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Probiotics and immune response
THE AMERICAN JOURNAL OF GASTROENTEROLOGY © 2000 by Am. Coll. of Gastroenterology Published by Elsevier Science Inc.
Vol. 95, No. 1, Suppl., 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(99)00813-8
Probiotics and Immune Response Susanna Cunningham-Rundles, Ph.D., Siv Ahrne´, Ph.D., Stig Bengmark, M.D., Ph.D, Rosemary Johann-Liang, M.D., Florence Marshall, M.D., Linda Metakis, B.S., Claudia Califano, B.A., Ann-Margaret Dunn, R.N., C.P.N.P., Claudia Grassey, R.N., C.P.N.P., Gilberto Hinds, R.N., and Joseph Cervia, M.D. Cornell Medical Center, New York, New York
ABSTRACT Current evidence supports the concept that oral administration of probiotic lactobacilli may be therapeutic in preventing antibiotic-associated diarrhea in children and in reestablishing normal flora in the gastrointestinal tract. Children with human immunodeficiency virus (HIV) infections may have episodes of diarrhea and frequently experience malabsorption associated with possible bacterial overgrowth; together these may interact to produce the growth abnormalities characteristic of this group. The overall objective of this investigation has been to determine whether oral administration of the probiotic Lactobacillus plantarum 299v could improve nutrient status and promote growth in children congenitally exposed to HIV. In addition, the possible beneficial effect of Lactobacillus plantarum 299v in modulating immune response was evaluated. In preliminary results described here, we report on the ability of Lactobacillus plantarum 299v to colonize children with HIV and to elicit specific systemic immune response after oral supplementation. (Am J Gastroenterol 2000;95(Suppl.): S22–S25. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION The development of an immune response toward foreign antigens of microbial and viral origin provides an essentially systematic way through which potentially pathogenic organisms are recognized and controlled. Although the gastrointestinal (GI) tract contains many prokaryotic organisms, which, based on cell number alone far outnumber all of the cells of the body, healthy blood is sterile, and deep tissues operate in the absence of bacteria. This environment is achieved and maintained by the physical structure of the GI tract, as well as the activity of the immune system in preventing bacterial overgrowth and translocation (1, 2). Whereas some microbes tend to produce toxins or compete with the host for common substrates, others produce nutrients, absorbable peptides, and vitamins essential for the host (3, 4). Furthermore, colonization of the GI tract with beneficial microbes may prevent overgrowth from more pathogenic ones if these bacteria have a selective advantage (5). In recent years the science of probiotics has emerged to
evaluate and to explore the possible significance of probiotic bacteria and how some of these organisms may be used therapeutically (6, 7). The present study was undertaken to determine whether dietary supplementation with a well-characterized probiotic, Lactobacillus plantarum 299v (DSM 9843) (8), might be able to colonize the GI tract and improve growth in children congenitally exposed to human immunodeficiency virus (HIV) infection who display clinical evidence of growth failure. Although growth failure is likely to be multifactorial in HIV disease, malabsorption and altered GI milieu are important contributing factors (9, 10). A recent study has shown that lactobacillus administration confers intestinal resistance to the opportunistic pathogen Cryptosporidium parvum in immunosuppressed mice (11). Preliminary data are presented here on immune response to L. plantarum 299v after oral supplementation, and we address potential safety issues in these profoundly immune-deficient children.
PROTOCOL Children were enrolled into the study after informed consent of the parent, in accordance with the protocol (approved by the Cornell University Medical College Internal Review Board). The first child to be studied received a juice formula (12) prepared by the Skånemejerier Dairy in Lund Sweden, whereas subsequent children received packets containing either L. plantarum 299v (8, 13) or an identical placebo prepared as a lyophilized powder in an oatmeal base. The lyophilized preparations were given by the parent to the child either orally by mixing the powder into a beverage, in formula once per day or, in one case, into the percutaneous enteral feeding tube. Colonization was not evaluated in the first case, whereas in the second phase of study colonization was directly tested by culture of rectal swabs onto Rugosa agar plates, a medium selective for lactobacilli. Lactobacilli that grew out were subsequently genetically typed for presence of L. plantarum 299v. Antigenic recognition of this lactobacilli was evaluated as proliferative response to heatinactivated whole organisms of L. plantarum 299v in a standardized microtiter plate system (14, 15).
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Figure 1. Weight change in response to L. plantarum 299v in congenital acquired immune deficiency.
RESULTS The first congenitally HIV-infected child to be given L. plantarum 299v was 11 yr of age at the time of study and received juice containing L. plantarum 299v every day for 1 month. At the beginning of the study, he was evaluated as having failure-to-thrive, observed to have persistent mouth ulcers associated with candidal esophagitis, and had chronic diarrhea. This child was a fussy eater, unable to finish meals, and was quoted by his grandmother as saying that he “would like to kill myself if I were not afraid to die.” He had virtual absence of CD4⫹ T cells in peripheral blood and was receiving azidothymidine (AZT). After 1 month of treatment with L. plantarum 299v, his growth improved (Fig. 1), and his mouth ulcers, candidiasis, and diarrhea resolved. The child’s appetite improved markedly; he ate three full meals each day, and he was able to enjoy playing again. The improvement in weight was statistically significant if the two pretreatment values were compared either with the first three posttreatment values (p ⬍ 0.03) over 34 days or over 142 days (p ⬍ 0.001). This dramatic response in one child led to the second phase of study, which was undertaken as a double-blind, placebo-controlled study of L. plantarum 299v supplementation in congenital HIV infection. Of 17 children who were enrolled into the study, no child was colonized with L. plantarum 299v before receiving the supplement orally, and no child who did not receive active product became colonized with L. plantarum 299v. Approximately 50% of swab cultures from these children grew out a few lactobacilli representing species other than L. plantarum 299v. All of the children tolerated the supplement, and no child was withdrawn from the study because of side effects or problems with taste. Although flatulence has sometimes been reported with use of L. plantarum 299v in adults, this was not observed in the present study. Colonization appeared to take place within 2 wk. Although it was not evaluated extensively, L. plantarum 299v colonization dis-
appeared after about 1 month or less after the end of supplementation. Mononuclear cells isolated from peripheral blood from 17 children congenitally exposed to HIV were evaluated for immune response in vitro to L. plantarum 299v before supplementation. Positive response was defined as mean (counts per minute of triplicate wells) of maximum response to any concentration of the L. plantarum 299v antigen ⱖ3.5 times the background response of unstimulated cells cultured in parallel. Ten children were evaluated as having positive response and seven children were considered nonresponsive. The mean difference in response between responders and nonresponders was significantly different (Fig. 2) and validated the use of this differentiation of response from background proliferation for this particular antigen in the setting of HIV infection. Responders to L. plantarum 299v did not have a statistically different higher response to phytohemagglutinin (PHA, a standard T-cell mitogen used as a positive lymphocyte activator) than did those who did not respond (data not shown). This result suggests that immune deficiency alone was not the reason for lack of response. Included among the group of nonresponsive children were two children who also did not respond to PHA. One of these, a child with marked growth failure born to an HIVpositive mother, was subsequently found to be HIV-negative by polymerase chain reaction (PCR) and eventually showed seroreversion of the previously positive HIV antibody reactivity. This child did show overall improvement in height and weight while receiving L. plantarum 299v, but this could not be directly attributed to L. plantarum 299v because of inadequate follow-up visits and limited baseline data close to the time of administration. Interestingly, a marked augmentation in immune response was observed (Fig. 3). This difference reflected a change from generalized anergy to normal immune response within 1 month and occurred in the absence of any other intervention or treatment.
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AJG – Vol. 95, No. 1, Suppl., 2000
Figure 2. Natural immune response to L. plantarum in congenital HIV exposure.
DISCUSSION Therefore, preliminary data from this study indicate that approximately 69% of HIV-positive children who have never received L. plantarum 299v do have significant preexisting immune response to this bacteria, probably due to cross-reactivity with other endogenous strains of L. plantarum or with strains of different lactobacilli species. The observation that immune response to L. plantarum 299v was not directly related to response to PHA is important for two reasons. First, absence of relationship suggests that lack of response was not a simple reflection of immune deficiency. Second, because antigen response is often lost early in HIV infection, the studies indicate that response to L. plantarum 299v may be specifically retained in HIV-positive children. As noted, all children tolerated this supplementation, there were no side effects, and children were not permanently colonized. These preliminary results suggest L. plantarum 299v may have the potential to improve growth and development. Furthermore, the data in one child indicate the possibility of
effects mediated through modulation of immune response after supplementation with L. plantarum 299v. This finding is indirectly supported by other studies in volunteers showing that oral supplementation with different lactobacilli did act to modulate and increase phagocytic activity in peripheral blood (16). Although there was no effect on lymphocyte response to mitogens in this study, this may not be surprising as immune response was already in the normal range in these healthy adults. These findings may indicate a potential for therapeutic use of lactobacilli in HIV infected children.
SUMMARY AND CONCLUSIONS This study examined the effect of L. plantarum 299v supplementation in children congenitally exposed to HIV. Results show that 69% of HIV-positive children had preexisting cross-reactive immune response to this antigen as measured by proliferative response to L. plantarum 299v before oral administration. No child was colonized with L. plantarum 299v at baseline, as shown by genetic typing of
Figure 3. Immune response after L. plantarum supplementation in HIV-exposed child with failure to thrive.
AJG – January, Suppl., 2000
lactobacilli grown from rectal swabs. Mononuclear cells from 10 confirmed HIV-positive children responded to L. plantarum 299v in vitro. Seven children did not react. Responders had a significantly higher response to L. plantarum 299v than did their nonreactive counterparts, but they did not have a significantly stronger response to PHA. These results demonstrate both that natural immune response to L. plantarum 299v occurs frequently and that this response is highly conserved in the HIV-positive child. The data suggest that L. plantarum 299v may be given safely to the immunocompromised host and may indeed have a positive effect on immune response. Reprint requests and correspondence: Susanna CunninghamRundles, Ph.D., Cornell Medical Center, 525 E. 68th Street, New York, NY 10021. Received Mar. 17, 1999; accepted Sep. 7, 1999.
REFERENCES 1. Prindull G, Ahmad M. The ontogeny of the gut mucosal immune system and the susceptibility to infections in infants of developing countries. Eur J Ped 1993;152:786 –92. 2. Cunningham-Rundles S. Malnutrition and gut immune function. Curr Op Gastroenterol 1994;10:664 –70. 3. Murry AC, Lewis RD, Amos HE. The effect of microbial phytase in a pearl millet-soybean meal diet on apparent digestibility and retention of nutrients, serum mineral concentration, and bone mineral density of nursery pigs. J Anim Sci 1997;75:1284 –91. 4. Torrallardona D, Harris CI, Coates ME, et al. Microbial amino acid synthesis and utilization in rats: Incorporation of 15N from 15NH4Cl into lysine in the tissues of germ-free and conventional rats. Br J Nutr 1996;76:689 –700. 5. Drago L, Gismondo MR, Lombardi A, et al. Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin. FEMS Microbiol Lett 1997;153: 455– 63.
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6. Bengmark S, Jeppsson B. Gastrointestinal surface protection and mucosa reconditioning. J Parenter Enteral Nutr 1995;19: 410 –5. 7. Majamaa H, Isolauri E. Probiotics: A novel approach in the management of food allergy. J Allergy Clin Immunol 1997; 99:179 – 85. 8. Johansson ML, Molin G, Jeppsson B, et al. Administration of different Lactobacillus strains in fermented oatmeal soup: In vivo colonization of human intestinal mucosa and effect on the indigenous flora. J App Environ Microbiol 1993;59:15–20. 9. Crocker KS. Gastrointestinal manifestations of the acquired immunodeficiency syndrome. Nurs Clin North Am 1989;24: 395– 406. 10. Kotler DP, Tierney AR, Culpepper-Morgan JA, et al. Effect of home total parenteral nutrition on body composition in patients with acquired immunodeficiency syndrome. J Parenter Enteral Nutr 1990;14:454 – 8. 11. Alak JI, Wolf BW, Mdurvwa EG, et al. Effect of Lactobacillus reuteri on intestinal resistance to Cryptosporidium parvum infection in a murine model of acquired immunodeficiency syndrome. J Infect Dis 1997;175:218 –21. 12. Johansson ML, Berggren A, Nobaek S, et al. Survival of Lactobacillus plantarum DSM 9843, and effect on the short chain fatty acid content in feces after ingestion of a rose hip drink with fermented oats. Int J Food Microbiol 1999 (in press). 13. Johansson ML, Quednau M, Ahrne S, et al. Classification of Lactobacillus plantarum by restriction endonuclease analysis of total chromosomal DNA, using conventional agarose gel electrophoresis. Int J Syst Bacteriol 1995;45:670 –5. 14. Cunningham-Rundles S, Yeger-Arbitman R, Edelson P, et al. Experimental approach to the study of immune function in children with possible human immunodeficiency virus, HIV, infection. J Clin Lab Analysis 1990;4:399 – 404. 15. Paxton H, Cunningham-Rundles S, O’Gorman MRG. Laboratory evaluation of the cellular immune system. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods. Philadelphia: WB Saunders and Co, 1995:887–912. 16. Schiffrin EJ, Rochat F, Link-Amster H, et al. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J Dairy Sci 1995;78:491–7.
Vol. 95, No. 1, Suppl., 2000 ISSN 0002-9270/00/$20.00 PII S0002-9270(99)00813-8
Probiotics and Immune Response Susanna Cunningham-Rundles, Ph.D., Siv Ahrne´, Ph.D., Stig Bengmark, M.D., Ph.D, Rosemary Johann-Liang, M.D., Florence Marshall, M.D., Linda Metakis, B.S., Claudia Califano, B.A., Ann-Margaret Dunn, R.N., C.P.N.P., Claudia Grassey, R.N., C.P.N.P., Gilberto Hinds, R.N., and Joseph Cervia, M.D. Cornell Medical Center, New York, New York
ABSTRACT Current evidence supports the concept that oral administration of probiotic lactobacilli may be therapeutic in preventing antibiotic-associated diarrhea in children and in reestablishing normal flora in the gastrointestinal tract. Children with human immunodeficiency virus (HIV) infections may have episodes of diarrhea and frequently experience malabsorption associated with possible bacterial overgrowth; together these may interact to produce the growth abnormalities characteristic of this group. The overall objective of this investigation has been to determine whether oral administration of the probiotic Lactobacillus plantarum 299v could improve nutrient status and promote growth in children congenitally exposed to HIV. In addition, the possible beneficial effect of Lactobacillus plantarum 299v in modulating immune response was evaluated. In preliminary results described here, we report on the ability of Lactobacillus plantarum 299v to colonize children with HIV and to elicit specific systemic immune response after oral supplementation. (Am J Gastroenterol 2000;95(Suppl.): S22–S25. © 2000 by Am. Coll. of Gastroenterology)
INTRODUCTION The development of an immune response toward foreign antigens of microbial and viral origin provides an essentially systematic way through which potentially pathogenic organisms are recognized and controlled. Although the gastrointestinal (GI) tract contains many prokaryotic organisms, which, based on cell number alone far outnumber all of the cells of the body, healthy blood is sterile, and deep tissues operate in the absence of bacteria. This environment is achieved and maintained by the physical structure of the GI tract, as well as the activity of the immune system in preventing bacterial overgrowth and translocation (1, 2). Whereas some microbes tend to produce toxins or compete with the host for common substrates, others produce nutrients, absorbable peptides, and vitamins essential for the host (3, 4). Furthermore, colonization of the GI tract with beneficial microbes may prevent overgrowth from more pathogenic ones if these bacteria have a selective advantage (5). In recent years the science of probiotics has emerged to
evaluate and to explore the possible significance of probiotic bacteria and how some of these organisms may be used therapeutically (6, 7). The present study was undertaken to determine whether dietary supplementation with a well-characterized probiotic, Lactobacillus plantarum 299v (DSM 9843) (8), might be able to colonize the GI tract and improve growth in children congenitally exposed to human immunodeficiency virus (HIV) infection who display clinical evidence of growth failure. Although growth failure is likely to be multifactorial in HIV disease, malabsorption and altered GI milieu are important contributing factors (9, 10). A recent study has shown that lactobacillus administration confers intestinal resistance to the opportunistic pathogen Cryptosporidium parvum in immunosuppressed mice (11). Preliminary data are presented here on immune response to L. plantarum 299v after oral supplementation, and we address potential safety issues in these profoundly immune-deficient children.
PROTOCOL Children were enrolled into the study after informed consent of the parent, in accordance with the protocol (approved by the Cornell University Medical College Internal Review Board). The first child to be studied received a juice formula (12) prepared by the Skånemejerier Dairy in Lund Sweden, whereas subsequent children received packets containing either L. plantarum 299v (8, 13) or an identical placebo prepared as a lyophilized powder in an oatmeal base. The lyophilized preparations were given by the parent to the child either orally by mixing the powder into a beverage, in formula once per day or, in one case, into the percutaneous enteral feeding tube. Colonization was not evaluated in the first case, whereas in the second phase of study colonization was directly tested by culture of rectal swabs onto Rugosa agar plates, a medium selective for lactobacilli. Lactobacilli that grew out were subsequently genetically typed for presence of L. plantarum 299v. Antigenic recognition of this lactobacilli was evaluated as proliferative response to heatinactivated whole organisms of L. plantarum 299v in a standardized microtiter plate system (14, 15).
AJG – January, Suppl., 2000
Probiotics and Immune Response
S23
Figure 1. Weight change in response to L. plantarum 299v in congenital acquired immune deficiency.
RESULTS The first congenitally HIV-infected child to be given L. plantarum 299v was 11 yr of age at the time of study and received juice containing L. plantarum 299v every day for 1 month. At the beginning of the study, he was evaluated as having failure-to-thrive, observed to have persistent mouth ulcers associated with candidal esophagitis, and had chronic diarrhea. This child was a fussy eater, unable to finish meals, and was quoted by his grandmother as saying that he “would like to kill myself if I were not afraid to die.” He had virtual absence of CD4⫹ T cells in peripheral blood and was receiving azidothymidine (AZT). After 1 month of treatment with L. plantarum 299v, his growth improved (Fig. 1), and his mouth ulcers, candidiasis, and diarrhea resolved. The child’s appetite improved markedly; he ate three full meals each day, and he was able to enjoy playing again. The improvement in weight was statistically significant if the two pretreatment values were compared either with the first three posttreatment values (p ⬍ 0.03) over 34 days or over 142 days (p ⬍ 0.001). This dramatic response in one child led to the second phase of study, which was undertaken as a double-blind, placebo-controlled study of L. plantarum 299v supplementation in congenital HIV infection. Of 17 children who were enrolled into the study, no child was colonized with L. plantarum 299v before receiving the supplement orally, and no child who did not receive active product became colonized with L. plantarum 299v. Approximately 50% of swab cultures from these children grew out a few lactobacilli representing species other than L. plantarum 299v. All of the children tolerated the supplement, and no child was withdrawn from the study because of side effects or problems with taste. Although flatulence has sometimes been reported with use of L. plantarum 299v in adults, this was not observed in the present study. Colonization appeared to take place within 2 wk. Although it was not evaluated extensively, L. plantarum 299v colonization dis-
appeared after about 1 month or less after the end of supplementation. Mononuclear cells isolated from peripheral blood from 17 children congenitally exposed to HIV were evaluated for immune response in vitro to L. plantarum 299v before supplementation. Positive response was defined as mean (counts per minute of triplicate wells) of maximum response to any concentration of the L. plantarum 299v antigen ⱖ3.5 times the background response of unstimulated cells cultured in parallel. Ten children were evaluated as having positive response and seven children were considered nonresponsive. The mean difference in response between responders and nonresponders was significantly different (Fig. 2) and validated the use of this differentiation of response from background proliferation for this particular antigen in the setting of HIV infection. Responders to L. plantarum 299v did not have a statistically different higher response to phytohemagglutinin (PHA, a standard T-cell mitogen used as a positive lymphocyte activator) than did those who did not respond (data not shown). This result suggests that immune deficiency alone was not the reason for lack of response. Included among the group of nonresponsive children were two children who also did not respond to PHA. One of these, a child with marked growth failure born to an HIVpositive mother, was subsequently found to be HIV-negative by polymerase chain reaction (PCR) and eventually showed seroreversion of the previously positive HIV antibody reactivity. This child did show overall improvement in height and weight while receiving L. plantarum 299v, but this could not be directly attributed to L. plantarum 299v because of inadequate follow-up visits and limited baseline data close to the time of administration. Interestingly, a marked augmentation in immune response was observed (Fig. 3). This difference reflected a change from generalized anergy to normal immune response within 1 month and occurred in the absence of any other intervention or treatment.
S24
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AJG – Vol. 95, No. 1, Suppl., 2000
Figure 2. Natural immune response to L. plantarum in congenital HIV exposure.
DISCUSSION Therefore, preliminary data from this study indicate that approximately 69% of HIV-positive children who have never received L. plantarum 299v do have significant preexisting immune response to this bacteria, probably due to cross-reactivity with other endogenous strains of L. plantarum or with strains of different lactobacilli species. The observation that immune response to L. plantarum 299v was not directly related to response to PHA is important for two reasons. First, absence of relationship suggests that lack of response was not a simple reflection of immune deficiency. Second, because antigen response is often lost early in HIV infection, the studies indicate that response to L. plantarum 299v may be specifically retained in HIV-positive children. As noted, all children tolerated this supplementation, there were no side effects, and children were not permanently colonized. These preliminary results suggest L. plantarum 299v may have the potential to improve growth and development. Furthermore, the data in one child indicate the possibility of
effects mediated through modulation of immune response after supplementation with L. plantarum 299v. This finding is indirectly supported by other studies in volunteers showing that oral supplementation with different lactobacilli did act to modulate and increase phagocytic activity in peripheral blood (16). Although there was no effect on lymphocyte response to mitogens in this study, this may not be surprising as immune response was already in the normal range in these healthy adults. These findings may indicate a potential for therapeutic use of lactobacilli in HIV infected children.
SUMMARY AND CONCLUSIONS This study examined the effect of L. plantarum 299v supplementation in children congenitally exposed to HIV. Results show that 69% of HIV-positive children had preexisting cross-reactive immune response to this antigen as measured by proliferative response to L. plantarum 299v before oral administration. No child was colonized with L. plantarum 299v at baseline, as shown by genetic typing of
Figure 3. Immune response after L. plantarum supplementation in HIV-exposed child with failure to thrive.
AJG – January, Suppl., 2000
lactobacilli grown from rectal swabs. Mononuclear cells from 10 confirmed HIV-positive children responded to L. plantarum 299v in vitro. Seven children did not react. Responders had a significantly higher response to L. plantarum 299v than did their nonreactive counterparts, but they did not have a significantly stronger response to PHA. These results demonstrate both that natural immune response to L. plantarum 299v occurs frequently and that this response is highly conserved in the HIV-positive child. The data suggest that L. plantarum 299v may be given safely to the immunocompromised host and may indeed have a positive effect on immune response. Reprint requests and correspondence: Susanna CunninghamRundles, Ph.D., Cornell Medical Center, 525 E. 68th Street, New York, NY 10021. Received Mar. 17, 1999; accepted Sep. 7, 1999.
REFERENCES 1. Prindull G, Ahmad M. The ontogeny of the gut mucosal immune system and the susceptibility to infections in infants of developing countries. Eur J Ped 1993;152:786 –92. 2. Cunningham-Rundles S. Malnutrition and gut immune function. Curr Op Gastroenterol 1994;10:664 –70. 3. Murry AC, Lewis RD, Amos HE. The effect of microbial phytase in a pearl millet-soybean meal diet on apparent digestibility and retention of nutrients, serum mineral concentration, and bone mineral density of nursery pigs. J Anim Sci 1997;75:1284 –91. 4. Torrallardona D, Harris CI, Coates ME, et al. Microbial amino acid synthesis and utilization in rats: Incorporation of 15N from 15NH4Cl into lysine in the tissues of germ-free and conventional rats. Br J Nutr 1996;76:689 –700. 5. Drago L, Gismondo MR, Lombardi A, et al. Inhibition of in vitro growth of enteropathogens by new Lactobacillus isolates of human intestinal origin. FEMS Microbiol Lett 1997;153: 455– 63.
Probiotics and Immune Response
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6. Bengmark S, Jeppsson B. Gastrointestinal surface protection and mucosa reconditioning. J Parenter Enteral Nutr 1995;19: 410 –5. 7. Majamaa H, Isolauri E. Probiotics: A novel approach in the management of food allergy. J Allergy Clin Immunol 1997; 99:179 – 85. 8. Johansson ML, Molin G, Jeppsson B, et al. Administration of different Lactobacillus strains in fermented oatmeal soup: In vivo colonization of human intestinal mucosa and effect on the indigenous flora. J App Environ Microbiol 1993;59:15–20. 9. Crocker KS. Gastrointestinal manifestations of the acquired immunodeficiency syndrome. Nurs Clin North Am 1989;24: 395– 406. 10. Kotler DP, Tierney AR, Culpepper-Morgan JA, et al. Effect of home total parenteral nutrition on body composition in patients with acquired immunodeficiency syndrome. J Parenter Enteral Nutr 1990;14:454 – 8. 11. Alak JI, Wolf BW, Mdurvwa EG, et al. Effect of Lactobacillus reuteri on intestinal resistance to Cryptosporidium parvum infection in a murine model of acquired immunodeficiency syndrome. J Infect Dis 1997;175:218 –21. 12. Johansson ML, Berggren A, Nobaek S, et al. Survival of Lactobacillus plantarum DSM 9843, and effect on the short chain fatty acid content in feces after ingestion of a rose hip drink with fermented oats. Int J Food Microbiol 1999 (in press). 13. Johansson ML, Quednau M, Ahrne S, et al. Classification of Lactobacillus plantarum by restriction endonuclease analysis of total chromosomal DNA, using conventional agarose gel electrophoresis. Int J Syst Bacteriol 1995;45:670 –5. 14. Cunningham-Rundles S, Yeger-Arbitman R, Edelson P, et al. Experimental approach to the study of immune function in children with possible human immunodeficiency virus, HIV, infection. J Clin Lab Analysis 1990;4:399 – 404. 15. Paxton H, Cunningham-Rundles S, O’Gorman MRG. Laboratory evaluation of the cellular immune system. In: Henry JB, ed. Clinical diagnosis and management by laboratory methods. Philadelphia: WB Saunders and Co, 1995:887–912. 16. Schiffrin EJ, Rochat F, Link-Amster H, et al. Immunomodulation of human blood cells following the ingestion of lactic acid bacteria. J Dairy Sci 1995;78:491–7.