Impact of Heat-Killed Lactobacillus casei Strain IMAU60214 on the Immune Function of Macrophages in Malnourished Children.

Malnutrition is commonly associated with immunological deregulation, increasing the risk of infectious illness and death. The objective of this work was to determine the in vitro effects of heat-killed Lactobacillus casei IMAU60214 on monocyte-derived macrophages (MDMs) from well-nourished healthy children, well-nourished infected children and malnourished infected children, which was evaluated by an oxygen-dependent microbicidal mechanism assay of luminol-increase chemiluminescence and the secretion of tumor necrosis factor (TNF-α), interleukin (IL-1ß), IL-6 and IL-10, as well as phagocytosis using zymosan and as its antibacterial activity against Salmonella typhimurium, Escherichia coli and Staphylococcus aureus. We found that reactive oxygen species (ROS), secretion cytokines (TNFα, IL-1ß, IL-6 and IL-10 levels), phagocytosis and bactericidal capacity increased in all groups after pre-treatment with heat-killed L. casei IMAU60214 at a ratio of 500:1 (bacteria:MDM) over 24 h compared with MDM cells without pre-treatment. The results could indicate that heat-killed L. casei IMAU60214 is a potential candidate for regulating the immune function of macrophages.

Immune Activation and Microbial Translocation Markers in HIV-Exposed Uninfected Malawian Infants in the First Year of Life.

BACKGROUND: HIV-exposed uninfected (HEU) infants show a high rate of morbidity. We aimed to investigate on biomarkers of immune activation/microbial translocation in HEU infants, evaluating the impact that infections/malnutrition can have on biomarker levels during the first year of life. METHODS: Clinical data of 72 Malawian infants were recorded monthly and correlated with levels of soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP) and intestinal fatty acid-binding protein (I-FABP), analyzed longitudinally. RESULTS: Levels of sCD14 and LBP showed a significant age-related increase. Higher levels of LBP (19.4 vs. 15.2 µg/ml) were associated with stunting, affecting 30% of the infants. The association remained statistically significant after adjusting for cytomegalovirus acquisition, malaria and respiratory infections (p = 0.031). I-FABP levels were significantly increased in infants experiencing gastrointestinal infections (1442.8 vs. 860.0 pg/ml, p = 0.018). CONCLUSION: We provide evidence that stunting is associated with an enhanced inflammatory response to microbial products in HEU children, suggesting that malnutrition status should be taken into consideration to better understand the alteration of the immune profile of HEU infants living in poor socioeconomic settings.

Biological effects of anti-RANKL antibody administration in pregnant mice and their newborns.

Denosumab, a fully human monoclonal antibody that neutralizes receptor activator of nuclear factor-κB ligand (RANKL) and blocks osteoclast differentiation, has received approval in Japan for use as an anti-resorptive drug for osteoporosis and skeletal-related events (SREs) in patients with solid cancer. Denosumab is contraindicated during pregnancy, though the effects of blocking RANKL activity on pregnant mothers and their newborns are unclear. We used mice to investigate the effects of an anti-RANKL antibody on maternal and newborn health. Mothers injected with the anti-RANKL antibody had increased bone mass as compared with the controls, while osteoclast number and the level of tartrate-resistant acid phosphatase (TRAP) in serum were increased at the end of pregnancy. Newborn mice exposed to the antibody in utero were normally born, but showed increased bone mass and died within 48 h after birth. None of the newborns were found to have milk in their stomachs, suggesting that they died due to a maternal defect in lactation. Consistent with this, anti-RANKL antibody-injected mothers displayed impaired mammary gland development. However, fostering by healthy surrogate mothers rescued only 33% of the antibody-exposed newborns, suggesting that neonatal mortality was due, at least in part, to an intrinsic defect in the newborns. Our findings show that anti-RANKL antibody administration during pregnancy results in not only an undesirable increase in bone mass, but also has harmful effects on newborn survival.

Validity of antibodies in lymphocyte supernatant in diagnosing tuberculosis in severely malnourished children presenting with pneumonia.

BACKGROUND: The diagnosis of tuberculosis (TB) in young children can be challenging, especially in severely malnourished children. There is a critical need for improved diagnostics for children. Thus, we sought to evaluate the performance of a technique that measures antibodies in lymphocyte supernatant (ALS) for the diagnosis of TB in severely malnourished children presenting with suspected pneumonia. METHODS: Children less than 5 years with severe acute malnutrition and radiological features of pneumonia admitted to the Dhaka Hospital of International Centre for Diarrhoeal Disease Research, Bangladesh, were enrolled consecutively following informed written consent. In addition to clinical and radiological assessment, samples taken for TB diagnosis included gastric lavage fluid and induced sputum for microbiological confirmation. ALS was measured from venous blood, and results were evaluated in children classified as "confirmed", "non-confirmed TB" or "not TB". RESULTS: Among 224 children who had ALS analysis, 12 (5.4%) children had microbiologically "confirmed TB", a further 41 (18%) had clinically diagnosed "non-confirmed TB" and the remaining 168 (75%) were considered not to have TB. ALS was positive in 89 (40%) and negative in 85 (39%) of children, with a large number (47 or 21%) reported as "borderline". These proportions were similar between the three diagnostic groups. The sensitivity and specificity of ALS when comparing "Confirmed TB" to "Not TB" was only 67% (95% CI: 31-91%) and 51% (95% CI: 42-60%), respectively. CONCLUSIONS AND SIGNIFICANCE: Our data suggest that ALS is not sufficiently accurate to improve the diagnosis of TB in children with severe malnutrition.

Challenges of malnutrition care among HIV-infected children on antiretroviral treatment in Africa.

More than 90% of the estimated 3.2 million children with HIV worldwide, at the end of 2013, were living in sub-Saharan Africa. The management of these children was still difficult in 2014 despite the progress in access to antiretroviral drugs. A great number of HIV-infected children are not diagnosed at 6 weeks and start antiretroviral treatment late, at an advanced stage of HIV disease complicated by other comorbidities such as malnutrition. Malnutrition is a major problem in the sub-Saharan Africa global population; it is an additional burden for HIV-infected children because they do not respond as well as non-infected children to the usual nutritional care. HIV infection and malnutrition interact, creating a vicious circle. It is important to understand the relationship between these 2 conditions and the effect of antiretroviral treatment on this circle to taking them into account for an optimal management of pediatric HIV. An improved monitoring of growth during follow-up and the introduction of a nutritional support among HIV-infected children, especially at antiretroviral treatment initiation, are important factors that could improve response to antiretroviral treatment and optimize the management of pediatric HIV in resource-limited countries.

Immunogenicity of poliovirus vaccines in chronically malnourished infants: a randomized controlled trial in Pakistan.

Reaching high population immunity against polioviruses (PV) is essential to achieving global polio eradication. Efficacy of oral poliovirus vaccine (OPV) varies and is lower among children living in tropical areas with impoverished environments. Malnutrition found as a risk factor for lower serological protection against PV. We compared whether inactivated polio vaccine (IPV) can be used to rapidly close the immunity gap among chronically malnourished (stunted) infants in Pakistan who will not be eligible for the 14 week IPV dose in routine EPI schedule. A phase 3, multicenter 4-arm randomized controlled trial conducted at five Primary Health Care (PHC) centers in Karachi, Pakistan. Infants, 9-12 months were stratified by length for age Z score into chronically malnourished and normally nourished. Infants were randomized to receive one dose of either bivalent OPV (bOPV) alone or bOPV+IPV. Baseline seroprevalence of PV antibodies and serum immune response to study vaccine dose were assessed by neutralization assay. Vaccine PV shedding in stool was evaluated 7 days after a bOPV challenge dose. Sera and stool were analyzed from 852/928 (92%) enrolled children. At baseline, the seroprevalence was 85.6% (n=386), 73.6% (n=332), and 70.7% (n=319) in malnourished children against PV types 1, 2 and 3 respectively; and 94.1% (n=448), 87.0% (n=441) and 83.6% (n=397) in the normally nourished group (p<0.05). Children had previously received 9-10 doses of bOPV (80%) or tOPV (20%). One dose of IPV+bOPV given to malnourished children increased their serological protection (PV1, n=201, 97.6%; PV2, n=198, 96.1% and PV3, n=189, 91.7%) to parity with normally nourished children who had not received IPV (p=<0.001). Seroconversion and boosting for all three serotypes was significantly more frequent in children who received IPV+bOPV than in those with bOPV only (p<0.001) in both strata. Shedding of polioviruses in stool did not differ between study groups and ranged from 2.4% (n=5) to 7.1% (n=15). In malnourished children the shedding was reduced after bOPV+IPV compared to bOPV only. Chronically malnourished infants were more likely to be unprotected against polioviruses than normal infants. bOPV+IPV helped close the immunity gap better than bOPV alone.

Neonatal overfeeding alters hypothalamic microglial profiles and central responses to immune challenge long-term.

The early life period is one of significant vulnerability to programming effects from the environment. Given the sensitivity of microglial cells to early life programming and to adult diet, we hypothesized overfeeding during the neonatal period would acutely alter microglial profiles within the developing brain, predisposing the individual to a lasting central pro-inflammatory profile that contributes to overactive immune responses long-term. We tested this idea by manipulating litter sizes in which Wistar rat pups were raised, so the pups were suckled in litters of 4 (neonatally overfed) or 12 (control). This manipulation induces obesity and susceptibility to lipopolysaccharide (LPS) long-term. We then examined microglial and central pro-inflammatory profiles during development and in adulthood as well as susceptibility to neuroimmune challenge with LPS. Neonatally overfed rats have evidence of microgliosis in the paraventricular nucleus of the hypothalamus (PVN) as early as postnatal day 14. They also show changes in hypothalamic gene expression at this time, with suppressed hypothalamic interleukin 1ß mRNA. These effects persist into adulthood, with basal PVN microgliosis and increased hypothalamic toll-like receptor 4, nuclear factor κB, and interleukin 6 gene expression. These neonatally overfed rats also have dramatically exacerbated microglial activation in the PVN 24h after an adult LPS challenge, coupled with changes in inflammatory gene expression. Thus, it appears neonatal overfeeding sensitizes PVN microglia, contributing to a basal pro-inflammatory profile and an altered response to a neuroimmune challenge throughout life. It remains to be seen if these effects can be reversed with early interventions.

T lymphocytes from malnourished infants are short-lived and dysfunctional cells.

To investigate T-cell functional molecules and inflammatory cytokines and to assess T-cell apoptosis in malnourished infants, 64 infants from undernourished women and 28 healthy control infants were recruited to the study. Malnourished infants showed a significant decrease in the levels of circulating IL-2 and IL-7 and increases in the levels of IL-1ß, IL-6, IL-10 and TNF-α, as measured by flow cytometry. There was a significant reduction in the number of CD3(+) T cells and an increase in apoptotic T cells, which was associated with an up-regulation of CD95 and PD-1 expression on CD3(+) T cells in malnourished compared to control infants. Significant reductions were also observed in the phosphorylation of AKT and STAT5 and in the expression of CCR7 and CXCR4 receptors in malnourished children, and these reductions were associated with a significant reduction in T-cell migratory capacity to their ligands CCL21 and CXCL12, respectively, as measured using an in vitro chemotaxis assay. Taken together, these data suggest that lymphocytes from malnourished infants are short-lived and dysfunctional.

Progress of enteral feeding practice over time: moving from energy supply to patient- and disease-adapted formulations.

Enteral nutrition comprises the delivery of a liquid formula beyond the esophagus via a feeding tube in a patient with insufficient oral intake, as well as the provision of specialized nutritional formula irrespective of the route of delivery. Pediatric formulae have been designed for different age groups, and for children with certain diseases; examples are special formulations for regurgitating infants, metabolic diseases, cow's milk or multiple food allergies, intestinal, pancreatic, renal, and hepatic insufficiency. Exclusive enteral nutrition is a therapeutic concept to induce remission in children and adolescents with active Crohn's disease. A new area of nutritional research in pediatrics is potential immunonutrition in critically ill children. Formulae are enriched with single components or a combination of key substrates that might play a crucial role during intermediary metabolism in sepsis, inflammation, tissue healing, and growth. For pharmaconutrition, single components are investigated in a scientific stepwise procedure in order to identify effective disease-dedicated nutrition therapy. Any new formula needs to be evaluated, if possible in comparison to a normal diet or the reference formulation to demonstrate its safety and efficacy (equal or superior to standard formula).

Sustainable clinical research, health economic aspects and medical marketing: drivers of product innovation.

Marketing-driven innovation in the field of pediatric nutrition, in particular in the infant formula segment is not sustainable. New benefits of products must be scientifically proven and safety and efficacy of new formulae established in clinical trials. The scientific innovation process of three infant formulae is described. Improvement in protein quality allowed to reduce the protein concentration in whey-based infant formula. Weight gain and BMI of infants fed those formulae corresponds to breastfed infants and is lower than in infants fed traditional formulae with higher protein concentration. A meta-analysis indicates associations between rapid weight gain in infancy and obesity later in life. If infants cannot be exclusively breastfed until 4-6 months of age, feeding low-protein formulae may contribute to positive long-term health outcome with potentially important health economic effects. A partially hydrolyzed whey based formula for prevention of allergic symptoms in children with hereditary risk for allergic diseases was developed more than 25 years ago. The most recent meta-analysis which included 15 randomized clinical trials indicates that the risk of all allergic diseases and atopic dermatitis/eczema is significantly reduced in infants at risk when the partially hydrolyzed formula is fed. The partially hydrolyzed formula had the same protective effect as casein-based high-degree extensively hydrolyzed formula. Because of substantial price differences between the two formulae, feeding the partially hydrolyzed whey formula is cost saving. Hypoallergenic claims can be made in many countries, and international nutrition committees have positively commented the preventive effect of those formulae. Acidified formulae have been widely used during the last decade in replacement feeding programs for infants whose mothers are HIV positive. The formula was innovated by improving whey protein quality and lowering protein concentration. The bacteriostatic properties of the new formula were proven in in vitro tests. Meta-analysis indicated that feeding the formula to immunocompromised infants resulted in growth similar to breastfeeding. The bacteriostatic effects of the acidified formula need to be communicated to health care professionals, but also the risks if replacement feeding is not acceptable, feasible, affordable, sustainable, and safe for mother and infant.

Evaluation of dietetic product innovations: the relative role of preclinical and clinical studies.

A variety of systems are used to establish efficacy of food ingredients. Immortal human cell lines have the advantage of rapid throughput and often have the ability to point to mechanisms of action. Transgenic and natural variants of animals (usually rats and mice) have proven to be extremely useful in elucidating effects in vivo, although extrapolation of results to humans has risks. Animal models are also useful in establishing safety and toxic levels of ingredients. Human trials have the most relevance to society. Types of evidence for efficacy rise from improved status level in subjects as a result of eating food (long-chain polyunsaturated fatty acid, levels in erythrocytes), change in surrogate markers as a result of eating food (plasma cholesterol or glutathione peroxidase activity), change in a physiological outcome (such as visual evoked potential acuity or heart rate variability) through to the highest level of evidence, a change in a clinical outcome (improved global development, reduction in infections) established in randomized controlled trials. Ultimately, there is a need for tests of pragmatic interventions that can easily be incorporated into usual dietary practices of the culture in which it is tested.

CD95 expression in white blood cells of malnourished infants during hospitalization and catch-up growth.

Malnutrition continues to be a major health burden in developing countries. Flow cytometric estimation of the apoptotic marker CD95 in peripheral neutrophils, lymphocytes and monocytes was done for 18 infants with non-oedematous protein energy malnutrition (PEM) and 12 oedematous ones, on hospital admission and after supervised nutritional rehabilitation, and compared with 12 matched controls. CD95 counts in the 3 types of white blood cells were significantly higher in PEM infants and showed improvement after nutritional rehabilitation yet not reaching the control values. Enhanced apoptosis in the leukocytes of peripheral blood of PEM patients may be a marker of increased infection and immune disturbances. This derangement reverses upon proper nutritional rehabilitation.

Perinatal malnutrition programs sustained alterations in nitric oxide released by activated macrophages in response to fluoxetine in adult rats.

OBJECTIVE: Nutritional restriction during lactation has long-term consequences on the functioning of neuroimmune systems. Receptors and transporter serotonin (5-HT) are present in macrophages and may influence their role. This study evaluated nitric oxide release by alveolar macrophages (AMs) in adult control rats and rats malnourished during lactation in response to different fluoxetine (FLX) concentrations and 5-HT(1A) and 5-HT(1B) agonists at different times. METHODS: Male Wistar rats were distributed into two groups according to maternal diet during lactation: a control group of 12 rats whose dams had received a 23% protein diet and a malnourished group of 12 rats whose dams had received an 8% protein diet. After weaning, all rats received a 23% protein diet. On the 90th day after birth, nitric oxide (NO) release kinetics was measured in supernatants of AMs cultured with FLX. The NO release following the adjunction of serotoninergic agonists was also quantified. RESULTS: The malnourished rats weighed less at weaning (control rats = 15.3 +/- 0.4 g, malnourished rats = 11.8 +/- 0.4 g); this difference persisted until 90 days of life (control rats = 355.4 +/- 8.6 g; malnourished rats = 267.8 +/- 7.9 g). In the presence of 10(-6)M FLX, NO release by AMs in control rats was lower. The addition of agonists did not interfere with NO release by AMs in control rats. NO release by AMs from malnourished rats was modified neither by FLX nor by agonists. As a consequence of malnutrition, there were lower numbers of cells and AMs in the bronchoalveolar lavage fluid, and cell viability and NO release by AMs were impaired. CONCLUSIONS: Nutritional manipulation in the perinatal period seems to interfere with the functional programming of macrophages; it also seems to affect their serotoninergic regulation through adulthood.

Neonatal programming of neuroimmunomodulation--role of adipocytokines and neuropeptides.

Programming is an epigenetic phenomenon by which nutrition, environment and stress acting in a critical period earlier in life change the organism's development. This process was evolutionarily selected as an adaptive tool for the survival of organisms living in nutritionally deficient areas and submitted to stressful conditions. Thus, perinatal malnutrition turns on different genes that provide the organism with a thrifty phenotype. In conditions of abundant supply of nutrients, those programmed organisms can be at risk of developing metabolic diseases (obesity, dyslipidemia, diabetes and hypertension). How nutrition or neonatal stress can program the immune system is less well known. Here, we discuss some of the hormonal and metabolic changes that occur in mothers and neonates and how those factors can imprint hormonal or metabolic changes that program neuroimmunomodulatory effects. Some of these changes involve thyroid hormones, leptin, insulin, glucocorticoids and prolactin as potential imprinting factors. Most of them can be transferred through the milk and may change with malnutrition or stress. We discuss the programming effects of these hormones upon body weight, body composition, insulin action, thyroid, adrenal and immune and inflammatory responses, with special emphasis on leptin, a cytokine that seems to play a central role in these events.

Expression of cytokine mRNA in lymphocytes of malnourished children.

INTRODUCTION: Protein-calorie malnutrition represents a significant worldwide health problem and is associated with an increased risk for infections. The purpose of this study was to evaluate possible changes in type 1/type 2 responses balance in malnourished children. RESULTS: The data obtained in the present study showed that the expression levels of tumor necrosis factor-alpha, interleukin (IL)-4, and IL-10 were more highly, in contrast IL-2, gamma interferon, and IL-6 genes were expressed less in all groups of malnourished children compared with the well-nourished infected children. It is important to indicate that the data collected in the present work agree with the results obtained by different authors, who showed differences in the production of cytokines in malnourished children. CONCLUSION: In conclusion, the results suggest that alterations in the balance of type 1/type 2 immune responses exist in malnourished children, and this could be the reason that the immunological system of the malnourished children is incapable of eradicating infections.

Stress/aggressiveness-induced immune changes are altered in adult rats submitted to neonatal malnutrition.

BACKGROUND/AIMS: Neonatal malnutrition induces metabolic and endocrine changes that have beneficial effects on the neonatal in the short term but, in the longer term, these alterations lead to maladaptations. We investigated the effect of neonatal malnutrition on immune responses in adult rats submitted or not to an aggressiveness test. METHODS: Male Wistar rats were distributed to one of two groups according to their mothers' diet during lactation: the well-nourished group (group C, n = 42, receiving 23% of protein) and the malnourished group (group MN, n = 42, receiving 8% of protein). After weaning, all rats received normoproteic diet. Ninety days after birth, each group was subdivided into three subgroups: control rats (n = 14, respectively), aggressive rats (n = 14, respectively) and rats receiving foot shock (FS; n = 14, respectively). Plasma corticosterone concentration was measured after FS sessions. Leukocyte counts and humoral immunity were evaluated. RESULTS: In neonatal malnourished animals, FS-induced stress reduced plasma corticosterone concentration. Intraspecific aggressiveness induced alterations in leukocyte counts and antibody titers 7 and 15 days after immunization. Neonatal malnourished animals showed no changes in the immune parameters evaluated. CONCLUSIONS: Expression of intraspecific aggressiveness activates the immune system. Neonatal malnutrition seems to have a long-lasting effect on components of both neuroendocrine and immune functions.

Flow cytometry study of lymphocyte subsets in malnourished and well-nourished children with bacterial infections.

Protein-energy malnutrition is the primary cause of immune deficiency in children across the world. It has been related to changes in peripheral T-lymphocyte subsets. The aim of the present study was to evaluate the effects of infection and malnutrition on the proportion of peripheral-lymphocyte subsets in well-nourished non-bacterium-infected (WN), well-nourished bacterium-infected (WNI), and malnourished bacterium-infected (MNI) children by flow cytometry. A prospectively monitored cohort of 15 MNI, 12 WNI, and 17 WN children was studied. All the children were 3 years old or younger and had only bacterial infections. Results showed a significant decrease in the proportion of T CD3(+) (P < 0.05 for relative and P < 0.03 for absolute values), CD4(+) (P < 0.01 for relative and absolute values), and CD8(+) (P < 0.05 for relative values) lymphocyte subsets in WNI children compared to the results seen with WN children. Additionally, B lymphocytes in MNI children showed significant lower values (CD20(+) P < 0.02 for relative and P < 0.05 for absolute values) in relation to the results seen with WNI children. These results suggest that the decreased proportions of T-lymphocyte subsets observed in WNI children were associated with infection diseases and that the incapacity to increase the proportion of B lymphocyte was associated with malnutrition. This low proportion of B lymphocytes may be associated with the mechanisms involved in the immunodeficiency of malnourished children.

Neonatal enteropathies: defining the causes of protracted diarrhea of infancy.

The underlying causes of chronic diarrhea beginning early in life are increasingly well defined. Infectious and post-infectious enteropathies and food sensitive/allergic enteropathy account for the majority of cases. Recent attention has focused on characterizing defined entities, which cause protracted diarrhea in infants and young children. Disorders of intestinal ion transport usually present at birth following a pregnancy complicated by polyhydramnios. Intestinal mucosal biopsies show normal architect with intact villus-crypt axis. Neonatal enteropathies, by contrast, are characterized by blunting of the villi. These include microvillus inclusion disease, tufting enteropathy, autoimmune enteropathy and IPEX syndrome - and it is these conditions that are the subject of the current review.