Effect of lactoferrin in oral nutrition supplement (ONS) towards IL-6 and IL-10 in failure to thrive children with infection

Background Growth failure due to infection in children is a major health problem throughout the world. It provokes a systemic immune response, with increased interleukin (IL)-6 and reduced IL-10. Lactoferrin (Lf) is a multifunctional iron-binding protein that can be found in whey protein inside formula milk such as oral nutrition supplement (ONS), which is able to upregulate anti-inflammatory cytokines (IL-10) and modulate pro-inflammatory cytokines. We conducted this study to investigate the effect of Lf supplementation in ONS on IL-6 and IL-10 levels in children with failure to thrive and infection. Methods We performed a quasi-experimental pre- and post-study in children aged 12–60 months old with failure to thrive due to infectious illness. The subjects received 400 ml of oral nutritional supplements (ONS, 1 ml equivalent to 1 kcal) each day for 90 days, and their parents received dietary advice and medication based on the underlying illness. Blood was drawn to measure IL-6 and IL-10 before and after the intervention. Results There were 75 subjects recruited and divided into group-1 and group-2 based on age. The incidence of undernutrition was 37.33%. Lf in ONS intervention improved body weight and body length. Lf also reduced IL-6, although there was not a significant difference before and after the intervention. However, the IL-6 reduction was significantly higher in subjects with undernutrition compared with subjects with weight faltering. Pre-intervention IL-6 levels were higher in children with stunting than in children with normal stature. There was a greater change in IL-6 in children with severe stunting than in children with normal stature or stunting. IL-10 was significantly reduced after the intervention. Conclusions In addition to improving body weight and length, Lf supplementation in ONS improved immune response homeostasis by balancing IL-6 and IL-10 levels and by improving the IL-6/IL-10 ratio. ClinicalTrials.gov number ID: NCT05289674, dated May 3 rd 2022.


Methods
We performed a quasi-experimental pre-and post-study in children aged 12-60 months old with failure to thrive due to infectious illness.The subjects received 400 ml of oral nutritional supplements (ONS, 1 ml equivalent to 1 kcal) each day for 90 days, and their parents received dietary advice and medication based on the underlying illness.Blood was drawn to measure IL-6 and IL-10 before and after the intervention.

Results
There were 75 subjects recruited and divided into group-1 and group-2 based on age.The incidence of undernutrition was 37.33%.Lf in Any reports and responses or comments on the article can be found at the end of the article.

Open Peer Review
ONS intervention improved body weight and body length.Lf also reduced IL-6, although there was not a significant difference before and after the intervention.However, the IL-6 reduction was significantly higher in subjects with undernutrition compared with subjects with weight faltering.Pre-intervention IL-6 levels were higher in children with stunting than in children with normal stature.There was a greater change in IL-6 in children with severe stunting than in children with normal stature or stunting.IL-10 was significantly reduced after the intervention.

Conclusions
In addition to improving body weight and length, Lf supplementation in ONS improved immune response homeostasis by balancing IL-6 and IL-10 levels and by improving the IL-6/IL-10 ratio.

Introduction
Growth failure is an important health problem, with weight-for-age z-score (WAZ) and length-for-age z-score (LAZ) declining during the golden period or golden 1,000 days (period during pregnancy until second years of life), 1 and insignificant growth thereafter. 2Nutritional intervention during this period will impact a child's growth, development and ability to thrive. 1 Infection in children causes growth failure by provoking a systemic immune response which affects the nutritional status, 3 especially as a result of a reduction of insulin-like growth factor 1 (IGF-1). 4dernutrition refers to children who are underweight, stunted, or wasted, or those with nutrient deficiencies, which render them vulnerable to infections. 5 Stunting, defined as a height-for-age z-score (HAZ) or length-for-age z-score (LAZ) less than -2 standard deviations (SD), 6,7 is a linear growth failure resulting from chronic malnutrition that is irreversible.Wasting, characterized by being too thin, is defined as a weight-for-height z-score (WHZ) less than -2 SD, representing acute malnutrition, while underweight is identified by a low weight-for-age z-score (WAZ) using the WHO child growth standards as the indicator. 8The prevalence of stunted and severely stunted children under two-years-old was 33.7% 9 and 45.4% in Nigerian children, 10 which was higher compared to this study.While the prevalence of underweight in two-to five-year-old children population in Gaza was 19.6%. 11The prevalence of stunted/severely stunted children was higher in group-1 compared to group-2 in our study, which was similar to the study conducted in Nigeria, accounting for 45.5% vs. 12.2%. 10However, a study in West Sulawesi, Indonesia found that children aged two to five years had a higher incidence of stunted/severely stunted growth compared to children aged one-to two-years-old, 33.64 vs. 23.12%. 12dernutrition leads to a chronic inflammation through immune defects, resulting in recurrent infections. 13This immune alteration is known to be associated with an increased mortality risk in undernourished children. 14The interaction between undernutrition and infections occurs in two ways: undernutrition heightens children's susceptibility to infection, and infection, in turn, contributes to undernutrition. 15In particular, infection inhibits or slows growth velocity, leading to stunting. 16Moreover, infection induces the acute phase response, suspected as a cause of stunting. 17The induction of the acute phase response and the production of proinflammatory cytokines by infection directly impact bone remodeling, crucial for long bone growth, 3 and also inhibit chondrogenesis. 18Proinflammatory cytokines such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) have been found to be elevated in stunted children, leading to increased leptin levels and a subsequent decrease in appetite. 4These proinflammatory cytokines also contribute to bone breakdown. 18IL-6, with both anti-and pro-inflammatory functions, stimulates hepatocytes in the liver to produce acute-phase proteins and cytokines via multiple signaling pathways when it binds with IL-6 receptors. 19terleukin-10 (IL-10) is an anti-inflammatory cytokine with essential roles. 20It functions to dampen and minimize damage resulting from the pro-inflammatory response 21 by inhibiting the activation of lymphocyte, thereby terminating the immune reaction. 22An animal study using malnutrition models indicated that IL-10 levels remain within the normal range in malnourished mice but are elevated in kwashiorkor mice, 23 which supports by another finding. 24Malnutrition downregulates type 1 cytokines (IL-2, IFNγ) but upregulates type 2 cytokines (IL-4 and IL-10), as observed in marasmic children. 22This condition will make undernourished children become more susceptible to infections, with the potential for fatality leading to death.The risk of infection correlates directly with malnutrition degree, in which children with WAZ or HAZ below -3 SD have a 37% risk of diarrhoea. 25e prevalence of tuberculosis (TB) is high in Indonesia, with an increase observed during 2017-2019, rising from 429,219 to 523,614 individuals, or from 167 per 100,000 to 196 per 100,000. 26The manifestation of TB in children REVISED Amendments from Version 2 Introduction: kwarshiorkhor, we changed it as suggested with kwarshiorkor.
Results, second paragraph.The sentences "Table 2 summarized the test of normality and homogeneity for each variable which was needed for further statistical test.It showed that body weight, body length/height, WAZ, LAZ/HAZ, and WLZ/WHZ had normal distribution and homogenous, so the independent sample T-test and oneway anova could be ruled out." was taken places at the last paragraph, we moved it to the last sentences of second paragraph, as the structure of tables were changed, as Reviewer's suggested.
Results: we changed the Tables' order, we moved Table 7 as Table 2.
Discussion: we added our study's limitation in the last paragraph, and several sentences needed grammar revisions.
Any further responses from the reviewers can be found at the end of the article varies, depending on the type of TB, such as the presence of chronic cough, fever, weight loss, or failure-to-thrive. 27The burden of TB in the pediatric population remains significant, with 1.2 million out of 10 million children affected by TB, resulting in a mortality rate of 16%. 28Urinary tract infection (UTI) contributes to the incidence of stunting in children, as it causes anorexia, leading to a stagnant or inadequate weight gain. 29al nutritional supplements (ONS), also known as food for special medical purposes, contains both macro-and micronutrients that are sufficient to meet daily nutritional needs for those at risk of malnutrition. 30ONS is prescribed to increase nutritional intake due to insufficiency in diets to meet daily nutritional requirements, 31 particularly protein and calories. 32ONS not only offers benefits for hospitalized patients, including a reduced length of hospital stay (LOS), lower inpatient costs, decreased complication rates, and rates of readmission, 33 but it also enhances lean body mass recovery.Furthermore, ONS contributes to improved energy intake and positively influences nutritional indicators such as body weight, length, and mid-arm circumference. 34For children, ONS is a dairy milk-based product, which is designed to provide an energy density of 1-1.5 kcal/ml, and is expected to be effective to improve growth. 35ctoferrin (Lf) concentration within the whey protein that is contained in the modified cow's milk formula is only 0.1 mg/ml. 36ONS contains 10.8 g of protein per 100 g, comprising 46% whey and 54% casein.The effect of Lf supplementation (dose 0.6 g/L and 1.00) compared to standard infant formula on body weight showed no significant difference in children until 12 months old.Lf acts as an innate immune regulator and defense mechanism due to its antimicrobial properties. 38As an immunodulatory, Lf acts by balancing the regulation of innate and adaptive immune cells (up-or down-regulate) to create the immune homeostasis. 38Lf can also interact with the immune system, such as influencing cytokine activity by upregulating anti-inflammatory cytokines (IL-4 and IL-10) or modulating proinflammatory cytokines. 39An in vitro study demonstrated that administering Lf at a dosage of 10 mg/mouse intravenously before thymectomy resulted in a 70% reduction in IL-6 and a 30% reduction in TNF-α levels four hours after the operation. 40A study in adults showed that Lf reduced systemic inflammatory biomarkers by 61%, improved immune function by 75%, changed immune cell activity by 40% and reduced respiratory tract infection outcome by 60%.In adults, lactoferrin has been shown to reduce IL-6 by 24.9 pg/mL. 41re, we investigate the effect of lactoferrin in ONS towards IL-6 and IL-10 in failure to thrive children with infections during a 90-day intervention.Our hypothesis is that lactoferrin influences the immune systems of children with failure to thrive and infections, particularly in relation to IL-6 and IL-10.

Ethical statement
The study passed ethical exemption and was declared to be ethically appropriate by the Health Research Ethics Committee, Airlangga University, Surabaya, Indonesia, number 226/EC/KEPK/FKUA/2021 on October 4 th 2021 and registered on ClinicalTrials.govnumber ID: NCT05289674, initial released on May 3 rd , 2022.

Participants
A quasi experimental pre-and post-study design was performed from October 2021 until July 2022 recruiting children aged between 1 years (12-months-old) and 5 years (60-months-old) with failure to thrive due to infectious illness (mainly urinary tract infection and tuberculosis (TB)) diagnosed by a paediatrician (the researcher) based on clinical and laboratory findings at Husada Utama Hospital outpatient unit, Surabaya, Indonesia.The subjects included in the study were excluded if they had fluid retention, organomegaly, a tumor mass, congenital abnormalities, cerebral palsy or hormonal disorders and syndromes.A written informed consent was signed by the parents as approval to participate in the study after the researcher explained the importance, the risks and the benefits of this study.

Sample size
The sample size was determined using the formula below: The sample size was 80 subjects; pre-and post-design needed for the study was 80 samples.

Interventions
The subjects were given an oral nutritional supplement or ONS with Lf (1 ml ~1 kcal, 400 ml/day), SGM Eksplor Gain Optigrow ® prescribed by the researchers for 15 days consumption (equal to 4 boxes of 400 g) for initial intervention to detect any adverse reaction.The authors used this formula due to its relatively cheaper cost compared to other high calorie formula (ONS) available in Indonesia.The parents also had dietary counseling, animal protein was provided and a medication plan was given according to the underlying disease.Parents were asked to report any side effects to the researcher's team by phone for further medical treatment.The parents were asked to visit the doctor after 14 days of ONS consumption for anthropometric measurements, compliance and side effect monitoring at day 15.While visiting, parents also received ONS for the next 15 days' consumption (day 16 to 30), and were asked to visit the doctor again on day 30, 60 and 90.At the 30-day visit, parents received ONS for two months' consumption (day 31 to day 60, and day 61 until day 90) (8 boxes of 400 g) and anthropometric measurements.
Blood was withdrawn via vena cubiti by a laboratory employee at Husada Utama Hospital to measure IL-6 (human IL-6 ELISA kit, code E0090Hu, BT Lab) and IL-10 (human IL-10 ELISA kit, code E0102Hu, BT Lab) before (day 0, when the parents agreed to participate in this study) and after the intervention (day 90).After the blood samples were collected, they were placed in a non-EDTA containing tube for micro-centrifugation to separate blood plasma from blood serum at 3000 rpm for 10 minutes.The supernatant was removed and placed in a PCR tube of 1.5 mL, then kept in a freezer at -4°C.Due to the researchers working during evening until night, the subjects were taken the blood at that time by the laboratory employee accompanied by the doctor's nurses without fasting.
An indirect sandwich ELISA was performed to analyse IL-6 and IL-10 levels before-and after nutritional intervention using blood serum.For the sandwich ELISA, all reagents (standard solution, wash buffer, substrate solution A, substrate solution B and stop solution) were brought to room temperature before use (27°C). 43eparation of standard solution A total of 120 μL standard solution (640 ng/ml) was diluted with 12 μL standard diluent to produce a 320 ng/L standard stock solution, and it was then allowed to rest for 15 minutes.Standard duplication points were made using a serial dilution of standard stock solution to produce 160 ng/L, 80 ng/L, 40 ng/L and 20 ng/L solutions.
Preparation for wash buffer solution Then 20 ml of wash buffer concentrate 25 Â was added to distilled water to yield 500 mL of 1 Â wash buffer.The wash buffer was mixed gently if crystals formed in the concentrate until the crystals had completely dissolved.

Assay procedure
The assay procedure was performed at room temperature after we determined the number of strips required for the assay, and then we inserted the strips in the frames for use.
1. 50 μL of the standard solution was added into all the sample wells.
2. Then 50 μL standard solution was added into the standard wells.
3. 40 μL of sample was added to the sample wells and then 10 μL of human IL-6 or IL-10 antibody was added.Then 50 μL streptavidin-HRP was added to sample wells and standard wells, but not the blank control well.Each of them was mixed before the wells were placed on the plate and then sealed for incubation at 37°C for 60 minutes.
4. After 60 minutes of incubation, the seals were removed, and the plates were washed 5 times with wash buffer; the wells were soaked in 300 μL of wash buffer for 30 seconds to 1 minute for each wash.
5. 50 μL of substrate solution A and 50 μL of substrate solution B were added to each well and the plate was covered and incubated for 10 minutes at 37°C in the dark.
6. 50 μL of stop solution was added to each well, so that the blue colour changed to yellow immediately.
We then determined the optical density (OD value) of each well immediately using a microplate reader set at 450 nm of wavelength within 30 minutes after the stop solution was added, and then the standard curve were made. 43dy weight was measured using a Seca 354 digital baby scale or a Seca 813 electronic flat scale) and body length/height was measured using a Seca 415 infantometer or Seca 213 stadiometer).Both measurements were taken twice by a trained nurse in the outpatient department of Husada Utama Hospital.The weight and length/height were the average value of the two measurements.When the subjects were weighed and measured, they wore light clothes without footwear or hair accessories.Anthropometry measurement for weight-for-age z-score (WAZ), length-for-age or height-for-age z-score or height-for-age z-score (LAZ/HAZ) and weight-for-length or weight-for-height z-score or weight-for-height z-score (WLZ/WHZ) were determined using WHO Anthro offline version 3.2.2.All the data was summarized in underlying data 44 and extended data. 45e hypotheses that:

Statistical analysis
• Lactoferrin in ONS improves body weight and length/height.
• Lactoferrin supplementation in ONS improves immune response homeostasis by balancing IL-6 and IL-10 levels, and by improving the IL-6/IL-10 ratio.
• At least there was a pair of groups of nutritional status had a significant difference of IL-6 and IL-10 levels, and the IL-6/IL-10 ratio.

Results
Seventy-five subjects were involved in the study and divided into two groups based on the age of the participant: group-1 (age 1-2 years, n=39) and group-2 (age 2-5 years old, n=36), as summarized in Figure 1.
Table 1 summarizes the characteristics of the subjects who participated in the study.The ratio of male/female was 12/13 and there was no significant difference in gender distribution in both groups (p = 0.108).There was no significant difference in the main complaint (p = 0.229), duration of complaints (p = 0.580), WAZ (p = 0.482) and WLZ/WHZ (p = 0.499).Age, ideal body weight and height age were lower in group-1 compared to group-2 (p <0.05).LAZ was lower in group-1 compared to group-2 (-1.95 AE 1.17 vs. -1.19AE 0.86, p = 0.002).
Table 2 summarized the test of normality and homogeneity for each variable which was needed for further statistical test.It showed that body weight, body length/height, WAZ, LAZ/HAZ, and WLZ/WHZ had normal distribution and homogenous, so the independent sample T-test and oneway anova could be ruled out.
The incidence of underweight and severely underweight children in group-1 and group-2 was 33.33% and 5.33% respectively, and there was no significant difference in WAZ categories in both groups (p = 0.874).The incidence of stunted and severely stunted children in group-1 and group-2 was 25.33% and 13.33%, respectively, demonstrating a higher prevalence of stunted/severely stunted children in group-1 compared to group-2 (56.41% vs. 19.45%,p = 0.004).However, the incidence of stunted/severely stunted children in group-1 was predominantly boys (6 boys vs. 1 girl).The incidence of wasted and severely wasted children in group-1 and group-2 were 12% and 2.67% (p = 0.486).
The effect of ONS on body weight and body length/height change is summarized in Table 3.Initial body weight before treatment was lower in group-1 compared to group-2 (p = 0.000).post intervention body weight was lower in group-1   There was significantly improvement in the IL-6/IL-10 ratio after the intervention (0.33 AE 0.22 vs. 0.44 AE 0.12, Δ = 0.11 AE 0.23, p = 0.000).The reduction in the IL-6/IL-10 ratio showed no significant difference in either group (0.11 AE 0.29 vs. 0.12 AE 0.17, p = 0.991).
The levels of IL-6 based on anthropometric categories are summarized in Table 5.Based on the LAZ categories, there was a significant difference in IL-6 levels pre-intervention (p = 0.045), in which the stunted group had higher levels of IL-6 compared to those with a normal stature (212.06AE 146.05 vs. 115.81AE 93.84 pg/mL, p = 0.037).Although there was no significant difference, IL-6 was higher in the stunted group compared to those who were severely stunted (212.06AE 146.05 vs. 85.45AE 89.06 cm, p = 0.057).There was no significant difference in post-intervention levels of IL-6 (p = 0.083); however, IL-6 was lower in normal stature children compared to stunted and severely stunted children.Although  Initial and late levels of IL-10, and changes of IL-10 based on the anthropometric categories are summarized in Table 6.
There was no significant difference in IL-10 before and after the intervention, or in changes of IL-10 (p <0.05) based on the LAZ/HAZ categories.A similar phenomenon was also seen in the WAZ and WLZ/WHZ categories (p <0.05).
The IL-6/IL-10 ratio based on anthropometric measurements is summarized in Table 7.The IL-6/IL-10 ratio based on the LAZ/HAZ categories showed no significant difference pre-and post-intervention.However, ONS supplementation increased the IL-6/IL-10 ratio in all LAZ/HAZ categories.In the WAZ categories, severely underweight children had a lower IL-6/IL-10 ratio compared to underweight children, even though there was no significant difference.The IL-6/IL-10 ratio increased after ONS therapy in all WAZ categories.A higher increment was seen in the severely underweight, but there was no significant difference.The WLZ/WHZ categories also showed no significant difference in the initial and late changes of the IL-6/IL-10 ratio.Note: WAZ = weight-for-age z-score; LAZ = length-for-age z-score; HAZ = height-for-age z-score; WLZ = weight-for-length z-score; WHZ = weight-for-height z-score. 1 One-way ANOVA.

Discussion
Stunted growth was found to be associated with age, and it was more prevalent in children aged less than 24-monthsold. 46Due to the incidence of stunted growth, which was higher in group-1, the LAZ value was significantly lower in group-1 compared to group-2.It was also found that children with stunted growth were significantly shorter in length/ height than the control group in another study. 47It was reported that children aged 12-23 months old had an increased risk of stunting by 1.8 times. 9,48 group-2, the incidence of stunted/severely stunted growth was predominant in males, aligning with the findings of Akombi et al. (2017), which identified male sex as one of the risk factors for stunting in children aged 0-5 years. 10This consistency with our study suggests that males may be more vulnerable to health inequalities. 49The biological reason is due to the sex difference in the immune and endocrine systems, and testosterone, luteinizing hormone and follicle stimulating hormone are suspected to play a role. 50Feeding practice preferences between boys and girls such as early weaning in boys, and a tendency for boys to consume more than one complementary feed meal within a 24-hour period, may also contribute to the observed patterns. 51S intervention in undernourished or at nutritional risk children aged nine months to 12-years-old improved body weight by 0.423 kg after six months of intervention and height gain was 0.417 cm compared to the control, with greater gains in weight in the first 7-10 days of intervention (0.089 kg). 52ONS improved growth in underweight children aged five-to 12-years-old after six and 12 months, 53 which was in line with this study, where both group-1 and group-2 gained weight.Formula feeding supplemented with lactoferrin is safe for infants under one year old with no difference in growth rate (g/day). 37ctoferrin intervention in children with diarrhoea aged 12-36 months old increased the LAZ/HAZ score (p = 0.03) compared to the placebo, 54 and the children also showed an increment in length/height.A similar result was also found in Vietnamese children aged 24-48 months old in a 12-month intervention.The intervention of 450 kcal of additional ONS during the first three months resulted in an increase in height of 1.62 cm, 55 which was lower than our results in a similar group (group-2).A higher calorie density intervention (2.4 kcal/ml vs. 1.5 kcal/ml) for 28 days increased the children's height by 0.87 [0.59-1.16]and 0.55 [0.17-0.93]cm, p = 0.007 in children aged greater than one year and less than 12 years old with growth faltering. 35ctoferrin is known to have a bacteriostatic or bactericidal effect and it can activate the immune response of an organism, 56 and limits tissue damage due to excessive pro-inflammatory response (chronic response) caused by the infection. 38It can therefore reduce the incidence of acute gastrointestinal symptoms and reduce the duration of respiratory symptoms in children under 12 months old due to viral or bacterial infection. 57Regarding the immunological profile, when comparing an infant who received Lf supplementation vs. non-Lf supplementation vs. standard formula, although there was no significant difference between groups, there was an increase in TGF-β1 (6.5 vs. 4.3 vs. 2.8 ng/mL), TGF-β2 (0.26 vs. 0.26 vs. 0.22 ng/mL) and IL-2 (0.21 vs. 0.5 vs. 0.4 pg/mL), but a decrease in TNF-α (-2.4 vs. -1.5 vs. -1.7 pg/mL) during a four-month intervention. 58A study that examined piglets with a 2 ml/day supplementation showed a decrease in bacterial colonies compared to those without Lf supplementation (1.109 x 10 7 vs. 3.6183 x 10 8 CFU) via an anal swab after a seven-day intervention. 59It was stated that Lf induced the development of T cell helper type 1 (Th1) immunity, thus created the balance of monocytic pro-and anti-inflammatory cytokines.In a dose-dependent manner, Lf enhanced pro-inflammatory response in vitro (splenocyte and adherent (F4/80 + ) splenocyte populations, bone marrow derived monocytes (BMM), and J774A.1 cultured cells) and induced IL-12 and IL-10 production and increased the ratio IL-12: IL-10 in lipopolysaccharide (LPS) stimulated cells. 60Studies of Mycobacterium tuberculosis infection treated with Bacillus Calmette-Guérin (BCG) and Lf emulsified with Freund's adjuvant in mice showed a decreased mycobacterial load in the lungs and spleen.It also increased the protection against M. tuberculosis, 37,61,62 via downregulation of proinflammatory mediators (TNF-α, IL-1β) by modulation of macrophages and dendritic cell ability to present antigens and stimulate T-cells.Lf also increased IFNγ, which was the specific response towards Th1. 38A study examining mice with urinary tract infection due to Escherichia coli showed that Lf intervention orally was able to decrease the number of bacteria in the kidneys and bladder after 24 h of Lf consumption, and reduced IL-6 by urinary leucocytes. 63A study conducted on Senegalese children receiving tetanus vaccine in stunted children aged one-to nine-years-old showed that the production of IFNγ was compromised. 2It was stated that undernutrition is related to immunodeficiency, even in mild cases, affecting both the innate and adaptive immune systems. 2 In our study, even though there was no significant difference in IL-6 levels before and after the intervention, Lf reduced IL-6, which is in line with other studies showing a reduction in undernutrition groups.It was found that IL-6 levels were lower in undernutrition compared to good nutrition groups (2.54 pg/mL vs. 6.02 pg/mL, p <0.0001). 64Genetic investigation showed that the IL-6 164 gene with a GG and GC genotype (mutant phenotype) was more frequent in undernourished children. 65hen the groups were examined based on the LAZ/HAZ categories, stunted subjects had higher IL-6 levels compared to normal stature and there was a significant difference compared to severely stunted.This is in-line with a study in Egyptian children, where IL-6 was higher in stunted compared to normal stature children (1.6 AE 0.2 vs. 1.5 AE 0.3 pg/mL), 66 but it was decreased in malnourished compared to normal children. 64This showed that when the children had an LAZ/HAZ score greater than or equal to -2 SD, IL-6 was increased but it decreased when children had an LAZ/HAZ score greater or equal to -3 SD.On malnutrition, the acute-phase response was attenuated, and the production of cytokines decreased.An animal study showed that IL-1β production decreased in malnourished guinea pigs induced with endotoxins. 19Stunting is a form of growth failure due to long term nutritional deficiency or it is caused by chronic malnutrition or recurrent undernutrition. 8,67After a six month intervention with food supplementation, stunted Bangladeshi children aged 12-18 months old experienced an IL-6 increment (from 0 [0-1.2] to 1.68 [0.83-4.7]pg/mL, p = 0.001), 68 which contradicts this study as IL-6 levels were reduced in stunted and normal stature children.However, IL-6 was found to have increased in severely stunted children, so the post-intervention levels of IL-6 were higher in severely stunted even-though there was no significant difference.Severely stunted children might undergo these immune alterations which are similar to severely acute malnutrition, so IL-6 levels were lowest at the outset but increased drastically after the intervention to surpass normal stature and stunted children.It was stated that immune function is an activity with high costs on energy demand, and in developing children the allocation of energy in immune functions may lead to a trade-off with physical growth, particularly those with exposure to infection. 69similar anomaly was also seen in the WAZ categories even though there was no significant difference.Being underweight has been used as an indicator of undernutrition due to a short term nutritional deficiency.8 However, an in vitro study using peripheral blood mononuclear cells (PBMC) taken from children suffering from protein energy malnutrition (PEM) contradicted this study, which showed an increment in IL-6 expression after stimulation with LPS, 70 even though it was expressed earlier, reached its peak earlier, and lasted longer than controls in rats.71 As the immune function is costly in terms of energy, it has negative effects on growth.In children with mildly elevated immune activity, they experience a growth reduction of up to 49%, 69 as seen in underweight children who experienced an increase in IL-6 due to the trade-off in body fat between immune function and growth.69 Regarding malnutrition, lymphatic tissue, particularly the thymus, experiences atrophy, leads to a reduction in delayedtype hypersensitivity responses, followed by a reduction in levels of antibodies in severely malnourished children (≥-3 SD of WLZ/WHZ WHO child growth standards), but it remains intact in moderate malnutrition (leucocyte and lymphocyte, high levels of immunoglobulin, particularly IgA, and acute phase response), and cytokine patterns are skewed towards a Th2-response.14 However, our study found that IL-6 started to reduce in wasted patients, with the lowest levels in those that were severely wasted. Wenling C57BL/6 J mice in a wasting models study which underwent 14 days of weight loss showed increases of IL-10 in the malnourished group at three and at 14 days.23 It was stated that malnutrition modifies the body's resistance against infection, particularly the immune response.Lipopolysaccharide (LPS) injection (1.25 μg i.v.) in a protein-energy malnutrition (PEM) mouse model, showed that the circulating levels of IL-10 were increased and high levels were found in bone marrow cells, which showed immunodeficiency.24 This finding was in-line with a study in children with marasmic-PEM, IL-10 was significantly higher compared to controls (19.08 AE 5.93 vs 10.46 AE 3.90 pg/mL; p = 0.000).22 This may be caused by the deficits of NF-kB activation. NFkB was the major transcription pathway for proinflammatory cytokine production.72 Using BMI as the parameter to determine malnutrition, subjects with severe malnutrition (BMI <16.5) had higher levels of IL-10 (8.0 AE 3.6 pg/mL) compared to those with moderate malnutrition (BMI = 16.5-18.4) (26 AE 4.3 pg/mL) and good nutrition (BMI ≥18.5) (2.8 AE 0.7 pg/mL) in adults, 73 which was similar to the WAZ category where IL-10 was slightly increased in those underweight, and increased drastically in those severely underweight.
Nutritional intervention increases IL-10 significantly in children aged 12-60 months old with moderate and severe malnutrition receiving curd (milk product) compared to leaf protein concentrate (LPC) (from 30.9 AE 29.5 to 67.4 AE 96.2 pg/mL vs. 29.2AE 25.8 to 31.5 AE 24.9 pg/mL).Based on Gomez criteria for malnutrition severity, children with mild malnutrition had lower IL-10 compared to children with severe malnutrition.It was higher in subjects aged more than two years old compared to two-to five-year-olds due to a balancing pro-inflammatory response to minimalize tissue damage. 74In malnourished children, IL-10 was found to be reduced, while in line with this study, IL-10 was depressed in severely wasted subjects. 75However, the level of IL-10 was still normal in severely stunted or severely underweight children.The reduction is due to a deficiency in the number and functional Th cells, which may be caused by incomplete differentiation of T lymphocyte precursors and steroid-induced lympholysis. 75Another study of malnourished children due to inadequate food intake (anorexia nervosa) and diarrhoea receiving nutritional intervention in the form of milk and yoghurt, showed increased IFNγ production post intervention, 76 which may be caused by incomplete differentiation of T lymphocyte precursors and steroid-induced lympholysis.In undernutrition subjects and weight faltering subjects, IL-10 tends to reduce, and a higher reduction was seen in undernutrition subjects, which showed that before intervention undernutrition subjects may experience immune alterations, as seen in the IL-6/IL-10 ratio, which was higher in undernutrition group-1, but lower in group-2.At post intervention, almost all the group had a similar value, ranging from 0.43 to 0.47.Adipose tissue is the main storage for nutrients, which can sense that nutrients are inadequate by releasing adipokines (particularly leptin) to control cellular metabolism and immune function.So, undernutrition has a direct impact on adipose tissue (volume and number), and directly influences the immune system.Leptin not only mediates glucose and lipid metabolism but also immune function, by stimulating activation, proliferation and production of pro-inflammatory cytokine (IL-6, TNF-α, monocytes, macrophages, dendritic cells, and NK cells).Leptin also promotes T-cell activation and development towards Th-1 and Th-17 cell subset which is proinflammatory.Regarding undernutrition, there was leptin depletion and in contrast adiponectin is produced, resulting in the polarization towards M2 or an alternative macrophage which then secretes IL-10 and IL-1Rα.This limits the activation of the NF-kB pathway, and reduces both T-cells or B-cells.Moreover, cortisol hormone restrains the generation of the proinflammatory immune response, so the ability of macrophages and neutrophils to infiltrate the infection site was also restrained.Proinflammatory cytokine production is also reduced, but anti-inflammatory cytokines (IL-10 and IL-33) are increased. 5-6 has been used as a potential biomarker to identify patients receiving anti-inflammatory therapies as it is secreted widely as a response to pathological states such as infection, inflammation and cancer.IL-10 acts as an anti-inflammatory response, it is secreted as a response to dampen pro-inflammatory bursts and minimize tissue damage.The balance of IL-6 and IL-10 is an important biomarker reflecting the homeostasis of the immune response.In Covid-19 patients, each point increment of the IL-6/IL-10 ratio was associated with a 5.6 times more severe outcome. 5In children with pneumonia, the IL-6/IL-10 ratio at 9.61 determines those with severe pneumonia to those with mild disease (sensitivity 76.5% and specificity 93%). 77r study had several limitations, as it did not account for the possibility of subjects having another infection.In this scenario, where all subjects are identified with a bacterial infection, it does not rule out the possibility of one having an additional infection.An acute infection that might have been caused by a viral agent could lead to alterations in the levels of IL-6 and IL-10.The increased IL-6 level may be followed by a decrease in IL-10, as it acts as a regulator.This could result in some subjects presenting with higher IL-6 and lower IL-10 levels than others.

Conclusions
Lactoferrin in ONS intervention improved immune response homeostasis by balancing IL-6 and IL-10 and improved the IL-6/IL-10 ratio, not only body weight but also body length.

Consent
Written informed consent for publication of the patients' details was obtained from the parents of the patients.

General Response:
We would like to thank the authors for their immense efforts to address our previous comments.We are thoroughly impressed with the improvements in flow and clarity.The concerns do not impact comprehension of the article's core results.However, we highly recommend the authors incorporate these suggestions into their manuscript.

Major Comments:
A major component that is missing from the methods was disease burden tracking/monitoring of the course of disease progression for the sample participants.If information on the resolution of viral infections was not collected, its absence should be included in the limitations section.As stated, all participants were identified as "[failing] to thrive due to infectious illness" but it was never mentioned whether any of the participants' infections were resolved over the course of the intervention.Since disease burden is the identifying variable for this population, this is critical information, especially since individuals whose infections cleared over the period of observation would likely make significantly higher gains in weight and length/height.It is important to not conflate normal growth gains due to aging/growth and development and reduced disease burden with the impact of the ONS. 1.

Minor Comments:
Statements regarding Nigerian, Gazan, and Indonesian children in the intro (paragraph 2) is still out of place, as it's discussing results.We recommend that the authors remove the following clauses and rephrase accordingly for clarity, as they have not discussed methods and should not be discussing generated results at this point: "....which was higher compared to this study" ; "the prevalence of stunted/severely stunted children was higher in group-1 compared to group-2 in our study..." The following clause should not stand alone as a sentence and should be rephrased: "While the prevalence of underweight in two-to five-year-old children population in Gaza was 19.6%."

○
Intro paragraph 4 -typo -",which supports by another finding" .The authors can either remove "by" or rephrase slightly to say "which is supported by" (or something similar in nature)

○
Unclear where this study is occurring until intro paragraph 5

○
Unclear statement in methods, before "interventions" -"The sample size was 80 subjects; pre-and post-design needed for the study was 80 samples."Does this mean there were 160 total samples?A pre-and post-intervention sample from each participant?Is this including participant withdrawals?
○ Typo in methods, before "results" -"We hypotheses that" -should be "we hypothesize that" ○ Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Our combined areas of expertise involve human milk, maternal-infant health, biomarkers of stress (e.g., cortisol) and immunity/immune responses (e.g., cytokines, antibodies), anthropometry, human skeletal growth and plasticity, and biological specimen collection (including blood draws, saliva and milk collections), as well as enzyme immunoassay estimations.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
As previously mentioned, hypothesis driven research should begin with clear hypotheses presented in the introduction.While the authors have added hypotheses, they have not been contextualized with appropriate background literature to support the proposed relationships.As currently presented, the hypotheses are actually results that have been rephrased as hypothetical statements which is not appropriate project formulation.If the authors did not already have hypotheses formulated prior to data collection and analyses, then the analyses needs to be restructured for an exploratory protocol and therefore rerun with appropriate tests for such a study [e.g., model selection/information theoretic approach using akaike information criterion corrected (AICc) or bayesian information criterion (BIC)].

2.
A major component that is missing from the methods was disease burden tracking for the sample participants.As stated, all participants were identified as "[failing] to thrive due to infectious illness" but it was never mentioned whether any of the participants' infections were resolved over the course of the intervention.Since disease burden is the identifying variable for this population, this is critical information, especially since individuals whose 3.
infections cleared over the period of observation would likely make significantly higher gains in weight and length/height.It is important to not conflate normal growth gains due to aging/growth and development and reduced disease burden with the impact of the ONS.
There needs to be a "Limitations" section to address some of the pitfalls of the study.For example, the authors could use this section to discuss the issue of doing non-fasting blood draws in the evening when the biomarkers of interest may be subject to dietary fluctuations or to diurnal variation.While this was addressed in the response to reviewers, it should be included in the text of the manuscript, so that readers are given a complete picture of the methods and the associated factors that may confound results.

4.
As mentioned in our first review of this manuscript, the results presented in the abstract and results/discussion sections of the manuscript seem to be conflicting.Without having a control (or a healthy population, or other predetermined baselines attributing to what should be expected), there is no way to determine whether the lactoferrin and ONS is responsible for "improving" the weight gains or immune responses.

5.
The authors directly addressed many of our previous comments from the original version of the manuscript in their response to the reviewer report.However, they neglected to revise those comments in the updated resubmission.For example: "Main Complaints" is still not addressed in the methods, so it is unclear how these complaints were documented/assessed (e.g., via survey at the beginning of study) and whether these complaints occurred during the course of the trial.Your response to us in the report "The complaints why the subjects visited the doctor were asked in the beginning by the nurse, "such as: what's wrong with the baby, ma'am"" should be clarified in the methods section.

Minor Comments:
Introduction paragraph 4 concludes by stating that infection correlates with malnutrition degree but uses WAZ or HAZ as a metric of malnutrition which is not an appropriate analogous metric (growth impairment is not a direct measure of malnourishment, rather, it is a symptom of it).Instead, risk of infection correlates with degree of growth impairment based on WAZ or HAZ.

1.
Improper citations: Paragraph 5 introduction -"Urinary tract infection (UTI) contributed to the incidence of stunting in children, as it causes anorexia which leads to a stagnant or inadequate weight gain.

Rachael Anyim
Anthropology, Binghamton University, Binghamton, New York, USA Mallory Peters Anthropology, Binghamton University, Binghamton, New York, USA Widjaja et al.'s manuscript highlights growth failure in children (diagnosed with infectious diseases) and whether lactoferrin in oral nutrition supplementation improves their immune responses, captured here via changes in pro-inflammatory and anti-inflammatory cytokines, interleukin-6 and interleukin-10, respectively.Participating children received lactoferrin in oral nutrition supplementation for a period of 90 days, which the authors claim resulted in increased body length and weight, as well as decreased interleukin-10 and interleukin-6 (though the latter was not statistically significant between pre-and post-intervention).Furthermore, children with severe growth stunting experienced a larger decrease in interleukin-6 compared to children of normal stature.
As a whole, the authors' manuscript is very detailed and well written, evidenced by their thorough references to the current literature.The methods for data collection are written clearly for replication and the study is well executed.However, the paper could benefit from some reorganizing.For example, "stunting" is defined on page 12 in the middle of paragraph 3 but should be discussed much earlier in the paper (i.e., in the introduction).The statistics presented also need a significant overhaul.See "Major Comments" and "Minor Comments" for additional details.

Major Comments:
The introduction section should be expanded to include functions/relevance of interleukin-10 (not just IL-6 and lactoferrin), definitions of stunting versus wasting, and outline the growth standards used.It could also benefit from highlighting the study scope and setting.For example, in the first paragraph in the discussion section (page 11), it is unclear why the authors discuss the prevalence of stunting in other countries when these have not been previously addressed.It might be better to move this information to the introduction to improve the background information presented and flesh out the current relevance to Indonesia in particular.It could be useful to state the rationale for conducting this study in Indonesia, what infectious disease burdens are prevalent in the area (e.g., urinary tract infections and tuberculosis are mentioned in the methods but then COVID-19 and pneumonia are in the discussion) and whether these may be confounding variables in analyses.If available within the data they collected, the authors could also address cultural considerations since part of the intervention includes dietary counseling and dietary supplementation of animal protein, as well as whether participants are still breastfeeding and how this might impact the generated results.

1.
While the authors have cited the literature, they have not stated what hypotheses may be driving their analyses and what results they expect to yield.These should be foregrounded in the introduction for readers.

2.
Methods for data collection, including anthropometrics and blood draws, seem appropriate.However, it would have been good to see a discussion on the volume of blood drawn and whether it was a fasting draw, as well as information on time of day for collections since some bioactive properties (e.g., IL-6) can exhibit diurnal variation, thereby confounding the results.The authors have only one sentence detailing their statistical analyses and superscripts in corresponding tables, which severely limits the replicability of their quantitative methods.It would be useful to expand on their methods here -including the results of normality and homogeneity tests and clearly describing which tests were performed on which variables, so that there is no confusion as to how analyses were conducted.

3.
Clarification on the results presented would be helpful; as currently written, there seem to be mismatches between the statistics and the authors' reports/concluding statements.We wonder if, perhaps, different tests are reported in the text versus in the tables; if so, the results yielded in those tests should match those in the tables/text.For example, in the abstract the authors suggest that IL-10 was significantly reduced after intervention.However, the closest value to statistical significance is p-value = 0.076 presented in Table 5.Additionally, they state that their intervention improved the IL-6/IL-10 ratio.However, there are no statistically significant differences in pre-and post-intervention ratios, as seen in Table 6.Convention is moving beyond solely using p < 0.05 as a threshold for data interpretation, so if that is the case here, the authors should disclose their cutoff point for determining "significant" results.Furthermore, the authors should remodel their tables to not only include p-values but also to include the comprehensive test results, test statistics, and effect sizes/magnitude of the effect.The p-values, means, and standard deviations presented do not convey a complete picture of the story written in the abstract and in the paper itself.Additionally, presenting SD as a +/-value suggests symmetry of data without actually testing for it.It could be useful if the authors instead did repeated measures ANOVA of pre-and post-intervention within group changes for IL-6 and IL-10, as well as present the tests of normality and homogeneity and the results of those tests.Additionally, clarification on the specific growth curves being used and how it was determined that Oral Nutrition Supplement intervention improved body weight and length/height could also be useful.For example, Table 1/paragraph 1 states that age, body weight, and height age were lower in group 1 versus group 2. However, the groups were broken up by age, so these changes could largely be innate and not necessarily linked to ONS supplementation.Table 2 presents a similar issue.Were the authors comparing these metrics to external growth standards?If so, those standards are not clearly identified here (aside from mentioning WHO Anthro).If not, how were the authors able to confirm whether the growth is an actual improvement of health due to the ONS intervention and not the result of normal growth over the three-month period?Perhaps, it may also be useful to add the rationale in age group cutoffs (i.e., group 1 vs. group 2).

Minor Comments:
Sentence Structure: In the introduction section's first paragraph (page 3), the authors can remove "still", as it presupposes the audience is aware of growth failure as a historic health 1.
problem.In paragraph two, the second to last sentence serves no clear purpose and should either be expanded or removed.The first 2 sentences in the third paragraph can be consolidated a bit.The first sentence in the last paragraph on page 11 ("Lactoferrin…act accordingly and limits tissue damage") doesn't seem to make sense with the last clause.This may be due to missing words or punctuation.
Typographical Errors and Points of Clarification: In the discussion section, "secrets" should be "secretes" in the twelfth paragraph (page 13)."Main Complains", in Table 1, should be "Main Complaints".Furthermore, "Main Complaints" were not addressed in the methods, so it is unclear how these complaints were documented/assessed (e.g., via survey at the beginning of study) and whether these complaints occurred during the course of the trial."Liquid pups" should also be corrected in the table.Weight faltering ("normo-weight") is unclearly defined in the table and in the text of the paper.In the far right column of Table 6, the column is labeled as ΔIL-10 but should be ΔIL-6/IL-10.It could also be useful if the authors clarified in the methods how animal protein supplementation was conducted, the rationale behind it, and how its application was measured.

2.
Overall, the study design and work presented have academic merit, as well as public health implications.If, for example, a relatively inexpensive intervention demonstrates positive health outcomes, this is a valid justification for using it in potentially low-income, nutrient impoverished, or high disease-burden settings.However, for the reasons stated above, it is a little difficult to accurately assess whether there is a reduction in IL-6 and if this is indeed directly related to the intervention.

Is the work clearly and accurately presented and does it cite the current literature? Partly
Is the study design appropriate and is the work technically sound?Yes

If applicable, is the statistical analysis and its interpretation appropriate? No
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Our combined areas of expertise involve human milk, maternal-infant health, biomarkers of stress (e.g., cortisol) and immunity/immune responses (e.g., cytokines, antibodies), anthropometry, human skeletal growth and plasticity, and biological specimen collection (including blood draws, saliva and milk collections), as well as enzyme immunoassay estimations.
We confirm that we have read this submission and believe that we have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however we have significant reservations, as outlined above.
Author Response 02 Sep 2023

Nur Aisiyah Widjaja
As a whole, the authors' manuscript is very detailed and well written, evidenced by their thorough references to the current literature.The methods for data collection are written clearly for replication and the study is well executed.However, the paper could benefit from some reorganizing.For example, "stunting" is defined on page 12 in the middle of paragraph 3 but should be discussed much earlier in the paper (i.e., in the introduction).
The statistics presented also need a significant overhaul.See "Major Comments" and "Minor Comments" for additional details.

Response: Paragraph 2 sentences 2, and soon
Major Comments: The introduction section should be expanded to include functions/relevance of interleukin-10 (not just IL-6 and lactoferrin), definitions of stunting versus wasting, and outline the growth standards used.Response: Noted: Paragraph 2 sentences 2 to 3.
It could also benefit from highlighting the study scope and setting.For example, in the first paragraph in the discussion section (page 11), it is unclear why the authors discuss the prevalence of stunting in other countries when these have not been previously addressed.It might be better to move this information to the introduction to improve the background information presented and flesh out the current relevance to Indonesia in particular.Response: Noted: Paragraph 2 sentences 4 until the last paragraph.
It could be useful to state the rationale for conducting this study in Indonesia, what infectious disease burdens are prevalent in the area (e.g., urinary tract infections and tuberculosis are mentioned in the methods Response: Noted.Paragraph 5. But then COVID-19 and pneumonia are in the discussion) and whether these may be confounding variables in analyses.Response: We add COVID-19 due to during the intervention, we found that our subjects were infected.As for pneumonia, we found this infection in several subjects.We thought those disease as the confounding factors, but after we analysed statistically, they didn't (p>0.05).
If available within the data they collected, the authors could also address cultural considerations since part of the intervention includes dietary counseling and dietary supplementation of animal protein, as well as whether participants are still breastfeeding and how this might impact the generated results.
Response: All of the subjects had breastfed predominantly (those under 2 years old), but the older ones consume growing up formula.
While the authors have cited the literature, they have not stated what hypotheses may be driving their analyses and what results they expect to yield.These should be foregrounded in the introduction for readers.Response: Noted: The last sentences in introduction, and hypotheses in the methods.
Methods for data collection, including anthropometrics and blood draws, seem appropriate.However, it would have been good to see a discussion on the volume of blood drawn and whether it was a fasting draw, as well as information on time of day for collections since some bioactive properties (e.g., IL-6) can exhibit diurnal variation, thereby confounding the results.Response: Noted: Paragraph 2 sub heading intervention in Method.Due to the researchers working during evening until night, the subjects were taken the blood at that time by the laboratory employee accompanied by the doctor's nurses without fasting.
The authors have only one sentence detailing their statistical analyses and superscripts in corresponding tables, which severely limits the replicability of their quantitative methods.It would be useful to expand on their methods here -including the results of normality and homogeneity tests and clearly describing which tests were performed on which variables, so that there is no confusion as to how analyses were conducted.Response: Noted: Table 7 Clarification on the results presented would be helpful; as currently written, there seem to be mismatches between the statistics and the authors' reports/concluding statements.We wonder if, perhaps, different tests are reported in the text versus in the tables; if so, the results yielded in those tests should match those in the tables/text.For example, in the abstract the authors suggest that IL-10 was significantly reduced after intervention.However, the closest value to statistical significance is p-value = 0.076 presented in Table 5.Additionally, they state that their intervention improved the IL-6/IL-10 ratio.However, there are no statistically significant differences in pre-and post-intervention ratios, as seen in Table 6.Convention is moving beyond solely using p < 0.05 as a threshold for data interpretation, so if that is the case here, the authors should disclose their cutoff point for determining "significant" results.Furthermore, the authors should remodel their tables to not only include p-values but also to include the comprehensive test results, test statistics, and effect sizes/magnitude of the effect.The p-values, means, and standard deviations presented do not convey a complete picture of the story written in the abstract and in the paper itself.Additionally, presenting SD as a +/-value suggests symmetry of data without actually testing for it.It could be useful if the authors instead did repeated measures ANOVA of pre-and post-intervention within group changes for IL-6 and IL-10, as well as present the tests of normality and homogeneity and the results of those tests.Additionally, clarification on the specific growth curves being used and how it was determined that Oral Nutrition Supplement intervention improved body weight and length/height could also be useful.For example, Table 1/paragraph 1 states that age, body weight, and height age were lower in group 1 versus group 2. However, the groups were broken up by age, so these

University of Agricultural Sciences, Dharwad, India
The study is novel and addresses the most important issue of poor nutritional status leading to infant mortality and morbidity in infants and toddlers.Appropriate statistical tool and methodology is adopted.However the title can be more specific as effect of lactoferrin on growth and stunting in children.And the sample size will be 80 and 160 as the same sample will be used for pre and post test.
The interpretation and discussion part needs to be rewritten as the present study is measuring only IL-6 and IL-10 levels in failure to thrive children.No where the results indicate the effect on immune system like reduction in morbidity status and increase in weight and length by morbidity status.More reviews can be added to support the effect of lactoferrin in oral nutrition on growth of children.By discussing the studies by other researchers on improvement of immune system cannot be used to conclude as" Lactoferrin in ONS intervention improved immune response homeostasis by balancing IL-6 and IL-10".Better to restrict to the results of present study indicate the effect on immune system like reduction in morbidity status and increase in weight and length by morbidity status.More reviews can be added to support the effect of lactoferrin in oral nutrition on growth of children.By discussing the studies by other researchers on improvement of immune system cannot be used to conclude as" Lactoferrin in ONS intervention improved immune response homeostasis by balancing IL-6 and IL-10".Better to restrict to the results of present study For growth parameters, we made other paper entitled "The Effect of High Calorie Formula on Weight, Height Increment, IGF-1 and TLC in Growth Faltering Children".in this paper we add total lymphocyte count (TLC) as the immunological parameters in these children.

We will include the suggestion in the third version.
Competing Interests: No competing interests were disclosed.
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5 .
Reviewer Report 30 August 2023 https://doi.org/10.5256/f1000research.142910.r197396© 2023 Anyim R et al.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Is the work clearly and accurately presented and does it cite the current literature?YesIs the study design appropriate and is the work technically sound?YesAre sufficient details of methods and analysis provided to allow replication by others?YesIf applicable, is the statistical analysis and its interpretation appropriate?PartlyAre all the source data underlying the results available to ensure full reproducibility?YesAre the conclusions drawn adequately supported by the results?PartlyCompeting Interests: No competing interests were disclosed.Reviewer Expertise: Assessment of children, Differently abled and elderly I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Table 1 .
Subject's characteristic during the study.

Table 2 .
Test of normality and homogeneity.

Table 3
. Body weight (in g) and body length/height (in cm) changes after intervention.ParametersGroup-1 (mean AE SD) Group-2 (mean AE SD) p Initial body weight 8,735.38AE 1,318.64 12,164.17AE 1,688.890.000 1 Late body weight 9,657.95AE 1,607.4813,019.72AE 1,768.91 0.000 1 Body weight change (Δ Body weight) 922.56 AE 671.28 855.55 AE 577.1 Independent sample T-test.IL-6 levels were higher in the stunted group compared to the severely stunted group, there was no significant difference between both groups (212.06AE 146.05 vs. 85.45AE 89.06 pg/mL, p = 0.057).Changes in IL-6 (ΔIL-6) based on the LAZ/HAZ categories showed no significant difference (p = 0.055), but the changes in severely stunted children were higher compared to the stunted group (47.33 AE 93.48 vs. -41.66AE 108.69 pg/mL, p = 0.036) and the normal stature group
(47.33 AE 93.48vs.-20.28AE77.36pg/mL,p = 0.031).This was due to an increase in IL-6 levels in severely stunted children, while stunted children and those of normal stature exhibited reduced IL-6 levels.The levels of IL-6 based on the WAZ categories showed no significant difference pre-intervention (p = 0.903) or postintervention (p = 0.173), but the change in IL-6 (ΔIL-6) showed a significant difference (p = 0.014), where the WAZ in severely underweight children increased, while the underweight and weight faltering decreased.Therefore, severely underweight children had higher changes of IL-6 compared to stunted children (78.14AE 32.06 vs. -44.19AE137.03 pg/mL, p = 0.012) and weight faltering/normal weight children (78.14 AE 32.06 vs. -16.09AE73.95 pg/mL, p = 0.001).The initial and late changes of IL-6 (ΔIL-6) based on WLZ/WHZ categories showed no significant difference (p >0.05).The initial level of IL-6 was higher in good nutritional status subjects compared to wasted and severely wasted.But after the intervention, IL-6 levels in good nutritional status subjects were reduced (-22.05AE 92.83 pg/ml), while the wasted and severely wasted group increased (9.73 AE 84.73 and 36.22AE 3.46 pg/ml respectively).

Table 1 :
Data for Manuscript Effect of Lactoferrin in Oral Nutrition Supplement (ONS) towards IL-6 and IL-10 in Failure to Thrive Childre.xlsxTREND checklist for 'Effect of Lactoferrin in Oral Nutrition Supplement (ONS) towards IL-6 and IL-10 in Failure to Thrive Children with Infection', https://www.doi.org/10.6084/m9.figshare.22210798.v3. 45Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0) ○ Result of OD + Code.pdf○Result of OD Excel.xls○Result of OD.pdf ○ Standard curve.pdf○Sample scheme & Standard.pdf•Data archive 2: Elisa IL-6 Post Intervention.rar○The concentration of IL-6, ng per L.pdf ○ Result of OD + Code.pdf○Result of OD Excel.xls○Result of OD.pdf ○ Standard curve.pdf○Sample scheme & Standard.pdf•Data archive 3: Elisa_IL-10 Pre Intervention.rar○Result of concentration IL-10, pg per ml.pdf ○ Result of OD + Code.pdf○Result of OD Excel.xls○Result of OD.pdf ○ Standard curve.pdf○Sample scheme & Standard.pdf•Data archive 4: Elisa_IL-10 Post Intervention.rar○Result of calculation concentration pg per ml.pdf ○ Result of OD + Code.pdf○Result of OD Excel.xls○ Result of OD.pdf ○ Standard curve.pdf○ Sample scheme & Standard.pdf68.Sakr Y, Dubois MJ, De Backer D, et al.: Persistent-microcirculatory alterations are associated with organ failure and death in patients with septic shock.Crit.Care Med.2004; 32(9): 1825-1831.Publisher Full Text 69.Urlacher SS, Ellison PT, Sugiyama LS, et al.: Tradeoffs between immune function and childhood growth among Amazonian forager-horticulturalists. Proc.Natl.Acad.Sci.U. S. A. 2018; 115(17): E3914-E3921.Reference Source 76.Solis B, Nova E, Gómez S, et al.: The effect of fermented milk on interferon production in malnourished children and in anorexia nervosa patients undergoing nutritional care.Eur.J. Clin.Nutr.2002; 56: S27-S33.PubMed Abstract|Publisher Full Text 77. de Brito RCCM, Lucena-Silva N, Torres LC, et al.: The balance between the serum levels of IL-6 and IL-10 cytokines discriminates mild and severe acute pneumonia.BMC Pulm.Med.2016; 16(1): 121-170.PubMed Abstract|Publisher Full Text|Free Full Text 28" in which reference 28 is "Mckenna L, Sari AH, Mane S, et al.:The headings for columns 4 in table 5 and table 6 seem to be incorrectly labeled.We previously flagged table 6 as needing to have a ΔIL6/IL10 ratio.Instead, it would appear that the authors have mistakenly labeled the Δ in table 5, which does not seem accurate.