TITLE OF THE INVENTION
PRO-GUT MATURATION AND ANTI-INFLAMMATORY EFFECTS OF LACTOBACILLUS AND LACTOBACILLUS SECRETED PROTEINS, CARBOHYDRATES AND LIPIDS
BACKGROUND OF THE INVENTION
This application claims priority to U.S. Provisional Patent Application Serial No.
60/147,792, filed August 9, 1999, the entire contents of which are incorporated herein by
reference.
Field of the Invention
The present invention relates to Lactobacillus secreted proteins, carbohydrates and
lipids and other anaerobic bacterial strains known to exert effects similar to Lactobacillus
strains and their use in the prevention and treatment of inflammation. The Lactobacillus
secreted proteins, carbohydrates and lipids of the invention can be used to protect intestinal
cells against injury caused by disease, infectious agents, toxins, chemicals and other
injurious substances. The Lactobacillus secreted proteins, carbohydrates and lipids of the
invention can be used, in particular, to prevent and treat neonatal necrotizing enterocolitis.
Background of the Prior Art
Digestive problems, which comprise the number one health problem in North
America, appear to be occurring with more frequency in recent years. One way to maintain
digestive health is to maintain proper intestinal flora. Like many groups of living things,
bacteria have "friendly" and "unfriendly" populations. Friendly bacteria play a major role in
balancing and counteracting the unfriendly bacteria. When friendly bacteria are not at
appropriate levels and when unfriendly bacteria dominate the intestinal flora, health problems
can result, including intestinal toxicity and malabsorption of nutrients.
Lactobacilli are one of the most important types of friendly bacteria found in the
digestive tract. The bacteria, which are named because they are able to turn milk sugar into
lactic acid, play a key role in producing fermented milk, yogurt and cheese. In the early
1900's, Elie Metchinkoff hypothesized that Lactobacilli would provide a hostile environment
to unfriendly bacteria in the intestinal environment. This hypothesis was later proven correct.
Lactobacilli have long been known to have positive effects in the intestine especially
in maintaining a healthy gut microflora. These organisms generally act when they are
available at the action site (intestine) live to exert their effects. These organisms are also
known to secrete antimicrobial substances known as bacteriocins, Le , substances that kill
closely-related strains of other bacteria.
Bacterial translocation, he., the passage of viable intestinal bacteria across the
intestinal epithelial cell layer into the normally sterile extra intestinal tissues, of few bacteria
is a normal process and the mucosal immune system (macrophages as first line of defense)
along with the consequent immune activation generally prevent detrimental translocation.
Secretory immunoglobulins may also prevent the attachment of the same bacteria to the
mucosal surface. Bacterial translocation has been suggested to play a role in the etiology of
posttraumatic infections and multiple organ failure. This is presumed to be due to breakdown
of the intestinal mucosal barrier, permitting pathogenic bacteria to pass into the blood stream.
Lactobacilli are known to prevent pathogenic microorganisms from colonizing on
body surfaces (colonization resistance). Administration of antibiotics has a profound effect
of the normal flora and can result in colonization with antibiotic-resistant organisms.
Antibiotic-mediated disruption on the normal flora can thus lead to infection and its sequele.
Commercial preparations of Lactobacilli have been used to restore normal intestinal flora
after imbalance created by antibiotic therapy.
Despite significant advances in recent neonatal practice, neonatal necrotizing
enterocolitis (NEC) remains a major cause of morbidity and mortality in premature infants.
Survivors of NEC can also have considerable long-term morbidity resulting from the disease,
including short-gut syndrome, failure to thrive, intestinal stricture and the need for repeated
surgery. Although 11% of premature infants born weighing less than 1500 g develop NEC,
the cause of the disease remains unclear and no specific treatments are available. A
reasonable hypothesis suggests that a combination of factors including prematurity, intestinal
ischemia and bacterial colonization lead to stimulation of an inflammatory cascade and a
resulting final common pathway of NEC.
Bacterial colonization of the neonatal gastrointestinal tract begins when the infant
encounters maternal cervical and vaginal bacteria during delivery. Brooke et al.. Aerobic and
anaerobic bacterial flora of the maternal cervix and newborn gastric fluid and conjunctiva: A
prospective study, Pediatrics, 63:451-455 (1979). By 10 days of age, the majority of healthy
full-term newborns are fully colonized with a variety of bacterial species. Long et al.,
Development of anaerobic fecal flora in healthy newborn infants, J. Pediatr., 91 :298-301
(1977). The gut of a premature infant, in contrast, does not provide for proper colonization of
the normally heterogeneous bacterial flora and rather demonstrates delayed colonization with
only a limited number of bacterial species. Gupta et al.. Endemic necrotizing enterocolitis:
lack of association with a specific infectious agent, Pediatr. Infect. Dis., 13:725-734 (1994).
It has been shown that the stool of preterm infants, with and without NEC, is colonized on the
average by fewer than 2.5 species of aerobic bacteria, compared to > 10 species in full terms.
Gupta et al. (1994). It is believed that limited friendly bacterial colonization at least in part
permits pathogenic bacterial overgrowth that could in turn initiate the cascade of events that
lead to NEC.
Human milk populates the intestine with Bifidobacteria and Lactobacilli, generating a
very different gut flora than that seen after formula feeding. Kevworth et al.. Development of
cutaneous microflora in premature neonates, Arch. Dis. Child, 67:792 (1992). A number of
investigators have found decreased numbers of Lactobacilli in preterm infants; the reduction
being correlated with antibiotic therapy and time spent in the incubator. Hall et al.. Factors
influencing the presence of fecal lactobacilli in early infancy, Arch. Dis. Child, 65:185-188
(1990).
A variety oϊin vitro studies indicate that endogenous intestinal bacteria can inhibit
pathogenic bacteria. For example, Sullivan et al. Inhibitions of growth of C. botulinum by
intestinal microflora isolated from healthy infants, Microbial. Ecology in Health and Disease,
1 :179-192 (1988), showed that gut isolates of Bifidobacteria, Lactobacilli, Proprionibacteria
and Enterococci inhibit C. botulinum in vitro. Numerous in vivo studies also lend support to
the ability of selected Lactobacilli to modify the intestinal microflora. Conway. Lactobacilli:
Fact and fiction, Ch. 16 in The regulatory and protective role of the normal flora, Grun,
Midvedt and Norm, eds., Stockton Press, pp. 263-281 (1988).
Studies indicate that it is possible to successfully modify the gut flora in preterm
infants by orally administering Bifidobacteria and Lactobacilli during and after antibiotic
therapy.
Copending U.S. Patent Application Ser. No. 08/818,995, the entire contents of which
are incorporated herein by reference, discloses that two strains of Lactobacillus, i.e.,
Lactobacillus acidophilus and Lactobacillus plantarum, reduced tissue injury and
inflammatory cell infiltration, suggesting that they are useful in prevention and/or treatment
ofNEC.
Rubaletti et al.. Probiotics Feeding Prevents Necrotizing Enterocolitis in Preterm
Infants: A Prospective Double-Blind Study, Pediatric Academic Societies and American
Academy of Pediatrics Joint Meeting (May 2000), discloses that Lactobacillus GG
supplementation reduces the occurrence of NEC and urinary tract infection in preterm infants.
U.S. Patent No. 5,413,785 discloses a method for reducing the quantity of endotoxin
in blood plasma that includes administering Lactobacillus and a biocompatible carrier into the gastrointestinal tract.
Thus, the clinical use of Lactobacillus to enhance intestinal defense against potential
luminal pathogens has been tested in vivo; however, an understanding of the mechanisms
responsible for the observed protection is lacking. There thus exists a need to understand the
underlying mechanisms responsible for Lactobacilli 's beneficial effects in preventing and
treating infection and inflammation.
SUMMARY OF THE INVENTION
Elevated levels of proinflammatory cytokines have been demonstrated in blood and
tissue samples from babies with NEC. It has previously been shown that adherent E. coli can
cause NEC-like injury in a rabbit ileal loop model and that Gram (+) bacteria can block such
injury.
The present inventor has surprisingly discovered that certain strains of Lactobacilli
have the ability to reduce or block pro-inflammatory cytokines and that these strains also
induce anti-inflammatory cytokines. Such effects have important ramifications in the host;
permitting the use of Lactobacilli to prepare vaccines against inflammatory disease, as well
as to prepare therapeutic agents to reduce inflammation if the disease has already been
established.
The present invention is also directed to unique proteins, carbohydrates and lipids
secreted by Lactobacilli. Although these secretions are capable of blocking bacterial
adherence, they exhibit very little or no anti-microbial activity.
The present inventor has also surprisingly discovered that Lactobacilli are able to
stimulate gut maturation.
The Lactobacillus strains, proteins, carbohydrates and lipids of the present invention
may be used to treat adult and pediatric patients in intensive care units under total parenteral
nutrition (intravenous feed) to avoid mucosal dysfunction and further bacterial translocation.
The Lactobacillus strains, proteins, carbohydrates and lipids of the present invention
may also be used to treat patients undergoing chemotherapy, irradiation and bone marrow
transplantation.
The Lactobacillus strains and secretions of the invention may be used to prevent and
treat food allergy and intolerance, where injury caused by an antecedent bacterial infection
allows the passage of food antigens through the gut mucosa and further triggers the
inflammatory process.
The Lactobacillus strains and secretions of the invention may also be used to prevent
and treat other GI disorders including but not limited to Celiac disease, where initial damage
to the gut mucosa allows the passage of the triggering antigen to gain access to deeper layers
of the intestine, which in turn, in concert with other immunologic, infective, or genetic factors
can cause the clinical disease.
The Lactobacillus strains, proteins, carbohydrates and lipids of the present invention
may also be used to prevent or treat other inflammatory diseases of the GI tract that may have
a bacterial etiologic component.
The Lactobacillus strains, proteins, carbohydrates and lipids of the present invention
may further be used in to treat fullterms, children, and adults, in GI dysfunctions of infective
and/or inflammatory origin where bacterial infection may act as a trigger or aid in disease
progression.
A preferred method of treating neonatal necrotizing enterocolitis comprises providing
the Lactobacillus strains, proteins, carbohydrates and lipids of the invention for reducing
inflammation caused by bacterial adherence, invasion and injury.
A preferred method of treating gastro-intestinal dysfunctions includes providing the
Lactobacillus strains, proteins, carbohydrates and lipids of the invention for improving
physiological functions.
These and further and other objects and features of the invention are apparent in the
disclosure, which includes the above and ongoing written specification, with the claims and
the drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1: Lactobacillus secreted factors from overnight growth in DMEM were
separated by PAGE (15%) and stained with Coomassie blue. M: molecular weight markers
in kilo Dalton; Lane 1 : DMEM (experimental medium) only; Lane 2: whole cell lysates of .
plantarum; Lane 3: secreted factors of L. plantarum; Lane 4: whole cell lysates ofZ.
salivarius spp salivarius; Lane 5: secreted factors of L. salivarius spp salivarius. A protein
with approximate molecular weight of 20 KD is identified in L. plantarum secretions (arrow).
Two proteins with approximate molecular weights of 48 KD and 30 KD are identified in the
secretions of L. salivarius spp salivarius (arrows).
Figure 2: Adherence assay using E. coli and L. plantarum-secτeted factors. E.M.:
experimental medium (DMEM only); CM: conditioned medium (from overnight culture of
L. plantarum); Pr.K: conditioned medium treated with proteinase-K before the adherence
assay. Oxidised: conditioned medium oxidized before the adherence assay; Live in EM: live
bacteria (L. plantarum) with E. coli. Note the reduction in activity (blocking E. coli) after
destroying the proteins and carbohydrates. These results suggest that the 20 KD protein and
additional factor(s), carbohydrate in nature mediate the effects of L. plantarum.
Figure 3: Adherence assay using E. coli and L. salivarius spp salivarius -secreted
factors. E.M.: experimental medium (DMEM only); CM: conditioned medium (from
overnight culture of L. salivarius); Pr.K: conditioned medium treated with proteinase-K
before the adherence assay. Amylase: conditioned medium treated with amylase to destroy
the carbohydrates; Am/PrK: conditioned medium treated with amylase plus proteinase-K.
Note a non-significant reduction in activity (blocking E. coli) after destroying the proteins
and carbohydrates. Contrary to the results in Figure 2, these data suggest that the E. coli
blocking effects of L. salivarius are mediated by lipids.
Figure 4: Induction of cytokine mRNA in rabbit ileal loops infected with E. coli and
a combination of E. coli and L. plantarum. There was a significant reduction of
proinflammatory cytokine IL-1 after co-infection with L. plantarum. Anti-inflammatory
cytokine IL-10 was induced only after co-infection with L. plantarum (E. coli alone did not
induce IL-10). Also seen was an increased induction of IL-1 RA (receptor antagonist) after
co-infection with L. plantarum .
Figure 5. Expression of small intestinal enzymes by Caco-2 cells. There was a
significant increase in the expression of maltase, sucrase, and glucoamylase after the cells
were treated with L. plantarum .
Figure 6. Expression of small intestinal enzymes by Caco-2 cells. Note the increase
in lactase and palatinase after treatment with L. plantarum .
Figure 7. TEER (trans epithelial electrical resistance) measurement after treatment
with L. plantarum. There was a significant increase in TEER after 1 and 2 hours treatment
with L. plantarum.
Figure 8. Long term TEER (trans epithelial electrical resistance) measurement after
treatment with L. plantarum. The increase in TEER was maintained over the 6 hr period after
treatment with L. plantarum.
DETAILED DESCRIPTION OF THE INVENTION
The present invention demonstrates that specific strains of Lactobacilli have the
ability to reduce or block pro-inflammatory cytokines and induce anti-inflammatory
cytokines. These effects have important ramifications in the host; and may be used as a
vaccine against inflammatory diseases, as well as a therapeutic agent to reduce inflammation
when the disease has already been established. The invention is also directed to unique
proteins, carbohydrates and lipids secreted by these organisms. The proteins, carbohydrates
and lipids, which are capable of blocking bacterial adherence and translocati on/invasion,
have very little or no antimicrobial activity.
The following Examples are provided for illustrative purposes only and are in no way
intended to limit the scope of the present invention.
Examples
Two Lactobacillus strains L. plantarum and L. salivarius spp salivarius (assigned
ATCC 202195 and ATCC 202196, respectively, by the American Type Culture Collection,
10801 University Blvd., Manassas, VA 20110-2209 USA) were used in the following
experiments:
Caco-2 cell Adherence Assay. A Caco-2 cell adherence assay was performed using
standard techniques, E. coli was used as the adherent candidate Gram (-) organism according
to Panigrahi et al.. Occurrence of necrotizing entrocolitis may be dependent on patterns of
bacterial adherence and intestinal colonization: Studies in Caco-2 tissue culture and weanling
rabbit models, Pediatr. Res., 36:115-121 (1994). Briefly, mono- and co-infections of Caco-2
cells were performed with Lactobacillus strains L. plantarum and L. salivarius spp salivarius
(109 organisms/ml) andE. coli (108 organisms/ml), followed by washing, fixing with ethyl
alcohol and incubation with hyperimmune rabbit serum against E. coli. After further
washings, monolayers were again incubated with FITC-anti-rabbit IgG (Fab specific). Figure
1 A shows the effect of concentrated media of Lactobacillus on adherence of 6-1 to Caco-2
cells. The results demonstrate that the number of E. coli adhering to Caco-2 cells was
drastically reduced after co-infection with two Lactobacillus strains, Le., L. plantarum and L.
salivarius spp salivarius.
Caco-2 Cell Transwell System. A Caco-2 cell transwell system was used in
accordance with Panigraphi et al.. Development of an in vitro model for study of non-01
Vibrio cholerae virulence using Caco-2 cells, Infect. Immun., 58:3415-3424 (1990) and
Panigraphi et al. (1994) to grow cells on a membrane allowing the measurement of bacteria
that translocate. Briefly, Caco-2 cells were grown on polycarbonate filters in transwell
clusters and TEER (trans-epithelial electrical resistance) was measured before and after
Lactobacillus treatment.
Lactobacillus Secretions. Lactobacillus was grown in DMEM overnight from fresh
plate cultures. The medium was centrifuged and clarified by passing through a 0.2 nm filter.
Adherence assays were performed as previously described. Figure 1 shows the proteins that
have been secreted from both strains.
Figure 2 shows the effect of conditioned media of L. plantarum on adherence of E.
coli to Caco-2 cells. The results show a significant reduction in E. coli adherence when
Lactobacillus secretions were used. Note the reduction in activity (blocking E. coli) after
destroying the proteins and carbohydrates. These results suggest that the 20 KD protein and
additional factor(s), carbohydrate in nature mediate the effects of L. plantarum.
Figure 3 shows the effect of conditioned media of L. salivarius spp salivarius on
adherence of E. coli to Caco-2 cells. Note a non-significant reduction in activity (blocking E.
coli) after destroying the proteins and carbohydrates. Contrary to the results in Figure 2,
these data suggest that the E. coli blocking effects of L. salivarius are mediated by lipids.
Anti-inflammatory Effects of Lactobacillus. The anti-inflammatory effects of
Lactobacillus were determined in the rabbit ileal loop experiments by RT-PCR. The
expression of pro-inflammatory cytokine IL-1 was significantly reduced after co-infection
with L. plantarum. Anti-inflammatory cytokine IL-10 was induced only after co-infection
with L. plantarum (E. coli alone did not induce IL-10). Also seen was an increased induction
of IL-1RA (receptor antagonist) after co-infection with . plantarum. The levels of these
mRNAs were increased after E. coli infection.
There was an increase in the expression of IL-10 and IX- IRA (anti-inflammatory
cytokines) and a decrease in the expression of pro-inflammatory cytokine IL-1 in these
experiments. These results suggest that Lactobacilli may exert their beneficial effect by
blocking bad cytokines and concurrently stimulating the expression of good cytokines.
Gut Maturation After Lactobacillus Treatment. Gut maturation after Lactobacillus
treatment studies were performed in cultured Caco-2 cells grown in petri dishes or in
transwell cultures. The trans-epithelial electrical resistance was significantly increased after
treatment of the monolayers with L. plantarum. Expression of brush border specific enzymes
(that are highly related to maturation) were assayed in control and Lactobacillus-tτeated cells.
There was a significant increase in the expression of maltase, sucrase, glucoamylase and
palatinase, and to a lesser extent lactase.
Weanling Rabbit Ileal Loop Experiment: Following previously described
protocols, (Panigrahi P. Gupta S. Gewolb IH, and Morris JG Jr. 1994. Occurrence of
Necrotizing Enterocolitis may be dependent on patterns of bacterial adherence and intestinal
colonization: Studies in Caco-2 tissue culture and weanling rabbit models. Ped. Res. 36
(1):115-121), weanling rabbit ileal loops were infected with either E. coli alone or in
combination with L. plantarum. After 12-16 hr, animals were euthanasized and segments of
the loops were frozen in liquid nitrogen. RNA was extracted from pulverized intestinal
segments, followed by cDNA synthesis and RT-PCR for cytokines.
Various other modifications will be apparent to and can be readily made by those
skilled in the art without departing from the scope and spirit of this invention. Accordingly, it
is not intended that the scope of the claims appended hereto be limited to the description as
set forth herein, but rather that the claims be broadly construed.