BACKGROUND
the ability to eat sufficient amounts orally is inversely associated with the extent of frailty and studies have indicated that nutritional intake is often compromised in the elderly  <cit> .
poor gut health and secondary health issues as a consequence are matters of nutritional attention for the elderly, infants and immune compromised persons.
diet is a factor that can be extremely varied in terms of quantity, quality, frequency and duration of meals in individuals, but that can also be modified with targeted dietary interventions to produce the best results for any individual  <cit> .
diets of the elderly are generally low in protein, low in energy and deficient in a range of micro-nutrients  <cit> .
high protein diets, in particular those based on whey proteins, are being marketed to improve the health status of the elderly  <cit>  as well as other target groups e.g.
athletes and babies.
furthermore micro-nutrients including anti-oxidants e.g.
vitamins a, c and zinc are also being used for fortification or as dietary supplements for healthy older patients  <cit> .
there are growing number of compositional foods under development, being patented or emerging in the market that target specific nutritional deficiencies beyond the traditional infant formula market.
recent examples include wo2007/ <dig> a high protein dietary supplement for treating various diseases associated with protein deficiency ; and wo2007/ <dig> compositions for enhancing vascular integrity, cellular survival and reducing apoptosis in ischema .
enprocal, a recently formulated supplementary food is designed to meet the nutritional needs of the frail elderly and to be delivered to them in common every day foods.
enprocal's formulation was developed by specialised aged care dieticians and a food scientist experienced in powder based food supplements.
the ingredients of enprocal such as dairy-based proteins , vitamins and minerals , vegetable oils and inulin  were selected to meet the nutritional profile of elderly specified though dietetic input .
with whey protein concentrate   as its key ingredient it provides i) the highest-quality protein , ii) rapid protein digestion, iii) essential amino acids, in particular cysteine, threonine and leucine that may be required in increased levels in the elderly  <cit> , iv) cysteine to improve glutathione status and muscle protein synthesis, v) high concentrations of branched chain amino acids, and vi) minor bioactive components such as immuno-globulins, growth factors, and anti-microbial proteins and peptides to assist in effective gut function  <cit> .
today, whey is a popular dietary protein supplement purported to provide antimicrobial activity, immune modulation, improved muscle strength and body composition, and to prevent cardiovascular disease and osteoporosis .
enprocal with wpc as its key ingredient therefore contains high levels of constituent proteins including beta-lactoglobulin, alpha-lactalbumin, lactoferrin, lactoperoxidase and glycomacropeptide which have been demonstrated a range of immune-enhancing properties  <cit> .
in addition, whey has the ability to act as an antioxidant, antihypertensive, antitumor, hypolipidemic, antiviral, antibacterial, and chelating agent  <cit> .
whey proteins have been reported to enhance digestion and gut function  <cit>  as well as glutathione production and immune function, hence increasing dietary availability may promote general health in a variety of ways  <cit> .
developing and maintaining a healthy intestinal tract is a pre-requisite for general health and aiding disease prevention.
the gut epithelial cells lining of the intestinal lumen are the first point of contact between intestinal contents and the rest of the body.
it is at this interface that nutrition, environment and genetics come together to determine gut health.
the intestinal mucosa is lined by a monolayer of intestinal epithelial cells joined together at their apical poles by tight junctions, and supported by an extensive and complex immune system and transcellular pathways for nutrient absorption  <cit> .
the mucosal surface of the gastrointestinal tract forms a barrier that separates the luminal contents from the effector immune cells underneath.
with the emergence of the concept of immunophysiology, it is clear that the function of the intestinal epithelium is affected by many immune cell types  <cit> .
any abnormal activation of such immune cells  overproduces the inflammatory cytokines e.g., tumor necrosis factor -α, interleukin -1β and il- <dig> that cause destructive cell damage to the intestinal epithelial monolayers and subsequent mucosal inflammation leading to inflammatory bowel disease  such as crohn's disease  and ulcerative colitis   <cit> .
cellular injury from reactive oxygen species  is implicated in a wide variety of gut diseases and pathologic conditions  <cit> .
there is a growing body of evidence that oxidative stress may contribute to gut inflammation, ulcers, inflammatory bowel disease, and leaky gut syndrome.
newborn and the elderly are more prone to oxidant injury  <cit> .
the present study was aimed to assess the potential of enprocal to improve gut and immune health functions.
we investigated specifically whether enprocal, after digestion with a cocktail of gut enzymes maintained gut cell proliferation and mucosal integrity, possessed immunomodulatory , anti-inflammatory  and anti-oxidative properties.
we sought to achieve conditions as close as possible to those of the human digestive tract by employing in vitro, most widely used human gut epithelial cells alone  or in coculture with human immune thp- <dig>  and jurkat  cells in transwell system.
fhs  <dig> int and caco- <dig> cell lines used worldwide by the researchers were employed as the models of human intestinal epithelial cells in the study.
fhs 74-int cells, used in the study were although derived from normal human fetal intestine but have been reported to show mature epithelial-like characteristics  <cit> .
the growth-promoting activities of egf, tgf-α igf-i and insulin were examined using the same intestinal cells  <cit> .
caco- <dig> cells  have gained enormous popularity as a reliable and high-throughput in vitro model system for the study of human intestinal permeability and evaluation of a large number of drug candidates for their intestinal absorption  <cit> .
for orally administered compounds, permeability through caco- <dig> cell monolayers correlates well with human in vivo absorption  <cit> .
caco- <dig> monolayers can display electrical properties typical of either small intestinal or colonic enterocytes  <cit> .
caco- <dig> cells are able to differentiate spontaneously in culture and display a small intestine-like  phenotype.
the differentiated cells polarise, form microvilli and secrete enzymes associated with the enterocyte brush border such as sucrase-isomaltase, alkaline phosphatase, alkaline phosphatase, lactase and aminopeptidase  <cit> .
this cell line thus represents an appropriate model for the study of transport mechanisms related to the intestinal barrier and for investigating diverse problems of nutrients bioavailability and absorption without concern for differences between human and animals  <cit> .
our study, provides the first report on the bioactivity of digested enprocal components which may be released through enzymatic action from its key ingredients such as whey protein concentrate for gut epithelial cell growth, integrity, absorptive, immunomodulatory, anti-inflammatory and anti-oxidative properties.
RESULTS
digested enprocal  maintains cell growth, enhances cell proliferation and shows no cytotoxicity in normal human intestinal epithelial cells
near complete digestion of enprocal and other digested milk product controls was achieved after  <dig> hours incubation with gut enzyme cocktail mixture .
in our preliminary experiments we treated enprocal with the gut enzyme cocktail at different enzyme cocktail: protein ratios at ph  <dig> – <dig> .
a 1: <dig> ratio of enzyme cocktail: protein at ph  <dig>  was found to be most optimal and complete digestion was achieved after  <dig> hours.
further it was deemed necessary to find a concentration range which would not kill/disrupt the cell monolayers needed to test in bioassays on permeability, immunomodulation, cell adhesion, and oxidative stress.
we treated caco- <dig> and fhs  <dig> int cells with digested and undigested samples of enprocal at different concentrations  to determine the effect on cell viability and growth determined by trypan blue exclusion assay .
with both enprocal undigested and digested samples, the caco- <dig> cell viability as compared to that of fhs  <dig> int was significantly reduced in  at concentrations  <dig> and  <dig> μg/ml.
there was visible reduction in caco- <dig> viability with enprocal d compared to undigested enprocal.
however, these levels did not reach any significance.
there was no effect of all concentrations tested in the range of 100– <dig> μg/ml on the cell viability of normal intestinal fhs  <dig> int cells.
this was further confirmed in the ldh released cytotoxicity assay .
there was no significant increase  in the cytotoxicity as compared to control  when fhs  <dig> int cells were treated with digested and undigested samples of enprocal from 100– <dig> μg/ml.
the enzyme cocktail showed no effect of its own on cell viability and cytotoxicity in both the cell lines.
although there was no significant difference between the cell viability and cytotoxicity effects of digested and undigested enprocal samples but to mimic the in vivo system we selected the digested form for bioassays.
from the cell viability and cell cytotoxicity results 500– <dig> μg/ml was selected as the optimal concentration range for the bioassays.
the other control milk product digests at these concentrations were tested for their effects on cell viability in trypan blue assay .
when compared to media only control  and enprocal d, heat smp digest samples reduced significantly  the caco- <dig> cell viability at  <dig> μg/ml.
the control milk products were further tested in ldh release assay , and cytotoxicity levels were below 10% and not significant  at  <dig> μg/ml concentration when compared with enprocal d and media only control.
in tunel and annexin-v-fluos assays, enprocal d did not significantly  increase the apoptotic index  and necrotic index , however heat smp induced significant  ai as compared to media only control in caco- <dig> cells .
in a dose dependent manner  enprocal d increased significantly  the proliferation of fhs  <dig> int cells , determined by mtt assay, as compared to untreated and positive control  cells.
when compared with other control products this effect was significant  except for p <dig>  where the difference was insignificant between enprocal and p <dig>  however the cell proliferation of caco- <dig> cells was decreased significantly following treatment with enprocal d .
enprocal d maintains the mucosal integrity, increases the expression of zo- <dig>  and macromolecular absorption
intestinal caco- <dig> monolayers grown on millipore cell culture inserts in transwell system were used to measure the transepithelial resistance  following treatments with enprocal d and other digested milk product controls at a concentration of  <dig> μg/ml .
at  <dig> hour before treatment, the caco- <dig> monolayers after  <dig> days of growth were found to be fully differentiated to enterocytes and polarized, continuous without any gaps and displayed teer values  <dig> ±  <dig> ohm.cm <dig> in control growth media.
figure 5a showed the teer values of cell monolayers following different treatments relative to control .
the most significant  decrease was observed in all treatments after the first  <dig> hours except for enprocal d and p <dig> .
both p <dig> and enprocal d were able to recover the teer values at  <dig> hours and the increase was maintained further till  <dig> hours duration  as compared to other control milk products.
however, teer resistance values for enprocal d were found to increase further after  <dig> hours.
p <dig> was found to decrease significantly  the teer after  <dig> hours.
the gut enzyme cocktail had no effect on the teer when compared with media only control.
the triton-x, known to disrupt the cell monolayer, was used as the positive control.
it reduced significantly  the teer resistance at  <dig> hours which were not recovered throughout the experimental period.
the monolayer integrity results were further confirmed by expression of tight junction protein -zo- <dig>  figure 5b shows an upregulation of zo- <dig> expression on caco- <dig> monolayers treated with  <dig> μg/ml of enprocal and p <dig> digests for  <dig> hours.
in horseradish peroxidase  flux assay, enprocal d aided macromolecular transport through the caco- <dig> cell monolayer .
hrp flow through increased with time after treatment with enprocal d and other milk product digests.
after  <dig> hours significant  hrp flux increase  as compared to media only control was observed in enprocal treated cells which increased further to  <dig> and  <dig>  fold after  <dig> and  <dig> hours respectively.
among other products  <dig> ,  <dig> ,  <dig>  and  <dig>  fold increase was observed for heat smp, wpc, p <dig> and p <dig> respectively at  <dig> hours while significant  increase,  <dig>   <dig> ,  <dig>  and  <dig>  fold occurred at  <dig> hours for heat smp, wpc, p <dig> and p <dig> respectively.
as expected the positive control triton x, showed  <dig>  fold increases at  <dig> hours which remained consistent till  <dig> hours.
the enzyme cocktail mixture had no effect when compared with untreated cells.
hrp flux results are shown in percentage for 4– <dig> hours determined in the basolateral compartment.
all samples were assayed in three independent assays each performed in triplicate and the mean for all experiments was calculated and presented as a mean ± sd values.
** indicates a highly significant p <  <dig>  value from the control with media only .
*indicates a significant p <  <dig>  value from the control with media only .
triton-x  <dig> is a known cell disruptor and act as a positive control in hrp flux assay
anti-oxidant activities of enprocal d
we examined the levels of aoe activities in caco- <dig> cells treated with enprocal d and compared with other control commercial milk product  digests.
there was no significant change in the activities of either cuznsod or mnsod in enprocal d and wpc treated cells as compared to vitamin c  treated cells .
this indicates that while enprocal d did not induce oxidative stress in the gut cells, p <dig> treatment led to highly significant  increase in the activities of these enzymes indicating that it is inducing an oxidative stress response.
increased sod activity if not accompanied by similar levels of other aoes may be harmful.
activity levels for cat were not significantly  effected by the treatments with enprocal, wpc and p <dig>  however activity levels for gpx increased significantly  after treatment with p <dig> digest .
enprocal d upregulates the surface expression of co-stimulatory cd <dig>  cd <dig>  cd <dig> mhc i & mhc ii on human macrophages and inhibits the monocyte/lymphocyte-epithelial cell adhesion
in a transwell coculture model of caco- <dig> and pma differentiated thp- <dig> macrophages, movement of bioactive components of with enprocal d and other digested milk product controls from mucosal  to serosal  induced up-regulation of cd <dig>  cd <dig>  cd <dig> and mhc-i & ii expression on thp- <dig> macrophages in a manner similar to that of immune stimulant ifn-γ .
although thp- <dig> macrophages showed no expression of activation markers in response to the negative control , they strongly up-regulated expression of cd <dig> , cd <dig> , cd <dig> , and mhc i & ii  on the cell surface upon treatment with enprocal d, other digested milk product controls compared to no treatment control.
the response observed was maximal with enprocal d.
enprocal d in a dose dependant manner significantly  inhibited the adhesion of lymphocytes and monocytes to caco- <dig> cells.
similar levels of inhibition of adhesion were observed with p <dig> .
enprocal d modulates the cytokine response in human immune cells
the levels of proinflammatory , both th <dig>  and th <dig>  cytokines and chemokine il- <dig> were measured in cell culture supernates of thp- <dig>  and jurkat e6- <dig> cells.
these immune cells were grown in lower  wells of transwell coculture models of caco- <dig> and thp- <dig> caco- <dig> and jurkat e6- <dig> cells.
as shown in table  <dig> the cytokine modulatory effect upon movement of bioactive components of enprocal d and other digested milk product controls from mucosal  to serosal  was evident.
all the cytokine levels in the growth media samples and untreated cells were below the detection limits of elisa.
the cytokines response induced in the lps only stimulated cells was highly significant  as compared to non-stimulated cells and considered 100%.
the levels of lps stimulated tnf-α, il-1β and il- <dig> were reduced dramatically by 91%, 57% and 44% respectively after treating cells with enprocal d as compared to lps only control.
wpc similarly reduced the levels by 85%, 75% and 84% for tnf-α, il-1β and il- <dig> respectively.
however, with p <dig> control product levels of tnf-α and il- <dig> were either negligible or below detection limits and it decreased the levels by 87% for il- <dig> β. there was no effect on il- <dig> production by any of the products tested as compared to maximal response.
when compared with the positive control  enprocal d treatment increased levels of ifn-γ by 29%, while both wpc and p <dig> decreased the levels of ifn-γ by 51% and 88% respectively in the supernates of jurkat e6- <dig> cells.
enprocal d treatment increased levels of il- <dig> by 48% compared with control pma stimulated cells and wpc and p <dig> increased il- <dig> by 26% and 15% respectively.
both enprocal d and wpc induced il- <dig> secretion in non-stimulated jurkat cells to nearly the same  levels as were achieved with pma stimulated cells  control .
with p <dig> digest treatment the il- <dig> secretion levels were decreased by 56% when compared with positive  control .
ud = undetectable levels.
aenprocal digested and other digested milk product controls were added to caco- <dig> cells in apical  wells and culture supernatants were collected from the thp- <dig>  cells grown in basolateral  chamber after  <dig> hours  and  <dig> hours  incubation.
benprocal digested and other digested milk product controls were added to caco- <dig> cells in apical wells and culture supernatants were collected from the jurkat cells grown in basolateral chamber.
supernatant from jurkat cells stimulated with pma was used as positive control for the measurement of il- <dig>  inf-γ and il- <dig> cytokines.
ccontrol thp- <dig> cells with enzyme cocktail and with lps stimulation.
dcontrol jurkat cells with enzyme cocktail and without pma stimulation.
concentration of cytokines in the culture medium was determined by elisa as described in the methods section.
cytokine concentrations in negative control  and growth media only were below the detection limits.
all samples were assayed in triplicate and three independent experiments were performed.
the mean for all experiment was calculated and presented as a mean ± sd values.
** indicates a highly significant p <  <dig>  value from the control with positive control  only.
*indicates a significant p <  <dig>  value from the control with positive control  only.
discussion
in the present study, we reported for the first time the potential of enprocal to improve gut and immune health.
in the past decade, it has become clear that much of the gastrointestinal tract maintains youthful function well into the eighth and ninth decade of life.
there is little difference between a  <dig> year-old and an  <dig> year-old in the quantity and quality of biliary, pancreatic and intestinal secretions, in the absorptive capacity of the small intestine, and general nutritional requirements  <cit> .
in practical terms in gut health, swallowing and defecation are the two most likely functions to be affected by aging  <cit> .
the areas at greatest risk of developing aging-related dysfunction are the upper gi tract, particularly the oropharynx and esophagus, and the distal tract .
the inability to eat sufficient amounts orally leads to an often compromised nutritional intake in the elderly.
their diets are low in protein, low in energy  and deficient in micronutrients  <cit>  and elderly gut slows the absorption of nutrients from the gut lumen have also been reported  <cit> .
enprocal is a formulated supplement developed to meet the nutritional profile of elderly and contains whey protein concentrate, skim milk powder, whole milk powder, vitamins and minerals , vegetable oils and inulin .
enprocal d helps in gut cell growth, maintains gut barrier function and mucosal-integrity
although not mimicking the ageing gut epithelium, fhs  <dig> int and caco- <dig> cell lines are employed world wide as suitable cell culture models displaying enterocyte like differentiation for studying the proliferation and gut integrity/permeability and immunomodulation at cellular level  <cit> .
we first determined a concentration range of enprocal, which was not cytotoxic  to the caco- <dig> cell monolayers, needed to test the downstream intestinal permeability assays, adhesion assays etc.
we used initially both undigested versus digested enprocal to determine if there was any difference between the effects of two upon treatment with gut cocktail mixture on cell viability.
both digested and undigested enprocal had no affect on caco- <dig> cell viability over a dose range of 500– <dig> μg/ml and this concentration range was chosen for bioassays.
since enprocal is a dietary supplement to be taken orally and to mimic conditions as close as possible to those of the human digestive tract following oral route, we conducted all the assays with digested samples only.
other digested milk product controls were also tested for their effects over this dose range on caco- <dig> cell viability and cell death .
at 500– <dig> μg/ml, caco- <dig> cells appeared to be resistant to the cell death/cytotoxic effects  by enprocal d after  <dig> hours as compared to other digested milk product controls.
in particular heat smp has significantly increased the apoptosis/necrotic index indicating the presence of unidentified potential anti-cancer bioactive peptides released upon its digestion  <cit> .
at higher doses  enprocal  induced cell death and loss of viability in caco- <dig> cells but not in normal fhs  <dig> int cells.
enprocal samples were found to provide normal intestinal epithelial cell protection as no cytotoxicity resulted towards fhs  <dig> int cells after treatment with enprocal  at 100– <dig> μg/ml tested in the ldh release assay.
it has been reported that an α-lactoalbumin complex from acid-precipitated human milk casein  induces apoptosis in tumor cells  and immature cells, but not in mature differentiated fhs  <dig> int cells  <cit> .
in mtt cell proliferation assay, enprocal d in a dose dependent manner stimulated the selective proliferation of normal fhs  <dig> int cells and this stimulation was significantly  more effective than positive control egf.
due to an absence of cytotoxicity effects against these cells our results suggest that enprocal is safe to be taken orally and may prove beneficial to premature infant gut growth.
further studies are needed to determine the mechanisms involved in growth-promoting effect of enprocal on fetal small intestinal cells.
also the anti-cancer property of enprocal in caco- <dig> cells at higher doses is a matter of an interesting future investigation.
the anti-cancer effect of enprocal may be attributed to the activity of its wpc' components such as lactoferrin, which have been shown to inhibit tumour cell growth both in vitro and in vivo  <cit>  or the synergistic effect of wpc components with vitamins and calcium  <cit> .
we further demonstrated that enprocal improved gut cell wall integrity, aided in macromolecular absorption.
disruption of the epithelial barrier in the gut is a hallmark of gut inflammation and diseases.
failure of barrier function in the gut epithelium is a key feature in the pathology of diseases, such as leaky gut syndrome, ibd, cancer etc.
<cit> .
healthy intestinal tract acts a barrier against colonization and/or translocation of pathogens and toxic compounds.
diarrhoeal diseases as a result of gut infection caused by escherichia coli, salmonella, shigella, campylobacter jejuni, entamoeba histolytica and rotavirus are one of the important causes of infant mortality and morbidity in developed and underdeveloped countries  <cit> .
to ensure monolayer integrity, teer of caco- <dig> polarised monolayers grown on millicell cell culture inserts  was measured using millicell-ers system voltohm meter which measured the monolayer resistance under aseptic conditions.
compared with other control products, high teer values were observed in monolayers' treated with enprocal d and p <dig> after  <dig> hours of treatment.
these values returned to baseline after  <dig> hours and after  <dig> hours teer values went further up with only enprocal d treatment and which were maintained till  <dig> hours.
these observations indicate that there was no difference between enprocal d and p <dig> with regards to the functional activities to gut cells following treatment at till  <dig> hours.
the gut cell integrity results were again confirmed by expression of tight junction protein zona occludin  on treated caco- <dig> monolayers.
enprocal d and p <dig> as compared to control products increased the expression of tight junction protein -zo- <dig>
in the gut permeability assay, enprocal d aided macromolecular transport studied by hrp-flux through the caco- <dig> monolayer.
the transport of nutrients across the intestinal epithelium may occur by one or more of  <dig> different routes: the passive transcellular and paracellular routes, the carrier mediated route or by transcytosis.
caco- <dig> monolayers have been world widely used to study drug transport by all  <dig> pathways  <cit>  and permeability through caco- <dig> cell monolayers correlates well with human in vivo absorption  <cit> .
the results of the hrp flux indicated that hrp flow through increases with time after treatment with enprocal d and other digested control milk products.
this was reflected in the increased flux of hrp across the monolayers by the  <dig> hour point.
all these results of teer, hrp-flux and zo- <dig> expression clearly indicates that enprocal maintains the gut cell integrity even after digestion and showed no side effect on its absorption and permeability through caco- <dig> monolayer.
enprocal treatment does not induce oxidative stress/injury
increases in the levels of reactive oxygen species  that may occur during periods of oxidative stress, appear to be detected by redox-sensitive regulatory molecules in the cell, triggering homeostatic responses to prevent cellular injury  <cit> .
among those responses is the regulation of aoe, as the levels and balance of aoe modulate the susceptibility of the cell to oxidative injury.
highly reactive free radicals formed by exogenous chemicals or endogenous metabolic processes in the human body or in food systems are capable of oxidizing biomolecules, resulting in cell death and tissue damage.
in the gut, variations in the activities of aoe have been reported under different inflammatory/pathologic conditions associated with free radical injury  <cit> .
we examined the specific aoes such as cuznsod, mnsod, gpx, and cat activities in caco- <dig> epithelial cell cultures treated with enprocal d and other digested milk product controls.
with enprocal d there were no significant changes in the induction of aoe activity levels and were similar in effect to natural anti-oxidant vitamin c used as a positive control.
these results indicated that no oxidative stress or free radical injury occurred to the cells and these findings can be explained on the basis of synergistic effects of enprocal product formulated with vitamin c, a, and zn, the known anti-oxidants.
enprocal up-regulates the surface expression of co-stimulatory  molecules and mhc i and ii on human macrophages
macrophages as antigen-presenting cells  play an important role in innate immune response in the gut and represent one of the first lines of nonspecific defence against bacterial invasion.
the macrophages are highly responsive to their environment  and relay messages instructing other arms of the immune system e.g., t cells and b cells, to respond in an appropriate way  <cit> .
these messages can be through costimulatory molecules  or soluble proteins .
even though cytokines play diverse roles in regulating immune functions, some cytokines, e.g., tumor necrosis factor -α, interleukin -1β and il- <dig> have received more attention than others because they have traditionally been classified as proinflammatory and as such are known to be associated with chronic inflammation including ibd  <cit> .
recently an in vitro coculture system has been reported that could be utilized for an assay to search for drugs or food substances, which could prevent intestinal inflammation  <cit> .
to study the cell-to-cell interaction between intestinal epithelial cells  and activated macrophages , we utilized the similar transwell model.
we used thp- <dig> cell line rather than primary human monocytes or dendritic cells  to minimize the variability due to sample collection from different subjects.
dcs are considered to be highly sensitive and professional apcs of the immune system  <cit> , but they are difficult to isolate and require careful handling to prevent them becoming activated before treatment with any bioactive.
thus, they can produce variable results  <cit> .
we found that digested enprocal when added to apical  wells containing caco- <dig>  up-regulated the expression of "activation markers"  in pma differentiated thp- <dig> macrophages cultured in bottom  wells.
this implies that as proposed in the hypothesis  in figure  <dig>  enprocal components moved from the 'mucosal' to 'serosal' side by any of the mechanisms shown and interact with 'apc' to induce upregulation of costimulatory molecules and aid gut-immune cell cross-talk.
the intestinal anti-inflammatory activity exerted by enprocal was characterized by a down-regulation of cell adhesion of thp- <dig> and jurkat e6- <dig> cells to caco- <dig> monolayers treated with digested samples of enprocal and other digested milk product controls in a static assay system.
although these adhesion assays do not replicate the in vivo conditions of dynamic gastrointestinal tract but these heterotypic assays have been used earlier for the development of immunomodulatory peptides  <cit> .
moreover it has been reported that there are peripheral  target sites for the biologically active milk-derived peptides that may be absorbed intact  and enter the circulation.
in that situation this anti-inflammatory activity of enprocal may hold promise and clearly more work needs to be done in adhesion assays using normal primary human immune and epithelial/endothelial cells.
the intestinal intraepithelial lymphocytes and monocytes are effector cells capable of secreting cytokines in response to stimulation  <cit> .
these cytokines include tumor necrosis factor-α , interferon-γ  and interleukin -1β, il- <dig>  il- <dig> and il- <dig>  and it has been suggested that ifn-γ and tnf-α can act directly on intestinal epithelial cells to mediate changes in the epithelial permeability and the capacity for electrogenic ion transport  <cit> .
the mucosal to serosal movement of enprocal d components' in our coculture system led to modulation of the cytokine secretion by monocytes and t-lymphocytes.
in enprocal d-treated thp- <dig> cell supernates, despite the levels of tnf-α and il-1β being reduced, il- <dig>  secretion was not reduced to the scale of wpc.
il- <dig>  secretion levels by jurkat e6- <dig> cells were significantly higher with enprocal treatment.
both il- <dig> and il- <dig> are important in the terminal differentiation of b-cells into plasma cells  <cit> .
enprocal d significantly enhanced the secretion of ifn-γ, and il- <dig> compared to the positive control pma, wpc and p <dig> treated t-lymphocytes.
il- <dig> is considered anti-inflammatory which inhibits the generation of cell mediated immune responses.
il- <dig> is an interesting multifunctional cytokine produced by t-cells, monocytes, and other cell types  <cit> .
il- <dig> enhances b-cell viability and augments the proliferation and differentiation of b-cells after stimulation through the ig receptor  <cit> , therefore associated with humoral or type  <dig> immune responses.
overall, enprocal d provided the most consistent up-regulation of anti inflammatory cytokines and was the second most prolific down-regulator of pro inflammatory cytokines.
enprocal, through down-regulation of proinflammatory cytokines such as tnf-α and il-1β , may prove beneficial in ibd, cd & uc.
tnf-α, in particular has been recognized as playing a central role in the pathogenesis of crohn disease.
nevertheless, reducing the production or effect of tnf-α in crohn disease patients is believed to be beneficial  <cit> .
in fact, different drugs capable of interfering with the activity of tnf-α cytokine have been successfully developed for ibd therapy  <cit> .
however, despite earlier hopes, the results from studies using tnf-α antagonists were disappointing, and there were some reports of severe complications  <cit> .
CONCLUSIONS
in conclusion, the schematic diagram shown in figure  <dig> proposes a model  on mechanism of enprocal's function in gut and immune health.
following oral administration and upon digestion of enprocal, amino acids and potentially bioactive di and tri-peptides, which may be released through enzymatic action from its key ingredients such as whey protein concentrate, aid in normal human intestinal cell proliferation with no cytotoxicity, maintain gut integrity, increase tight junction protein expression, and enhance activation of immune responses.
through modulation of the th <dig> and th <dig> cytokines responses enprocal may aid the production of antibodies and gut immune functions.
these observed effects of enprocal however may arise not only from the presence of dairy proteins but also from synergistic relationships with its other components in particular the micro-nutrients.
strong gut cell integrity and activation of immune cells with enprocal may prevent infections caused by pathogens or the invasion or entry of allergens.
thus in future the detailed studies should be carried out on efficacy of enprocal in reducing individual pathogens and gut cell interactions.
stimulation of fetal intestinal cell proliferation without cell cytotoxicity by enprocal indicates that it may also be given as infant food particularly for premature babies.
although gut epithelial stem cells show no intrinsic limit to their proliferative capacity but recent evidence  indicates that they suffer important functional impairments associated with an altered pattern of cellular response to dna damage during the course of ageing.
in future studies, it would also be worthwhile to determine whether consumption of enprocal is effective in preventing such functional damage to the stem cells in the aged gut.
finally, our in vitro findings by employing well characterized currently available and widely used cell models may be relevant for gut and immune health of consumer groups with special needs, such as infants and the elderly.
however future studies are needed to reveal the potential of enprocal as a formulated supplement that produces defined effects on the human gut and immune health.

 the ability to eat sufficient amounts orally is inversely associated with the extent of frailty and studies have indicated that nutritional intake is often compromised in the elderly. poor gut health and secondary health issues as a consequence are matters of nutritional attention for the elderly, infants and immune compromised persons. diet is a factor that can be extremely varied in terms of quantity, quality, frequency and duration of meals in individuals, but that can also be modified with targeted dietary interventions to produce the best results for any individual. diets of the elderly are generally low in protein, low in energy and deficient in a range of micro-nutrients. high protein diets, in particular those based on whey proteins, are being marketed to improve the health status of the elderly as well as other target groups e.g. athletes and babies. furthermore micro-nutrients including anti-oxidants e.g. vitamins a, c and zinc are also being used for fortification or as dietary supplements for healthy older patients. there are growing number of compositional foods under development, being patented or emerging in the market that target specific nutritional deficiencies beyond the traditional infant formula market. recent examples include wo2007/ a high protein dietary supplement for treating various diseases associated with protein deficiency; and wo2007/ compositions for enhancing vascular integrity, cellular survival and reducing apoptosis in ischema. enprocal, a recently formulated supplementary food is designed to meet the nutritional needs of the frail elderly and to be delivered to them in common every day foods. enprocal's formulation was developed by specialised aged care dieticians and a food scientist experienced in powder based food supplements. the ingredients of enprocal such as dairy-based proteins, vitamins and minerals, vegetable oils and inulin were selected to meet the nutritional profile of elderly specified though dietetic input. with whey protein concentrate as its key ingredient it provides i) the highest-quality protein, ii) rapid protein digestion, iii) essential amino acids, in particular cysteine, threonine and leucine that may be required in increased levels in the elderly, iv) cysteine to improve glutathione status and muscle protein synthesis, v) high concentrations of branched chain amino acids, and vi) minor bioactive components such as immuno-globulins, growth factors, and anti-microbial proteins and peptides to assist in effective gut function. today, whey is a popular dietary protein supplement purported to provide antimicrobial activity, immune modulation, improved muscle strength and body composition, and to prevent cardiovascular disease and osteoporosis. enprocal with wpc as its key ingredient therefore contains high levels of constituent proteins including beta-lactoglobulin, alpha-lactalbumin, lactoferrin, lactoperoxidase and glycomacropeptide which have been demonstrated a range of immune-enhancing properties. in addition, whey has the ability to act as an antioxidant, antihypertensive, antitumor, hypolipidemic, antiviral, antibacterial, and chelating agent. whey proteins have been reported to enhance digestion and gut function as well as glutathione production and immune function, hence increasing dietary availability may promote general health in a variety of ways. developing and maintaining a healthy intestinal tract is a pre-requisite for general health and aiding disease prevention. the gut epithelial cells lining of the intestinal lumen are the first point of contact between intestinal contents and the rest of the body. it is at this interface that nutrition, environment and genetics come together to determine gut health. the intestinal mucosa is lined by a monolayer of intestinal epithelial cells joined together at their apical poles by tight junctions, and supported by an extensive and complex immune system and transcellular pathways for nutrient absorption. the mucosal surface of the gastrointestinal tract forms a barrier that separates the luminal contents from the effector immune cells underneath. with the emergence of the concept of immunophysiology, it is clear that the function of the intestinal epithelium is affected by many immune cell types. any abnormal activation of such immune cells overproduces the inflammatory cytokines e.g., tumor necrosis factor -α, interleukin -1β and il- that cause destructive cell damage to the intestinal epithelial monolayers and subsequent mucosal inflammation leading to inflammatory bowel disease such as crohn's disease and ulcerative colitis. cellular injury from reactive oxygen species is implicated in a wide variety of gut diseases and pathologic conditions. there is a growing body of evidence that oxidative stress may contribute to gut inflammation, ulcers, inflammatory bowel disease, and leaky gut syndrome. newborn and the elderly are more prone to oxidant injury. the present study was aimed to assess the potential of enprocal to improve gut and immune health functions. we investigated specifically whether enprocal, after digestion with a cocktail of gut enzymes maintained gut cell proliferation and mucosal integrity, possessed immunomodulatory, anti-inflammatory and anti-oxidative properties. we sought to achieve conditions as close as possible to those of the human digestive tract by employing in vitro, most widely used human gut epithelial cells alone or in coculture with human immune thp- and jurkat cells in transwell system. fhs int and caco- cell lines used worldwide by the researchers were employed as the models of human intestinal epithelial cells in the study. fhs 74-int cells, used in the study were although derived from normal human fetal intestine but have been reported to show mature epithelial-like characteristics. the growth-promoting activities of egf, tgf-α igf-i and insulin were examined using the same intestinal cells. caco- cells have gained enormous popularity as a reliable and high-throughput in vitro model system for the study of human intestinal permeability and evaluation of a large number of drug candidates for their intestinal absorption. for orally administered compounds, permeability through caco- cell monolayers correlates well with human in vivo absorption. caco- monolayers can display electrical properties typical of either small intestinal or colonic enterocytes. caco- cells are able to differentiate spontaneously in culture and display a small intestine-like phenotype. the differentiated cells polarise, form microvilli and secrete enzymes associated with the enterocyte brush border such as sucrase-isomaltase, alkaline phosphatase, alkaline phosphatase, lactase and aminopeptidase. this cell line thus represents an appropriate model for the study of transport mechanisms related to the intestinal barrier and for investigating diverse problems of nutrients bioavailability and absorption without concern for differences between human and animals. our study, provides the first report on the bioactivity of digested enprocal components which may be released through enzymatic action from its key ingredients such as whey protein concentrate for gut epithelial cell growth, integrity, absorptive, immunomodulatory, anti-inflammatory and anti-oxidative properties.  digested enprocal maintains cell growth, enhances cell proliferation and shows no cytotoxicity in normal human intestinal epithelial cells near complete digestion of enprocal and other digested milk product controls was achieved after hours incubation with gut enzyme cocktail mixture. in our preliminary experiments we treated enprocal with the gut enzyme cocktail at different enzyme cocktail: protein ratios at ph –. a 1: ratio of enzyme cocktail: protein at ph was found to be most optimal and complete digestion was achieved after hours. further it was deemed necessary to find a concentration range which would not kill/disrupt the cell monolayers needed to test in bioassays on permeability, immunomodulation, cell adhesion, and oxidative stress. we treated caco- and fhs int cells with digested and undigested samples of enprocal at different concentrations to determine the effect on cell viability and growth determined by trypan blue exclusion assay. with both enprocal undigested and digested samples, the caco- cell viability as compared to that of fhs int was significantly reduced in at concentrations and μg/ml. there was visible reduction in caco- viability with enprocal d compared to undigested enprocal. however, these levels did not reach any significance. there was no effect of all concentrations tested in the range of 100– μg/ml on the cell viability of normal intestinal fhs int cells. this was further confirmed in the ldh released cytotoxicity assay. there was no significant increase in the cytotoxicity as compared to control when fhs int cells were treated with digested and undigested samples of enprocal from 100– μg/ml. the enzyme cocktail showed no effect of its own on cell viability and cytotoxicity in both the cell lines. although there was no significant difference between the cell viability and cytotoxicity effects of digested and undigested enprocal samples but to mimic the in vivo system we selected the digested form for bioassays. from the cell viability and cell cytotoxicity results 500– μg/ml was selected as the optimal concentration range for the bioassays. the other control milk product digests at these concentrations were tested for their effects on cell viability in trypan blue assay. when compared to media only control and enprocal d, heat smp digest samples reduced significantly the caco- cell viability at μg/ml. the control milk products were further tested in ldh release assay, and cytotoxicity levels were below 10% and not significant at μg/ml concentration when compared with enprocal d and media only control. in tunel and annexin-v-fluos assays, enprocal d did not significantly increase the apoptotic index and necrotic index, however heat smp induced significant ai as compared to media only control in caco- cells. in a dose dependent manner enprocal d increased significantly the proliferation of fhs int cells, determined by mtt assay, as compared to untreated and positive control cells. when compared with other control products this effect was significant except for p where the difference was insignificant between enprocal and p however the cell proliferation of caco- cells was decreased significantly following treatment with enprocal d. enprocal d maintains the mucosal integrity, increases the expression of zo- and macromolecular absorption intestinal caco- monolayers grown on millipore cell culture inserts in transwell system were used to measure the transepithelial resistance following treatments with enprocal d and other digested milk product controls at a concentration of μg/ml. at hour before treatment, the caco- monolayers after days of growth were found to be fully differentiated to enterocytes and polarized, continuous without any gaps and displayed teer values ± ohm.cm in control growth media. figure 5a showed the teer values of cell monolayers following different treatments relative to control. the most significant decrease was observed in all treatments after the first hours except for enprocal d and p. both p and enprocal d were able to recover the teer values at hours and the increase was maintained further till hours duration as compared to other control milk products. however, teer resistance values for enprocal d were found to increase further after hours. p was found to decrease significantly the teer after hours. the gut enzyme cocktail had no effect on the teer when compared with media only control. the triton-x, known to disrupt the cell monolayer, was used as the positive control. it reduced significantly the teer resistance at hours which were not recovered throughout the experimental period. the monolayer integrity results were further confirmed by expression of tight junction protein -zo- figure 5b shows an upregulation of zo- expression on caco- monolayers treated with μg/ml of enprocal and p digests for hours. in horseradish peroxidase flux assay, enprocal d aided macromolecular transport through the caco- cell monolayer. hrp flow through increased with time after treatment with enprocal d and other milk product digests. after hours significant hrp flux increase as compared to media only control was observed in enprocal treated cells which increased further to and fold after and hours respectively. among other products,, and fold increase was observed for heat smp, wpc, p and p respectively at hours while significant increase,, and fold occurred at hours for heat smp, wpc, p and p respectively. as expected the positive control triton x, showed fold increases at hours which remained consistent till hours. the enzyme cocktail mixture had no effect when compared with untreated cells. hrp flux results are shown in percentage for 4– hours determined in the basolateral compartment. all samples were assayed in three independent assays each performed in triplicate and the mean for all experiments was calculated and presented as a mean ± sd values. ** indicates a highly significant p < value from the control with media only. *indicates a significant p < value from the control with media only. triton-x is a known cell disruptor and act as a positive control in hrp flux assay anti-oxidant activities of enprocal d we examined the levels of aoe activities in caco- cells treated with enprocal d and compared with other control commercial milk product digests. there was no significant change in the activities of either cuznsod or mnsod in enprocal d and wpc treated cells as compared to vitamin c treated cells. this indicates that while enprocal d did not induce oxidative stress in the gut cells, p treatment led to highly significant increase in the activities of these enzymes indicating that it is inducing an oxidative stress response. increased sod activity if not accompanied by similar levels of other aoes may be harmful. activity levels for cat were not significantly effected by the treatments with enprocal, wpc and p however activity levels for gpx increased significantly after treatment with p digest. enprocal d upregulates the surface expression of co-stimulatory cd cd cd mhc i & mhc ii on human macrophages and inhibits the monocyte/lymphocyte-epithelial cell adhesion in a transwell coculture model of caco- and pma differentiated thp- macrophages, movement of bioactive components of with enprocal d and other digested milk product controls from mucosal to serosal induced up-regulation of cd cd cd and mhc-i & ii expression on thp- macrophages in a manner similar to that of immune stimulant ifn-γ. although thp- macrophages showed no expression of activation markers in response to the negative control, they strongly up-regulated expression of cd, cd, cd, and mhc i & ii on the cell surface upon treatment with enprocal d, other digested milk product controls compared to no treatment control. the response observed was maximal with enprocal d. enprocal d in a dose dependant manner significantly inhibited the adhesion of lymphocytes and monocytes to caco- cells. similar levels of inhibition of adhesion were observed with p. enprocal d modulates the cytokine response in human immune cells the levels of proinflammatory, both th and th cytokines and chemokine il- were measured in cell culture supernates of thp- and jurkat e6- cells. these immune cells were grown in lower wells of transwell coculture models of caco- and thp- caco- and jurkat e6- cells. as shown in table the cytokine modulatory effect upon movement of bioactive components of enprocal d and other digested milk product controls from mucosal to serosal was evident. all the cytokine levels in the growth media samples and untreated cells were below the detection limits of elisa. the cytokines response induced in the lps only stimulated cells was highly significant as compared to non-stimulated cells and considered 100%. the levels of lps stimulated tnf-α, il-1β and il- were reduced dramatically by 91%, 57% and 44% respectively after treating cells with enprocal d as compared to lps only control. wpc similarly reduced the levels by 85%, 75% and 84% for tnf-α, il-1β and il- respectively. however, with p control product levels of tnf-α and il- were either negligible or below detection limits and it decreased the levels by 87% for il- β. there was no effect on il- production by any of the products tested as compared to maximal response. when compared with the positive control enprocal d treatment increased levels of ifn-γ by 29%, while both wpc and p decreased the levels of ifn-γ by 51% and 88% respectively in the supernates of jurkat e6- cells. enprocal d treatment increased levels of il- by 48% compared with control pma stimulated cells and wpc and p increased il- by 26% and 15% respectively. both enprocal d and wpc induced il- secretion in non-stimulated jurkat cells to nearly the same levels as were achieved with pma stimulated cells control. with p digest treatment the il- secretion levels were decreased by 56% when compared with positive control. ud = undetectable levels. aenprocal digested and other digested milk product controls were added to caco- cells in apical wells and culture supernatants were collected from the thp- cells grown in basolateral chamber after hours and hours incubation. benprocal digested and other digested milk product controls were added to caco- cells in apical wells and culture supernatants were collected from the jurkat cells grown in basolateral chamber. supernatant from jurkat cells stimulated with pma was used as positive control for the measurement of il- inf-γ and il- cytokines. ccontrol thp- cells with enzyme cocktail and with lps stimulation. dcontrol jurkat cells with enzyme cocktail and without pma stimulation. concentration of cytokines in the culture medium was determined by elisa as described in the methods section. cytokine concentrations in negative control and growth media only were below the detection limits. all samples were assayed in triplicate and three independent experiments were performed. the mean for all experiment was calculated and presented as a mean ± sd values. ** indicates a highly significant p < value from the control with positive control only. *indicates a significant p < value from the control with positive control only. discussion in the present study, we reported for the first time the potential of enprocal to improve gut and immune health. in the past decade, it has become clear that much of the gastrointestinal tract maintains youthful function well into the eighth and ninth decade of life. there is little difference between a year-old and an year-old in the quantity and quality of biliary, pancreatic and intestinal secretions, in the absorptive capacity of the small intestine, and general nutritional requirements. in practical terms in gut health, swallowing and defecation are the two most likely functions to be affected by aging. the areas at greatest risk of developing aging-related dysfunction are the upper gi tract, particularly the oropharynx and esophagus, and the distal tract. the inability to eat sufficient amounts orally leads to an often compromised nutritional intake in the elderly. their diets are low in protein, low in energy and deficient in micronutrients and elderly gut slows the absorption of nutrients from the gut lumen have also been reported. enprocal is a formulated supplement developed to meet the nutritional profile of elderly and contains whey protein concentrate, skim milk powder, whole milk powder, vitamins and minerals, vegetable oils and inulin. enprocal d helps in gut cell growth, maintains gut barrier function and mucosal-integrity although not mimicking the ageing gut epithelium, fhs int and caco- cell lines are employed world wide as suitable cell culture models displaying enterocyte like differentiation for studying the proliferation and gut integrity/permeability and immunomodulation at cellular level. we first determined a concentration range of enprocal, which was not cytotoxic to the caco- cell monolayers, needed to test the downstream intestinal permeability assays, adhesion assays etc. we used initially both undigested versus digested enprocal to determine if there was any difference between the effects of two upon treatment with gut cocktail mixture on cell viability. both digested and undigested enprocal had no affect on caco- cell viability over a dose range of 500– μg/ml and this concentration range was chosen for bioassays. since enprocal is a dietary supplement to be taken orally and to mimic conditions as close as possible to those of the human digestive tract following oral route, we conducted all the assays with digested samples only. other digested milk product controls were also tested for their effects over this dose range on caco- cell viability and cell death. at 500– μg/ml, caco- cells appeared to be resistant to the cell death/cytotoxic effects by enprocal d after hours as compared to other digested milk product controls. in particular heat smp has significantly increased the apoptosis/necrotic index indicating the presence of unidentified potential anti-cancer bioactive peptides released upon its digestion. at higher doses enprocal induced cell death and loss of viability in caco- cells but not in normal fhs int cells. enprocal samples were found to provide normal intestinal epithelial cell protection as no cytotoxicity resulted towards fhs int cells after treatment with enprocal at 100– μg/ml tested in the ldh release assay. it has been reported that an α-lactoalbumin complex from acid-precipitated human milk casein induces apoptosis in tumor cells and immature cells, but not in mature differentiated fhs int cells. in mtt cell proliferation assay, enprocal d in a dose dependent manner stimulated the selective proliferation of normal fhs int cells and this stimulation was significantly more effective than positive control egf. due to an absence of cytotoxicity effects against these cells our results suggest that enprocal is safe to be taken orally and may prove beneficial to premature infant gut growth. further studies are needed to determine the mechanisms involved in growth-promoting effect of enprocal on fetal small intestinal cells. also the anti-cancer property of enprocal in caco- cells at higher doses is a matter of an interesting future investigation. the anti-cancer effect of enprocal may be attributed to the activity of its wpc' components such as lactoferrin, which have been shown to inhibit tumour cell growth both in vitro and in vivo or the synergistic effect of wpc components with vitamins and calcium. we further demonstrated that enprocal improved gut cell wall integrity, aided in macromolecular absorption. disruption of the epithelial barrier in the gut is a hallmark of gut inflammation and diseases. failure of barrier function in the gut epithelium is a key feature in the pathology of diseases, such as leaky gut syndrome, ibd, cancer etc.. healthy intestinal tract acts a barrier against colonization and/or translocation of pathogens and toxic compounds. diarrhoeal diseases as a result of gut infection caused by escherichia coli, salmonella, shigella, campylobacter jejuni, entamoeba histolytica and rotavirus are one of the important causes of infant mortality and morbidity in developed and underdeveloped countries. to ensure monolayer integrity, teer of caco- polarised monolayers grown on millicell cell culture inserts was measured using millicell-ers system voltohm meter which measured the monolayer resistance under aseptic conditions. compared with other control products, high teer values were observed in monolayers' treated with enprocal d and p after hours of treatment. these values returned to baseline after hours and after hours teer values went further up with only enprocal d treatment and which were maintained till hours. these observations indicate that there was no difference between enprocal d and p with regards to the functional activities to gut cells following treatment at till hours. the gut cell integrity results were again confirmed by expression of tight junction protein zona occludin on treated caco- monolayers. enprocal d and p as compared to control products increased the expression of tight junction protein -zo- in the gut permeability assay, enprocal d aided macromolecular transport studied by hrp-flux through the caco- monolayer. the transport of nutrients across the intestinal epithelium may occur by one or more of different routes: the passive transcellular and paracellular routes, the carrier mediated route or by transcytosis. caco- monolayers have been world widely used to study drug transport by all pathways and permeability through caco- cell monolayers correlates well with human in vivo absorption. the results of the hrp flux indicated that hrp flow through increases with time after treatment with enprocal d and other digested control milk products. this was reflected in the increased flux of hrp across the monolayers by the hour point. all these results of teer, hrp-flux and zo- expression clearly indicates that enprocal maintains the gut cell integrity even after digestion and showed no side effect on its absorption and permeability through caco- monolayer. enprocal treatment does not induce oxidative stress/injury increases in the levels of reactive oxygen species that may occur during periods of oxidative stress, appear to be detected by redox-sensitive regulatory molecules in the cell, triggering homeostatic responses to prevent cellular injury. among those responses is the regulation of aoe, as the levels and balance of aoe modulate the susceptibility of the cell to oxidative injury. highly reactive free radicals formed by exogenous chemicals or endogenous metabolic processes in the human body or in food systems are capable of oxidizing biomolecules, resulting in cell death and tissue damage. in the gut, variations in the activities of aoe have been reported under different inflammatory/pathologic conditions associated with free radical injury. we examined the specific aoes such as cuznsod, mnsod, gpx, and cat activities in caco- epithelial cell cultures treated with enprocal d and other digested milk product controls. with enprocal d there were no significant changes in the induction of aoe activity levels and were similar in effect to natural anti-oxidant vitamin c used as a positive control. these results indicated that no oxidative stress or free radical injury occurred to the cells and these findings can be explained on the basis of synergistic effects of enprocal product formulated with vitamin c, a, and zn, the known anti-oxidants. enprocal up-regulates the surface expression of co-stimulatory molecules and mhc i and ii on human macrophages macrophages as antigen-presenting cells play an important role in innate immune response in the gut and represent one of the first lines of nonspecific defence against bacterial invasion. the macrophages are highly responsive to their environment and relay messages instructing other arms of the immune system e.g., t cells and b cells, to respond in an appropriate way. these messages can be through costimulatory molecules or soluble proteins. even though cytokines play diverse roles in regulating immune functions, some cytokines, e.g., tumor necrosis factor -α, interleukin -1β and il- have received more attention than others because they have traditionally been classified as proinflammatory and as such are known to be associated with chronic inflammation including ibd. recently an in vitro coculture system has been reported that could be utilized for an assay to search for drugs or food substances, which could prevent intestinal inflammation. to study the cell-to-cell interaction between intestinal epithelial cells and activated macrophages, we utilized the similar transwell model. we used thp- cell line rather than primary human monocytes or dendritic cells to minimize the variability due to sample collection from different subjects. dcs are considered to be highly sensitive and professional apcs of the immune system, but they are difficult to isolate and require careful handling to prevent them becoming activated before treatment with any bioactive. thus, they can produce variable results. we found that digested enprocal when added to apical wells containing caco- up-regulated the expression of "activation markers" in pma differentiated thp- macrophages cultured in bottom wells. this implies that as proposed in the hypothesis in figure enprocal components moved from the'mucosal' to'serosal' side by any of the mechanisms shown and interact with'apc' to induce upregulation of costimulatory molecules and aid gut-immune cell cross-talk. the intestinal anti-inflammatory activity exerted by enprocal was characterized by a down-regulation of cell adhesion of thp- and jurkat e6- cells to caco- monolayers treated with digested samples of enprocal and other digested milk product controls in a static assay system. although these adhesion assays do not replicate the in vivo conditions of dynamic gastrointestinal tract but these heterotypic assays have been used earlier for the development of immunomodulatory peptides. moreover it has been reported that there are peripheral target sites for the biologically active milk-derived peptides that may be absorbed intact and enter the circulation. in that situation this anti-inflammatory activity of enprocal may hold promise and clearly more work needs to be done in adhesion assays using normal primary human immune and epithelial/endothelial cells. the intestinal intraepithelial lymphocytes and monocytes are effector cells capable of secreting cytokines in response to stimulation. these cytokines include tumor necrosis factor-α, interferon-γ and interleukin -1β, il- il- and il- and it has been suggested that ifn-γ and tnf-α can act directly on intestinal epithelial cells to mediate changes in the epithelial permeability and the capacity for electrogenic ion transport. the mucosal to serosal movement of enprocal d components' in our coculture system led to modulation of the cytokine secretion by monocytes and t-lymphocytes. in enprocal d-treated thp- cell supernates, despite the levels of tnf-α and il-1β being reduced, il- secretion was not reduced to the scale of wpc. il- secretion levels by jurkat e6- cells were significantly higher with enprocal treatment. both il- and il- are important in the terminal differentiation of b-cells into plasma cells. enprocal d significantly enhanced the secretion of ifn-γ, and il- compared to the positive control pma, wpc and p treated t-lymphocytes. il- is considered anti-inflammatory which inhibits the generation of cell mediated immune responses. il- is an interesting multifunctional cytokine produced by t-cells, monocytes, and other cell types. il- enhances b-cell viability and augments the proliferation and differentiation of b-cells after stimulation through the ig receptor, therefore associated with humoral or type immune responses. overall, enprocal d provided the most consistent up-regulation of anti inflammatory cytokines and was the second most prolific down-regulator of pro inflammatory cytokines. enprocal, through down-regulation of proinflammatory cytokines such as tnf-α and il-1β, may prove beneficial in ibd, cd & uc. tnf-α, in particular has been recognized as playing a central role in the pathogenesis of crohn disease. nevertheless, reducing the production or effect of tnf-α in crohn disease patients is believed to be beneficial. in fact, different drugs capable of interfering with the activity of tnf-α cytokine have been successfully developed for ibd therapy. however, despite earlier hopes, the results from studies using tnf-α antagonists were disappointing, and there were some reports of severe complications.  in conclusion, the schematic diagram shown in figure proposes a model on mechanism of enprocal's function in gut and immune health. following oral administration and upon digestion of enprocal, amino acids and potentially bioactive di and tri-peptides, which may be released through enzymatic action from its key ingredients such as whey protein concentrate, aid in normal human intestinal cell proliferation with no cytotoxicity, maintain gut integrity, increase tight junction protein expression, and enhance activation of immune responses. through modulation of the th and th cytokines responses enprocal may aid the production of antibodies and gut immune functions. these observed effects of enprocal however may arise not only from the presence of dairy proteins but also from synergistic relationships with its other components in particular the micro-nutrients. strong gut cell integrity and activation of immune cells with enprocal may prevent infections caused by pathogens or the invasion or entry of allergens. thus in future the detailed studies should be carried out on efficacy of enprocal in reducing individual pathogens and gut cell interactions. stimulation of fetal intestinal cell proliferation without cell cytotoxicity by enprocal indicates that it may also be given as infant food particularly for premature babies. although gut epithelial stem cells show no intrinsic limit to their proliferative capacity but recent evidence indicates that they suffer important functional impairments associated with an altered pattern of cellular response to dna damage during the course of ageing. in future studies, it would also be worthwhile to determine whether consumption of enprocal is effective in preventing such functional damage to the stem cells in the aged gut. finally, our in vitro findings by employing well characterized currently available and widely used cell models may be relevant for gut and immune health of consumer groups with special needs, such as infants and the elderly. however future studies are needed to reveal the potential of enprocal as a formulated supplement that produces defined effects on the human gut and immune health. 
our results indicate that enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. effects of enprocal were compared with whey protein concentrate, heat treated skim milk powder, and other commercially available milk derived products. we studied the potential of enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. enprocal and digested selectively enhanced cell proliferation in normal human intestinal epithelial cells and showed no cytotoxicity. stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies. in cytokine secretion analyses, enprocal d down-regulated the secretion of proinflammatory cytokines and up-regulated ifn-γ, il- and il- furthermore, through downregulation of proinflammatory cytokines, enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease. enprocal d upregulated the surface expression of co-stimulatory, mhc i and mhc ii molecules on pma differentiated thp- macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte -epithelial cell adhesion. digested enprocal maintained the intestinal integrity in transepithelial resistance assay, increased the permeability of horseradish peroxidase and did not induce oxidative stress to the gut epithelial cells. in a dose dependent manner enprocal induced cell death in caco- cells.
 enprocal is a high-protein micro-nutrient rich formulated supplementary food designed to meet the nutritional needs of the frail elderly and be delivered to them in every day foods. we studied the potential of enprocal to improve gut and immune health using simple and robust bioassays for gut cell proliferation, intestinal integrity/permeability, immunomodulatory, anti-inflammatory and anti-oxidative activities. effects of enprocal were compared with whey protein concentrate, heat treated skim milk powder, and other commercially available milk derived products.  enprocal and digested selectively enhanced cell proliferation in normal human intestinal epithelial cells and showed no cytotoxicity. in a dose dependent manner enprocal induced cell death in caco- cells. digested enprocal maintained the intestinal integrity in transepithelial resistance assay, increased the permeability of horseradish peroxidase and did not induce oxidative stress to the gut epithelial cells. enprocal d upregulated the surface expression of co-stimulatory, mhc i and mhc ii molecules on pma differentiated thp- macrophages in coculture transwell model, and inhibited the monocyte/lymphocyte -epithelial cell adhesion. in cytokine secretion analyses, enprocal d down-regulated the secretion of proinflammatory cytokines and up-regulated ifn-γ, il- and il-  our results indicate that enprocal creates neither oxidative injury nor cytotoxicity, stimulates normal gut cell proliferation, up regulates immune cell activation markers and may aid in the production of antibodies. furthermore, through downregulation of proinflammatory cytokines, enprocal appears to be beneficial in reducing the effects of chronic gut inflammatory diseases such as inflammatory bowel disease. stimulation of normal human fetal intestinal cell proliferation without cell cytotoxicity indicates it may also be given as infant food particularly for premature babies. 