Isoleucine Added ORS in Children With Diarrhoea
Condition: Acute Infectious Diarrhoea in Children
MeSH terms: Dysentery; Diarrhea
Study type: Interventional
Phase: Phase 2

Summary: 
      Efforts are continuing to improve the efficacy of oral rehydration solution in terms of
      reducing the severity (stool volume) and duration of diarrhoea (enhancement of recovery).
      Antimicrobial peptides, produced by the epithelial cells, represent an important component of
      the innate immunity of all epithelial surfaces of the body including intestine. Induction of
      expression of antimicrobial peptides on epithelial cell surface, may thus hasten recovery
      from infectious diarrhoea. Isoleucine is an essential amino acid that is easily available and
      not very expensive, and its addition to oral rehydration solution might help early clearance
      of diarrhoeal pathogen by inducing secretion of antimicrobial peptide by the small intestinal
      epithelial cells. Additionally, it is expected to hasten recovery from diarrhoea by
      reestablishing the commensal bacteria. The aim of this pilot study is to assess if addition
      of isoleucine to oral rehydration solution induces secretion of antimicrobial peptide, help
      establish normal bacterial populations in the intestine, and favourably impacts the severity
      and duration of diarrhoea in young children. If the results are found encouraging, a
      therapeutic trial with adequate sample size would be justified.
    

Detailed description: 
      Globally, diarrhoea still accounts for 1.6 to 2.5 million deaths annually, and children in
      the developing world experience an average of 3 episodes of diarrhoea each year. Despite the
      decline in diarrhoeal mortality, it remains a leading cause of childhood morbidity and deaths
      [1]. Prevention of dehydration using appropriate home-made fluids or ORS solution, and
      correction of dehydration using ORS solution or intravenous fluid as indicated followed by
      maintenance of hydration using ORS solution, along with continuation of usual food including
      breastfeeding are most important elements of management of acute diarrhea. Antimicrobial
      therapy is recommended in the management of a few specific etiologic diarrhoeas such as
      severe cholera, shigellosis, invasive intestinal amoebiasis, and symptomatic giardiasis. The
      cost of therapy and more importantly the emergence of resistance pathogens are the major
      concerns for antimicrobial therapy for diarrhoea as for other bacterial infections.

      ORS plays a major role in the treatment diarrhoeal diseases; however, it does neither reduce
      the severity nor the duration of diarrhoea, its major limitations. Efforts are continuing to
      overcome these limitations by developing newer formulations and delivery mechanisms e.g.

        1. Changing/replacing the substrates (e.g. addition of alanine or glycine to glucose or
           replacing glucose by multodextrin and rice powder)

        2. Reducing the concentration of glucose and sodium and thus osmolarity of ORS solution.

      Alanine/glycine and rice powder containing ORS demonstrated some reduction in the stool
      output [2-5]. However, none of the formulations developed and tested till date demonstrated
      any beneficial effect on the duration of diarrhoea. Similarly, reduced osmolarity ORS has
      some beneficial effect on reducing the stool output and vomiting, and reducing failure of ORT
      mainly in children with non-cholera diarrhea, but did not have any effect on the diarrhoea
      duration [6]. The World Health Organization (WHO) and the United Nations Children Fund
      (UNICEF) have recently recommended routine use of the reduced-osmolarity ORS in the
      management of diarrhoeal diseases [7]; however, there are scopes to further improve its
      efficacy.

      Antimicrobial peptides and innate immunity

      Antimicrobial peptides represent an important component of the innate immune defenses of
      organisms ranging from plants to insects to humans. They are broad-spectrum, surface-active
      agents that kill microbes by forming pores in their membranes. Most have broad spectrum of
      activities against bacteria, viruses and fungi. In mammals, two major classes of
      antimicrobial peptides have been described- defensins and cathelicidins (LL-37). The
      defensins are a subclass of antimicrobial peptides, and in mammals, they are present in
      neutrophil granules where they are necessary for the non-oxidative killing of phagocytised
      microbes [8]. It is now well established that defensins are also produced at virtually all
      epithelial surfaces of mammals, including those of the skin, airways, gut, and urogenital
      tracts [9, 10]. Expression of some epithelial defensins is constitutive and contributes to a
      non-inflammatory antimicrobial barrier at the epithelial surface. Other defensins are
      inducible and highly expressed at sites of inflammation or infection [9, 11, 12]. Impairment
      of defensin functions increases susceptibility to infection of the airway in cystic fibrosis
      [13] and to enhanced Salmonella infection in the mouse intestinal tract [14]. In addition to
      their direct antimicrobial activities, Beta-defensins are chemotactic for memory T cells and
      dendritic cells, suggesting that they play an important role in the integration of the innate
      and acquired immune responses [15].

      The molecular mechanisms underlying induction of epithelial defensins remain largely
      unexplored. Pattern-recognition receptors [16] likely play a critical role in this process,
      as has been shown in CD-14-mediated induction of Beta-defensins by bacterial
      lipopolysaccharide [17]. Whole heat-killed bacteria and fungi induce human Beta-defensins-2
      in human keratinocytes, but the molecular basis of this response is not understood [11].
      Inflammatory cytokines such as tumor necrosis factor Alpha (TNF) and IL-10 also induces
      Beta-defensins [18, 19]. Because pharmacological induction of defensins at epithelial
      barriers may have therapeutic utility, a search for novel molecules that could induce
      production of epithelial defensin, assessed by using cell-based assays, have long been
      underway. It has recently been observed that L-isoleucine and its analogues are highly
      specific Beta-defensin inducers in epithelial cells [20]. It has also been observed that when
      isoleucine is administered to animals (chicken), the gut flora shifts to the probiotic
      species, it stimulates paneth cells to secrete antimicrobial peptides in human intestinal
      tissue, and it causes a shift in gut flora leading to an increased numbers of Lactobacilli.
      (Manuscript, Ashida T & M Zasloff et al) in treatment of patients with Crohn's disease.

      The above observations suggest a potential role of L-isoleucine in the management of
      infections of mucosal tissues including those in the gastrointestinal tract.

      Antimicrobial peptides in Shigella infections

      In 2001, researchers from ICDDRB and Karolinska Institute (BA) reported in Nature Medicine a
      down regulation of LL-37 and human Beta-defensin-1 (HBD-1) in the epithelial cells in
      diarrhoea caused by Shigella dysenteriae type I and other enteric bacteria [21]. This down
      regulation could be reproduced in vitro by using the cell lines U937 of monocyte origin and
      HT-29 of colonic epithelial origin, when infected with Shigella or plasmid DNA derived from
      Shigella. The down regulation can be an important immune escape mechanism for pathogens to
      avoid potent mucosal effector molecules such as antimicrobial peptides. Since antimicrobial
      factors drench the mucosal surfaces, it is conceivable that pathogenic bacteria down-regulate
      the front line defenses of humans as a part of their invasive process. Therefore, a novel
      approach will be to block this down-regulation or to take a therapeutic approach to
      up-regulate the production of endogenous antimicrobials. Recent studies have shown that short
      chain fatty acids (SCFA) can up-regulate expression of LL-37 in colonic epithelial cells, and
      among them sodium butyrate was the best inducer [22, 23]. Moreover, butyrate induces colonic
      cell differentiation that has been reported to be a key determinant of LL-37 expression in
      human colon epithelium [22]. However, it was shown that pathways other than those involved in
      cell differentiation are involved in the up-regulation of LL-37 with butyrate [23]. Butyrate
      is produced in the colon by fermentation of dietary fibre. Thus, there is a link between the
      normal gut flora and the expression of endogenous antibiotics. Additionally, SCFAs including
      butyrate has been shown to reduce clinical symptoms, and alter microbiological and
      pathological features in experimental Shigella infections in rabbit models [24]. Thus, a
      relationship between the severity of Shigella infections and mucosal levels of antimicrobial
      peptides is conceivable. Oral administration of sodium butyrate to rabbits infected with
      Shigella showed up-regulation of CAP-18 (rabbit homologue of LL-37) in the colonic epithelial
      cells with a concomitant reduction in shigella load in stool and clinical recovery (Raqib et
      al, manuscript accepted). Preventing down-regulation or enhancing antimicrobial peptide
      expression could provide an alternative treatment in protecting humans and animal livestock
      against bacterial pathogens. These studies may provide a basis for therapeutic manipulation
      of endogenous antimicrobial peptides including LL-37 expression in vivo using dietary
      substances or butyrate to strengthen the epithelial defense barrier. Therefore, we have
      initiated a clinical study in ICDDRB to evaluate the efficacy of butyrate enema in the
      treatment of shigellosis.

      Butyrate has been shown to induce antimicrobial peptides in the colon; however; it can't be
      administered orally as such. Moreover, most of the acute diarrhoeal diseases are caused by
      infection of the small intestine. Isoleucine, an essential amino acid, might be a reasonable
      alternative for enhancing secretion of antimicrobial peptides from the paneth cells and other
      defense molecules from the upper small intestine (Manuscript, Ashida T & M Zasloff et al).
      This may help clear the pathogens, shift gut flora to a more "normal" commensal species
      distribution, and enhance recovery from infectious diarrhoea. Therefore, we hypothesize that
      use of L-isoleucine supplemented ORS solution will induce antimicrobial peptides and enhance
      clinical recovery of children with acute watery diarrhoea.
    

Intervention name(s): ORS + Isoleucine; ORS without Isoleucine
Intervention type(s): Other; Other
Intervention description(s): ORS with Isolelucine for treatment of diarrhoeal in children; ORS without Isoleucine for the treatment of diarrhoea in children
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Quadruple (Participant, Care Provider, Investigator, Outcomes Assessor)
Primary purpose: Treatment
Keywords: Diarrhoea; L-isoleucine; children; ORS; Antibacterial peptide