EP1212066A1

EP1212066A1 - Pro-gut maturation and anti-inflammatory effects of lactobacillus and lactobacillus secreted proteins, carbohydrates and lipids

Info

Publication number: EP1212066A1
Application number: EP00952657A
Authority: EP
Inventors: Pinaki Panigrahi
Original assignee: University of Maryland Baltimore; University of Maryland College Park
Current assignee: University of Maryland Baltimore; University of Maryland College Park
Priority date: 1999-08-09
Filing date: 2000-08-09
Publication date: 2002-06-12
Also published as: WO2001010448A1; EP1212066A4; CA2378620A1

Abstract

Strains of Lactobacilli having the ability to reduce or block pro-inflammatory cytokines and also to induce anti-inflammatory cytokines are disclosed. These strains may be used to prepare therapeutic agents that reduce inflammation. Lactobacillus secreted proteins, carbohydrates and lipids are also disclosed. The Lactobacillus secretions, which block translocation of bacterial agents such as Gram (-) bacteria, other infectious agents, toxins, chemicals and injurious substances, may be used in the prevention and treatment of inflammation caused by bacterial translocation and injury and in treating gastrointestinal dysfunctions.

Description

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.

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.

may also be used to prevent or treat other inflammatory diseases of the GI tract that may have

a bacterial etiologic component.

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

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.

Claims

WHAT IS CLAIMED IS:

1. Lactobacillus secretions, said Lactobacillus secretions being proteinaceous,

said Lactobacillus secretions block epithelial cell bacterial adherence and translocation.

2. Lactobacillus secretions, said Lactobacillus secretions being carbohydrates,

3. Lactobacillus strains and secretions, said Lactobacillus strains and secretions

have anti-inflammatory activity by blocking pro-inflammatory cytokines and by inducing

anti-inflammatory cytokines.

4. Lactobacillus strains and secretions, said Lactobacillus strains and secretions

induce gut maturation.

5. A method of preventing necrotizing tissue injury in the gastrointestinal tract

comprising orally administering Lactobacillus secretions selected from the group consisting

of Lactobacillus proteinaceous secretions and Lactobacillus carbohydrate secretions.

6. The method of claim 5, further comprising protecting tissues along the

gastrointestinal tract by blocking bacterial translocation with said Lactobacillus secretions.

7. The method of claim 5, further comprising protecting tissues along the

gastrointestinal tract with the administered Lactobacillus secretions thereby blocking

translocation of bacterial agents, other infectious agents, toxins, chemicals, and injurious substances.

8. The method of claim 6, wherein the blocking bacterial translocation comprises blocking Gram (-) bacteria.

9. A method of treating neonatal necrotizing enterocolitis wherein inflammation caused by bacterial translocation and injury is reduced comprising administering

Lactobacillus secretions selected from the group consisting oϊ Lactobacillus proteinaceous

secretions and Lactobacillus carbohydrate secretions.

10. A method of preventing gastrointestinal dysfunction characterized by infection

or inflamation comprising administering Lactobacillus secretions selected from the group

consisting oϊ Lactobacillus proteinaceous secretions and Lactobacillus carbohydrate

secretions.

11. A vaccine prepared using the Lactobacillus secretions of Claim 1.