing at about 75%. The precipitous fall at 3 4 months is associated with the introduction of weaning Figure 2. Seasonal changes in weight z scores (top) and gut permeability (bottom) in Gambian infants from study entry at age 2 months until study end (age 15 months). Each bar represents mean values for infants (mean age, 8 months). z score bars are means of differences between respective monthly data less values from previous month. L/M, lactulose:mannitol.

3 JID 2000;182 (Suppl 1) Vitamin A and Gut Immunity S25 example, C-reactive protein and is therefore a useful measure of chronic or subclinical infection [10]. Figures 2 and 3 illustrate that from April to June subclinical infection was at its lowest, growth was least impaired, and gut integrity was at its best. These months correspond with the mango season in The Gambia, and mangoes are the principal source of VA for the whole year [16]. Hence, it was important to determine if VA alone influenced gut integrity or whether other seasonal factors were more important. Figure 4 shows results from two VA-intervention studies in Indian infants. In the community study, the infants who were supplemented with VA (16,700 IU) once a week for 8 weeks showed a significant improvement in the permeability ratio at both 4 and 8 weeks (28 and 56 days) when compared with the baseline values. In comparison, in the placebo-treated group, there was no significant change with time during the study; furthermore, the mean L:M ratio of the VA- and placebotreated groups did not differ from one another (t test) at any time point, probably because 8 weeks of intervention was too Figure 3. Seasonal changes in mean values of a-1-antichymotrypsin (ACT) ( SE) in Gambian infants. Data are shown from study entry (age 2 months) until study end (age 15 months). Each bar shows mean values for infants (mean age, 8 months). iest time of the year, but respiratory disease and rotovirus do occur during this period. Rain falls from June to September, a period of greatest work output and agricultural effort. Food is in short supply between June and September (hungry season), and miscellaneous leaves from the surrounding fields may be used to make sauces to accompany the main staple. These sauces probably supply some VA, but by far the largest supply of VA is obtained during the mango season from April to June. During the rainy season, the prevalence of diarrheal disease is highest and malaria becomes a problem [16]. Figure 2 shows the close relationship between age-standardized weight gain and gut integrity in Gambian infants. It is apparent that most children in this age group are losing weight (see also figure 1) and that the greatest weight loss occurs between May and August when food is in shortest supply and when gut integrity of the infants is at its worst. Figure 3 shows mean values for the ACT biomarker by month. ACT responds rapidly to infection, but remains elevated for longer than, for Figure 4. Changes in lactulose:mannitol (L:M) gut permeability index ( SE) after vitamin A (VA) supplementation in 2 studies of Indian infants. In a community study, 80 infants were randomly assigned to receive either 16,700 IU of retinyl palmitate ( ) or placebo ( ) orally weekly for 8 weeks. Mean L:M ratios fell between baseline and 4 weeks and between 4 and 8 weeks ( P!.05 for both). There was no change in the placebo group. In a hospital study, children admitted with diarrhea or respiratory disease were randomly assigned to 3 groups. Groups 1 and 2 received 200,000 IU of retinyl palmitate in peanut oil orally on admission ( ) or at discharge ( ; 5 days). Placebo group ( ) received peanut oil at discharge. L:M ratios in both VA groups were significantly lower than in placebo group 10 and 30 days after treatment. At 30 days, L:M ratios in all 3 hospitalized groups were significantly lower than at baseline, but only VA-treated infants were lower at 10 days. In both studies, repeated measures analysis of variance and group tests were used to test for significance of differences ( P!.05).

4 S26 Thurnham et al. JID 2000;182 (Suppl 1) short. However, because all children had minor ailments at the outset of the study and received some medical attention, there was some evidence of a fall in the mean L:M ratio in the placebo group, but this was not significant. The significant fall in L:M ratios in the VA group demonstrates that gut morphology improved, but it was still not normal when compared with data for healthy Cambridge (UK) infants [8], whose mean L:M ratio was 0.12 ( SE 0.02). In the second study, a repeated measures analysis of variance (ANOVA) indicated that at both 10 and 30 days after treatment, L:M ratios were significantly lower in both VA-treated groups than in the control group. However, no differences were detectable in the response to VA between those treated at admission or discharge or between those with diarrheal or respiratory disease at the outset (not shown). Discussion VA and gut integrity. In The Gambia, April is associated with wide availability of mangoes, the principal dietary source of VA for the whole year [16]. Figure 1 illustrates how the growth of Gambian infants deteriorates from ages 4 12 months relative to Western standards, and the z scores in figure 2 show that there is less deterioration in growth during the dry season (November to May) than in the rainy season (June to October). November follows the annual harvest of the main dietary staples, rice and peanuts, so food is fairly plentiful during the dry season, but there is very little VA in the diet until April when mangoes are in great abundance [16]. The role of retinoic acid in cell differentiation and development links VA to growth, and a growth-promoting function of retinoic acid has been identified, where it activates the gene responsible for producing growth hormone [17, 18]. The question arises: What is responsible for what in these data? Food alone probably stimulates growth and helps to reduce the weight loss in the dry season, and the increase in dietary VA may be particularly important in reducing the level of infection as shown by the ACT concentrations. The monthly distribution of mean ACT values mirrors those of the L:M ratios. The lowest monthly mean ACT value was in April, at the height of the mango season, and the values for April, May, and June were not significantly different (figure 3). Thus, food and VA may stimulate growth hormone production and growth, but their effects are tempered by intermittent bouts of rotovirus during the dry season and high levels of infection in the rainy season (June to October). The mechanism by which dietary VA influences infection is not clear. A direct effect of the VA on immune cells is possible; but, as gut tissue is unique in being able to use nutrients both from the serosal side of the mucosa and from the lumen, it is possible that the dietary VA may act directly on the gut to restore integrity, thereby reducing bacterial translocation and the risk of further infection. VA deficiency is not considered to be a clinical problem in The Gambia, but several studies have shown large seasonal fluctuations both in plasma concentrations [19] and in dietary intake of VA [16]. In well-nourished populations, plasma retinol concentrations are closely regulated and are not influenced by fluctuations in dietary intake. Thus, the fluctuating plasma values in Gambian infants indicate that VA status may not always be adequate and that poor status may play a role in the deteriorating permeability ratios in the postweaning period. For there to be a loss of epithelial integrity, there must be a breakdown in surface structure, and this may be due to physical rupture of the tissue wall, as might be caused by parasites such as hookworm or be due to cellular changes that facilitate easier passage between or through the epithelial surface. In both populations studied, damage from gut parasites will be minimal, since gut parasites mainly affect children only when they become mobile. Giardia intestinalis, however, is transmitted by water, and most infants have been exposed by age 8 months. Nevertheless, Lunn et al. [20], who studied Gambian infants longitudinally between ages 2 and 8 months, could find no evidence that growth was related to the level of giardia-specific antibody titers during this period. In contrast, a role for VA in maintaining gut integrity is compatible with the known functions of the vitamin in controlling the growth and differentiation of epithelial tissues. Many years ago, Wolbach and Howe [21] demonstrated the importance of VA for the maintenance of epithelial structure, and others have shown the necessity of VA to maintain tissue differentiation and mucous production in epithelial tissues [22, 23]. A lack of VA causes changes in the differentiation pathway: The production of mucous-secreting ciliated epithelium is reduced, while keratinized squamous cells increase in a process termed squamous metaplasia [24]. Ross [25] suggested that the critical role that VA plays in regulating cellular differentiation provides a common mechanism that would at least partly explain its influence on epithelial barriers, immune competence, healing resistance, and recovery. Alexander [4] also pointed out that other substances that increase mucous production (e.g., prostaglandins E 1 or E 2 analogues and sucralfate) also reduce bacterial translocation. In both studies in India, mean L:M ratios in the different groups were abnormal at the outset. It is clear, however, that the infants in the community study had lower L:M ratios, that is, less mucosal damage than those of infants who were hospitalized. Infants in the community study received 16,700 IU of retinyl palmitate weekly for 8 weeks (in total, 134,000 IU). However, at 8 weeks, mean L:M ratios were still 2 3 times higher than those of UK infants, and the rate at which mucosal integrity improved was slow even in the VA-treated group. In the hospital study, many L:M ratios of children treated with VA were still higher than in the VA-treated children in the community 30 days after discharge, but the rate of improvement was much higher in the previously hospitalized children than that in the community study. Furthermore, at 30 days (25 days after discharge), the mean L:M ratios in the hospitalized group

5 JID 2000;182 (Suppl 1) Vitamin A and Gut Immunity S27 who received the placebo treatment was still higher than that of children in the community group at the start of treatment, suggesting that the physical damage in the small intestine, which produces the impaired permeability index, is still very abnormal in the convalescing children. The rate of change in permeability index in the VA-treated hospitalized children appeared greater than in children in the community study. However, it is not known whether the rate of change in L:M ratios is related to the dose of VA or to the initial L:M ratio. Factors responsible for the continuation of impaired gut integrity. In the Indian studies, gut integrity was more impaired in the hospitalized infants than in children in the community. After medical treatment alone in the hospital study, there was improvement in gut integrity. Likewise, in the Gambian study, the monthly change in gut integrity showed great similarity to the monthly changes in ACT concentrations. Therefore, infection clearly contributes to the loss of gut integrity. In addition, in the Indian studies, treatment with small or large amounts of VA increased the rate of repair of the gut damage during convalescence. In the initial Gambian study, the mean values for the acute-phase ACT protein were distributed in the same pattern as those of the L:M ratios, indicating a close relationship between gut integrity and infection. In the absence of additional VA, the rate of repair was reduced. The study was of insufficient length to learn whether L:M ratios similar to those found in Western infants would eventually be achieved. It is likely that a new bout of infection would interrupt the process and precipitate further mucosal damage and its consequent effects on growth. The Indian studies were too short to detect any effects on growth. Such effects are difficult to measure over a short period and in any case may remain suboptimal until normal gut integrity is attained. If impaired gut integrity facilitates bacterial translocation, then a high L:M ratio indicates increased susceptibility to infection. Thus, the slow recovery of mucosal integrity in the absence of VA may also may facilitate reinfection. Furthermore, the presence of infection will impair release of VA into the circulation, and low plasma retinol concentrations coupled with low dietary VA may further delay the repair of gut integrity. The Indian data suggest that dietary VA can disrupt or halt the vicious cycle but that the VA supply needs to be regular. Factors responsible for the initial impairment in gut integrity. It is more difficult to know what causes the initial loss of gut integrity in infants in The Gambia. Before the introduction of the first weaning foods, Gambian infants have normal gut integrity, and growth rates are also nearly normal, but both rapidly deteriorate thereafter [8]. The deterioration may be due to the onset of infection introduced by contaminated foods. In the first 18 months of life before the acquired immune system is fully established, the infant is more vulnerable to infection and more dependent on the cells of the immune system (e.g., macrophages, neutrophils, and natural killer [NK] cells) to rapidly clear large numbers of pathogenic organisms [26, 27]. Likewise, the mechanical integrity of the gut is strongly influenced by the number of bacteria in the gut [28], since a high concentration of bacteria in the gut can increase the risk of bacteria binding to the enterocyte, which promotes a breakdown in gut integrity. VA status may influence these mechanisms. Ross [29] reported that severe VA deficiency in rats impairs antibody responses to bacterial antigens and the maintenance of NK cells. Severe VA deficiency also has a markedly adverse effect on the mucosal IgA antibody responses in rats [30]. However, even marginal VA status may influence IgA antibody responses, since changes in serum IgA were inversely related with changes in plasma retinol values in a 3-month iron supplementation study in Pakistani infants [31]. Impaired IgA antibody responses could lead to bacterial overgrowth with its consequent detrimental effects on gut integrity. Alternatively, food antigens, possibly dietary lectins, can have potent antinutritional properties, influencing the structure and function of both enterocytes and lymphocytes [32]. Lectins are glycoproteins that occur in common dietary staples such as cereal grains and legumes. Lectins in general can bind to the surface glycans on gut brush border epithelial cells, causing damage to the base of the villi, disarrangement of the cytoskeleton, increasing endocytosis, and shortening of microvilli [32]. The structural changes induced by lectins upon epithelial cells elicit functional changes, including increased permeability [33], which may then facilitate the passage of undergraded antigens and pathogenic bacteria into the system circulation [34]. Under normal circumstances, luminal concentrations of intact dietary proteins is low, and absorbed protein will generally elicit minimal allergic response because of the limiting influence of T suppressor cells. However, the introduction of locally prepared weaning foods to an immature infant may initiate tissue damage due the presence of poorly cooked and digested lectins or, alternatively, in combination with increased bacterial contamination, inflammation and increased intestinal permeability may result, facilitating the translocation of bacteria and an increased risk of systemic infection. Because few cases of abnormal gut integrity are found in Western infants and bacterial contamination of weaning food is much lower than in the developing world, contamination of foods may be the more important factor, but this needs to be established. In conclusion, gut integrity in infants in developing areas is often abnormal, and the abnormality is closely linked to growth impairment. We found that biochemical measurements of gut integrity were best at that time of year when dietary VA was most abundant. In addition, intervention with VA supplements accelerated the improvement in gut integrity in sick infants both in the community and in those admitted to hospital. The mechanism by which VA improves gut integrity is not known. There may be a direct effect of VA on the gut cells, or the increased intake may increase circulating concentrations of retinol and have systemic effects on many systems.

6 S28 Thurnham et al. JID 2000;182 (Suppl 1) Acknowledgments We are grateful to Hoffmann La Roche for financial assistance and for the VA supplement used in the Indian studies. References 1. McCollum EV. The supplementary dietary relationships among our natural foodstuffs. JAMA 1917;68: McCullough FSW, Northrop-Clewes CA, Thurnham DI. The effect of vitamin A on epithelial integrity. Proc Nutr Soc 1999;58: Berg RD. Translocation of indigenous bacteria from the intestinal tract. In: Eentges DJ, ed. Human intestinal microflora in health and disease. New York: Academic Press, 1983: Alexander JW. Bacterial translocation during enteral and parenteralnutrition. Proc Nutr Soc 1998;57: Noone C, Menzies IS, Banatvala JE, et al. Intestinal permeability and lactose hydrolysis in human rotoviral gastroenteritis assess simultaneouslybynoninvasive differential sugar permeation. Eur J Clin Invest 1986;16: Elia M, Behrens R, Northrop CA, et al. 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