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Experimental Biology and Medicine 232:454-460 (2007)
© 2007 Society for Experimental Biology and Medicine

ORIGINAL RESEARCH ARTICLE

Cryptosporidiosis Induces a Transient Upregulation of the Oligopeptides Transporter (PepT1) Activity in Neonatal Rats

Perrine Marquet*, Laurence Barbot*,{dagger}, Aurélia Planté*, Jean François Huneau{dagger}, Jean Gérard Gobert* and Nathalie Kapel*,{dagger},1

* EA209 "Eucaryotes Pathogènes," Faculté des Sciences Pharmaceutiques et Biologiques, Université René Descartes, 75006 Paris, France; {dagger} Service de Coprologie Fonctionnelle, Groupe-Hospitalier Pitié-Salpêtrière, 75013 Paris, France; and {ddagger} UMR INRA/INA-PG Physiologie de la Nutrition et du Comportement Alimentaire, Institut National Agronomique Paris-Grignon, 75005 Paris, France

1To whom requests for reprints should be addressed at KAPEL Equipe EA209 "Eucaryotes pathogènes," UFR des Sciences Pharmaceutiques et Biologiques Paris 5, 4 avenue de l’Observatoire, 75006 Paris, France. E-mail: nathalie.kapel{at}univ-paris5.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Cryptosporidium parvum is a parasitic protozoa increasingly appreciated as a cause of intestinal malabsorptive syndrome leading to malnutrition and/or growth failure. Because a major mechanism for apical peptide absorption by small intestine is via the proton-coupled transporter PepT1, we investigated the expression and functionality of this transporter in our model of acute cryptosporidiosis. Four-day-old Sprague-Dawley rats were inoculated by gavage with 5 x 105 oocysts of C. parvum and killed at Day 12 (peak of the infection) or Day 21 (spontaneous clearance of the parasite). PepT1 expression and functionality were quantified in the distal small intestine, preferential site of C. parvum implantation, and in the proximal small intestine, free of parasite, using Western blot and Ussing chambers, respectively. No difference in total PepT1 protein expression or in glycyl-sarcosine fluxes was observed in C. parvum–infected rats compared with controls either on Day 12 or on Day 21, both in the proximal and in the distal small intestine. However, a significant decrease of apical membrane protein expression of PepT1 was observed in C. parvum–infected enterocytes compared with controls. This maintained dipeptide transport observed despite villous atrophy and decreased expression of the protein at the brush-border membrane strongly suggest a transient upregulation of PepT1 activity, probably related to {gamma}-interferon regulation.

Key Words: PepT1 • cryptosporidiosis • neonatal rats • malnutrition


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Cryptosporidium parvum is a protozoan parasite from the Apicomplexa phylum, capable of infecting humans by ingestion of oocysts. Once ingested, the parasite anchors the apical membrane of the host enterocytes, leading to a reorganization of the host-cell actin cytoskeleton and the formation of a parasitophorous vacuole with an intracellular but extracytoplasmic location, where the parasite undergoes further development. Although first recognized as an opportunistic pathogen in human, leading to life-threatening diarrhea in immunocompromised patients, C. parvum is now regarded as one of the most serious and refractory causes of waterborne-related diarrhea (1, 2). Epidemiologic reports from developing countries have also established the lasting impact of cryptosporidial infection on nutritional status, growth, fitness, and cognitive development in immunocompetent children, thus emphasizing the functional consequences of cryptosporidial infection, particularly when acquired during infancy (3–7). Many antimicrobial drugs have been evaluated to combat C. parvum infection; however, none has proved effective in treating the disease. Therefore, much attention should be paid to alleviate the adverse effects of cryptosporidiosis on child development and growth (8).

We previously developed a suckling rat model of acute cryptosporidiosis in which C. parvum transiently infests the distal part of the small intestine, with a maximum parasite load on Day 12 and an almost complete clearance of the parasite by Day 16–18 (9). In this model, C. parvum is responsible for failure to thrive associated with a dramatic reduction in amino acid absorption at the peak of infection. This malabsorption involves the whole small intestine, whereas only the distal part is parasitized, and is not counterbalanced by any upregulation of amino acid transport, even after spontaneous clearance of the parasite, thus preventing any catch up in growth rate (10).

Absorption of di- and tripeptides in the small intestine occurs mainly through the proton-coupled transporter PepT1 (11). PepT1 is evenly distributed along the small intestine, and this pattern remains similar from Day 4 until adulthood in rats (12). In a preliminary study, we showed that PepT1 mRNA level was maintained in infected intestine and even transiently overexpressed at the peak of infection, in contrast to mRNA coding for amino acid transporters, such as EAAT3 (13). These results suggest a transcriptional upregulation of the peptide transporter in response to parasite development and C. parvum–induced malnutrition. However, because the parasite development induces a rearrangement of the brush-border actin network of the enterocyte, post-translational regulation in PepT1 transport activity could not be ruled out. The purpose of the present study was thus to investigate whether or not C. parvum infection alters PepT1 expression and activity in our model of acute cryptosporidiosis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Parasites.
C. parvum oocysts (genotype 2) (gift from M. Naciri, INRA, Nouzilly, France) were initially purified from the feces of a patient infected with human immunodeficiency virus and maintained by regular passage in suckling newborn rats (9). This strain is responsible for a failure to thrive in infected animals (14). The pooled colonic perfusates were kept in 150 mM phosphate-buffered saline (PBS; pH 7.2) at 4°C in the presence of penicillin (100 IU·ml–1), streptomycin (100 µg·ml–1), and amphotericin B (0.25 µg·ml–1) until use.

Experimental Design.
A total of 22 litters of specific-pathogen-free (SPF) outbred Sprague-Dawley suckling rats were used in this study: 11 were parasitized with C. parvum, and 11 were used as controls. In each group, six litters were used for Western blot analysis, three were dedicated to fluxes measurements, and two were used for {gamma}-interferon (IFN-{gamma}) assays.

Female rats with 4-day-old litters were obtained from Charles River (L’Arbresle, France) and handled according to the regulations of the French Ministry of Agriculture. Food (M20 diet, Special Diets Services, Witham, UK) and water were available ad libitum. Each single dam was housed with its litter (adjusted to 10–11 pups) in a separate cage and maintained in an appropriate animal facility. At Day 4, pups were infected by gavage with 100 µl of colonic perfusate containing 5 x 105 cryptosporidia. Rats from the control litters were inoculated by gavage on Day 4 with 100 µl of colonic perfusate obtained from 12-day-old SPF rats free from C. parvum.

Animals were killed either on Day 12 (peak of infection) or Day 21 (after spontaneous clearance of the parasite) by intraperitoneal pentobarbital injection. The parasite load of the animals used in each experiment was checked at the time of killing on a 1-cm-long segment of distal ileum, which was homogenized in 10 volume of PBS and then stained with the Ziehl-Neelsen reagent, according to Henriksen’s method (15).

Ex-Vivo Measurements of Oligopeptides (Glycyl-Sarcosine) Fluxes Across the Ileal Mucosa.
Mucosal to serosal fluxes of glycyl-sarcosine across the intestinal mucosa were determined at Day 12 and Day 21, using ileal sheets mounted in Ussing chambers as previously described (10). Eleven rats originating from different litters were used at each time point in each group. Briefly, a 3-cm segment of ileum, adjacent to the ileo-cecal junction, was rinsed free of intestinal contents, opened along the mesenteric border, and mounted between two halves of a custom-made Ussing chamber with an aperture of 0.39 cm2 (Marty Technologie, Marcilly sur Eure, France). The mucosal and serosal compartments of the chambers were filled with 3 ml of MES/Tris buffer (pH 6) containing (in mM) 25 MES, 140 NaCl, 5.4 KCl, 1.8 CaCl2, 0.8 MgSO4, and 5 glucose. The tissue was oxygenated by a gas lift of O2/CO2 (95:5) and the temperature maintained at 37°C throughout the experiment. Transepithelial fluxes of glycyl-sarcosine across the ileal mucosa were measured by adding 100 µM of glycyl-sarcosine (Sigma Chemicals, St. Louis, MO) and 1 µCi of [3H]glycyl-sarcosine (Hartmann Analytics, Braunschweig, Germany) to the MES/Tris buffer in the mucosal compartment of the Ussing chambers. A 20-µl sample was taken from the mucosal compartment 10 min after the addition of radiolabeled glycyl-sarcosine, and 500-µl samples were withdrawn from the serosal compartment every 15 mins for 60 mins and replaced with an equivalent volume of fresh MES/Tris buffer. The amount of radioactivity was measured in the different samples by liquid scintillation counting using a Liquid Scintillation Analyzer 2200 CA (Packard Instrument, Rungis, France) and used to calculate peptide fluxes across the ileal mucosa (expressed as nmol·cm–2·min–1) for the 15- to 60-min period.

Western Blot Analysis.
Preparation of Intestinal Epithelial Cells.
Controls and infected rats were sacrificed on Day 12 (n = 43 and n = 34 for controls and infected rats, respectively) and Day 21 (n = 6 for control and infected rats, respectively). Epithelial cells were isolated from intestinal epithelium, as described by Déchelotte et al. (16). After opening the abdominal cavity, the whole small intestine was removed and divided into three segments of equal length. The proximal and distal parts were rinsed free of intestinal content with saline, ligatured to one end, filled with 150 mM NaCl containing 2.5 mM EDTA, ligatured at the other end, and placed in 150 mM NaCl for 45 mins at 4°C. Luminal content, containing isolated epithelial cells, was drained, mixed with 4 ml of ice-cold 150 mM NaCl, and centrifuged for 3 min at 4°C and 1600 g, and the cellular pellet was washed twice with 150 mM NaCl. For 12-day-old rats, cell pellets isolates from three rats of the same litter were pooled together to gather a sufficient amount of material. Cell samples were sonicated using a 50-W Vibracell 72434 (Bioblock, Illkirch, France) with 30 pulses, setting 40%, in an ice bath, centrifuged at 9000 g for 30 mins at 4°C. The supernatant was added with antiproteases (aprotinin 2 µg·ml–1, PMSF 1mM) and stored at –20°C. Protein concentrations were determined using the bicinchoninic acid kit (Sigma Chemicals) with bovine serum albumin as protein standard.

Preparation of Brush-Border Membrane Vesicles.
Controls (n = 20) and C. parvum–infected rats (n = 12) were sacrificed on Day 12. Epithelial cells were isolated from the distal part of the intestine as described before and maintained at 4°C. Samples from three to five rats per litter were pooled and used to prepare brush-border membrane vesicles (BBMV) by the method of Kessler et al. (17) but using MgCl2 precipitation instead of CaCl2 to avoid activation of proteases as described by Van Voorhis et al. (18). BBMV were added with antiproteases, lysed by sonication for 30 secs in an ice bath, and stored at –20°C before immunoblot analyses. Aliquots were used for protein determination and measurement of sucrase activity.

Sucrase Measurement.
Sucrase activity in samples can be considered as representative of brush-border enrichment and cell maturity because it is located in the brush border of epithelial cells in the villi, with an increasing gradient from the crypt-villous junction to villus tip, as previously described for PepT1 (12). This PepT1 gradient was maintained during C. parvum infection (13). Briefly, aliquots of epithelial cells samples were incubated at 37°C for 1 hr in the presence of {alpha}1-glucopyranosyl-ß2-fructofuranoside, the sucrase substrate, and glucose production was measured with glucose oxidase (19).

Immunoblot Analyses.
The abundance of PepT1 protein in the isolated epithelial cells and in the BBMV was measured by quantitative Western blot analysis using an affinity-purified rabbit anti-rat PepT1 (1 mg·ml–1) produced against the synthetic peptide VGKENPYS-SLEPVSQTNM (Agro Bio, La Ferté St Aubin, France) according to Miyamoto et al. (20). Specificity of the rabbit anti-rat PepT1 and the molecular weight of the PepT1 band were controlled by performing immunoblotting with and without preincubation of the anti-rat PepT1 antibody with the synthetic peptide for 1 hr at room temperature.

Proteins from sonicated cell samples or sonicated BBM vesicles (10–25 µg per well) were resolved on a 9% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions and transferred on polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 10% nonfat dry milk in TBS-T (20 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.05% Tween 20) and probed for 1 hr with the affinity-purified rabbit anti-rat PepT1 (final concentration: 1 mg·ml–1). The membranes were washed five times in TBS-T and probed with peroxidase-conjugated goat anti-rabbit IgG 1:10000 (Sigma Chemicals) for 1 hr, both steps at room temperature. After five more washings in TBS-T and TBS (TBS-T without Tween 20), the bound antibodies were detected with an enhanced chemiluminescence immunoblotting system (ECL Plus, Amersham Life Science, Arlington Heights, IL). The intensity of the signal was quantified using a digital imaging system (Alpha Innotech Corporation, San Leandro, CA). PepT1 expression in intestinal epithelial cell was standardized for the amount of protein in the cell lysate. To take into account the differences in brush-border membrane enrichment from one sample to another, sucrase activity was used to normalize the amount of PepT1 in BBMV fractions.

IFN-{gamma} Assay.
Control (n = 6) and C. parvum–infected (n = 6) rats originating from different litters were sacrificed on Day 12 and Day 21. Blood was collected by intra-peritoneal cardiac puncture then centrifuged at 1600 g for 10 mins at 4°C. The serum was stored at –80°C until use. After opening the abdominal cavity, the whole small intestine was removed and divided into three segments of equal length. The proximal and distal parts were rinsed free of intestinal content with 150 mM PBS (pH 7.2), homogenized in five volumes of 150 mM PBS (pH 7.2) with 2 µg·ml–1 aprotinin and 1 mM PMSF using a glass tissue grinder in an ice bath, and then centrifuged at 9000 g for 30 mins at 4°C. The supernatants were stored at –80°C until use. Before carrying out the assay, cells in proximal and distal intestinal homogenates were sonicated using VibraCell 72434 as previously described and then centrifuged at 9000 g for 10 mins at 4°C. IFN-{gamma} levels in serum and intestinal homogenates were measured in duplicate by means of a sandwich enzyme-linked immunoabsorbent assay (ELISA) (Rat Interferon-{gamma} ELISA kit, Biosource, Nivelles, Belgium). The sensitivity of the ELISA was <13 pg·ml–1 in serum and intestinal homogenate without dilution of samples. Results were expressed as pg of IFN{gamma} per ml for serum and as pg of IFN-{gamma} per mg of total protein for intestinal homogenates.

Statistical Analysis.
All data in the figures are expressed as mean ± standard deviation. Statistical analysis of the results was performed by analysis of variance followed by Fisher’s protected least significant difference and Student’s t test for PepT1 and IFN-{gamma} quantification and oligopeptides fluxes analysis, respectively, using the statistical analysis software (Statview, Abacus Concepts Inc., Berkeley, CA). P < 0.05 was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oocysts were never detected in controls rats. In C. parvum–infected rats, the parasite load was at least 50 oocysts per 50 high-power microscopic field (x400) on Day 12, whereas C. parvum oocysts were no longer detected on Day 21.

Ex-Vivo Measurements of Glycyl-Sarcosine Fluxes Across the Ileal Mucosa.
The accumulation of glycyl-sarcosine in the serosal compartments of Ussing chambers increased linearly between 15 and 60 mins, in both control and C. parvum–infected rats at Day 12 and Day 21. Transepithelial dipeptide fluxes were therefore calculated from the slope of the serosal accumulation during this period. Although lower at Day 21 compared with Day 12 in both control and infected animals, the difference in ileal fluxes of glycyl-sarcosine was significant only in controls (P < 0.05). Interestingly, fluxes were similar in C. parvum–infected and controls rats, both on Day 12 (0.0760 ± 0.0281 and 0.0784 ± 0.0354 nM·cm–2·min–1, respectively) and Day 21 (0.0551 ± 0.0160 and 0.0459 ± 0.0164 nM·cm–2·min–1, respectively) (Fig. 1Go). No interaction between age and infection was observed.


Figure 1
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  		Figure 1. Time course and fluxes of glycyl-sarcosine transfer from the mucosal to the serosal side of C. parvum–infected ({blacksquare}, n = 11) and control ({circ}, n = 11) ileal mucosa at Day 12 and Day 21. In each animal, [3H]glycyl-sarcosine transport per cm2 was determined over the 15- to 60-min period. The bar graphs represent the fluxes. Results are expressed as mean ± SD. Ileal fluxes were significantly decreased at Day 21 compared with Day 12 in controls (P < 0.05) but not in C. parvum–infected rats. No significant variation was observed between infected and control tissues at Day 12 or at Day 21.

 
Expression of Total PepT1 Protein in the Isolated Epithelial Cells and Brush-Border Membrane.
To further investigate the level of regulation of peptide transport, expression of the PepT1 protein was assessed using Western blot analysis.

As depicted in Figure 2Go, competitive immunoblotting analysis showed that the affinity-purified rabbit anti-rat PepT1 recognizes a band of 97 kDa, which was specifically blocked by preabsorbing the primary antibody with the immunizing peptide. PepT1 expression in epithelial cell did not vary significantly with age or with the proximo-distal localization in the small intestine and was unaffected by C. parvum infection on both Day 12 and Day 21 (Fig. 2Go).


Figure 2
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  		Figure 2. Expression of total PepT1 protein in the isolated epithelial cells and brush-border membrane. (A) Competitive immunoblotting performed with and without preincubation of the affinity-purified rabbit anti-rat PepT1 with the synthetic peptide VGKENPYSSLEPVSQTNM for 1 hr at room temperature. The band for PepT1 is at 97 kDa. (B) Expression of total PepT1 transporter in proximal and distal epithelial cell lysates isolated from C. parvum–infected rats ({blacksquare}, n = 23 and 6 on Day 12 and Day 21, respectively) and controls ({square}, n = 22 and 6 on Day 12 and Day 21, respectively). For 12-day-old rats, cell isolates from three rats per litter were pooled. Twenty-five (distal) or 20 (proximal) µg of proteins were subjected to 9% SDS-PAGE. Resolved proteins were transferred to PVDF membranes and incubated for 1 hr at room temperature with the anti-PepT1 antibody. Densities of Western blot bands were quantified in regard with total protein of the sample (AU/µg of proteins). (C) Expression of PepT1 protein in BBMV prepared with epithelial cells isolated at Day 12 from distal part of the small intestine in C. parvum–infected rats ({blacksquare}, n = 12) and controls ({square}, n = 20). Cell isolates from three to four infected rats and from five control animals were pooled. Ten µg of proteins were subjected to 9% SDS-PAGE, transferred to PVDF membranes and then incubated 1 hr at room temperature with the anti-PepT1 antibody. Densities of the bands were quantified in regard with sucrase activity of the sample (AU/U sucrase).

 
The subcellular distribution of PepT1 was further investigated at the time peak of infection (Day 12) by measuring its expression in brush-border membranes prepared from isolated epithelial cells. Sucrase activity, which is known to specifically locate in the brush-border membrane, was used to normalize PepT1 expression. There was no difference in sucrase activity between epithelial cells isolated from control and C. parvum–infected rats on Day 12 (data not shown).

In contrast to total protein, expression of PepT1 protein in the brush-border membranes was dramatically decreased (–80%, P < 0.05) at Day 12 in samples originating from C. parvum–infected rats compared with controls (Fig. 2Go).

IFN-{gamma} Level in Mucosa and Serum.
Serum IFN-{gamma} level concentration was increased by 400% at Day 12 in infected compared with control rats (51.7 ± 32.8 pg·ml–1 and 9.5 ± 0.5 pg·ml–1, respectively; P < 0.02). IFN-{gamma} level in the distal part of the small intestine was 3-fold increased in infected compared with control rats (5.2 ± 1.3 pg·mg–1 and 1.8 ± 0.3 pg·mg–1 respectively; P < 0.001). Although nonsignificant, IFN-{gamma} level was also increased in the proximal part of the small intestine at the peak of infection compared with controls (7.0 ± 1.4 pg·mg–1 of protein and 4.1 ± 3.5 pg·mg–1 of protein, respectively). No more difference was observed at Day 21.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We have previously shown that C. parvum infection in neonatal rats is responsible for a major amino acid malabsorption, leading to undernutrition, and growth failure that is not counterbalanced by any upregulation, even after spontaneous clearance of the parasite (10, 14). In contrast, the present study shows that peptide transport across the small intestine is unaffected during cryptosporidiosis, despite villous atrophy and a reduced expression of PepT1 in the brush-border membrane, thus suggesting a transient post-translational upregulation of the oligopeptide transport activity. These data suggest a specific role for the ditripeptide transporter PepT1 in the metabolic adaptation to cryptosporidiosis-associated malnutrition.

Using the same rat model, we have previously shown that PepT1 mRNA expression was upregulated in the small intestine of C. parvum–infected rats at the peak of infection (13). This result agreed with those of other studies reporting an increase in PepT1 transcripts during malnutrition and fast-induced intestinal injury (21–25). However, no increase was observed in total protein expression in our model. This discrepancy should be due to the mucosal secretion of IFN-{gamma} observed during C. parvum infection, which has been recently shown to activate proteasome activity (26). Moreover, the present results show no evidence for an upregulation of glycyl-sarcosine transport across the intestinal mucosa of C. parvum–infected rats, which remained similar to that of control animals, thus suggesting a more complex regulation of peptide transport in this pathology. Indeed, the results of Western blot experiments show that total PepT1 expression in intestinal epithelial cell was similar in control and infected rats, both in the proximal and distal part of the small intestine, but the amount of PepT1 protein recovered in the brush-border membrane was significantly decreased at the peak of infection in C. parvum–infected rats. This suggests that PepT1 targeting to the brush-border membrane is impaired during cryptosporidiosis and that parasite infestation results in transporter segregation within an intracellular pool. This is consistent with the results of a previous immunohistochemical experiment, showing an accumulation of PepT1 immunoreactivity within the enterocyte of C. parvum–infected rats at the peak of infection, whereas the staining was restricted to the brush-border membrane in control rats (13). The existence of an intracellular pool of peptide transporters has been reported in Caco-2 cells and rat intestine (27, 28).

Despite the impaired trafficking of peptide transporter to the apical membrane of epithelial cell and the long recognized marked villous and microvillous atrophy responsible for a decrease in intestinal absorption surface, peptide transport across the small intestine of C. parvum–infected rats was similar to that measured in control rats. This suggests the existence of additional mechanisms resulting in an increased activity of the transporter during C. parvum infection, thus counteracting the consequences of a reduction in the number of transporters available for peptide absorption.

Most attention has been given to the transcriptional regulation of peptide transport in the small intestine (25), and little is known about its post-translational regulation. Buyse et al. (29) showed that IFN-{gamma} dose dependently upregulates dipeptide transport through an increase in intracellular pH, thus enhancing the proton electrochemical gradient across the brush-border membrane, which represents the driving force for PepT1. An IFN-{gamma}–dependent regulation of PepT1 was recently confirmed by Vavricka et al. (30), who showed that IFN-{gamma} and tumor necrosis factor-{alpha} upregulated PepT1 expression and activity in Caco-2 cells. Mucosal IFN-{gamma} is known to play a key role in immune response to C. parvum (31–33). In the present study, we showed a 3-fold increase in IFN-{gamma} level in the distal small intestinal mucosa of infected rats at the peak of infection compared with controls. The large increase in IFN-{gamma} may activate the transporter, through the enhancement of the inward proton gradient or alternative mechanisms that remain to be investigated, thus counterbalancing the effect of a reduction in the amount of transporter.

Overall, the present data demonstrate that in contrast to glucose and amino acids, peptide transport across the small intestine is preserved during experimental cryptosporidiosis. The mechanisms responsible for the maintenance of this transport are complex, as illustrated by the increase in the level of transcripts coding for PepT1, the expression of the PepT1 protein at a normal level in the intestinal epithelium, and the decrease in the amount of PepT1 protein recovered in the brush-border membrane. Altogether, these results strongly suggest the involvement of post-translational mechanisms, probably related to the secretion of IFN-{gamma} during this infection.

These results are particularly important for children with cryptosporidiosis and children infected with human immunodeficiency virus, who could benefit from nutritional support to break the vicious circle of malnutrition/immunodepression/cryptosporidiosis.

Received for publication September 14, 2006. Accepted for publication October 24, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Current WL, Garcia LS. Cryptosporidiosis. Clin Microbiol Rev 4: 325–358, 1991.[Abstract/Free Full Text]
  2. Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA, Peterson DE, Kazmierczak JJ, Addiss DG, Fox KR, Rose JB, Davis JP. A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply. N Engl J Med 331: 161–167, 1994.[Abstract/Free Full Text]
  3. Molbak K, Andersen M, Aaby P, Hojlyng N, Jakobsen M, Sodemann M, Da Silva AP. Cryptosporidium infection in infancy as a cause of malnutrition: a community study from Guinea-Bissau, West Africa. Am J Clin Nutr 65:149–152, 1997.[Abstract/Free Full Text]
  4. Checkley W, Gilman RH, Epstein LD, Suarez M, Diaz JF, Cabrera L, Black RE, Sterling CR. Asymptomatic and symptomatic cryptosporidiosis: their acute effect in weight gain in Peruvian children. Am J Epidemiol 45:156–163, 1997.
  5. Checkley W, Epstein LD, Gilman RH, Black RE, Cabrera L, Sterling CR. Effects of Cryptosporidium parvum infection in Peruvian children: growth faltering and subsequent catch-up growth. Am J Epidemiol 148: 497–506, 1998.[Abstract/Free Full Text]
  6. Agnew DG, Lima AAM, Newman RD, Wuhib T, Moore RD, Guerrant RL, Sears CL. Cryptosporidiosis in Northeastern Brazilian children: association with increased diarrhea morbidity. J Infect Dis 177: 754–760, 1998.[Medline]
  7. Guerrant DI, Moore SR, Lima AA, Patrick PD, Schorling JB, Guerrant RL. Association of early childhood diarrhea and cryptosporidiosis with impaired physical fitness and cognitive function four-seven years later in a poor urban community in northeast Brazil. Am J Trop Med Hyg 61:707–713, 1999.[Abstract]
  8. Farthing MJ. Clinical aspects of human cryptosporidiosis Contrib Microbiol 6:50–74, 2000.
  9. Capet C, Kapel N, Huneau JF, Magne D, Laikuen R, Tricottet V, Benhamou Y, Tome D, Gobert JG. Cryptosporidium parvum infection in suckling rats: impairment of mucosal permeability and Na(+)-glucose cotransport. Exp Parasitol 91:119–125, 1999.[Medline]
  10. Topouchian A, Huneau JF, Barbot L, Rome S, Gobert JG, Tome D, Kapel N. Evidence for the absence of an intestinal adaptive mechanism to compensate for C. parvum-induced amino acid malabsorption in suckling rats. Parasitol Res 91:197–203, 2003.[Medline]
  11. Fei YJ, Kanai Y, Nussberger G, Ganapathy V, Laibach FH, Romero MF, Singh SK, Boron WF, Hediger MA. Expression cloning of a mammalian proton-coupled oligopeptide transporter. Nature 368: 563–566, 1994.[Medline]
  12. Rome S, Barbot L, Windsor E, Kapel N, Tricottet V, Huneau JF, Reynes M, Gobert JG, Tomé D. The regionalization of PepT1, NBAT and EAAC1 transporters in the small intestine of rats are unchanged from birth to adulthood. J Nutr 132:1009–1011, 2002.[Abstract/Free Full Text]
  13. Barbot L, Windsor E, Rome S, Tricottet V, Reynès M, Topouchian A, Huneau JF, Gobert JG, Tome D, Kapel N. Intestinal peptide transporter PepT1 is over-expressed during acute cryptosporidiosis in suckling rats as a result of both malnutrition and experimental parasite infection. Parasitol Res 89:364–370, 2003.[Medline]
  14. Topouchian A, Kapel N, Huneau JF, Barbot L, Magne D, Tome D, Gobert JG. Impairment of amino-acid absorption in suckling rats infected with Cryptosporidium parvum. Parasitol Res 87:891–896, 2001.[Medline]
  15. Henriksen SA, Pohlenz JF. Staining of cryptosporidia by a modified Ziehl-Neelsen technique. Acta Vet Scand 22:594–596, 1981.[Medline]
  16. Dechelotte P, Sabouraud A, Sandouk P, Hackbarth I, Schwenk M. Uptake, 3-, and 6-glucuronidation of morphine in isolated cells from stomach, intestine, colon, and liver of the guinea pig. Drug Metab Dispos 21:13–17, 1993.[Abstract]
  17. Kessler M, Acuto O, Storelli C, Murer H, Muller M, Semenza G. A modified procedure for the rapid preparation of efficiently transporting vesicles from small intestinal brush border membranes. Biochim Biophys Acta 506:136–154, 1978.[Medline]
  18. Van Voorhis K, Said HM, Ghishan FK, Abumrad NN. Transport of glutamine in rat intestinal brush-border membrane vesicles. Biochim Biophys Acta 978:51–55, 1989.[Medline]
  19. Dahlquist A. Method for assay of intestinal disaccharidases. Analyt Biochem 7:18–25, 1964.
  20. Miyamoto K, Shiraga T, Morita K, Yamamoto H, Haga H, Taketani Y, Tamai I, Sai Y, Tsuji A, Takeda E. Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter. Biochim Biophys Acta 1305:34–38, 1996.[Medline]
  21. Walker D, Twaites DT, Simmons NL, Gilbert HJ, Hirst BH. Substrate up-regulation of the human small intestinal peptide transporter, hPepT1. J Physiol 507:697–706, 1998.[Abstract/Free Full Text]
  22. Shiraga T, Miyamoto KI, Tanaka H, Yamamoto H, Taketani Y, Morita K, Tamai I, Tsuji A, Takeda E. Cellular and molecular mechanisms of dietary regulation on rat intestinal H+/peptide transporter PepT1. Gastroenterology 116:354–362, 1999.[Medline]
  23. Thamotharan M, Bawwani SZ, Zhou X, Adibi SA. Functional and molecular expression of intestinal oligopeptides transporter (PepT-1) after a brief fast. Metabolism 48:681–684, 1999.[Medline]
  24. Ihara T, Tsujikawa T, Fujiyama Y, Bamba T. Regulation of PepT1 peptide transporter expression in the rat small intestine under malnourished conditions. Digestion 61:59–67, 2000.[Medline]
  25. Adibi SA. Regulation of expression of the intestinal oligopeptides transporter (PepT1) in health and disease. Am J Physiol Liver Physiol 285:G779–G788, 2003.
  26. Leblond J, Hubert-Buron A, Bole-Feysot C, Ducrotté P, Déchelotte P, Coëffier M. Regulation of proteolysis by cytokines in the human intestinal epithelial cell line HCT-8: role of IFN{gamma}. Biochimie 88: 759–768, 2006[Medline]
  27. Thamotharan M, Bawani SZ, Zhou X, Adibi S. Hormonal regulation of oligopeptide transporter PepT-1 in a human intestinal cell line. Am J Physiol 276:C821–C826, 1999.
  28. Buyse M, Berlioz F, Guilmeau S, Tsocas A, Voisin T, Péranzi Gl, Merlin D, Laburthe M, Lewin MJM, Roze C, Bado A. PepT1-mediated epithelial transport of dipeptides and cephalexin is enhanced by luminal leptin in the small intestine. J Clin Invest 108:1483–1494, 2001.[Medline]
  29. Buyse M, Charrier L, Sitaraman S, Gewirtz A, Merlin D. Interferon-gamma increases hPepT1-mediated uptake of di-tripeptides including the bacterial tripeptide fMLP in polarized intestinal epithelia. Am J Pathol 163:1969–1977, 2003.[Abstract/Free Full Text]
  30. Vavricka SR, Musch MW, Fujiya M, Kles K, Chang L, Eloranta JJ, Kullak-Ublick GA, Drabik, Merlin D, Chang FB. Tumor necrosis factor-alpha and interferon-gamma increase PepT1 expression and activity in the human colon carcinoma cell line Caco-2/bbe and in mouse intestine. Pfugers Arch Eur J Physiol 452:71–80, 2006.
  31. Ungar BL, Kao TC, Burris JA, Finkelman FD. Cryptosporidium infection in an adult mouse model: independent roles for IFN-gamma and CD4+ T lymphocytes in protective immunity. J Immunol 147: 1014–1022, 1991.[Abstract]
  32. Kapel N, Benhamou Y, Buraud M, Magne D, Opolon P, Gobert JG. Kinetics of mucosal ileal gamma-interferon response during cryptosporidiosis in immunocompetent neonatal mice. Parasitol Res 82: 664–667, 1996.[Medline]
  33. Pollock R, Farthing M, Bajaj-Elliott M, Sanderson J, McDonald V. Interferon gamma induces enterocytes resistance against infection by the intracellular pathogen Cryptosporidium parvum. Gastroenterology 120:99–107, 2001.[Medline]




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