Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat

Tal Koppelmann
Yulia Pollak
Jorge Mogilner
Jacob Bejar
Arnold G Coran
Igor Sukhotnik
Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat
R E S E A R C H A R T I C L E Open Access
Background: Arginine (ARG) and nitric oxide maintain the mucosal integrity of the intestine in various intestinal disorders. In the present study, we evaluated the effects of oral ARG supplementation on intestinal structural changes, enterocyte proliferation and apoptosis following methotrexate (MTX)-induced intestinal damage in a rat.Methods: Male rats were divided into four experimental groups: Control rats, CONTR-ARG rats, were treated with oral ARG given in drinking water 72 hours before and 72 hours following vehicle injection, MTX rats were treated with a single dose of methotrexate, and MTX-ARG rats were treated with oral ARG following injection of MTX. Intestinal mucosal damage, mucosal structural changes, enterocyte proliferation and enterocyte apoptosis were determined 72 hours following MTX injection. RT-PCR was used to determine bax and bcl-2 mRNA expression.Results: MTX-ARG rats demonstrated greater jejunal and ileal bowel weight, greater ileal mucosal weight, greater ileal mucosal DNA and protein levels, greater villus height in jejunum and ileum and crypt depth in ileum, compared to MTX animals. A significant decrease in enterocyte apoptosis in the ileum of MTX-ARG rats (vs MTX) was accompanied by decreased bax mRNA and protein expression and increased bcl-2 protein levels.Conclusions: Treatment with oral ARG prevents mucosal injury and improves intestinal recovery following MTXinjury in the rat.CONTR-control, MTX-methotrexate, ARG-arginine * P < 0.05 All groups vs CONTR rats, † P < 0.05 MTX-ARG vs MTX rats. Koppelmann et al. BMC Gastroenterology 2012, 12:41  Figure 4  Effect of MTX and dietary arginine on b-catenin, p-ERK, bax and bcl-2 protein levels in ileum (Western blotting). Values are presented as a ratio to ERK protein level and are expressed as a percentage of the control group. CONTR-control, MTX-methotrexate, ARGarginine.Koppelmann et al. BMC Gastroenterology 2012, 12:41

Background

Mucositis is a debilitating, dose-limiting, and costly side effect of cancer therapy. Mucositis occurs in 40% of cancer patients after standard doses of treatment and in almost 100% of patients treated with high doses of chemotherapy [1] and can affect the entire gastrointestinal tract causing discomfort, nausea, vomiting, bloating, diarrhoea, ulceration, bleeding and in some cases result in septicaemia [2]. The mechanisms of mucositis are poorly understood. It has been suggested that proinflammatory cytokines, e.g., TNF-α and IL-1β, may be involved in the amplification phase of intestinal mucositis [3].

Arginine is a non-essential amino acid processed metabolically by the urea cycle and involved in multiple metabolic and biologic processes including release of several hormones, immune response, the regulation of inflammation, collagen synthesis during wound healing, and tumor biology [4]. L-Arginine is the precursor for the formation of nitric oxide (NO), an important signaling molecule involved in neurotransmission, vascular homeostasis, immune regulation, and host defense [5]. In addition, arginine is an essential metabolic substrate for immune cells and required for normal lymphocyte function [6]. Arginine and NO are critical to the normal physiology of the gastrointestinal tract. Several studies have suggested that endogenous formation of nitric oxide maintains the mucosal integrity of the intestine and protects the gut from injuries from blood-borne toxins and tissue destructive mediators [7,8]. In a recent study, we have shown that oral arginine decreases intestinal injury caused by lipopolysacharide endotoxemia in a rat [9]. In another study, we have demonstrated that oral arginine administration improves mucosal recovery following intestinal ischemia/reperfusion injury in the rat [10]. In a recent review, Drover et al. have described that perioperative administration of arginine supplemented diets in elective surgical patients results in a substantial reduction in infectious complications and shorter hospital length of stay, with no overall effect on mortality compared with standard care [11].

The purpose of this study was to evaluate the effectiveness of dietary L-arginine in prevention of methotrexate (MTX) induced intestinal damage in a rat model and to investigate the mechanisms involved in the stimulating effect of arginine on enterocyte turnover including its effects on cell proliferation and cell death via apoptosis.

Methods

Animals

The experimental protocol was approved by the "Guide for the Care and Use of Laboratory Animals," Rappaport Faculty of Medicine, Technion (Haifa, Israel). Male Sprague-Dawley rats weighing 250-300 g were acclimatized at 21°C on 12-h day and night cycles for a minimum of 1 week before experimentation. The rats had free access to water and were pair-fed with standard chow.

Experimental design

Animals were randomly assigned to one of four experimental groups of 8 rats each: (1) sham rats (Group A), (2) sham-ARG rats were pretreated with ARG given in drinking water (2%) for 7 days (Group B), (3) MTX rats, which underwent methotrexate injury by a single intraperitoneal injection of 20 μg/kg and were sacrificed 3 days later (Group C), and (4) MTX-ARG animals, which were pretreated with ARG given in drinking water 3 days before and 3 days after MTX injection (Group D).

Intestinal morphology analysis

The small intestine was removed and divided into two segments: proximal jejunum (10 from the Treitz ligament) and terminal ileum (10 cm from ileo-cecal junction). Each segment was weighed, mucosa was scraped off, weighed and snap frozen in liquid nitrogen. Mucosal samples (100 mg) were homogenized using Kontes Tenbroeck Tissue Grinder. DNA and protein were extracted using TRIZOL reagent as described by Chomczynski [12]. The DNA concentrations were recorded spectrophotometrically and calculated per cm of bowel length. Final protein concentration was measured spectrophotometrically using a commercially available kit (Bio-Rad, Protein Assay) and was calculated per cm of bowel length. Histological sections were prepared from the jejunal and ileal remnants. Pieces of proximal jejunum and terminal ileum near the ileo-cecal junction were placed in 5% phosphate-buffered formalin, washed with absolute alcohol, and embedded in paraffin, cut to 5 (m thickness, and stained with hematoxylin-eosin. Fifteen of longest villi and crypts were selected for the microscopic analysis, using a 10 × 4 magnifying lens.

Enterocyte proliferation and apoptosis

Crypt cell proliferation was assessed using a biotinylated monoclonal anti-BrdU antibody system provided in a kit form (Zymed Laboratories, Inc, San Francisco, CA). An index of proliferation was determined as the ratio of crypt cells staining positively for BrdU per 10 crypts. Additional 5 μm thick sections were prepared to establish the degree of enterocyte apoptosis. Immunohistochemistry for Caspase-3 (Caspase-3 cleaved concentrated polyclonal antibody; dilution 1:100; Biocare Medical, Walnut Creek, CA) was performed for identification of apoptotic cells using a combination of streptovidin-biotin-peroxidase method and microwave antigen retrieval on formalinfixed, paraffin-embedded tissues according to the manufacturers' protocols. For each group, the number of stained cells was counted in at least ten villi. The apoptotic index (AI) was defined as the number of apoptotic cells per ten villi. All measurements were performed by a qualified pathologist blinded as to the source of intestinal tissue.

Expression of bax and bcl-2 genes (real-time PCR)

Expression of Bax and Bcl-2 levels was determined by quantitative real-time PCR (7500 Real-Time PCR System, Applied Biosystems, USA) on cDNA samples using Cyber Green Master Mix (ROVALAB, Germany) with the exception of template and primers. Primers for Rattus norvegicus Bax and Bcl-2 were synthesized by Syntezza Bioscience ltd. Israel, and 18 s rRNA Control kit from Eurogentec, EGT Group.

Western blotting

Tissue from jejunum and ileum was homogenized in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 2 mM EDTA, supplemented with a cocktail of protease and phosphatase inhibitors. Protein concentrations were determined by Bradford reagent according to the manufacturer's instructions. Samples containing equal amounts of total protein (30 μg) were resolved by SDS-PAGE under reducing conditions. After electrophoresis, proteins were transferred to a PVDF membrane and probed with various primary antibodies to anti-Bcl-2 antibody (1:1000 dilution, sc-7382), anti-Bax antibody (1:200 dilution, sc-493), anti-phospho-ERK antibody (1:2500 dilution, sc-7383), anti-β-catenin antibody (1:1000 dilution, sc-7199) and anti-ERK2 antibody (1:1000 dilution, sc-56899). Horseradish peroxidase-conjugated secondary antibody was purchased from Jackson Immu-noResearch Laboratories Inc. (West Grove, PA) and an enhanced chemiluminescent substrate from Biological Industries (Kibbutz Beth HaEmek, Israel). The optical density of the specific protein bands was quantified by using a densitometer (Vilber Lourmat, Lion, France).

Statistical analysis

The data are expressed as the mean ± SEM. A one-way ANOVA for comparison, followed by Tukey's test for pair-wise comparison was used for statistical analysis. Prism software was used (GraphPad Software, Inc., San Diego, CA) and statistical significance was defined as P < 0.05.

Results

Water, arginine and energy intake

Total water intake was 15-18 ml/rat/day. There was no significant difference in fluid intake between all experimental groups (15 ml/rat/day in control animals and 18 ml/rat/day in MTX-groups). The amount of additional protein was 300 mg/rat/a day and additional calorie intake was 1.2 kcal/rat/day.

Body weight changes

MTX (Group C) rats demonstrated a significant decrease in final body weight (100 ± 2.6 vs 106 ± 0.4%initial weight, p < 0.05) compared to control animals (Group A). Although treatment with oral arginine resulted in a trend toward increase in final body weight in both sham (Group B) and MTX (Group D) animals, this trend did not achieve statistical significance.

Intestinal mucosal parameters

Oral arginine administration in control animals (Group B) resulted in a mild decrease in bowel weight and mucosal weight in jejunum and ileum, as well as mucosal DNA content (33%, p < 0.05) in jejunum compared to control animals ( Table 1). MTX-induced intestinal damage (Group C) was associated with a significant decrease in bowel weight in jejunum (26%, p < 0.05) and ileum (20%, p < 0.05), mucosal weight in jejunum (two-fold decrease, p < 0.05) and ileum (two-fold decrease 25%, p < 0.05), mucosal DNA in jejunum and ileum (two-fold decrease, p < 0.05), and mucosal protein in jejunum (38%, p < 0.05) and ileum (41%, p < 0.05) compared to control animals (Group A). Following dietary L-arginine administration (Group D), MTX-rats demonstrated a significant increase in bowel weight in jejunum (12%, p < 0.05), bowel (24%, p < 0.05) and mucosal (30%, p < 0.05) weight in ileum, ileal mucosal DNA (two-fold increase, p < 0.05) and

Microscopic bowel appearance

MTX rats showed severe villous atrophy, epithelial flattening, extensive crypt loss and signs of crypt remodeling, which was accompanied by marked cellularity and an increased number of blood vessels in the stroma ( Figure 1A). The proliferative zone in MTX-rats moved progressively upwards in the crypts toward the crypt-villus junction ( Figure 1B). At the same time, the proliferative zone of MTX-ARG rat was only mildly affected, showing a slight shift upwards within the crypts. Although the mucosa was still severely damaged, MTX-ARG rats showed the presence of newly formed crypts and regeneration. Villus height and crypt depth significantly decreased in jejunum (28% and 39%, correspondingly, p < 0.05) and ileum (42% and 37%, correspondingly, p < 0.05) in MTXrats compared to control animals (Table 1). Dietary L-arginine exerted different effects on the microscopic intestinal appearance in control and MTX animals. In control rats, oral arginine (Group B) resulted in a mild but significant decrease in villus height and crypt depth in jejunum and ileum compared to control animals (Group A). In MTX rats, dietary L-arginine (Group D) attenuated the negative effect of methotrexate on mucosal architecture. MTX-ARG rats showed greater villus height in jejunum (32%, p < 0.05) and ileum (38% increase, p < 0.05), and crypt depth in ileum (30% increase, p < 0.05) compared to MTX-animals (Group C).

Cellular proliferation and apoptosis

CONTR-ARG rats showed a trend toward a decrease in cell proliferation; however, this trend was not statistically significant (Figure 1). MTX intestinal damage (Group C) resulted in a significant decrease in enterocyte proliferation rates in jejunum (128 ± 12 vs 197 ± 14 BrdU positive cells/10crypts, p < 0.05) and ileum (133 ± 21 vs 210 ± 22 BrdU positive cells/10crypts, p < 0.05) when compared to control animals (Group A). Although treatment with dietary arginine of MTX-animals (Group D) resulted in a 10% increase in cell proliferation rates in jejunum and ileum, this change was not statistically significant.

The frequency of apoptotic cells was increased in the animals following MTX-induced damage in jejunum (1.8 ± 0.2 vs 0.8 ± 0.37 apoptotic cells/1-villi, p < 0.05) and ileum (1.5 ± 0.3 vs 0.6 ± 0.13 apoptotic cells/1-villi, p < 0.05) compared to control rats (Figure 1). Treatment with dietary arginine of MTX-animals (Group D) resulted in a significant two-fold decrease in cell apoptosis in both jejunum and ileum (p < 0.05) compared to MTX-nontreated animals (Group C).

Expression of apoptosis related genes

CONTR-ARG (Group B) rats showed a small but significant increase in bax mRNA (pro-apoptotic factor) expression in jejunum (33%) and ileum (53%) compared to control animals ( Figure 2). MTX rats (Group C) demonstrated a two-fold increase in bax mRNA expression in jejunum and ileum (p < 0.05) as well as a two-fold decrease in bcl-2 gene (anti-apoptotic protein) expression (p < 0.05) in jejunum compared to control animals (Group A). Treatment with arginine resulted in a significant decrease in bax mRNA expression in jejunum (38%, p < 0.05) and ileum (41%, p < 0.05) and a concomitant decrease in bcl-2 gene expression in ileum (44%, p < 0.05) compared to MTX animals ( Figure 3).

Western blotting

Although western blot analysis of proliferation and apoptosis markers has demonstrated a similar tendency both in jejunum and ileum, more representative changes were observed in the terminal ileum. Western blot analysis ( Figure 4) illustrated a significant decrease in bcl-2 protein levels as well as a significant increase in bax protein in MTX rats compared to control animals. These findings correlate with augmented cell apoptosis in MTX-animals compared to control rats. Decreased cell proliferation in this group coincided with decreased β-catenin levels. Treatment with arginine resulted in a significant increase in bcl-2 protein levels as well as a significant decrease in bax protein levels without changes in p-ERK and β-catenin levels compared to MTX rats. These qualitative and quantitative changes are in agreement with the mRNA expression data and with decreased apoptosis and unchanged proliferation in ileum following ARG administration.

Discussion

In the last two decades, arginine has attracted major interest since it has been identified as the natural substrate of nitric oxide, and is now recognized to play a major role in many regulation processes. Nitric oxide is a multifunctional intercellular messenger molecule that plays an important role in a variety of physiological processes. Nitric oxide is synthesized from L-arginine by the inducible nitric oxide synthase (iNOS). Arginine and nitric oxide are critical to the normal physiology of the gastrointestinal tract and maintain the mucosal integrity of the intestine in various intestinal disorders. Arginine stimulates intestinal cell migration and intestinal protein synthesis through a focal adhesion kinase dependent mechanism in vitro [13]. Arginine supplementation enhances intestinal growth and development in weanling piglets [14]. Dietary supplementation with arginine stimulates small intestinal mucosal recovery following experimental radiation enteritis [15] and accelerates ulcer healing in experimental ulcerative ileitis [16]. In a recent study, we have shown that oral arginine decreases intestinal injury caused by lipopolysacharide endotoxemia in a rat [9]. In another study, we have demonstrated that oral arginine administration improves mucosal recovery following intestinal ischemia-reperfusion injury in a rat [10]. The mechanisms of these positive effects are still unclear; however, a stimulating effect of appropriate amounts of NO on enterocyte proliferation and a suppressive effect on enterocyte death via apoptosis may be considered as one of them.

The role of arginine in prevention of chemotherapyinduced intestinal damage is unclear. Hämäläinen Met al have shown recently that chemotherapy inhibites iNOS expression, and subsequent NO production, in a dosedependent manner at therapeutically achievable drug concentrations in a human colon epithelial cell line [17]. In a recent experiment, Gulgun et al. have demonstrated that proanthocyanidin, arginine and glutamine supplementation had a positive effect in the protection of the small intestine from methotrexate-induced injury [18].  [19]. Methotrexate is the most commonly used anti-metabolite agent in clinical oncology practice. Since ARG maintains the mucosal integrity of the intestine in various intestinal disorders, we hypothesized in the present study that this amino acid could prevent intestinal mucosal injury or/and improve intestinal recovery following MTX-induced mucositis. Alterations in bowel and mucosal weights, mucosal DNA and protein content and histological appearance were measured. BrdU was used in our experiment to determine an index of crypt   * that the smaller mucosal mass of MTX animals can be attributed to cellular hypoplasia. Histologically, villus height decreased in response to MTX administration, suggesting decreased absorptive surface area. Mucosal DNA content as well as the enterocyte proliferation index decreased significantly in both jejunum and ileum following MTX administration. MTX is an analog of folic acid and works by attaching to dehydrofolate reductase to inhibit DNA production. As a result crypt proliferation in the small bowel is inhibited. In addition, the proliferative zone in MTX-rats moved progressively upwards in the crypts toward the crypt-villus junction. The mechanism responsible for this effect is poorly understood. Verburg and coworkers have shown in MTX-treated rats that BrdU-positive cells are not restricted to the crypts but are also found in up to onethird of the length of the villi due to migration of the cells [20]. Our data suggest that MTX animals had lower β-catenin levels which fits with decreased cell proliferation. Extensive experimental evidence suggests that Wnt/β-catenin signaling plays a central role in maintaining the intestinal stem-cell niche and in regulating differentiation of stem cells within the intestinal epithelium toward either enterocytes or one of three secretory cell lineages (Paneth, goblet, or enteroendocrine cells [21]. Cell loss in the small intestine MTX-induced mucositis is mainly regulated by programmed cell death. Our results show that the intrinsic pathway, with its regulation by the bcl-2 family of proteins, was altered by MTX consistent with changes in cell apoptosis: the mRNA levels of the pro-apoptotic gene bax increased, while those of the antiapoptotic bcl-2 gene decreased. With regard to the protein level, qualitative and quantitative changes were in agreement with mRNA expression. Bax protein levels were significantly up-regulated while bcl-2 protein levels were down regulated in MTXtreated rats compared to control animals. These changes correlate with the enhanced enterocyte apoptosis during MTX-induced mucositis.

Results of the present study show that dietary arginine protects the intestinal mucosa from damage caused by MTX. While MTX rats showed severe villous atrophy, epithelial flattening, extensive crypt loss and signs of crypt remodeling, marked cellularity and an increased number of blood vessels in stroma, arginine-treated rats showed more preserved architecture as well as the presence of newly formed crypts and regeneration. While the proliferative zone in MTX-rats moved progressively upwards in the crypts toward the crypt-villus junction, the proliferative zone of MTX-ARG rats was only mildly affected, showing a slight shift upwards within the crypts. In addition, exposure to oral arginine significantly enhanced intestinal recovery following MTXinduced damage. This is evident from the significant increase in bowel and mucosal, increased DNA and protein content in ileum. Increased mucosal weight without changes in mucosal surface area may suggest that mucosal hyperplasia rather than bowel enlargement or intestinal muscle hypertrophy is responsible for the increased intestinal mass. Increase in mucosal DNA and protein along with hypertrophy of the individual cells which we have demonstrated morphometrically is characteristic of tissues undergoing increased cell proliferation or repair. Histologically, marked increases in villus height in both jejunum and ileum suggest increased absorptive surface area and closely correlate with increased cell mass. The present data suggest that arginine did not change significantly mucosal proliferation in functioning intestine, but decreased significantly cell apoptosis rate, which may represent the main mechanism that maintains mucosal structure following MTX-induced damage. Our results show that the intrinsic pathway, with its regulation by the bcl-2 family of proteins, was altered by arginine in accordance with changes in cell apoptosis: the mRNA and protein levels of the pro-apoptotic bax decreased, while those of the antiapoptotic bcl-2 protein levels increased. Correspondingly, bax/bcl-2 ratio decreased in MTX-ARG rats compared to MTX animals, suggesting increased enterocyte survival. Further investigation is needed to define the regulation of this special apoptotic state with respect to the Fas/Fasl-mediated extrinsic pathway.

The mechanism of the positive effects of arginine is unclear. In non-operated, non-stressed animal, total food intake is 20-25 g/rat/day, total protein intake is about 6 g/rat/day, and total energy intake is 100 kcal/ rat/day [22]. We do not believe that an additional 300 mg/rat/day of protein (vs 6 g of total protein) as well as additional energy of 1.2 kcal/rat (vs total 100 kcal/rat/ day) may have influenced the results. The limitation of the current experiment is a lack of isonitrogenous MTX group. Therefore, this study is not able to prove that arginine supply limits intestinal damage. It was previously reported that other amino acids (like glutamine, glycine or histidine) have protective effects on intestinal mucosa during chemotherapy or inflammatory conditions. An additional MTX-isonitrogenous amino acid group will be added in future study to determine whether other amino acids have similar effects.

Conclusions

In a rat model of methotrexate-induced mucositis, administration of dietary arginine prevents intestinal damage, decreases cell death via apoptosis and accelerates intestinal recovery. These preliminary observations suggest that oral arginine may have clinical value in preventing or reducing the severity of chemotherapyinduced mucositis.

Figure 1
1The mechanisms of this effect were not investigated. In a A. Staining with H@E. Intestinal injury caused by MTX. MTX rats demonstrated a significant epithelial atrophy (thick arrows) and signs of crypt remodeling (thin arrows) which were accompanied by marked cellularity and an increased number of blood vessels (arrowheads) in the stroma. Following ARG administration rats showed less significant epithelial atrophy and crypt remodeling compared to MTX rats. B. BrdU staining (cell proliferation). Control rats show normal crypt compartment. The proliferative zone in MTX-rats moved progressively upwards in the crypts toward the crypt-villus junction. The proliferative zone of MTX-ARG rat was only mildly affected showed the presence of newly formed crypts and signs of regeneration. CONTR-control, MTX-methotrexate, ARG-arginine. trial, Izaola et al. have shown that arginine and glutamine enhanced formula decreases the rate of radiotherapy-induced oral mucositis in patients with head and neck cancer

Figure 2
2Effects of MTX and arginine on crypt cell proliferation and apoptosis. The number of labeled cells in 10 well-oriented, longitudinal crypts per section from each rat was determined using light microscopy. Identification of apoptotic cells was performed using immunohistochemistry for Caspase-3. The apoptotic index is expressed as the percentage of apoptotic cells per 10 villi. Values are mean ± SEM. CONTR-control, MTX-methotrexate, ARG-arginine. * P < 0.05 MTX and MTX-ARG vs CONTR rats, † P < 0.05 MTX-ARG vs MTX rats.

Figure 3
3Effect of arginine on bax and bcl-2 mRNA expression in gut mucosa following methotrexate induced intestinal damage. Results are expressed as the ratio of the investigated bax and bcl-2 mRNA to ERK. Values are mean ± SEM. CONTR-control, MTX-methotrexate, ARG-arginine. * P < 0.05 MTX and MTX-ARG vs CONTR rats, † P < 0.05 MTX-ARG vs MTX rats.

cell proliferation. Immunohistochemistry for caspase-3 was used to characterize enterocyte apoptosis. We have demonstrated that a single dose of MTX caused a severe mucosal injury in the small bowel, indicated by severe villous atrophy, epithelial flattening, intensive crypt loss and signs of crypt remodeling, which was accompanied by marked cellularity and an increased number of blood vessels in stroma. In addition, treatment with MTX resulted in significant mucosal hypoplasia. A decrease in bowel and mucosal weight, mucosal DNA and protein, and decrease in villus height support this conclusion. Parallel decreases in mucosal DNA and protein indicate

Table 1
1Intestinal mucosal parametersCONTR
CONTR-ARG
MTX
MTX-ARG

Bowel weight

(mg/cm/100 mg body weight)

Jejunum
23 ± 1
21 ± 1
17 ± 0.3 *
19 ± 1 * †

Ileum
21 ± 1
19 ± 1
17 ± 1 *
21 ± 1 * †

Mucosal weight

(mg/cm/100 mg body weight)

Jejunum
9.4 ± 0.5
7.6 ± 0.5 *
5.1 ± 0.6 *
6.2 ± 0.6

Ileum
9.1 ± 0.5
6.7 ± 0.5 *
4.7 ± 0.5 *
6.1 ± 0.6 * †

Mucosal DNA

(μg/cm/100 mg body weight)

Jejunum
45 ± 7
30 ± 4 *
22 ± 5 *
25 ± 3 *

Ileum
47 ± 6
52 ± 4
19 ± 4 *
38 ± 9  †

Mucosal protein

(μg/cm/100 mg body weight)

Jejunum
94 ± 21
60 ± 11
58 ± 10 *
58 ± 5 *

Ileum
54 ± 10
44 ± 8
32 ± 5 *
51 ± 8  †

Villus height (μm)

Jejunum
434 ± 29
345 ± 56
314 ± 33 *
413 ± 18  †

Ileum
371 ± 26
287 ± 19 *
217 ± 52 *
300 ± 23 * †

Crypt depth (μm)

Jejunum
183 ± 17
131 ± 8 *
112 ± 4 *
126 ± 11

Ileum
161 ± 16
120 ± 8 *
101 ± 15 *
131 ± 10 * †

Koppelmann et al. BMC Gastroenterology 2012, 12:41 http://www.biomedcentral.com/1471-230X/12/41
AcknowledgementsAuthors' contributions TK was responsible for conception and participation in design, experimental work and collection of data, analysis and interpretation of results, drifting and substantial editing the manuscript. YP was responsible for experimental work and collection of data, analysis and interpretation of results. JM was responsible conception and participation in design, analysis and interpretation of results. JB was responsible for experimental work and collection of data, analysis and interpretation of results. AGC participated actively in the conceptualization of the paper, analyses plan and provided critical reviews on the manuscripts. IS participated conceptualization, interpretation of the findings, drafting and editing of the manuscript. All authors read and approved the final manuscript.Competing interestsThe authors declare that they have no competing interests.
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Pre-publication history The pre-publication history for this paper can be accessed here. 10.1186/1471-230X-12-41Pre-publication history The pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/1471-230X/12/41/prepub doi:10.1186/1471-230X-12-41

Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution. Koppelmann, BMC Gastroenterology. 1241Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat. Submit your manuscript at www.biomedcentral.com/submitCite this article as: Koppelmann et al.: Dietary L-arginine supplementation reduces Methotrexate-induced intestinal mucosal injury in rat. BMC Gastroenterology 2012 12:41. Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color figure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit


The impact of lipid-based nutrient supplementation on anti-malarial antibodies in pregnant women in a randomized controlled trial

Upeksha P Chandrasiri
FreyaJ I Fowkes
Jack S Richards
Christine Langer
Yue-Mei Fan
Steve M Taylor
James G Beeson
Kathryn G Dewey
Kenneth Maleta
Stephen J Rogerson
The impact of lipid-based nutrient supplementation on anti-malarial antibodies in pregnant women in a randomized controlled trial
10.1186/s12936-015-0707-2R E S E A R C H
Background: Malaria and undernutrition frequently coexist, especially in pregnant women and young children. Nutrient supplementation of these vulnerable groups might reduce their susceptibility to malaria by improving immunity. Methods: Antibody immunity to antigens expressed by a placental-binding parasite isolate, a non-placental binding parasite isolate, merozoites and schizonts at enrolment (before 20 gestation weeks) and at 36 gestation weeks were measured in 1,009 Malawian pregnant women receiving a daily lipid-based nutrient supplement, multiple micronutrients or iron and folic acid, who were participants in a randomized clinical trial assessing the effects of nutrient supplementation on pregnancy outcomes and child development(registration ID: NCT01239693). Results: Antibodies to placental-binding isolates significantly increased while antibodies to most merozoite antigens declined over pregnancy. Overall, after adjustment for covariates, the type of supplementation did not influence antibody levels at 36 gestation weeks or the rate of change in antibody levels from enrolment to 36 weeks. A negative association between maternal body mass index and opsonizing antibodies to placental-binding antigens (coefficient (95% CI) -1.04 (−1.84, −0.24), was observed. Similarly, women with higher socioeconomic status had significantly lower IgG and opsonizing antibodies to placental-binding antigens. Neither of these associations was significantly influenced by the supplementation type.Conclusions: In the current cohort nutrient supplementation did not affect anti-malarial antibody responses, but poor and undernourished mothers should be a priority group in future trials.

Background

It is estimated that about 125 million pregnancies worldwide are at risk of malaria annually, significantly increasing maternal and child morbidity and mortality [1]. Undernutrition is prevalent in regions where women are at high risk of malaria in pregnancy [2]. This coexistence of undernutrition and malaria markedly increases the risk of adverse birth outcomes, such as intrauterine growth restriction, compared to malaria or undernutrition alone [3,4]. The complex relationship between malaria and nutrition has been studied [5,6], but understanding of its mechanisms remains limited. Malaria in pregnancy predisposes women to anaemia, and may result in reduced nutrient intake due to febrile illness and anorexia. Malaria also increases susceptibility to other infections through its immunosuppressive effects [7][8][9], whilst on the other hand both macronutrient and micronutrient deficiencies may increase the risk of infections including malaria [6]. This increased susceptibility is believed to be due to impairment of host immune defences [10] such as abnormalities in complement activation [11] and impaired cell mediated immunity [12] leading to reduced antibody production.

In endemic regions, pregnant women naturally acquire antibodies to malaria with repeated exposure to infectious mosquito bites prior to and during pregnancy. During first pregnancies, women frequently acquire antibodies to the pregnancy-specific antigens, primarily to VAR2CSA of Plasmodium falciparum erythrocyte membrane protein-1 family of variant surface antigens (VSA). These antibodies help protect women against adverse clinical outcomes in subsequent pregnancies [13]. However, undernourished women may have difficulty effectively maintaining or acquiring antibodies against malaria antigens.

In non-malaria-related studies both macro-and micronutrient supplementation have been shown to significantly improve pregnancy outcomes and maternal health [14,15]. Lipid-based nutrient supplements (LNS) are multiple micronutrient-fortified lipid-rich products that can be beneficial as prenatal supplements in vulnerable groups [16][17][18]. Prenatal LNS has been shown to improve birth length [16] and reduce weight loss in HIVinfected mothers [17], and in young HIV-exposed infants LNS is suitable as a breast milk replacement [19]. In addition, LNS may improve linear growth outcomes in young children [20,21].

The only study to date that has assessed the effects of maternal nutrient supplementation on malaria antibody levels reported that vitamin A prenatal supplements were associated with a reduction in antibody responses to a placental-binding isolate EJ-24, but no significant changes in antibody responses to non-pregnancy related parasite isolates were observed [22].

In areas with food insecurity and high malaria transmission, nutritional supplements could improve pregnancy outcomes and may also lead to stronger acquired immune responses to malaria. To investigate this, antibody immunity was measured to antigens expressed by placental-binding and non-placental-binding parasite isolates, merozoite antigens and schizont extract in pregnant women from Mangochi, Malawi enrolled in a randomized controlled trial receiving daily LNS, multiple micronutrients (MMN -multi vitamin and minerals combined supplement) or iron and folic acid supplements (IFA -60 mg of iron and 400 μg of folic acid). The primary aim was to determine whether LNS supplementation improved antibody responses to malaria in pregnancy compared to IFA or MMN.

Methods

Study participants

From February 2011, 1391 pregnant women attending four antenatal clinics in Mangochi District, Malawi were recruited to a single-blinded randomized controlled trial of nutrient supplementation to improve pregnancy outcomes and child development (registration ID: NCT01239693 [23]). Mangochi District experiences holoendemic malaria transmission and a high prevalence of stunting (low height-for-age Z-score) and low weight-for-age among infants. Women <20 gestation weeks (gw) pregnant by ultrasound dating, aged >15 years of age and without any chronic health conditions were enrolled in the study following informed consent. They were randomly assigned to receive one tablet of IFA, one tablet of MMN or 20 g of LNS (containing 20 mg iron and 400 μg of folic acid) daily [23]. The final maternal visit of the trial was completed in February 2013. All participants received two doses of sulphadoxine-pyrimethamine (SP) malaria intermittent preventative treatment at enrolment and at 28-34 gestation weeks [23]. The follow up of the children of the trial is currently ongoing with the last visit expected to complete by December 2015; for further details [23,24].

Ethics approval

The trial was approved by the College of Medicine Research and Ethics Committee of Malawi, and by Tampere University Hospital Ethics Committee, Finland. Laboratory studies were approved by the Melbourne Health Human Research Ethics Committee.

Plasma sample collection and malaria detection

Blood plasma samples were collected from participants at study entry and at 36 gw. Malaria parasitaemia was diagnosed by light microscopy slide positivity, PCR or rapid diagnostic test (RDT) for asexual P. falciparum and/or mixed Plasmodium infections at enrolment and at 36 gw (PCR and RDT only). Parasitized red blood cells (pRBCs) were counted microscopically against 200 leucocytes under 100 X magnification. PCR was performed as previously described [25]. Briefly, genomic DNA was extracted from dried blood spots using Chelex®100 (Bio-Rad, CA, USA) and a quantitative realtime PCR assay was performed to amplify the P. falciparum lactate dehydrogenase (pfldh) gene. The samples were assayed in duplicate using a Bio-Rad CFX384 Touch Real-Time PCR detection system. Concentration of PfLDH was estimated using standard curves computed from amplification curves of a series of 10 g DNA extracts of parasite line 3D7 that were included in each assay. RDT was performed using Clearview® Malaria Combo (British Biocell International Ltd., Dundee, UK) which detects the proteins PfLDH and histidine-rich protein 2. The prevalence of malaria in the current cohort was 23.2% by RDT at baseline [23].

Preparation of recombinant malaria antigens and schizont extract

Recombinant merozoite surface protein-1, MSP-1 19 kD (3D7 clone) and full-length MSP-2 (FC27 clone) antigens [26] were kindly provided by Paul Gilson (Burnet Institute, VIC, Australia) and Robin Anders (La Trobe University, Australia) respectively. In addition region III-V of erythrocyte binding antigen 175 (EBA 175) [27], P. falciparum reticulocyte binding homologue 2 (PfRh2) (construct PfRh2-2030) [28] and the full length MSP-3 recombinant proteins were prepared following established protocols. The schizont extract was prepared following the protocol described in [29].

Measuring IgG and IgG subclass antibodies to merozoite and schizont antigens, and quantitating total plasma IgG In brief, 0.5-2 μg/ml of each recombinant antigen was coated on to 384 well high protein binding black Optiplates (PerkinElmer Inc., Massachusetts, USA). The plates were washed with PBS/Tween 20 and non-specific binding blocked with 0.1% casein on the following day. The plates were then incubated with participant sera diluted at 1:1,000 in 0.1% casein and assayed in triplicates. Alexa Fluor 488-conjugated goat anti-human IgG (Life technologies, Australia) was used at 1:2,000 dilution to detect antigen-bound IgG. The fluorescence intensity (FI) of each well, which is proportional to the amount of IgG bound, was measured using BMG POLARstar Omega fluorimeter (BMG Labtech, Germany) in the 485-1 excitation and 520 emission spectra range.

Plasmas collected at enrolment and at 36 gw from 150 participants (50 from each supplementation group) were chosen by selecting alternate pairs of samples from 4 boxes containing equal numbers of samples from the three supplementation arms, blind to participant clinical data. These samples were used to measure IgG subclass antibodies to the above antigens. Antigens were coated at the same concentration on 384 well NUNC maxisorp plates (Thermo Fisher Scientific, VIC, Australia) and were incubated in triplicates with plasma on the following day at 1:250 dilution for IgG1and IgG3, and 1:50 for IgG2 and IgG4. Following incubation the plates were washed and incubated with respective mouse anti-human secondary antibodies (IgG1 clone HP6069, IgG2 clone HP6002, IgG3 HP6047 [Life Technologies, Australia] and IgG4 clone HP6023 (Merck Millipore, Darmstadt, Germany) at 1:1,000 dilution. Tertiary antibody horseradish peroxidase-conjugated goat anti-mouse IgG was added at 1:2,500 dilution followed by development of the assay using ABTS (2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid); SIGMA-Aldrich). The reaction was stopped using 1% sodium dodecyl sulphate, and absorbance was read on the BMG POLARstar Omega fluorimeter at 410 nm wavelength.

The total plasma IgG in the samples of the above 150 pregnant women were measured using the fluorescence assay method described above. Gamma-chain specific anti-human IgG capture antibody (SIGMA-Aldrich) was coated on to the plates at 0.5 μg/ml concentration and incubated with participants' plasma diluted at 1:50,000. The concentration of IgG in each sample was determined using a purified human IgG isotype control (Life technologies) standard curve (1 μg/ml -0.016 μg/ml).

Serial samples were run on the same assay. The mean FI (MFI) or optical density (OD) were reported as a percentage of the positive control standard curve following adjustment for inter and intra plate variability. The positive control was a pool of plasma collected from malaria-immune individuals. The positivity for IgG was determined as greater than mean plus three standard deviations of the negative control (60 plasma samples collected from Melbourne adults).

Parasites and cell culture

Placental-binding P. falciparum isolate, CS2 which binds to chondroitin sulphate A (CSA) expressed in placenta tissue with upregulated expression of var2csa [30] and non-placental binding P. falciparum isolate, E8B which binds to ICAM-1 and CD36 [31] were cultured as previously described [32]. Pro-monocytic THP-1 cells were obtained from ATCC (Manassas, VA, USA; catalogue number TIB-202™) and maintained as previously described [33].

Opsonic phagocytosis assay for measuring functional antibodies to placental-binding and non-placentalbinding malaria variant surface antigens

In order to measure antibodies that opsonize pRBCs for phagocytic clearance a high throughput assay previously established in the current laboratory was used [33], with minor modifications [34], as described [32]. The percentage phagocytosis was determined relative to the positive control (pRBCs incubated with a pool of sera from patients with high levels of IgG to VSA). A participant was considered seropositive for opsonizing antibodies if the percentage phagocytosis of the samples was greater than the sum of the average and three standard deviations of the percentage phagocytosis of the negative controls.

Measuring IgG to variant surface antigens expressed by placental-binding parasite isolate

Using published methods [32,35] IgG to VSA expressed by the placental-binding isolate was measured. Alexa Fluor® 647-conjugated donkey anti-rabbit antibody (Life Technologies) was used as the tertiary antibody at 4 μg/ml concentration in contrast to the previous methods. The geometric MFI of the samples were measured and adjusted for intra and inter-plate variability. The adjusted MFI for all samples were reported as a percentage of the positive control. Seropositivity to placental-binding VSA was calculated as geometric MFI greater than the mean and three standard deviations of the negative control.

Statistical analyses

All statistical analyses were performed in Stata version 13.0 (SataCorp LP, Texas, USA). Figures were created using GraphPad Prism 5. Statistical analyses were performed according to the pre-planned analyses for the current study approved and published in [24]. Antibody levels were compared between supplementation groups LNS versus IFA and LNS versus MMN using Mann Whitney U test. Differences between antibody levels at the two time points were determined using Wilcoxon matched-pair test. The rate of change in antibody levels was calculated as difference between antibody levels at 36 gw and antibody levels at enrolment divided by the number of weeks of the study. A rate of change of 0, suggests no change in antibody levels per week of gestation. The rate of change in antibody levels was compared between LNS and IFA, and LNS and MMN, where IFA and MMN were the respective reference groups, by performing linear regression analysis.

Levels of antibody of each IgG subclass to merozoite and schizont antigens were compared between supplementation groups LNS versus IFA and LNS versus MMN using Mann Whitney U test. Comparisons were also made between antibody levels at enrolment and at 36 weeks using Wilcoxon matched-pair test.

The covariates of the association between supplementation type and malaria antibody immunity were determined based on their biological relevance. Covariates chosen were gravidity, maternal age, HIV infection, malaria infection, residence, socioeconomic status (SES), body mass index (BMI) and antibody levels at enrolment. Gravidity, maternal age, HIV and malaria infection were previously shown to associate with malaria antibody immunity [36,37]. Participants' SES was calculated based on a scoring system for household assets (HHA) adapted from [38]. Participants whose HHA adjusted zscore was below the median were considered to have low SES. Body mass index (BMI) at enrolment was categorized into three groups as low (<18.5 kg/m 2 ); normal (18.5-25 kg/m 2 ) and increased (>25 kg/m 2 ). All the variables were treated as categorical except for antibody levels at enrolment which was treated as a continuous variable. Likelihood ratio test was used to test whether the association between supplementation type and antibody levels at 36 weeks varied with covariates chosen for the study.

Nevertheless, all of the above variables (gravidity, maternal age, HIV, malaria infection at enrolment, location of residence, SES, BMI and antibody levels at enrolment were included in the multivariate regression models. Multivariate linear regression analyses for continuous outcomes (antibody levels at 36 weeks and the rate of change in antibody levels) and multivariate logistic regression analyses for categorical outcomes (seropositivity to malaria antigens). Differences in antibody levels among women with different BMI and SES groups were determined following multivariate linear regression analysis, adjusting for covariates including supplementation group, as detailed in the table footnotes. Associations were considered statistically significant if the p value is <0.05.

Results

Participant characteristics

Overall 1,391 women were enrolled in the iLiNS-DYAD-M study. Malaria immunity was only measured in the 1,009 enrolment and 36 weeks paired plasma samples that were available. The baseline characteristics of the participants across the supplementation arms were comparable ( Table 1). The mean (SD) gestational age in weeks at enrolment was 16.5 (2.1) for IFA, 16.5 (2.2) for MMN and 16.6 (2.2) for LNS groups (Table 1). Approximately 60.2% of women were multigravidae. Proportion of women with BMI <18.5 kg/m 2 was 5.7% and MUAC < 23cm was 6.8%, who were identified as undernourished women in the current cohort. Thirteen percent of women were HIV infected. The proportion of participants lost to follow up between the supplementation groups was similar with an overall 6.04% lost to follow up [23].

Changes in antibody levels between enrolment and 36 weeks

Opsonizing antibodies that promote phagocytic clearance and IgG against the VSA expressed by placentalbinding isolate were significantly higher at 36 weeks compared to the levels at enrolment; median relative antibody levels, 49.7% compared to 32.7% at enrolment (p < 0.0001) and 42.3% compared to 15.0% at enrolment (p < 0.0001), respectively ( Figure 1A). However, opsonizing antibodies to non-placental-binding isolate significantly declined, 29.8% at 36 weeks compared to 35.4% at enrolment (p < 0.0001) ( Figure 1A). Antibody levels to MSP-1 19kD, MSP-2, MSP-3, EBA-175 and schizont extract were significantly reduced from enrolment to 36 weeks; median difference in relative antibody levels, 0.7% (p = 0.0006), 3.9% (p < 0.001), 0.2% (p = 0.019), 1.3% (p < 0.0001) and 6.5% (p < 0.0001), respectively ( Figure 1B). The plasma IgG levels also significantly declined from enrolment to 36 weeks; median relative antibody levels, 6.9% to 4.6% (p < 0.0001) (See Additional file 1), similar to the decline in antibody responses to merozoite antigens and non-placentalbinding parasite isolate VSA.

Effect of nutrient supplementation on antibody immune responses to malaria

When categorized by the supplementation groups, no significant differences in the levels of antibody to placentalbinding or non-placental-binding isolates were observed at 36 weeks (Figure 2A). There were also no significant differences in antibodies to merozoite or schizont antigens, when stratified by supplementation type ( Figure 2B). There were no differences in the prevalence of detectable antibody responses at 36 weeks by supplementation group (See Additional file 3). More than 85% of the pregnant women had antibodies to placentalbinding VSA.

Effect of supplementation on the rate of change in antibodies to malaria antigens Significant differences in the rate of change in antibodies to parasite isolate VSA were not found between supplementation groups (Table 2), except for MSP-2. The rate of change in MSP-2 antibodies was significantly negatively associated with LNS group compared to MMN −0.25 (−0.46, −0.04) suggesting a faster decline in MSP-2 antibody responses in the LNS group ( Table 2).

Modification of the association between malaria antibody levels and supplementation group by covariates

Factors that influenced the relationship between antibody levels at 36 weeks and supplementation groups were investigated. This relationship was only affected by the antibody levels at enrolment (see Additional file 4). Levels at enrolment of opsonizing antibodies to nonplacental binding and antibodies to MSP-2, MSP-3 and PfRh2 at enrolment significantly influenced the relationship between supplementation type and the levels of the respective antibodies at 36 weeks (0.001 < p < 0.006; Additional file 4). The effect modification by MSP-3 antibodies at enrolment was significant on the association between antibodies to MSP-3 at 36 weeks between LNS and IFA groups 3.44 (0.14, 6.75).

Effect of nutrient supplementation on IgG subclass responses to malaria

For all the antigens tested, the highest subclass response was for IgG3 followed by IgG1 and IgG2 with almost undetectable IgG4 responses (See Additional file 5). When categorized by the supplementation groups, subclass responses were not significantly different. For many subclass responses, antibody levels declined significantly between enrolment and 36 weeks, in keeping with the decline in IgG to each antigen observed; this was most apparent for IgG3 antibodies.

Influence of maternal nutrition status and socioeconomic status on malaria antibody responses

Overall, higher levels of malaria antibodies were observed for women with low BMI compared to the other BMI groups ( Figure 3A-C). This difference was significant for opsonic antibodies to both placental-binding; median (pvalue), 52.8% (p = 0.005) and non-placental-binding isolates 41.0% (p = 0.001) ( Figure 3B-C), and was not affected by the supplementation type. When antibody responses were stratified by supplementation groups, significant differences in antibody levels between BMI groups within the same supplementation group were observed, although these associations were not strongly consistent (Figure 3). Following adjustment for covariates, opsonizing antibodies to the placental-binding isolate, antibodies to PfRh2 and schizont extract were significantly negatively associated with BMI, a unit (1 kg/m 2 ) increase in BMI is associated with a decrease in relative antibody levels by −1.04% (95% CI: −1.84, −0.24), −0.66% (−1.32, −0.004) and −1.22% (−2.08, −0.37) respectively (Table 3).

In a univariate analysis, women with a low SES had significantly higher antibody levels to VSA compared to women with high SES, including both opsonizing antibodies and IgG to placental-binding VSA (p < 0.001),  (Table 3).

Discussion

The association between nutrient supplementation and malaria immunity was evaluated in 1,009 pregnant women enrolled in a randomized controlled trial comparing LNS, MMN and IFA. Ingestion of LNS was not associated with significant increases in malaria antibody levels, a faster rate of change of antibody levels from enrolment to 36 weeks, or likelihood of seropositivity to malaria antigens. This study is the first to investigate the relationship between nutrient supplementation and malaria immunity in detail. Antibodies were measured against VSA expressed by a placental-binding and non-placental-binding parasite isolate, merozoite antigens and schizont extract. Antibodies to placental-binding VSA reduced the risk of placental malaria by inhibiting cytoadhesion to receptors [37], and by promoting their phagocytic clearance [39]. Antibodies to merozoite antigens can prevent invasion of pRBCs [29] inhibit intra-erythrocytic development and opsonize merozoites for phagocytic clearance [40,41] contributing to the protection against clinical malaria.

A strong increase in both total and opsonizing antibodies to placental-binding VSA from study entry to 36 weeks was observed while antibodies to merozoite anti6gens and non-placental-binding isolates declined. Several explanations are possible. First, most parasite isolates infecting pregnant women express antigens that are unique and are exclusively expressed during pregnancy [42]. Antibodies induced against such antigens may have a longer half-life than those directed against merozoite antigens [43]. Second, reduction in total plasma IgG at 36 weeks suggests haemodilution may have an impact on antibody levels to non-placental-binding malaria antigens; however, antibodies to tetanus and measles show very little decline during pregnancy [43] suggesting haemodilution is not always a significant factor. Third, antibodies to antigens expressed by a placental-binding isolate appear to be maintained from previous pregnancies, and the majority of women in the cohort were multigravidae with high levels of these antibodies at study entry; exposure to new infections might rapidly boost antibody responses. By contrast, antibodies to merozoite antigens have been reported to fluctuate markedly with exposure during pregnancy [43]. Of note, all participants received repeated doses of SP; parasite resistance to this agent in Malawi is common, but not high-grade [44]. Parasites with SP resistance demonstrate mutations in the genes encoding P. falciparum dihydrofolate reductase (DHFR) and dihydropteroate (DHPS). In Malawian pregnant women quintuple mutations of DHFR and DHPS are highly prevalent, however the prevalence of more severe sextuple mutant DHPS 581 which is associated with increased placental inflammation is uncommon [45]. The type of supplementation had no strong or consistent influence on malaria antibody levels, however a regression analysis showed a decline in antibody responses to MSP-2 and PfRh2 from enrolment to 36 weeks, and an increased rate of reduction in antibody levels to MSP-2, in women who received LNS compared to MMN. Given the large number of comparisons reported, these differences in the adjusted models could be due to chance. Similar differences in other anti-merozoite antibodies were not observed, and the significance of the changes is unknown. The current study also reported the effects of nutrient supplementation on antibody responses in subsets of women at high risk of undernutrition and infection; those with poor nutritional status and low SES. A negative relationship was observed between malaria antibody levels and maternal BMI and SES. Overall, women with a low BMI at study entry had higher antibody levels to VSA than did women with normal or increased BMI. Supplementation type did not influence the relationship between antibody responses and BMI. Of all the antibody types being tested the opsonizing antibodies to VSA were the most strongly related to BMI and SES in the current cohort, suggesting that these women might be exposed to more malaria (Nkhoma M et al., unpublished work) during the current pregnancy, thereby eliciting pregnancyspecific immunity. Poorer women could have more   exposure to malaria due to poor housing that allows entry of mosquitoes, and they may be less likely to own insecticide-treated bed nets. In addition poverty could lead to poor nutrient intake due to inability to afford food especially during the hunger season. The study had several limitations. The timing of nutritional interventions may be critical; supplementation starting prior to conception and continuing through out pregnancy and lactation may offer more benefits. In the current study the intervention started on average at 16.5 gw, following malaria peak prevalence, at around 12-17gw. Therefore the maximal benefits of the supplementation on malaria antibody acquisition may have been missed. Primigravidae constituted only 20% of participants, limiting our ability to examine acquisition of pregnancy-specific immunity in this group. Samples were missing for 382 of 1,391 women enrolled. Potential causes include failure to attend the 36-week visit, or pre-term deliveries (prior to 37 weeks). In addition unavailability of participant data for some of the covariates may have reduced the power of the multiple regression analyses. For analysis of BMI and malaria immunity, stratification of participants reduced the study power: for instance only 57 pregnant women were in the low BMI group.

Conclusions

Overall the current study concluded that nutrient supplementation of pregnant women was not associated with broad changes in antibody responses to malaria. This study is the first to investigate interactions between nutrition and malaria immunity in pregnancy and further studies must be conducted to determine the role of nutrient supplementation on other components of host immune system such as cell-mediated immunity, to determine the overall influence of nutrient supplementation on malaria susceptibility and malaria immunity.   Percentage antibody levels at 36 gestation week adjusted for gravidity, maternal age, HIV, malaria infection at enrolment, bed net use, location of residence, antibody levels at enrolment, body mass index at enrolment, supplementation group. Statistical test: Multivariate linear regression analysis. Data reported as regression coefficient (95% confidence interval) and p-values. *Significant associations.

Following adjustment for covariates, the levels of antibody to MSP-2 and PfRh2 at 36 weeks were significantly lower in the LNS group compared to MMN −3.98 (−7.59, −0.37) and −4.44 (−8.85, −0.03), respectively (See Additional file 2).

Figure 1
1Antibodies to malaria antigens at enrolment and at 36 gestation weeks. (A) Antibodies to variant surface antigens expressed by placental-binding and non-placental-binding parasite isolates. (B) Antibodies to merozoite antigens and schizont extract. Data presented as box plots with the Y axis representing antibody levels as a percentage of the positive control. The whiskers denote the 10 th and the 90 th percentiles with outliers. Wilcoxon matched pairs test performed for antibody comparisons between enrolment and 36 gestation weeks. N = 1,009. and opsonizing antibodies to non-placental-binding VSA (p = 0.005) (Figure 3D-F). Both IgG and opsonizing antibodies to the placental-binding isolate (but not other responses) remained significantly associated with SES following adjustment for covariates, coefficients −9.54 (−14.74, −4.35) and −5.47 (−10.43, −0.50) respectively

Figure 2
2Antibodies to malaria antigens at 36 weeks categorized by supplementation type. (A) Antibodies to variant surface antigens expressed by placental-binding and non-placental-binding parasite isolates. (B) Antibodies to merozoite antigens and schizont extract. Data presented as box plots with the Y axis representing antibodies as a percentage of the positive control. The whiskers denote the 10 th and the 90 th percentiles with outliers. Mann Whitney U test performed for antibody comparisons between supplementation groups LNS versus IFA and LNS versus MMN. N = 1,009.

Figure 3
3Antibodies at 36 weeks stratified by maternal body mass index and socioeconomic status. (A-C) Antibodies to variant surface antigens of placental-binding and non-placental-binding parasite isolates stratified by body mass index (BMI) groups and supplementation groups, (D-F) stratified by socioeconomic status (SES) and supplementation groups. BMI groups < 18.5 kg/m 2 , n = 57; 18.5-25 kg/m 2 , n = 822; >25 kg/m 2 , n = 122. High SES = 441 and low SES n = 560.

.9 (−18.25, −9.58)* −9.54 (−14.74, −4.35)* <0.001* Opsonizing antibodies to placental-binding isolate VSA −0.86 (−1.65, −0.06)* −1.04 (−1.84, −0.24)* 0.011* −14.7 (−19.18, −10.27)* −5.47 (−10.43, −0.50)* 0.031* Opsonizing antibodies to non-placental-binding isolate VSA −0.53 (levels at 36 gestation week adjusted for gravidity, maternal age, HIV, malaria infection at enrolment, bed net use, location of residence, antibody levels at enrolment, socioeconomic status, supplementation group. b

Table 1
1Baseline characteristics of study participantsData presented as median (Interquartile range), or number (%) unless stated.Socioeconomic status calculated based on a scoring system for house hold assets. Low socioeconomic status is defined as house hold Z-score < median −0.387. Abbreviations: SD: standard deviation; RDT: rapid diagnostic test; BMI: body mass index; MUAC: mid-upper arm circumference, PCR: Polymerase chain reaction.Characteristic
IFA (n = 325)
MMN (n = 347)
LNS (n = 337)
All

Gestation weeks at enrolment, mean (SD)
16.5 (2.12)
16.5 (2.23)
16.6 (2.24)
16.5 (2.20)

Age, years
23 (20-28)
24 (20-29)
24 (20-28)
24 (20-28)

Gravidity

-Primigravidae
64 (19.7%)
67 (19.2%)
68 (20.1%)
199 (19.8%)

-Secundigravidae
77 (23.7%)
64 (18.4%)
62 (18.3%)
202 (20.1%)

-Multigravidae
184 (56.6%)
214 (61.5%)
208 (61.5%)
606 (60.2%)

BMI, kg/m 2 ;
21.5 (20.4-23.3)
21.6 (20.1-23.5)
21.7 (20.3-23.7)
21.6 (20.3-23.5)

-BMI <18.5 kg/m 2 , n (%)
18 (5.6%)
18 (5.3%)
21 (6.3%)
57 (5.7%)

MUAC (cm),
26.0 (24.7-27.5)
25.7 (24.2-27.7)
26.1 (24.6-27.8)
26.0 (24.5-27.7)

-MUAC < 23cm, n (%)
23 (7.1%)
21 (6.1%)
24 (7.1%)
68 (6.8%)

Malaria parasitaemia

-Microscopy
34 (10.5%)
36 (10.4%)
38
(11.3%)

108 (10.7%)

-PCR
81 (25.7%)
79 (23.4%)
86
(25.7%)

246 (24.9%)

-RDT
71 (21.8%)
83 (24.1%)
75
(22.5%)

229 (22.8%)

HIV infection, n (%)
47 (14.5%)
37 (10.7%)
46 (13.6%)
130 (13.0%)

Low Socioeconomic status, n (%) a
184 (56.7%)
187 (53.9%)
189 (56.1%)
560 (55.9%)

a

Table 2
2Association between nutrient supplementation and the rate of change in antibody levelsRate of change in antibody levels (Percentage antibody level change per gestation week, arbitrary
units)

LNS (n = 337) compared to IFA (n = 325)
LNS (n = 337) compared to MMN (n = 347)

Unadjusted
Adjusted
Adjusted
p-value

Unadjusted
Adjusted
Adjusted
p-value

IgG to placental-binding isolate VSA −0.12 (−0.34, 0.11)
−0.08 (−0.38, 0.22)
0.599
0.13 (−0.09, 0.36)
0.26 (−0.04, 0.55)
0.089

Opsonizing antibodies to
placental-binding solate VSA

−0.02 (−0.24, 0.19)
−0.11 (−0.41, 0.19)
0.469
0.06 (−0.14, 0.27)
0.02 (−0.26, 0.30)
0.864

Opsonizing antibodies to
non-placental-binding isolate VSA

Results presented as regression coefficients (95% confidence interval) calculated by comparing the percentage rate of change in antibody levels in LNS group compared to IFA (reference group) and LNS group compared to MMN (reference group). The percentage rate of change in antibody levels from enrolment to 36 gw were adjusted for gravidity, maternal age, HIV, malaria infection at enrolment, malaria infection at 36 weeks, bed net use, body mass index at enrolment, socioeconomic status and location of residence. Statistical test: Multivariate linear regression analysis. Data reported as regression coefficient (95% confidence interval) and p-values. *Significant associations.Schizont extract
−0.03 (−0.28, 0.23)
0.13 (−0.21, 0.48)
0.450
0.003 (−0.26, 0.27)
0.10 (−0.26, 0.47)
0.583

Table 3
3Relationship of maternal nutritional status and socioeconomic status to malaria antibody immunity at 36 weeks IgG to placental-binding isolate VSABody mass index at enrolment (kg/m 2 )
Socioeconomic status (high/low)

Unadjusted
Adjusted a
Adjusted p-value a Unadjusted
Adjusted b
Adjusted p-value b

AcknowledgementsWe thank the study participants, the local communities, the health service staff and our research personnel at the study sites as well as members of the trial's data safety and monitoring board, and the International Lipid-based Nutrient Supplementation (iLiNS)-Project Steering Committee (http://www.ilins.org/) for their positive attitude, support and help in all stages of the study. In addition we would like to thank Steven Meshnick, Kyaw L Thwai, Wina Hasang and Philippe Boeuf for advice and technical support.Additional filesAdditional file 1: Plasma IgG at enrolment and at 36 gestation weeks. Data presented as box plot with the whiskers representing 10 th and the 90 th percentiles with outliers. Y axis presents plasma IgG concentration in g/L calculated from the standard curve created by purified human IgG. Wilcoxon matched pair test performed for comparison of antibody levels between enrolment and 36 weeks. Total plasma IgG significantly reduced (p < 0.0001) by 36 weeks compared to the levels at enrolment. Plasma IgG concentration was measured in a subset of women (n = 150).Additional file 2: Association between nutrient supplementation and antibodies to malaria at 36 gestation weeks.Additional file 3: Seropositivity to malaria antigens at 36 gestation weeks.Additional file 4: Antibody levels at enrolment as effect modifiers of the relationship between antibody levels at 36 weeks and supplementation group.Additional file 5: IgG subclass measurements for merozoite antigens and schizont extract at enrolment and at 36 weeks. Antibody IgG subclasses presented as optical density (OD) measured at 410nm, categorized by supplementation groups for antigens MSP-1 19kD, MSP-2, MSP-3, PfRh2, EBA-175 and schizont extract (A-F). Kruskal Wallis test performed to determine subclass level differences between supplementation groups (IFA and LNS, MMN and LNS). Wilcoxon matched-pair test was performed for comparing subclass levels between enrolment and 36 gw. No significant differences in subclass levels across the supplementation groups were observed. Subclass levels at enrolment and 36 weeks were significantly different, denoted as P < 0.05 *, p < 0.005 **, p < 0.0001 ***. IFA (n = 50), MMN (n = 50), LNS (n = 50).Competing interestsThe authors declared that they have no competing interests.Authors' contributions UPC carried out all the malaria antibody level measurements, statistical analyses and drafting of the manuscript. FJIF assisted in statistical analyses and interpretation. JSR implemented immunofluorescence ELISA and IgG subclass level measurement techniques and assisted in data analysis, CL assisted in the preparation of recombinant merozoite antigens, YF was involved in sample collection and storage logistics, SMT conducted real-time PCR runs targeting pfldh gene, JGB provided the lab facilities and materials required for recombinant merozoite protein preparation, KGD and KM were involved in the setting up of the trial and are the co-principal investigators of the trial, PA is the primary investigator of the clinical trial who was involved in study design, supervision of the clinical trial, sample and data collection. SJR supervised the laboratory work, contributed to the data analyses and interpretation and was involved in the preparation of the manuscript. All authors read and approved the final manuscript.Submit your next manuscript to BioMed Central and take full advantage of:
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