Lactic Acid Bacteria Strains Useful Against Urogenital Pathogens And Compositions Containing Same

Graf; Federico ;   et al.

Patent Application Summary

U.S. patent application number 11/738102 was filed with the patent office on 2008-01-24 for lactic acid bacteria strains useful against urogenital pathogens and compositions containing same. Invention is credited to Dominique Brassart, Federico Graf, Philipp Grob.

Application Number20080019954 11/738102
Document ID /
Family ID35534903
Filed Date2008-01-24

United States Patent Application 20080019954
Kind Code A1
Graf; Federico ;   et al. January 24, 2008

LACTIC ACID BACTERIA STRAINS USEFUL AGAINST UROGENITAL PATHOGENS AND COMPOSITIONS CONTAINING SAME

Abstract

The invention relates to a method for preventing or treating urogenital infections in females, which comprises administering a pharmaceutical preparation comprising, in combination with a suitable galenical carrier, a therapeutically effective amount of at least one lactic acid bacteria strain of the genus L. acidophilus, L. crispatus, L. gasseri, L. helveticus and L. jensenii selected for their ability to kill urogenital and/or gastrointestinal pathogens and their ability to inhibit internalization of urogenital and/or gastrointestinal pathogens within vaginal epithelial cells, as well as for their ability to modulate a humoral and/or cellular immune response at the vaginal and/or gastrointestinal level, in particular to inhibit the inflammatory syndrome as well as the infectious syndrome.


Inventors: Graf; Federico; (Zurich, CH) ; Grob; Philipp; (Zurich, CH) ; Brassart; Dominique; (Chavannes/Renens, CH)
Correspondence Address:
    WINSTON & STRAWN LLP;PATENT DEPARTMENT
    1700 K STREET, N.W.
    WASHINGTON
    DC
    20006
    US
Family ID: 35534903
Appl. No.: 11/738102
Filed: April 20, 2007

Related U.S. Patent Documents

Application Number Filing Date Patent Number
PCT/EP05/11150 Oct 17, 2005
11738102 Apr 20, 2007

Current U.S. Class: 424/93.45 ; 435/252.9
Current CPC Class: A61K 9/48 20130101; A61P 15/02 20180101; A61K 2035/115 20130101; A61K 35/747 20130101; A23L 33/135 20160801; A61K 9/0053 20130101; A61P 13/10 20180101; A61K 9/0034 20130101; A23L 2/382 20130101; A61K 9/02 20130101; A61P 15/00 20180101; A61P 29/00 20180101; A61P 31/00 20180101; A61P 37/04 20180101; Y02A 50/473 20180101; A61P 31/10 20180101; A61P 13/02 20180101; A23V 2002/00 20130101; A61P 1/00 20180101; C12R 1/225 20130101; C12R 1/23 20130101; A61P 37/02 20180101; Y02A 50/30 20180101; A61P 31/04 20180101; A61P 13/00 20180101; A61K 35/747 20130101; A61K 2300/00 20130101; A23V 2002/00 20130101; A23V 2200/32 20130101; A23V 2200/3204 20130101
Class at Publication: 424/093.45 ; 435/252.9
International Class: A01N 63/00 20060101 A01N063/00; A01P 15/00 20060101 A01P015/00; C12N 1/20 20060101 C12N001/20

Foreign Application Data

Date Code Application Number
Oct 22, 2004 EP PCT/EP04/11980

Claims



1. A method for treating urogenital infections in females which comprises administering to a female in need of such treatment a pharmaceutical preparation comprising a therapeutically effective amount of at least one lactic acid bacteria strain selected from the group consisting of L. crispatus KS 116.1 (CNCM 1-3483), L. crispatus KS 119.4 (CNCM 1-3484), L. crispatus 127.1 (CNCM 1-3486), L. gasseri KS 114.1 (CNCM 1-3482), L. gasseri KS 120.1 (CNCM 1-3218), L. gasseri KS 123.1 (CNCM 1-3485), L. gasseri KS 124.3 (CNCM 1-3220), L. helveticus KS 300 (CNCM 1-3360), L. jensenii KS 119.1 (CNCM 1-3217) and L. jensenii KS 121.1 (CNCM 1-3219) in combination with a pharmaceutically acceptable galenical carrier.

2. The method of claim 1 wherein the galenical carrier is designed for local administration.

3. The method of claim 1 wherein the galenical carrier is designed for oral administration.

4. A method for inhibiting adhesion, colonization and/or growth of pathogeris in the urogenital tract of females which comprises administering to a female in need of such treatment a pharmaceutical preparation comprising a therapeutically effective amount of at least one lactic acid bacteria strain selected from the group consisting of L. crispatus KS 116.1 (CNCM 1-3483), L. crispatus KS 119.4 (CNCM 1-3484), L. crispatus 127.1 (CNCM 1-3486), L. gasseri KS 114.1 (CNCM 1-3482), L. gasseri KS 120.1 (CNCM 1-3218), L. gasseri KS 123.1 (NCM 1-3485), L. gasseri KS 124.3 (NCM 1-3220), L. helveticus KS 300 (CNCM 1-3360), L. jensenii KS 119.1 (CNCM 1-3217) and L. jensenii KS 121.1 (CNCM 1-3219) in combination with a pharmaceutically acceptable galenical carrier.

5. The method of claim 4 wherein the galenical carrier is designed for local administration.

6. The method of claim 4 wherein the galenical carrier is designed for oral administration.

7. Lactic acid bacteria strains selected from-the group consisting of L. crispatus KS 116.1 (CNCM 1-3483), L. crispatus KS 119.4 (CNCM 1-3484), L. crispatus 127.1 (CNCM 1-3486), L. gasseri KS 114.1 (CNCM 1-3482), L. gasseri KS 120.1 (CNCM 1-3218), L. gasseri KS 123.1 (CNCM 1-3485), L. gasseri KS 124.3 (CNCM 1-3220), L. helveticus KS 300 (CNCM 1-3360), L. jensenii KS 119.1 (CNCM 1-3217) and L. jensenii KS 121. 1 (CNCM 1-3219).
Description



CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of international application PCT/EP2005/011150 filed October 17, 2005, the entire content of which is expressly incorporated herein.

FIELD OF THE INVENTION

[0002] This invention refers to the treatment of infectious troubles caused by various pathogens in humans, more specifically to the prevention and/or the treatment of urogenital infections in females.

BACKGROUND OF THE INVENTION

[0003] Several experimental and clinical studies have assessed already the potential of certain lactobacilli in the prevention or treatment of certain genito-urinary tract infections such as bacterial vaginitis, vaginosis, or urinary tract infections and relevant therapy is applied for many decades already.

[0004] Urogenital infections remain a common problem, particularly in the female population. Bacterial adherence to the urogenital epithelium is recognized as an important mechanism in the initiation and pathogenesis of urinary tract infections (UTI) and, in particular, of vaginal infections. The urogenital pathogens originate predominantly in the intestinal tract and initially colonize the per-urethral region and ascend into the bladder, resulting in symptomatic or asymptomatic bacterial uria. Alternatively, these bacteria invade and then colonize the vagina causing there various types of symptomatic as well as asymptomatic vaginal infections. Thereafter, depending on host factors and bacterial virulence factors, the organisms may further ascend and give rise to pyelonephritis, respectively ascending infections of the genital tract in women. Urogenital pathogens express virulence characteristics that enable them to resist the normally efficient host defense mechanisms.

[0005] The use of bacteria of the autochthonous flora, such as lactobacilli, to exclude urogenital pathogens from colonizing the urogenital tract is an established concept studied rather extensively since years ago (see e.g. Cadieux et al.--Lactobacillus strains and vaginal ecology--Jama. 287:1940-41/2002; Butler B C, Beakley J W. Bacterial flora in vaginitis. Am J Obstet Gynaecol 1960;78:432-40; Eschenbach D A, Davick P R, Williams B L, Klebanoff S J, Young-Smith K, Critchlow C M et al. Prevalence of hydrogen peroxide-producing Lactobacillus species in normal women and women with bacterial vaginosis. J Clin Microbiol 1989;27:251-6; Sobel J D, Cook R L, Redondo-Lopez V. Lactobacilli: a role in controlling the vaginal microflora? in Horowitz B J, Mardh P-A, eds. Vaginitis and Vaginosis, pp 47-53. New York: Wiley-Liss, 1991; Lauritzen C, Graf F, Mucha M. Restoration of the physiological vaginal environment with Doederlein bacteria and estriol. Frauenarzt 1984; 4). Lactobacilli, when used in this context, are believed to contribute to the control of the urogenital micro flora by different mechanisms such as the production of lactic acid, antagonistic substances like bacteriocins, and H.sub.2O.sub.2.

[0006] The main goal of therapy with bio therapeutic agents should be to prevent overgrowth of pathogens until such a time that the normal microflora can be re-established. In addition, bacterial therapy is considered as "natural" and without side effects in contrast with conventional pharmaceutical treatments. Nowadays, despite of the progresses which have already been made concerning the intimate knowledge of lactic acid bacteria (LAB) strains, their properties and their potential use in the pharmaceutical area, there still remains the need to propose more convenient and more efficient LAB strains to the medical community.

[0007] The purpose of this invention is to provide new and useful LAB strains particularly efficient in the treatment of infections caused by various pathogens, namely infections of the urogenital tract in females, or in the restoration of a balanced and healthy urogenital flora after e.g. severe medical treatments like those performed with antibiotics. This invention provides as well new methods of prophylactic or therapeutic treatment of such infections which involve specifically selected LAB strains.

SUMMARY OF THE INVENTION

[0008] The present invention now provides lactic acid bacteria (LAB) strains of the genus L. acidophilus, L. crispatus, L. gasseri, L. helveticus and L. jensenii selected for their ability to kill urogenital and/or gastrointestinal pathogens and their ability to inhibit internalization of urogenital and/or gastrointestinal pathogens within vaginal epithelial cells.

[0009] These bacteria are further selected for their ability to modulate a humoral and/or cellular immune response in the urogenital and/or gastrointestinal mucous membrane cells and to inhibit the inflammatory syndrome within said mucous membrane cells.

[0010] The invention also provides a method for establishing, maintaining or restoring a healthy urogenital flora in females throughout life, which comprises administering a pharmaceutical preparation comprising, in combination with a suitable galenical carrier, a therapeutically effective amount of at least one of the selected LAB strains.

[0011] The invention further refers to a method for preventing or treating urogenital infections in females which comprises administering a pharmaceutical preparation comprising, in combination with a suitable galenical carrier, a therapeutically effective amount of at least one of said LAB strains.

[0012] The invention still further refers to a method for preventing or inhibiting adhesion, colonization and/or growth of pathogens in the urogenital tract of females which comprises administering a pharmaceutical preparation comprising, in combination with a suitable galenical carrier, a therapeutically effective amount of at least one of said LAB strains.

[0013] The invention also refers to compositions, in particular pharmaceutical compositions useful within the above mentioned frame, which comprise a therapeutically effective amount of at least one of the selected LAB strain in combination with a suitable galenical carrier.

[0014] Eventually, this invention refers to the use of one of said LAB strains in the preparation of compositions, more particularly pharmaceutical compositions mentioned here above.

DETAILED DESCRIPTION OF THE INVENTION

[0015] The LAB strains of this invention have been first selected for their ability to adhere to epithelial cells such as cervix HeLa or Caco-2 which were chosen as models. Cell adhesion is indeed a prerequisite selection feature as it conditions the capacity of the said LAB strains to colonize epithelial tissues, e.g. that of the urogenital tract, and then to compete with, inhibit or exclude pathogens adhesion from that specific location.

[0016] The said LAB strains have been further selected for their additional ability to inhibit adhesion, growth and even survival of pathogens, namely urogenital and gastrointestinal pathogens from epithelial cells. Gram-negative as well as Gram-positive pathogens, such as those mentioned here after are representative of those which are significantly affected by the LAB strains of this invention in terms of adhesion, growth or pathogenic activity: Salmonella species, like S. enterica serovar Typhimurium, E. coli, Streptococcus species, e.g. S. agalactiae, Staphylococcus species like S. aureus, Gardnerella species, e.g. G. vaginalis, Prevotella species, e.g. P. bivia; this enumeration is of course not exhaustive.

[0017] The LAB strains of the present invention have also the ability to ability to kill and/or to inhibit internalization of urogenital and/or gastrointestinal pathogens within vaginal epithelial cells, in particular urogenital or gastrointestinal gram-negative or gram-positive pathogens such as those referred to here above. The most efficient LAB within that frame are those which express high amounts of both hydrogen peroxide (H.sub.2O.sub.2) and lactic acid in situ where both factors act synergistically. The latter have indeed proved highly efficient against anaerobic urogenital pathogens like e.g. Gardnerella and Prevotella species.

[0018] The LAB strains of this invention, eventually, exhibit a further important feature, i.e. the ability to modulate the immune response of immune competent cells, namely cells of the gastrointestinal or urogenital mucous membrane as well as epithelial cells, in other words the ability to initiate, stimulate or reinforce the immune response of said cells when infected by Gram-negative or Gram-positive pathogens like those mentioned here above, in particular urogenital pathogenic E. coli. Due to their specific feature the said LAB strains have consequently the capacity to inhibit the inflammatory syndrome of the gastrointestinal or the urogenital mucous membrane, or of the vaginal epithelial cells when exposed to pathogen contamination.

[0019] Quite interestingly that specific feature performs the modulation of the immune response referred to here above using two distinct routes, i.e. via the induction of either pro- or anti-inflammatory cytokines like IL10, respectively, IL12, TNF or IFN. It has been further observed that some LAB strains of this invention exhibit a high IFN.gamma. induction potential, namely L. crispatus KS 116.1 and L. gasseri KS 124.3, a feature which favors the use of same as anti-infectious agents.

[0020] This strain specificity provides consequently to the man skilled in the art the possibility to select the most appropriate strains or combination of strains for performing the medical treatment which is at stake.

[0021] Among the LAB strains which exhibit these properties preferred species according to this invention are listed here after: L. jenseii KS 109, L. gasseri KS 114.1, L. crispatus KS 116.1, L. jensenii KS 119.1, L. crispatus KS 119.4, L. gasseri KS 120.1, L. jensenii KS 121.1, L. jensenii KS 122.1, L. gasseri KS 123.1, L. gasseri 124.3, L. gasseri KS 126.2, L. crispatus 127.1, L. jensenii KS 129.1, L. jensenii KS 130.1, L. helveticus KS 300 and L. acidophilus KS 400. Most of these strains are representative of the healthy human vaginal micro flora.

[0022] As particularly preferred species, one can further cite the following strains: [0023] L. gasseri KS 114.1 (CNCM1-3482): gram positive--catalase negative--oxidase negative--lactic acid production 10.5 g/l--H.sub.2O.sub.2 production 10 mg/I [0024] API 50 CHI test: positive for GAL, GLU, FRU, MNE, NAG, ESC, SAL, CEL, MAL, SAC, TRE and GEN [0025] negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARE, LAC, MEL, INU, MLZ, RAF, AMD, GLYG, XLT, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0026] L. crispatus KS 116.1 (CNCM 1-3483): gram positive--catalase negative--oxidase positive--lactic acid production 9.6 g/l--H.sub.2O.sub.2 production 2 mg/l [0027] API 50 CHI test: positive for GAL, FRU, MNE, NAG, ESC, SAL, MAL and SAC [0028] negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, GAL, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, CEL, LAC, MEL, TRE, INU, MLZ, RAF, AMD, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0029] L. jensenii KS 119.1 (CNCM 1-3217): gram positive--catalase negative--oxidase negative--lactic acid production 7.4 g/l--H.sub.2O.sub.2 production 20 mg/l [0030] API 50 CHI test: positive for GLU, FRU, MNE, NAG, AMY, ESC, SAL, CEL, MAL, MEL, SAC, GEN and TAG--variable for: RIB [0031] negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, GAL, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, ARB, LAC, TRE, INU, MLZ, RAP, AMD, GLYG, XLT, TUR, LYX, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0032] L. crispatus KS 119.4 (CNCM 1-3484): gram positive--catalase negative--oxidase positive--acid production 10.3 g/l--H.sub.2O.sub.2 production negative [0033] API 50 CHI test: positive for GAL, GLU, FRU, MNE, NAG, ESC, MAL, LAC, SAC and AMD [0034] negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, SAL, CEL, MEL, TRE, INU, MLZ, RAF, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0035] L. gasseri KS 120.1 (CNCM 1-3218): gram positive--catalase negative--oxidase negative--lactic acid production 10.6 g/l--H.sub.2O.sub.2 production 1 mg/l [0036] API 50 CHI test: positive for: GAL, GLU, FRU, MNE, AMY, ESC, SAL, CEL, MAL, LAC, SAC, THE and AMD [0037] negative for: KON, GLY, ERY, DARA, LARA, RIB, DXYL, LXYL, ADO, MDX, SBE, RHA, DDL, INO, MAN, SOR, MDM, MDG, NAG, ARE, MEL, INU, MLZ, RAF, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT, 2KG and 5KG [0038] L. jensenii KS 121.1 (CNCM1-3219): gram positive--catalase negative--oxidase negative--lactic acid production 10.6 g/l--H.sub.2O.sub.2 production 1 mg/l [0039] API 50 CHI test: positive for: GAL, GLU, FRU, MNE, AMY, ARE, ESC, SAL, CEL, MAL, SAC and AMD--variable for: RIB, NAG, LAC, RAF and LFUC [0040] negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, MEL, TRE, INU, MLZ, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, DARL, LARL, GNT, 2KG and 5KG [0041] L. gasseri KS 123.1 (CNCM 1-3485): gram positive--catalase negative--oxidase negative--lactic acid production 8.5 g/l--H202 production 10 mg/l [0042] API 50 CHI test: positive for: GLU, MNE, NAG, ESC, MAL and SAC--variable for RIB and 5KG [0043] negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, GAL, FRU, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, SAL, CEL, LAC, MEL, TRE, INU, MLZ, RAF, AMD, GLYG, XLT, GEN, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT and 2KG [0044] L. gasseri KS 124.3 (CNCM 1-3220): gram positive--catalase negative--oxidase negative--lactic acid production 17.0 g/l--H.sub.2O.sub.2 production 20 mg/l [0045] API 50 CHI test: positive for: GAL, GLU, FRU, MNE, NAG, ESC, SAL, MAL, SAC and TRE--variable for: RIB, AMD, GEN and 5KG [0046] negative for: KON, GLY, ERY, DARA, LARA, DXYL, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MAN, SOR, MDM, MDG, AMY, ARB, CEL, LAC, MEL, INU, MLZ, RAF, GLYG, XLT, TUR, LYX, TAG, DFUC, LFUC, DARL, LARL, GNT and 2KG [0047] L. crispatus KS 127.1 (CNCM 1-3486): gram positive--catalase negative--oxidase positive--lactic acid production 16.7 g/l--H.sub.2O.sub.2 production negative [0048] API 50 CHI test: positive for RIB, GAL, GLU, FRU, MNE, MAN, SOR, NAG, AMY, ESC, SAL, CEL, MAL LAC, SAC, TRE, MLZ, AMD, GLYG, GEN, TAG and GNT--variable for GLY and DXYL [0049] negative for: KON, ERY, DARA, LARA, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MDM, MDG, ARB, MEL, INU, MLZ, RAF, XLT, TUR, LYX, DFUC, LFUC, DARL, LARL, 2KG and 5KG [0050] L. helveticus KS 300 (CNCM1-3360): gram positive--lactic acid production 10.45 g/kg--H.sub.2O.sub.2 production 1 mg/l [0051] API 50 CHI test--positive for: GAL, GLU, FRU, MNE, AMY, ARE, ESC, SAL, GEL, MAL, LAC, SAC, TRE and AMD [0052] negative for: RIB, MAN, GLY, SOR, KON, ERY, MLZ, DARA, LARA, LXYL, ADO, MDX, SBE, RHA, DUL, INO, MDM, MDG, MEL, INU, RAF, TAG, GNT, XLT, TUR, LYX, DFUC, LFUC, DARL, LARL, 2KG and 5KG.

[0053] These strains have been duly registered at the Pasteur Institute, Paris (France) in accordance with the Budapest Treaty.

[0054] According to the present invention, due to their specific antipathogen activity, the LAB strains can be used advantageously for preventing or treating urogenital infections in females and for preventing or inhibiting the colonization and/or growth of pathogens in the urogenital tract of females as well, i.e. in a context wherein said LAB strains proved quite efficient.

[0055] Also, the said LAB strains can be used in a quite efficient way for maintaining or restoring a healthy urogenital flora in females throughout life, more particularly for establishing, maintaining or restoring a healthy urogenital flora in females prior to, during and after pregnancy, or for restoring a balanced and healthy flora after severe medical treatments like those performed with antibiotics.

[0056] The corresponding therapeutic or prophylactic treatments are performed by administering either orally or locally a pharmaceutical preparation comprising, in combination with a suitable galenical excipient, support or carrier a therapeutically effective amount of LAB strains of this invention.

[0057] Pharmaceutical compositions or preparations suitable to perform the above treatments can further comprise the usual LAB growth factors. When designed for an oral administration said compositions can be provided as e.g. food supplements, preferably in the form of ingestible capsules comprising lyophilized microorganisms, or in the form of edible milk based powders, suspensions or emulsions or in the form of edible fermented milk products, e.g. yogurts.

[0058] Compositions as those mentioned here above can comprise mixtures of LAB strains of this invention and mixtures of at least one of these strains together with one or several strains of the prior art as well. Also, these compositions may contain additional pharmaceutically active ingredients like chemical compounds specifically selected.

[0059] The compositions or preparations referred to here above may contain the selected microorganisms in amounts which can range from about 106 cfu (colony forming units), preferably from about 10.sup.8 to about 10.sup.11 cfu per g or dose or unit, usually in a dehydrated form that keeps their viability and their specificity intact, e.g. in a lyophilized form. The ultimate details of said compositions as well as their dosage shall depend eventually on the specific application and the target organ they are intended for, the age or health status of the patients or persons to be treated, the nature of the pathogen contamination. It is within current skills and expertise of the medical community to adjust all the relevant parameters.

[0060] When compared to prior known reference strains (see examples below) the LAB strains of the present invention have shown either similar or higher antipathogens activity depending on the experimental model which has been selected therefore.

[0061] The following examples illustrate only some of the embodiments of this invention and so are not intended to constitute any limitation or restriction thereof.

EXAMPLES

[0062] TABLE-US-00001 Material and Methods Tested Strain Code L. jensenii 109 KS 109 L. crispatus 116.1 KS 116.1 L. jensenii 119.1 KS 119.1 L. gasseri 120.1 KS 120.1 L. jensenii 121.1 KS 121.1 L. jensenii 122.1 KS 122.1 L. gasseri 124.3 KS 124.3 L. gasseri 126.2 KS 126.2 L. jensenii 129.1 KS 129.1 L. jensenii 130.1 KS 130.1 L. helveticus 300 KS 300 L. acidophilus 400 KS 400

[0063] The control adhering lactobacilli strain are the L. casei rhamnosus strain GG (ATCC Accession no 53103), the L. rhamnosus strain GR-1 (ATCC Accession no 55826) and the L. fermentum strain RC-14 (ATCC Accession no 55845).

[0064] All the lactobacilli strains were grown in De Man, Rogosa, Sharpe (MRS) broth (Biokar Diagnostic, Beauvais, France) for 18 h at 37.degree. C.

[0065] Bacterial pathogens. Salmonella enterica serovar Typhimurium strain SL 1344 was a gift of B.A.D. Stocker (Stanford, Calif.). Bacteria were grown overnight for 18 h at 37.degree. C. in Luria broth (Difco Laboratories).

[0066] Uropathogenic diffusely-adhering Escherichia coli strains IH 11128 and 7372, and diarrheagenic strain C1845 were gifts from B. Nowicki (Texas University, Galvestone) and S. Moseley (Seattle University). Strain 7372 carries the class II papG allele, the hly gene (haemolysin) and the Dr operon. Strain IH11128 carries the Dr operon. Strain C1845 carries the Daa operon. All bacterial strains were maintained on LB plates and prior to infection, bacteria were grown in LB broth at 37.degree. C. for 18 h.

[0067] Strains of Streptococcus agalactiae DSM 2134, Gardnerella vaginalis DSM 4944, Prevotella bivia Cl-I (DSM 20514) and Candida albicans DSM 1386 were from Medinova Ltd, Zurich, Switzerland). Bacteria were grown overnight at 37.degree. C. in TSA broth (Difco Laboratories). Streptococcus agalactiae strain was grown overnight at 37.degree. C. in BHI broth (Difco Laboratories).

[0068] Candida albicans strain was grown overnight at 37.degree. C. in Sabouraud broth. Gardnerella vaginalis and Prevotella bivia were grown on Gardnerella agar plates purchased from Bio Merieux France.

[0069] Bacteria were suspended in buffered sodium chloride-peptone solution pH 7.0 to about 10.sup.6 colony forming unit (CFU/ml). 500 ul or the prepared suspensions were spread out on the agar plate. The inoculated plates were dried under sterile laminar air flow conditions. The agar plates were then incubated under anaerobic conditions using a sealed anaerobic jar (Becton Dickinson, USA) at 37.degree. C. for 36 h in maximum. Before use, the Gardnerella vaginalis strain was sub-cultured in BHI supplemented with yeast extract, maltose and horse serum, under anaerobic conditions using a sealed anaerobic jar at 37.degree. C. for 36 h in maximum.

[0070] Before use, bacterial cultures were centrifuged at 5.500.times.g for 5 mm at 4.degree. C. The culture medium was discarded and the bacteria were washed once with phosphate-buffered saline (PBS) and re suspended in PBS.

[0071] Cell lines and cultures. Human cervical HeLa cells were cultured at 37.degree. C. in a 5% CO.sub.2-95% air atmosphere in RPMI 1640 with L-glutamine (Life Technologies) supplemented with 10% heat-inactivated (30 min, 56.degree. C.) foetal calf serum (FCS; Boehringer, Mannheim, Germany), as previously described. Cells were used for infection assays before confluence, i.e., after 5 days in culture.

[0072] The human intestinal cell line used was the TC7 clone (Caco-2/TC7), established from the parental Caco-2 cell line. Cells were routinely grown in Dulbecco modified Eagle's minimal essential medium (DMEM) (25 mM glucose) (Invitrogen, Cergy, France), supplemented with 15% heat-inactivated (30 min, 56.degree. C.) foetal calf serum (Invitrogene) and 1% non-essential amino acids (Invitrogene) as previously described. For maintenance purposes, cells were passaged weekly using 0.02% trypsin in Ca.sup.2+Mg.sup.2+-free PBS containing 3 mM EDTA. Experiments and maintenance of the cells were carried out at 37.degree. C. in a 10% CO.sub.2/90% air atmosphere. The culture medium was changed daily. Cells were used at post-confluence after 15 days of culture (fully differentiated cells) for infection assay of S. enterica serovar Typhimurium.

[0073] Adhesion assays. The adhesion of lactobacilli strains onto cervix HeLa cells and intestinal Caco-2/TC7 cells was examined according to the following steps: the cells monolayers were washed twice with phosphate-buffered saline (PBS). For each adhesion assay, 0.5 ml of the Lactobacillus suspension (bacteria with spent broth culture supernatant) was mixed with DMEM (0.5 ml), and then added to each well of the tissue culture plate (24 wells) which was then incubated at 37.degree. C. in 10% CO.sub.2/90% air. The final concentrations of bacteria examined were 1.times.10.sup.8, 2.times.10.sup.8, 1.times.10.sup.9, and 2.times.10.sup.9 bacteria per ml. After 1 h incubation, the monolayers were washed five times with sterile PBS, fixed with methanol, stained with Gram stain, and then examined microscopically under oil immersion. Each adhesion assay was conducted in duplicate with cells from three successive passages. For each assay, the number of adherent bacteria was determined in 20 random microscopic areas (adhesion score: 0 to 5). Moreover, the level of viable adhering lactobacilli was determined by quantitative determination of bacteria associated with the infected cell monolayers. After being infected, cells were washed twice with sterile PBS and lysed with sterilized H.sub.2O. Appropriate dilutions were plated on tryptic soy agar (TSA) to determine the number of viable cell-associated bacteria by bacterial colony counts.

[0074] Each cell-association assay was conducted at least in triplicate, with three successive cell passages. Results were expressed as CPU/ml of cell-associated bacteria.

[0075] Activity against the growth of pathogens. A culture medium containing MRS (5 ml) and specific pathogen culture medium (5 ml) was inoculated with 0.1 ml of a cultivated pathogen and 0.1 ml of cultured Lactobacillus strain. Control was a culture medium inoculated with 0.1 ml of a cultivated pathogen and 0.1 ml of non-cultivated MRS adjusted to pH 4.5. At indicated time-points, aliquots were removed, serially diluted and plated on tryptic soy agar to determine bacterial colony counts of pathogen. Each assay was conducted at least in triplicate. Results were expressed as CFU/ml.

[0076] Activity against the viability of pathogens. Colony count assays were performed by incubating 10.sup.8 CFU/ml pathogen (0.5 ml) with the lactobacilli culture (10.sup.8 CFU/ml, 0.5 ml) at 37.degree. C. Control was non-cultivated MRS adjusted to pH 4.5. Initially and at predetermined 15 intervals, aliquots were removed, serially diluted and plated on tryptic soy agar to determine bacterial colony counts of pathogen. Each assay was conducted at least in triplicate. Results were expressed as CFU/ml.

[0077] Inhibition of uropathogenic E. coli adhesion onto epithelial HeLa cells. For cell monolayer infection, pathogens were cultured at 37.degree. C. for 18 h in appropriate culture media as described above. Prior to infection, the cell monolayers, prepared in twenty four-well TPP tissue culture plates (ATGC, Paris, France), were washed twice with PBS. Infecting bacteria were suspended in the culture medium and a total of 0.5 ml DMEM+0.25 ml culture pathogen (1.times.10.sup.8 CFU/ml)+0.25 ml lactobacilli culture (1.5.times.10.sup.9 CFU/ml) were added to each well of the tissue culture plate. The plates were incubated at 37.degree. C. in 10% CO.sub.2/90% air for different time of infection as indicated and then were washed three times with sterile PBS and lysed with sterilized H.sub.2O. Appropriate dilutions were plated on tryptic soy agar to determine the number of viable cell-associated bacteria by bacterial colony counts. Each assay was conducted hi triplicate with three successive passages of HeLa cells.

[0078] Analysis. Results are expressed as means.+-.standard error to the mean. For statistical comparisons, Student's t test was performed.

Results

Example 1

1. Adhesion Capacity of L. Jensenii KS 119.1 and KS 130.1, L. Crispatus KS 116.1 and L. Gasseri KS 124.3 onto HeLa and Caco-2/TC7 Cells.

[0079] The level of adhesion of the above strains was determined after the cells were inoculated with four concentrations of lactobacilli (5.times.10.sup.7; 1.times.10.sup.8; 5.times.10.sup.8; 1.times.10.sup.9 CPU/well). Generally, a concentration-dependent adhesion was observed.

[0080] In cervix HeLa cells, adhesion levels observed show that all the tested strains are adhering. The L. jensenii KS 119.1 and KS 130.1 strains appeared the best adhering strains (7.5 log CFU/ml at 5.times.10.sup.8 CPU/well) as compared with the control adhering strains, L. casei rhamnosus GG and L. rhamnosus GR1 strains.

[0081] In intestinal Caco-2/TC7 cells, adhesion levels observed show that all the Medinova strains are adhering. The L. crispatus KS 116,1, L. jensenii 119.1,129.1 and KS 130.1, L. gasseri 124.3 strains appeared the best adhering strains (7.5-8 logs CFU/ml at 5.times.10.sup.8 CPU/well) as compared with the control adhering strains, L. casei rhamnosus GG and L. rhamnosus GR1 strains.

[0082] As observed by scanning electron microscopy, all the "invention lactobacilli strains" appeared adhering in close contact with the HeLa and Caco-2/TC7 cells.

[0083] On the basis of their adhering properties, the L. crispatus KS 116.1 and L. jensenii 119.1 have been selected for the following studies of antibacterial activities against urovaginal and intestinal pathogens.

2. Activity of KS 116.1 and KS 119.1 on the growth of Urogenital and Intestinal Pathogens.

[0084] It has been examined whether the above mentioned strains are active on the growth of Staphylococcus aureus, Streptococcus agalactiae, uropathogenic and diarrheagenic E. coli, and diarrheagenic Salmonella enterica serovar Typhimurium. The growth of pathogens was measured at 5, 8, 18 and 24 h.

[0085] For Staphylococcus aureus, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 inhibited the growth of bacteria. Similarly, L. crispatus KS 116.1 and L. jensenii 119.1 inhibited the growth of Staphylococcus aureus and showed a decrease in the viable bacteria number. When activities of lactobacilli strains were compared, the L. jensenii 119.1 appeared the most active strain.

[0086] For uropathogenic E. coli strains IH11128 and 7372, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 inhibited the growth of the bacteria. Similarly, L. crispatus KS 116.1 and L. jensenii KS 119.1 inhibited the growth of E. coli. When activities of lactobacilli strains were compared, the L. jensenii 119.1 appeared the most active strain.

[0087] For diarrheagenic E. coli strain C1845, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 inhibited the growth of the bacteria. Similarly, L. crispatus KS 116.1 and L. jensenii KS 119.1 inhibited the growth of E. coli. When activities of lactobacilli strains were compared, the same activity was found for all the lactobacilli strains examined.

[0088] For diarrheagenic S. enterica serovar Typhimurium strain SL1344, the control L. rhamnosus strain GR-1 and L. fermentuin strain RC-14 inhibited the growth of the bacteria. Similarly, L. jensenii 119.1 inhibited the growth of S. enterica serovar Typhimurium. When activities of lactobacilli strains were compared, the same activity was found for the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 and L. jensenii KS 119.1. In contrast, the L. crispatus KS 1116.1 showed a lower activity.

[0089] For Candida albicans no activity was found for the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. crispatus KS 116.1 and L. jensenii KS 119.1.

3. Killing Activity of KS 116.1 and KS 119.1 Against Urogenital and Intestinal Pathogens.

[0090] It has been examined whether said lactobacilli are active on the viability of Staphylococcus aureus, Streptococcus agalactiae, uropathogenic and diarrheagenic E. coli, and diarrheagenic Salmonella enterica serovar Typhimurium. The effect on viability of pathogens was measured at 2, 3, and 4 h.

[0091] For Staphylococcus aureus, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. jensenii 1 19.1 decreased for 2 logs the viability of bacteria. In contrast, the L. crispatus KS 116.1 showed no activity.

[0092] For Streptococcus agalactiae, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. jensenii 119.1 and L. crispatus KS 116.1 showed no activity.

[0093] For uropathogenic E. coli strains IH11128 and 7372, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 showed 4 logs of decrease in viability of bacteria. L. crispatus KS 116.1 and L. jensenii 119.1 were not active showing only one log of decrease in viability of the bacteria.

[0094] For diarrheagenic E. coli strain C 1845, both of the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. crispatus KS 116.1 and L. jensenii 119.1 showed a low activity on the viability of C1845 bacteria (2 logs of decrease).

[0095] For diarrheagenic S. enterica serovar Typhimurium strain SL1344, both of the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. crispatus KS 116.1 and L. jensenii 119.1 showed a great activity by decreasing the viability of SL1344 bacteria (5 logs of decrease).

[0096] For Gardnerella vaginalis, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. fermentum strain RC-14, and L. jensenii I 19.1 decreased for 2 logs the viability of Gardnerella. In contrast, the L. crispatus KS 116.1 showed no activity.

[0097] For Candida albicans no activity was found for the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. crispatus KS 116.1 and L. jensenii KS 119.1.

4. Inhibition of the Adhesion of Uropathogenic E. coli Strain IH11128 Strain onto HeLa Cells by KS 116.1 and KS 119.1.

[0098] It has been examined whether said lactobacilli are able to inhibit the adhesion of uropathogenic E. coli strain IH11128 onto HeLa cells. The effect of the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. jensenii 119.1 and L. crispatus KS 116.1 was measured at three concentrations: 1.times.10, 5.times.10.sup.8, and 1.times.10.sup.9 bacteria per well.

[0099] A 30 to 40% of inhibition of IH11128 adhesion was found at a concentration of 1.times.10.sup.8 bacteria per well for the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14. At this concentration, the L. jensenii KS 119.1 and L. crispatus KS 116.1 were inactive. Inhibition of IH11128 adhesion was effective at a concentration of 5.times.10.sup.8 bacteria per well for L. jensenii 119.1 and L. crispatus KS 116.1 and a similar inhibition that those obtained with the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 was observed. A similar high inhibition level of IH11128 adhesion was observed with the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14, and L. jensenii KS 119.1 and L. crispatus KS 116.1 at the concentration of 1.times.10.sup.9 bacteria per well.

Example 2

1. Activity of L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilus KS 400 on the Growth of Urogenital and Intestinal Pathogens.

[0100] It has been examined whether the strains referred to here above are active against the growth of Staphylococcus aureus, Streptococcus agalactiae, Candida albicans and uropathogenic and diarrheagenic E. coli strains IH11128 and 7372. The growth of pathogens was measured at 5, 8, 18 and 24 h.

[0101] No activity was developed against Streptococcus agalactiae and Candida albicans by L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilus KS 400 as well as by the control strains GR-1 and RC-14.

[0102] Concerning Staphylococcus aureus, the control L. rhamnosus strain GR-1 and L. fermentum strain RC-14 efficiently inhibited the growth of the bacteria. Similarly, L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilus KS 400 inhibited the growth of Staphylococcus aureus and showed a decrease in the viable bacteria number. When activities of lactobacilli strains were compared, the L. helveticus KS 300 appeared the most active strain.

[0103] For uropathogenic E. coli strains IH11128, the control strains L. rhamnosus GR-1 and L. fermentum RC-14 efficiently inhibited the growth of the bacteria. Similarly, L. helveticus KS 300 efficiently inhibited the growth of E. coli. When activities of lactobacilli strains were compared, a lower activity appeared for L. gasseri KS 124.3 and L. acidophilus KS 400.

[0104] For uropatho genie E. coli strain 7372, both control strains L. rhamnosus GR-1 and L. fermentum RC-14 strains inhibited the growth of bacteria. Similarly L. helveticus KS 300 inhibited the growth of said bacteria whereas L. acidophilus KS 400, however, was active only at 25 hours.

2. Killing Activity of KS 124.3, KS 300 and KS 400 against Urogenital and Intestinal Pathogens.

[0105] It has been examined whether said lactobacilli are active on the viability of Staphylococcus aureus, Strteptococcus agalactiae, Candida albicans, uropathogenic E. coli IH11128 and 7372, diarrheagenic E. coli C1845 and Gardnerella vaginalis. The effect on viability of pathogens was measured at 2, 3, and 4 h.

[0106] For Staphylococcus aureus, the control strains L. rhamnosus GR-1 and L. fermentum RC-14, and L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilus KS 400 decreased for 2-3 logs the viability of bacteria.

[0107] Concerning Streptococcus agalactiae and Candida albicans the two control strains and L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilus KS 400 showed no activity.

[0108] For uropathogenic E. coli strains IH11128, the control strains L. rhamnosus strain GR-1 and L. fermentum RC-14 and L. helveticus KS 300 as well showed 3 logs of decrease in viability of the bacteria. L. acidophilus KS 400 and L. gasseri KS 124.3 were not active.

[0109] Concerning uropathogenic E. coli strains 7372, the control strains showed 2 logs of decrease in viability of the bacteria. L. helveticus KS 300 showed 3 logs of decrease whereas L. acidophilus KS 400 and L. gasseri KS 124.3 were not active within the same conditions.

[0110] For Gardnerella vaginalis, both control strains, L. acidophilus KS 400 and L. gasseri KS 124.3 showed 3 logs of decrease in viability of the bacteria. A rapid and efficient activity was observed for L. helveticus KS 300, higher than that found for the above control strains.

[0111] For diarrheagenic E. coli strain C 1845, both of the control strains L. rhamnosus GR-1 and L. fermentum RC-14 killed the bacteria showing a 3 log decrease in the viability of same. Similar effect was observed for L. gasseri KS 124.3 whereas no activity was detected concerning L. acidophilus KS 400. L. helveticus KS 300 exhibits a killing which is definitely higher that that observed for the above control strains.

Example 3

1. Killing Activity of L. jensenii KS 121.1 and KS 122.1, L. gasseri KS 120.1 and L. helveticus KS 300 Against Urogenital and Intestinal Pathogens.

[0112] It has been examined whether said lactobacillus strains are active on the viability of Streptococcus agalactiae, Candida albicans, uropatho genie E. coli H11128, Gardnerella vaginalis, Prevotella bivia and Salmonella enterica Typhimurium. The effect on viability of pathogens was measured at 4 h of contact. Concerning both Streptococcus agalactiae and Candida albicans none of the tested lactobacilli showed an activity.

[0113] For uropathogenic E. coli strains IH11128, L. jensenii KS 121.1 and KS 122.1 showed no activity whereas, in contrast, L. gasseri KS 120.1 decreased efficiently (4 logs) the viability of E. coli in unshaken conditions. L. helveticus KS 300 and the L. fermentum RC-14 control strain decreased of 2 logs the viability of E. coli in unshaked conditions only.

[0114] Concerning Gardnerella vaginalis, both L. jensenii KS 121.1 and KS 122.1 showed no activity. In contrast L. gasseri KS 120.1 decreased efficiently (6 logs) the viability of Gardnerella vaginalis in unshaken conditions; L. helveticus KS 300 showed similar efficiency (4 logs of decrease) in unshaken conditions also, whereas control strain showed a 3 log of decrease only.

[0115] For Prevotella bivia, L. gasseri KS 120.1, L. jensenii 122.1, L. helveticus KS 300 and the control strain L. fermentum RC-14 decreased viability of the bacteria for 2 logs, in unshaken conditions. L. jensenii KS 121.1 which was highly active against Prevotella bivia in unshaken conditions had lost its activity when tested in shaken conditions.

[0116] Concerning Salmonella Typhimurium, L. gasseri KS 120.1 (3 logs), L. jensenii KS 121.1 and KS 122.1, L. helveticus KS 300 and the control strain L. fermentum RC-14 were quite active (6 logs of decrease) in unshaken conditions. L. gasseri KS 120.1 remained active even in shaken conditions.

2. Activity of KS 120, KS 121.1, KS 122.1 and KS 300 on the growth of Gardnerella Vaginalis and Prevotella Bivia

[0117] The tests have been perforrned in both shaken and unshaken conditions.

[0118] In unshaken conditions L. jensenii KS 121.1 and KS 122.1 inhibited the growth of Gardnerella vaginalis whereas L. gasseri 120.1, L. helveticus and the control strain L. fermentum RC-14 inhibited said activity at still a higher level.

[0119] In shaked conditions L. jensenii KS 121.1 and KS 122.1 and L. helveticus as well have lost their activity, whereas L. gasseri 120.1 remains active (2 logs of decrease) against Gardneralla vaginalis.

[0120] In both shaken and unshaken conditions L. gasseri KS 120.1, L. jensenii KS 121.1 and KS 122.1, L. helveticus KS 300 and the control strain inhibited the growth of Prevotella bivia at a high level.

3. Inhibition of Adhesion of Gardnerella Vaginalis and Prevotella bivia onto HeLa Cells KS 120.1, KS 121.1 and KS 300

[0121] The effect of L. gasseri KS 120.1, L. helveticus KS 300 of the control strains L. fennentum RC-14 as well as L. casei rhamnosus GG was measured at the concentration of 1.times.10.sup.9 bacteria per well.

[0122] The control L. fermentum RC-14 strain and L. jensenii KS 121.1 decreased for 2 logs the level of adhesion of Gardnerella vaginalis on the tested cells. L. gasseri KS 120.1 and L. helveticus KS 300 decreased said adhesion for 4 logs.

[0123] L. jensenii KS 121.1 decreased adhesion of Prevotella bivia for 1 log only, whereas L. gasseri KS 120.1, L. helveticus KS 300 as well as the control strain RC-14 decreased said adhesion for 2 logs.

4. Inhibition of Adhesion and Internalization of Uropathogenic E. coli strain IH11128 Strain onto HeLa Cells by KS 120.1, KS 121.1 and KS 300

[0124] A strategy often used by extra-intestinal pathogens like E. coli to evade host defense mechanism is to establish a local reservoir within epithelial cells (M. A. Muvlea in Eschrichia coli. Cell. Microbiol. 4, 257-271-2002) and cell entry by IH1128 strain appears to be an effective mechanism for promoting prolonged persistence these pathogens in the urinary tract.

[0125] The effect of L. gasseri KS 120.1, L. helveticus KS 300 and of the control strains RC-14 and GG strain was examined concerning the above uropathogenic E. coli.: L. jensenii 121.1 decreased for 2 logs the level of viable internalized E. coli, whereas L. gasseri 120.1, L. helveticus and both the control strains have shown a 4 logs of decrease of the internalized E. coli.

Example 4

Modulation of the Immune Response (in vivo Test Using Human PMBC)

[0126] The following strains have been tested within the conditions set hereafter concerning their ability to induce or modulate or affect an immune response, more specifically their ability to induce the secretion of cytokines and the like: L. crispatus KS 116.1, L. jensenii 119.1, L. jensenii KS 121.1 and KS 122.1, L. gasseri KS 120.1, L. gasseri KS 124.3, L. helveticus KS 300 and L. acidophilus KS 400.

[0127] The detection of the induction of cytokines was made by means of a test for in vitro stimulation of isolated peripheral blood mononuclear cells (PBMC). Among the cytokines induced during these tests, there are interleukins 10 and 12 (IL10 & IL12), .gamma.-interferon (.gamma.-IFN) and tumor necrosis factor .alpha. (TNF.alpha.).

Experimental Procedures

[0128] PMBC preparation: Fresh human blood obtained for healthy subjects (four donors) was diluted at a 1:2 ratio with PBS-Ca (GIBCO) and purified on a Ficoll gradient (GIBCO). After 5 centrifugation at 400.times.g for 30 min at 20.degree. C. the peripheral blood monocular cellular cells (PMBC's) formed an interphase ring layer in the serum. PMBC's were aspired carefully, suspended to a final volume of 50 ml using PBD-Ca and washed three times in the same buffer with centrifugation steps at 350.times.g for 10 min at 20.degree. C.

[0129] PMBC's were subsequently resuspended using complete RPMI medium (GIBCOP), supplemented with 10% w/v L-glutamine (GIBCO) and gentamycin (150 jig/ml) (GIBCO). PBMC's were counted under the microscope and adjusted at a concentration of 2.times.10.sup.6 cells/ml and distributed (in 1 ml aliquots) in 24-well tissues culture plates (Corning, Inc.).

[0130] Bacteria preparation: overnight LAB cultures were washed twice with PBS buffer, pH 7.2 before being resuspended in PBS at concentration of 2.times.10.sup.9 cfu/ml.

[0131] PMBC incubation: from these suspensions 10 .mu.l was transferred into wells of the PMBC plates which were incubated at 37.degree. C. in a 5% CO.sub.2/95% air atmosphere. After 24 hours incubation the supernatant was aspirated, centrifuged at 2000 rpm and the supernatant removed and stored at -20.degree. C. The control consisted of bacteria-free buffer.

[0132] Cytokine quantification: cytokine expression levels have been determined by ELISA tests (<<Enzyme linked immuno sorbent assay>>). ELISA plates are coated with anti-cytokine antibody (overnight procedure) and the antibody is blocked with PBS/BSA 1%. A proper standard was prepared with known concentrations of cytokines, covering the detection range of 15.62 to 2000 pg/ml (incubated overnight).

[0133] The anti-cytokine detection and quantification was performed with the streptavidine reaction on substrate (TMB Pharmigen). The commercial kits Pharmigen have been used according to the manufacturer's description. Four cytokines were determined: the pro-inflammatory/Th 1 cytokines TNF.alpha., IFN.gamma., IL 12 and the anti-inflammatory/Th 2 cytokine IL10. TABLE-US-00002 TABLE I IL 10 IL 12 TNF.alpha. IFN.gamma. IL10/IL12 Control 31.25 31.25 31.25 31.25 1 KS 120.1 1228.67 176.32 17698.83 3513.36 6.96840971 KS 121.1 2297.87 47.66 14180.66 897.65 48.2138061 KS 116.1 2856.26 167.6 33569.91 7209.33 17.0540573 KS 400 3177.49 103.85 26799 6949.13 30.5969186 KS 300 2290.47 59.7 18703.66 10047.75 38.3663317 KS 119.1 307.13 198.47 6693.3 9192.74 1.54748829 KS 124.3 2969.02 660.98 31307.71 16985.56 4.49184544

Observations [0134] a high level of TNF.alpha. induction for all the tested LAB strains [0135] a relatively low level of IFN.gamma. concerning L. jensenii KS 121.1 [0136] the highest IL10 induction potential concerning L. crispatus KS 116.1 and KS 400 [0137] in contrast to the two L. jensenii strains the two L. gasseri strains have shown a similar profile, especially when considering the ratio's in IL10/IL12 and in TNF.alpha./IFN.gamma..

[0138] Within the above testing frame it is clear that the cytokine induction profile is strain specific.

Example 5

Determination of the Anti-Inflammatory Activity (in vivo Test Using an Animal Model)

[0139] An acute model of mice has been adapted from Camoglio et al. (see Eur. J. Immunol. 2000) where the animals have been fed from day-5 to day +2 with selected lactic acid bacteria strains, at a rate of 10.sup.8 bacteria per mouse per day. TNBS was then injected on day zero, at a rate of 120 mg/kg mice in order to induce acute colitis and the animals have been sacrificed at day +2 and eventually subjected to both macroscopic (Wallace score--Table II) and histological (Ameho score--Table III) scoring.

[0140] These tables clearly show that the selected lactic acid bacteria strains exhibit a significant anti-inflammatory effect when compared to reference strains.

Example 6

6.1 Composition for Local Administration (Vaginal Capsules)

[0141] Samples of the LAB strains of this invention (see above) have been cultured for min. 24 hours in conditions similar to those mentioned here above. The cultured strains have been isolated, washed and lyophilized individually, individually suspended in a lactose/MSK powder mixture and eventually divided into unit doses each of them containing about 10.sup.8-10.sup.9 cfu (colony forming units).

[0142] Said unit doses have been then poured into gelatin vaginal capsules each of them comprising about 10.sup.8-10.sup.9 cfu of selected LAB strains of this invention.

6.2 Composition for Local Administration (Vaginal Suppositories)

[0143] Soft vaginal suppositories have been prepared using the following ingredients: [0144] buffered lactic acid solution [0145] lactose [0146] PEG 4000 [0147] PEG 600

[0148] The adequate amount of selected lyophilized LAB strains of this invention has been then added to unit doses to afford vaginal suppositories each comprising about 10.sup.8-10.sup.9 cfu.

Example 7

7.1 Composition for Oral Administration (Edible Capsules)

[0149] Edible cellulose capsules (hydroxypropyl methyl cellulose) each comprising about 10.sup.8-10.sup.9 cfu of selected LAB strains of this invention have been manufactured using filler comprising the following ingredients: [0150] dehydrated yogurt powder [0151] anhydrous dextrose [0152] potato starch microcrystalline cellulose [0153] selected lyophilized LAB strain 7.2 Composition for Oral Administration (Yogurt)

[0154] Portions of a so called "Yogurt Nature Light" have been prepared using the following process: to a batch of standardized 1.5% fat milk there was added 3% of skimmed milk powder (MSK) and the whole was then pasteurized at 90.degree. C. for 30 minutes. 1% volume of commercial starter cultures of L. bulgaricus and S. thermophilus have been added to the pasteurized milk; then the whole was gently stirred at room temperature, disposed in 100 ml containers which were all eventually incubated at 40.degree. C. during around 4 hours to afford the desired pH.

[0155] Then portions of selected lyophilized LAB strains of this invention were added to the yogurt cans in such an amount to have about 10.sup.8-10.sup.9 cfu per yogurt can and a further incubation was carried out for about 30 min. until to afford a pH of about 4.5 to 4.7. These yogurt portions can be stored at 4.degree. C. before consumption. TABLE-US-00003 TABLE II TNBS colitis induced at day zero Wallace score ##STR1##

[0156] TABLE-US-00004 TABLE III TNBS colitis induced at day zero Ameho score ##STR2##

* * * * *


uspto.report is an independent third-party trademark research tool that is not affiliated, endorsed, or sponsored by the United States Patent and Trademark Office (USPTO) or any other governmental organization. The information provided by uspto.report is based on publicly available data at the time of writing and is intended for informational purposes only.

While we strive to provide accurate and up-to-date information, we do not guarantee the accuracy, completeness, reliability, or suitability of the information displayed on this site. The use of this site is at your own risk. Any reliance you place on such information is therefore strictly at your own risk.

All official trademark data, including owner information, should be verified by visiting the official USPTO website at www.uspto.gov. This site is not intended to replace professional legal advice and should not be used as a substitute for consulting with a legal professional who is knowledgeable about trademark law.

© 2024 USPTO.report | Privacy Policy | Resources | RSS Feed of Trademarks | Trademark Filings Twitter Feed