Temporal changes in permeability and structure of piglet ileum after site-specific infection by cryptosporidium parvum

GASTROENTEROLOGY 1995;108:1030-1039

Temporal Changes in Permeability and Structure of Piglet Ileum After Site-Specific Infection by Cryptosporidium parvum RONDA MOORE,* SAUL TZIPORI, *'§ JEFFREY K. GRIFFITHS, t'§'ll KEITH JOHNSON,*'* LINA DE MONTIGNY,* and INNA LOMAKINA*'* *Department of Pathology, School of Medicine and Veterinary Medicine, and *Department of Comparative Medicine, School of Veterinary Medicine, Tufts University, Boston; IIDivision of Infectious Diseases, St. Elizabeth's Medical Center of Boston, Boston; and §Division of Geographic Medicine and Infectious Diseases, Tufts/New England Medical Center, Boston, Massachusetts

Background/Aims: Cryptosporidiosis is an important enteric infection associated with diarrhea in humans. The structural and functional basis for diarrhea is poorly understood. The aim of the study was to determine the structural and functional basis of diarrhea in cryptosporidiosis during evolving host cell-parasite interactions in the intestine, Methods: We used the piglet model for temporal studies of alterations in intestinal epithelial structure and function that occurred 1 2 - 4 8 hours postinoculation, Segments of intestine were directly inoculated in vivo, harvested, and studied in vitro using Ussing chamber techniques. Results: Villus architectural alterations corresponded to the extent of infection. Increased numbers of lamina propria inflammatory cells were evident at 36 hours postinoculation. Solute and macromolecular permeability was not increased. Glucose-responsive short-circuit current was diminished at 48 hours postinoculation. The short-circuit current response to phlorizin was attenuated. The shortcircuit current response to theophylline was the same in control and infected tissues. Conclusions: We conclude that passive solute and macromolecular permeability in infected tissues is not significantly increased during parasite-host cell interactions 1 2 - 4 8 hours postinoculation. Electrogenic glucose stimulated Na ÷ absorption, a function principally of villus absorptive cells, is impaired, and electrogenic CI- secretion, a function of crypt epithelial cells, remains the same. These findings parallel structural observations that include loss of the Na*/glucose-transporting villus epithelium without loss of crypt epithelium.

lor many years, the coccidia, Cryptosporidiumparvum, was chiefly recognized as an enteric pathogen of animals. I Cryptosporidiumhas since gained notoriety because it is now known that the significant morbidity that occurs in the human population infected with the human immunodeficiency virus is the result of enteric infection by this parasite. 2-4 Experimentally infected neonatal piglets and calves5-7

F

as well as humans, chiefly patients with acquired immunodeficiency syndrome (AIDS), with cryptosporidiosis 2 show a severe watery diarrhea as the chief clinical manifestation of disease. The underlying cause of diarrhea in cryptosporidiosis is poorly understood. A rational approach to the discovery of new therapies for cryptosporidiosis logically includes studies that will enhance our understanding of the evolving response of the intestinal epithelium to the life stages of this parasite. Several prospective studies in the Cryptosparidium-infected neonatal piglet 8'9 and retrospective studies of human intestinal biopsy or autopsy material from naturally infected patients 1'1°-12 showed striking alterations in permeability and structure of the intestinal epithelium 7 2 96 hours or longer after oral inoculation with the parasite. The evolution of altered intestinal epithelial function and structure from the earliest stages of infection to these later time points, in which striking changes in the epithelium are evident, has not been rigorously studied. To this end, we used the Cryptosporidium-infected neonatal piglet as a model. For these studies, we specifically inoculated the distal ileum of neonatal piglets with a known quantity of excysted oocysts. Infection was allowed to occur at this site in vivo, and the intestine was harvested at sequential postinoculation time points for in vitro studies of intestinal permeability and transport function. In this report, we will correlate functional alterations with structural ones in intestinal epithelium after sitespecific infection. Epithelial architecture is dramatically altered as early as 3 6 - 4 8 hours postinoculation. These changes are dependent on the size of the parasite inoculum. Epithelial solute permeability paralleled striking morphological changes in the intestinal villus. At the cellular level, macromolecular permeability is unchanged Abbreviations used in this paper: AIDS, acquired immunodeficiency

syndrome; HRP, horseradish peroxidase, © 1995 by the American Gastroenterological Association 0016-5085/95/$3.00

April 1995

in regions adjacent to infected enterocytes. Thus, infected enterocytes maintain their ability to regulate tight junctions. N e t electrogenic transport processes are profoundly diminished at time points paralleling marked morphological alterations in the infected intestinal villus, alt h o u g h these alterations do not parallel the appearance of recruited polymorphonuclear inflammatory ceils. W e report that alterations in electrogenic secretory processes are most likely caused by a loss of coupled Na+/substrate cotransport. The results of our studies do not support the notion of Cryptosporidium-induced, 5'-cyclic adenosine monophosphate ( c A M P ) - m e d i a t e d C1- secretion. Lastly, the rapid appearance of structural and functional alterations in infected epithelium occurred 1 - 2 days earlier than previously reported in Cryptosporidium-infected intestinal epithelium. 8'9

Materials and Methods Experimental Design Segments of intestine harvested from control and Cryptosporidium-infected neonatal piglets were studied in Ussing chambers at 12, 24, 36, and 48 hours postinoculation. Ileal mucosa was inoculated in vivo by injection of known concentrations of excysted oocysts into the lumen of the distal 10 cm of the small intestine. At each time point studied, transepithelial electrical resistance, short-circuit current, and transepithelial flux of the small inert sugar, mannitol, were measured in Ussing chambers. Chambered tissues were subsequently harvested for morphological studies to enable structure-function correlations. In addition, some tissues were additionally used for extracellular macromolecular tracer studies. Finally, the electrogenic ion transport response of infected and control epithelium to inhibitors of coupled Na+/glucose cotransport and to agonist of cAMP-mediated CI- secretion was assessed.

C. parvum Oocysts

C. parvum oocysts used for these studies were isolated from a patient with AIDS (GCH1, Grafton Cryptosporidium human strain 1), propagated in calves, and isolated as previously described. 13'14 Oocysts were treated with 10% bleach at 4°C for 10 minutes and washed twice with sterile distilled water. Oocysts were excysted in 0.15% taurocholate for 30 minutes at 37°C, washed, and counted in a hematocytometer. Excystation rate for 108 oocysts was approximately 10%.

Animals Piglets used in these studies were healthy, 3-7-dayold, colostrum-fed conventional Yorkshire-cross obtained from the farm at Tufts University School of Veterinary Medicine and removed from their dam 24 hours before the studies began. Food was witheld, and animals were supplemented with 80 mL/kg Travasol per day (Clintec Nutrition Co., Deerfield, IL) administered intraperitoneally in divided doses.

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Site-Specific Inoculation With Excysted Oocysts Piglets were anesthetized with 11 mg/kg ketamine HC1 and 2 mg/kg xylazine administered intramuscularly. A 3 - 4 - c m midline ventral abdominal incision was made under aseptic conditions. The distal small intestine was isolated, and a loose ligature was tied with a slipknot around the distal ileum. An inoculum of 1.0 mL of excysted oocyts or t.0 mL of saline was injected into the lumen. The ends of the slipknot were brought through the distal end of the incision, and the abdomen was closed. The piglet was maintained under anesthesia for 2 additional hours, the ligature was removed, and the animal was allowed to recover from anesthesia.

Tissues for Ussing Chamber Studies At 12, 24, 36, or 48 hours postinoculation, piglets were anesthetized, the abdomen was opened through a ventral midline incision, and the inoculated and a 1 0 - 1 5 - c m proximal segment were removed. The proximal segment was examined histologically for preexisting disease. Functional data from studies in which the proximal segment had histopathologic evidence of disease or the inoculated segment was coinfected with other pathogens were excluded. Segments of intestine for Ussing chamber studies were opened longitudinally along the mesenteric border, outer muscle layers were stripped off, and the remaining mucosa and submucosa were mounted between two halves of an Ussing chamber equipped with two voltage-sensitive calomel electrodes and two AgCI current passing electrodes as previously described. 15-~7 Briefly, buffer in the mucosal and serosal reservoirs was oxygenated with 95% O j 5 % CO2, recirculated by a gas-lift mechanism and maintained at 37°C by water-jackets. Ten milliliters of a buffer of the following composition was added to the mucosal and serosal reservoirs (in mmol/L): 114 NaC1, 5 Na2HPO4, 0.3 NaH2PO4, 25 NaHCO3, 1.1 MgSO4, 1.25 CaC12, and 15 glucose. To increase tissue viability, Oxypherol E.T. (Alpha Therapeutics, Los Angeles, CA), a perfluorocarbon of high oxygen carrying capacity was added to the mucosal reservoir. Twenty minutes after tissues were mounted, electrical resistance and short-circuit current were measured as previously described. 15-1v Measurements were repeated 4 - 5 times at 15-minute intervals. The mean of 3 - 4 repeated measurements was used as data points. Unidirectional serosal-to-mucosal flux of [3H]mannitol was determined by the addition of trace amounts of 3[H]mannitol to the serosal reservoir, followed by a 1 5 - 2 0 minute equilibration period and 15-minute flux periods. In flux experiments, 5 mmol/L "cold" mannitol was added to the serosal reservoir to increase diffusional activity. Identical quantities of solute were added to the mucosal reservoir to eliminate a transepithelial solute gradient. In some studies, 2.5 mmol/L phlorizin was used in the mucosal bath to show that changes in short-circuit current are specifically caused by the apical Na + glucose cotransporter. In other studies, the epithelial response to cAMP-dependent CI-

1032 MOOREET AL.

secretion was determined by assessing the change in shortcircuit current in the presence of theophylline.

Morphological Studies After studies in Ussing chambers were completed, tissues were fixed in chambers with 2.5% glutaraldehyde at 37°C, followed by postfixation in Karnovsky's solution at 4°C, washed with 0.1 mol/L Na cacodylate buffer, and processed for plastic embedding as previously described) 5-1v Toluidine blue-stained, 1.0-btm thick sections were obtained and examined, and representative thin sections were cut with a diamond knife, mounted on copper-mesh grids, and stained with uranyl acetate and lead citrate except as noted below. To determine the number of cells lining the intestinal villus, enterocytes were counted along the long axis of the villus from tip to base in well-oriented, plastic-embedded, 1.0-btm sections of epithelium. For each data point, 3 - 9 villi from each of 2 3 tissue blocks from each of 3 piglets were used. In some studies, the extracellular macromolecular tracers horseradish peroxidase (HRP) and microperoxidase were used for morphological studies to assess the ability of the epithelium to maintain a barrier to macromolecular permeation. One to four blocks of intestinal epithelium from each of a total of 10 piglets 12-48 hours postinoculation were examined for HRP reaction product. Five infected piglets were similarly used for microperoxidase studies. From each block, a minimum of 10 junctions was examined per section, 2 - 3 sections per block. For these studies, 0.5% HRP (Sigma Chemical Co., St. Louis, MO) or 0.25% microperoxidase (Sigma Chemical Co.) was added to the mucosal reservoir. Three to four-centimeter sections of mucosa were embedded in 5% agar, and tissue was chopped at 200 gtm. HRP reaction product was developed as previously described. ~8 Tissues were processed for electronmicroscopy as detailed above, excluding en bloc uranyl acetate and postembedding lead citrate staining.

Statistical Analysis Data were analyzed for differences between means of control and infected tissues by application of Student's t test or nonparametric Mann-Whimey test using two tails of the distribution. InStat (GraphPad, San Diego, CA) statistical software was used for these analyses.

Results Temporal Alterations in Epithelial Architecture and Extent of Infection 1 2 - 4 8 Hours After Inoculation Infection of inoculated intestine was determined by light microscopic examination of toluidine b l u e stained, 1.0-btm thick, plastic-embedded sections of intestinal epithelium. As shown in Figure 1, infection of villus epithelial cells was evident as early as 12 hours postinoculation. The degree of infection was quantitatively assessed by counting the number of parasites per 100 epithelial ceils. Infection increased 1 2 - 3 6 hours

GASTROENTEROLOGYVol. 108, No. 4

postinoculation (Figure 2). The extent of infection at 36 hours postinoculation was similar after infection with 1 × 108 and 5 × 108 excysted oocyts (Figure 2). Crypt epithelial cells rarely were infected at any postinoculation time point (Figure 1). W e also noted that, surprisingly, only 15 % of infected exfoliated enterocytes had originated from the side of the villus. The majority of infected cells (85%) was extruded at the villus tip. At 36-48 hours postinoculation, all stages of parasite development from trophozoite to macrogamont were present in the epithelium (Figure 3). The stages of parasite in extruded cells at the villus tip reflected the heterogeneous population of parasite stages in the villus epithelium (Figure 3). As shown in the photomicrographs of infected epithelium (Figure 1), at 1 2 - 3 6 hours postinoculation, villi are somewhat shorter than villi in time-matched control tissues. A moderate change in epithelial architecture is first evident at 36 hours postinoculation; here villi are significantly shorter compared with villi in time-matched control tissues. The alteration in villus height at each time point is quantitatively expressed by counting the number of epithelial cells along the long axis of the villi in well-oriented, 1.0-~m, plastic-embedded sections of intestinal epithelium. The number of villus epithelial cells dramatically decreases by nearly 50% at 48 hours postinoculation (36 + 6, 45 + 4, 30 + 4, and 18 + 2 cells/villi for 12, 24, 36, and 48 hours postinoculation with 1 × 108 excysted oocysts, respectively; P < 0.009 for comparisons of 48 hours with earlier time points) and at 36 hours postinoculation with 5 × 108 excysted oocysts (19 cells/villi, P < 0.009). Thus, the villus height relates in an inverse manner to the extent of infection (see related time points in Figure 1). Lastly, we quantitated the number of inflammatory cells in villi from infected and time-matched control tissues by counting mononuclear and polymorphonuclear cells in the lamina propria and epithelium. These data are expressed as the number of cells per villus in Table 1. Compared with control tissues, the number of lamina propria polymorphonuclear inflammatory ceils was not increased until 48 hours postinoculation. Lamina propria mononuclear cells were significantly increased in number at 36 hours postinoculation. Intraepithelial inflammatory cells were not different in number between control and infected tissues. At 12 and 24 hours postinoculation, inflammatory cells in the epithelium and lamina propria of infected tissues were similar to control tissues (data not shown).

Alterations in Epithelial Macromolecular Permeability

Cryptosporidium is an intracellular parasite that localizes to the apical cytoplasm 19 near the perijunctional

April 1995

INTESTINAL PERMEABILITY IN CRYPTOSPORIDIOSIS 1033

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Figure 1. Photomicrograph of plastic-embedded, toluidine blue-stained, 1.0-pm sections of chambered intestinal epithelium. (A) Uninfected epithelium, (B) 12 hours postinoculation, (C) 24 hours postinoculation, (D) 36 hours postinoculation with 1 x 108 excysted oocysts, (E) 36 hours postinoculation with 5 × 108 excysted oocysts, and (F) 48 hours postinoculation with 1 x 108 excysted oocysts. Arrowheads indicate Cryptosporidium in apical cytoplasm of enterocytes along the villus epithelium (original magnification 30ON).

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actin-myosin ring, the region of the cytoskeleton that putatively regulates tight junction f u n c t i o n . 2°-22 To determine cellular sites at which alterations in tight junction permeability may occur after infection, we used the macromolecules H R P (40,000 daltons) and microperoxidase (1900 daltons) as extracellular tracer molecules. As shown in multiple electronmicrographs of infected epithelium (Figure 4) examined at each time point, HRP and microperoxidase were restricted to the luminal side of the junctional complex. Regulation of tight junction function as shown by the ability of the cell to restrict these extracellular tracer macromolecules to the lumen does not seem to be altered even in epithelial cells grossly distorted by an intracellular form of the parasite in the apical cytoplasm (Figure 4). Finally, multiple 1.0-[.tm sections of infected epithelium at 12, 36, and 48 hours postinoculation were examined for the presence of defects in the continuity of contiguous villus epithelial cells at the villus tip. Surprisingly, between 12-48 hours postinoculation, such defects were found in only 2% of infected villi (data not shown).

Time Course of Alterations in Epithelial Solute Permeability We used in vitro Ussing chamber studies to determine functional alterations in epithelial permeability that occurred at each postinoculation time point. When evaluated together with the above morphological data, these studies permit correlations to be drawn between alterations in epithelial structure and epithelial function

during the evolution of Cryptosporidium infection in native intestinal epithelium. We compared the permeability characteristics of chambered, time-matched control tissues with infected tissues by analysis of transepithelial flux of the simple inert sugar mannitol (182 daltons). Mannitol approximates the size of glucose and permeates native intestinal epithelium at a very low rate. 15 Transepithelial mannitol flux is thereby a sensitive means for detecting enhanced epithelial permeability. Mannitol flux in infected tissues at 12, 24, 36, and 48 hours postinoculation with 1 X 108 excysted oocysts is consistently but not significantly higher than flux in control tissues (P > 0.05 for comparisons of infected tissues with controls) (Figure 5). To further assess the ability of the epithelium to restrict passive solute movement across the epithelium, we used measurements of transepithelial electrical resistance, a highly sensitive, direct electrophysiological measurement of passive ion permeation across epithelia. 23 Compared with control tissues, resistance in infected tissues is not significantly different at 1 2 - 3 6 hours postinoculation with 1 × 108 excysted oocysts (P > 0.05 for all comparisons of infected tissues with controls) (Figure 6). Thus, at these time points, even infected epithelium retains the ability to restrict passive movement of the

Figure 3. Electronmicrograph of villus tip 36 hours postinoculation. Enterocytes at the villus tip are located at the bottom of the electronmicrograph and are labeled with an asterisk. The small arrow indicates the microvilli of an enterocyte, and the large arrow indicates an extruded cell containing two parasite forms. Numerous parasites are present at the villus tip where enterocytes normally are exfoliated as senescent cells. Many stages of parasite development including early trophozoite (1), macrogamete (2), schizont (3), and late trophozoite (4 and 5) are present (original magnification 4752×).

April 1995

INTESTINAL PERMEABILITY IN CRYPTOSPORIDIOSIS 1035

Table 1. I n f l a m m a t o r y Cells in M u c o s a of Control and Infected Tissues Intraepithelial

Lamina propria

Postinoculation (h)

Polymorphonuclear cells (control/infected)

Mononuclear cells (control/infected)

Polymorphonuclear cells (control/infected)

Mononuclear cells (control/infected)

36 48 36 a

0/0 0/2 0/1

0/1 0/1 0/1

2/9 1/15 b 1/6

20/35 c 17/40 b 20/33 b

NOTE. Values are expressed as the number of cells per villi. For each data point for control and infected villi, the number of cells was counted by the examination of 3 villi in 1.0-~Lm thick sections from plastic-embedded tissues in 2 - 3 blocks from each of 2 - 3 piglets. alnfected tissues inoculated with 5 x 108 excysted oocytes. bCompared with control; P < 0.001. cCompared with control; P < 0.01.

major ions in the mucosal buffer, Na + and CI , across the epithelium. In contrast, at 48 hours postinoculation with 1 X 108 excysted oocysts (P < 0.002 compared with control tissue) and at 36 hours postinoculation with 5 X 108 excysted oocysts (P < 0.02 compared with control tissues), electrical resistance was significantly higher compared with resistance of time-matched control tissues (Figure 6). In addition to passive transepithelia] ion movement that is assessed by measurements of electrical resistance, numerous active electrogenic ion transport processes are also present in native intestinal epithelium. 24 Net electrogenic ion transport processes can be measured across intestinal epithelium mounted in Ussing chambers by

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Figure 4. Electronmicrographs of three infected enterocyLes labeled with asterisks at the villus tip at 36 hours postinoculation with 1 x 108 excysted oocysts. Tissue was exposed to 0.5% mucosal HRP for 60 minutes in Ussing chambers. HRP reaction product was developed by incubation of 200-pm-thick tissue sections in 0.005% diaminobenzidine/O.01% H202 solution. Sections were embedded in plastic and processed for electronmicroscopy. Thin sections were cut and examined unstained. Dark HRP reaction product is indicated by arrows along the plasma membrane of microvilli and the cell plasma membrane that envelops an intracyLoplasmic trophozoite (T). HRP is restricted to the luminal aspect of the tight junction and does not penetrate into the intercellular space (between the arrowheads) (original magnification 13,392x).

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Figure 5. Column graph representing mannitol flux across the epithelium in infected tissues ([~) and time-matched control tissues (1~) studied in Ussing chambers at 12, 24, 36, and 48 hours postinocuiation with 1 x 108 excysted oocysts and at 36 hours postinoculation with 5 x 108 excysted oocysts (Hi inoculum). Data points represent the mean of four readings obtained at 15-minute intervals from 1 - 4 chamber experiments with the intestinal epithelium from each of 3 4 controls and 3 - 5 infected piglets for each time point studied. Bars represent standard errors. At each time point, results from control tissues compared with infected tissues do not show a statistically significant difference; P > 0.05.

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Figure 6. Column graphs representing transepithelial electrical resistance of infected tissues expressed as percent change from resistance of time-matched control tissues. Mean resistance of normal neonatal piglet intestine was 75 ~ x cm 2. Raw data points were the means of four readings obtained at 15-minute intervals from 2 - 4 chamber experiments with the intestinal epithelium from each of 3 4 control and 3 - 5 infected piglets for each of the time points studied. The resistance of infected tissues compared with time-matched control tissues was statistically different at 48 hours postinoculation with 1 × 108 excysted oocysts (P < 0.002) and at 36 hours postinoculation with 5 x 108 excysted oocysts (P < 0,02).

measurements of short-circuit current, i.e., the amount of remaining current when all passive ion movement is clamped t o z e r o . 25 We found that the short-circuit current in infected tissues was not different from control tissues 1 2 - 3 6 hours postinoculation with 1 × 108 excysted oocysts (P > 0.05 compared with control tissues). However, at 48 hours postinoculation with 1 × 108 excysted oocysts (P < 0.001 compared to control tissues) and 36 hours postinoculation with 5 × 108 excysted oocysts (P < 0.01 compared to control tissues), shortcircuit current was significantly diminished compared with control tissues (Figure 7). The incremental change in short-circuit current in control tissues (-25.6) was significantly greater than in infected tissues (-12.4) (Table 2) in the presence of phlorizin, an inhibitor of apical Na+/glucose cotransport, a function unique to villus absorptive ceils in intestinal epithelium. 26-28 Under theophylline-stimulated conditions, the incremental change in short-circuit current in tissues 48 hour postinoculation with 1 × 108 excysted oocysts is identical to control tissues (Table 1) (+15.1 btA vs. +15.2 ~A for infected and control tissues, respectively; not significant).

Discussion T h e g o a l o f o u r studies was to a s s e s s e v o l v i n g changes in epithelial structure and function during the period in which the parasite establishes infection in vivo. We noted in numerous published studies of cryptosporidiosis in animal models that the data relative to epithe-

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Figure 7. Column graphs representing short-circuit current of infected tissues expressed as percent change from short-circuit current of time-matched control tissues. Raw data points were the means of four readings obtained at 15-minute intervals from 2 - 4 chamber experiments with the intestinal epithelium from each of 3 - 4 control and 3 - 5 infected piglets for each time point studied. The short-circuit current of infected tissues compared with time-matched control tissues was statistically different at 48 hours postinoculation with 1 x 108 excysted oocysts (P < 0.001) and at 36 hours postinoculation with 5 × 10 ~ excysted oocysts (P < 0.01).

lial structure and function was acquired after massive architectural changes in the epithelium 3 - 4 days after oral inoculation with oocysts,s'9 To our knowledge, our studies are the first to address the evolving structural and functional response of native intestinal epithelium to infection during the first 48 hours after infection. We are the first to report that under conditions of massive parasite infection of villus epithelial cells, macromolecular permeability assessed at the cellular level is not enhanced even at sites where the enterocyte apical cytoplasm is grossly distorted by the presence of parasite life forms. Mannitol flux, a more sensitive measurement of overall epithelial permeability, was consistently but not significantly higher in infected tissues than in control tissues. The largest increase in flux in infected tissues compared with time-matched control tissues was 20%, a trivial increase compared with other models of intestinal

Table 2. Theophylline and Phloridzin Response in Control and Infected Tissues 48 Hours Postinoculation

Theophylline response Phloridzin response

Control tissues a

Infected tissues b

(A ,LC4)

(A #A)

+15.2 -25.6

+15.1 -12.4

NOTE. Comparison of theophylline control to infected data: NS. Comparison of phloridzin control to infected data: P < 0.005. Unpaired t test with two tails. an (animals) = 2; n (tissues) = 2 - 3 . On (animals) = 3; n (tissues) = 4 - 6 .

April 1 9 9 5

epithelial injury in which mannitol flux was increased by 300% after minor, superficial injury to the tip of the intestinal villus. 15 Measurement of transepithelial electrical resistance, the reciprocal of conductivity, is a direct electrophysiological measurement of passive ion flow of Na + and C1-, the major ions in the mucosal buffer, and is an additional means to assess subtle changes in solute permeability. At 36 hours, postinoculation resistance was greater in infected tissues compared with controls. The major route of passive solute movement across this epithelium is via the paracellular pathway. 2° Because shorter villi were observed at time points corresponding to increased transepithelial resistance and have fewer enterocytes remaining along the villus axis, the number of available sites for passive ion permeation between ceils is diminished. Thus, the loss of sites for passive movement of ions across this epithelium corresponds with an increase in transepithelial electrical resistance. Our data also show that net electrogenic ion transport processes across infected epithelium are markedly diminished at time points corresponding to altered villus architecture. Under the glucose-stimulated conditions used in these studies, it is likely that the decrement in shortcircuit current observed in infected tissues is caused by a loss of electrogenic glucose-coupled Na + cotransport processes that are exclusively located in the apical membrane of villus epithelial cells or by a loss in the insulating characteristics of the epithelium itself. The latter possibility can be excluded by our resistance data because transepithelial resistance, a measurement that reflects the insulating characteristics of epithelia, was increased (Figure 6) in infected tissues at time points in which the short-circuit current was diminished. Furthermore, as discussed above, by morphological assessment, the integrity of the overlying epithelium was maintained at all postinoculation time points. To ascertain whether loss of active Na+/glucose-coupled cotransport processes was maximally diminished, we compared the short-circuit current response of infected and control tissues at 48 hours postinoculation to phlorizin, an inhibitor of apical Na+/glucose cotransport, which is a function unique to villus absorptive ceils in intestinal epithelium. 26-28 The short-circuit current response was significantly attenuated in infected tissues. These findings suggest that the specific contribution of Na+/glucose-coupled cotransport to net electrogenic processes in infected epithelium is significantly diminished at 48 hours postinoculation. Loss of baseline electrogenic C1- secretion, putatively a function of crypt epithelium, 29'3° could alternatively explain the decrement in short-circuit current in infected tissues. Based on analysis of dual bidirectional flux studies reported by Argenzio et al., *9 intestinal epithelium

INTESTINAL PERMEABILITY IN CRYPTOSPORIDIOSIS

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of the neonatal piglet does not show intestinal baseline C1- secretion. Moreover, as detailed above, crypt ceils are rarely infected by Cryptosporidium. The notion of a C1--dependent enterotoxin has been tendered by recent studies showing that a Cl--dependent increase in the short-circuit current is induced in human intestines after exposure to stool supernatant from calves with cryptosporidiosis. 31 In contrast, we find that short-circuit current is diminished in infected epithelium. Nevertheless, a decrease in other electrogenic ion transport processes, such as coupled Na+/glucose cotransport, could mask an increase in active C1- secretion. To test whether crypt cells in infected tissues were stimulated to secrete CI-, we applied theophylline, the C1--dependent, cAMP-mediated secretagogue, to tissues mounted in Ussing chambers and measured the maximal short-circuit current response. The maximal short-circuit current response after theophylline stimulation was identical in infected and control tissues. Thus, infected tissues maintain the ability to maximally secrete C1- in response to a cAMP-mediated secretagogue. Indeed, our data are in agreement with other studies in which alterations in C1- secretion at later time points were not observed in infected animal 9 or human 12 intestinal epithelium. The above changes in epithelial architecture, permeability, and transport in infected intestine that were assessed in our studies occurred within 48 hours postinoculation 2 4 - 4 8 hours earlier than previously reported. Our data are in complete agreement with the studies by Argenzio et al. of infected neonatal piglet intestine that suggest coupled Na÷/glucose cotransport is diminished. 8 W e show that altered Na+/glucose cotransport occurs earlier than previously reported. W e also show that altered Na+/glucose cotransport parallels alterations in villus height and is related inversely to the extent of infection along the intestinal villus. We observed increased polymorphonuclear and mononuclear ceils in the lamina propria and epithelium 3 6 - 4 8 hours postinoculation. At this time point, the short-circuit current was attenuated. Thus, from our data, it does not seem that agents released from recruited inflammatory cells enhance active transport processes in infected epithelium as assessed by short-circuit current and the short-circuit current response to phlorizin and theophylline. Our studies support the notion that the loss of villus epithelium and corresponding coupled Na÷/glucose cotransport contributes to diarrhea in cryptosporidiosis. Similar morphological alterations were observed in patients with AIDS, however, that do not have cryptosporidiosis nor present with diarrhea. 4'32 On the surface, these observations would tend not to support our conclusions. However, in our studies, the region of altered intes-

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tinal epithelium was confined to the 10-cm region of the intestine that was inoculated with excysted oocysts. Infected piglets in our studies rarely showed diarrhea at any time. Therefore, it is evident that altered epithelial morphology and function that is limited to a small region of the intestinal mucosa may not result in clinically apparent diarrhea. Evaluation of the intestinal mucosa in the above study on patients with AIDS was limited to examination of endoscopic biopsy specimens of the duodenal mucosa. It is also of interest that the intestinal morphology of rodents with cryptosporidiosis is altered, although these animals do not show diarrhea. 1,4 However, the extent of epithelial alteration is not clear in these animals. Furthermore, the effect of luminal substrate, such as glucose in the diet of rodents, on net absorptive and secretory processes in intestinal epithelium must also be considered. The density and turnover rate of the apical glucose cotransporter differs from species to species, from region to region of the intestine, and with varying diets. 33'34 Therefore, an important caveat to these studies is that the lack of clinical diarrhea cannot be interpreted as unequivocally indicative of normal intestinal function. Logically, structure-function correlates are highly meaningful when functional studies are specifically conducted on the region of epithelium with altered morphology, as we have done in these studies. We found it remarkable that only 2% of villus tips examined at 48 hours postinoculation showed a disrupted epithelial monolayer, i.e., regions in which epithelial cells were not contiguous. We also noted that although enterocytes along the sides of the villi were frequently infected, enterocytes were rarely found to exfoliate along the sides of the villus. Exfoliating, parasite-infected enterocytes were found almost exclusively at the villus tip, even at the latest time points studied. The rate of cell extrusion at the villus tip may be accelerated in infected epithelium or perhaps cell migration rates along the villus do not meet the demands of villus tip enterocyte loss in infected epithelium. Further studies might address whether or not infection by this parasite delays epithelial cell migration from crypt to villus tip or whether crypt cell proliferation is diminished in the first few days after infection.

References 1. Fayer R, Ungar B. Cryptosporidium sp. and cryptosporidiosis. Microbiol Rev 1986;50:458-483, 2. Navin T, Juranek D. Cryptosporidiosis: clinical, epidemiologic, and parasitologic review. Rev Infect Dis 1984;6:225-229. 3. Guerrant R, Petri W, Weikel C. Parasitic causes of diarrhea. Lebenthal E, Duffey M, eds. Textbook of secretory diarrhea. New York: Raven, 1990:273-280. 4. Sears C, Guerrant R. Cryptosporidiosis: the complexity of intestinal pathophysiology. Gastroenterology 1994;106:252-267.

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5. Tzipori S, McCartney E, Lawson G, Rowland A, Campbell I. Experimental infection in piglets with cryptosporidium. Res Vet Sci 1981; 31:358-368. 6. Tzipori S. Cryptosporidiosis in animals and humans. Microbiol Rev 1983;47:84-96. 7. Tzipori S. Cryptosporidiosis in perspective. Baker J, Mullen R, eds. Advances in parasitology. Volume 27. New York: Academic, 1988:63-129. 8. Argenzio R, Liacos J, Levy M, Meuten D, Lecce J, Powell D. Villous atrophy, crypt hyperplasia, cellular infiltration, and impaired glucose-NA absorption in enteric cryptosporidiosis of pigs. Gastroenterology 1990; 98:1129-1140. 9. Argenzio R, Lecce J, Powell D. Prostanoids inhibit intestinal NaCI absorption in experimental porcine cryptosporidiosis. Gastroenterology 1993; 104:440-447. 10. Soave R, Danner R, Honig C. Cryptosporidiosis in homosexual men. Ann Intern Med 1984;100:504-511. 11. Meiset J, Perera D, Melign C, Rubin C. Overwhelming watery diarrhea associated with a cryptosporidium in an immunosuppressed patient. Gastroenterology 1976; 70:1156-1160. 12. Kelly P, Thillainayagam A, Keating J, Smithson J, Forbes A, Gazzard B, Farthing M. HIV-related cryptosporidial diarrhea: water and electrolyte transport in human jejunum (abstr). Gastroenterology 1994; 106:A709. 13. Dubey J, Speer C, Fayer R. Techniques and laboratory maintenance of Cryptosporidium. Current W, ed. Cryptosporidiosis of man and animals. Boca Raton, FL: CRC, 1990:41-44. 14. Tzipori S, Brand W, Griffiths J, Widmer G, Crabb J. Evaluation of an animal model system for cryptosporidiosis: the therapeutic efficacy of paromomycin and hyperimmune bovine colostrum-immunoglobulin. Clin Diagn Lab Immunol 1994; 1:450-463. 15. Moore R, Carlson S, Madara J. Rapid barrier repair in an in vitro model of intestinal epithelial injury. Lab Invest 1989;60:237244. 16. Moore R, Carlson S, Madara J. Villus contraction aids repair of intestinal epithelium after injury. Am J Physiol 1989;257:G274G283. 17. Moore R, Carlson S, Madara J. C. difficiletoxin A increases intestinal permeability and induces Cl- secretion. Am J Physiol 1990; 259:G165-G172. 18. Karnovsky M. The ultrastructural basis of capillary permeability studied with peroxidase as a tracer. J Cell Biol 1967;35:213236. 19. Marcial M, Madara J. Cryptosporidium: Cellular localization, structural analysis of absorptive cell-parasite membrane-membrane interactions in guinea pigs, and suggestion of protozoan transport by M cells. Gastroenterology 1986;90:583-594. 20. Madara J, Trier J. Functional morphology of the mucosa of the small intestine. Johnson L, ed. Physiology of the gastrointestinal tract. Volume 2. 2nd ed. New York: Raven, 1986:1209-1250, 21. Madara J, Pappenheimer J. Structural basis for physiologic regulation of paracellular pathways in intestinal epithelia. J Membr Biol 1987; 100:149-164. 22. Madara J. Tight junction dynamics: is paracellular transport regulated? Cell 1988;53:497-498. 23. Marcial M, Madara J. Analysis of absorptive cell occluding junction: structure-function relationships in a state of enhanced permeability. Lab Invest 1987;56:424-434. 24. Armstrong W. Cellular mechanisms of ion transport in the small intestine. Johnson L, ed. Physiology of the gastrointestinal tract, Volume 2. 2nd ed. New York: Raven, 1987:1251-1265. 25. Ussing H, Zerahn K. Active transport of sodium as the source of electric current in the short-circuited isolated frog skin. Acta Physiol Scand 1951;23:110-127. 26. Toggenburger G, Kessler M, Hosang M, Semenza G. Phlorizin as a probe of small intestinal Na ÷, D-glucose co-transporter. A model. Biochim Biophys Acta 1982;688:557-571.

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27. Diedrich D. Competitive inhibition of intestinal glucose transport by phlorizin analogs. Arch Biochem Biophys 1966;117:248256. 28. Atisook K, Carlson S, Madara J. Effects of phlorizin and sodium on glucose-elicited alterations of cell junctions in intestinal epithelia. Am J Physiol 1990;258:C77-C85. 29. Welsh M, Smith P, Fromm M, Frizzell R, Crypts are the site of intestinal fluid and electrolyte secretion. Science 1982;218: 1219-1221. 30. Marcial M, Madara J. Partitioning of paracellular conductance along the ileal crypt-villus axis: a hypothesis based on structural analysis with detailed consideration of tight junction-structure relationships. J Membr Biol 1984;80:59-70. 31. Guarino A, Canani RB, Pozio E, Terracciano L, Albano F, Mazzeo M. Enterotoxic effect of stool supernatant of Cryptosporidiuminfected calves on human jejunum. Gastroenterology 1994; 106:28-34.

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32. Greenson J, Belitsos P, Yardley J, Bartlett J. AIDS enteropathy: occult enteric infections and duodenal mucosal alterations in chronic diarrhea. Ann Intern Med 1991;114:366-372. 33. Ferraris R, Diamond J. Use of phlodzin binding to demonstrate induction of intestinal glucose transporters. J Membr Biol 1986; 94:77-82. 34. Ferraris R, Lee P, Diamond J, Origin of regional and species differences in intestinal glucose uptake. Am J Physiol 1989; 257:G689-G697. Received July 21, 1994. Accepted December 20, 1994. Address requests for reprints to: Ronda Moore, D.V.M., Department of Pathology, School of Medicine and Veterinary Medicine, Tufts University, 136 Harrison Avenue, Boston, Massachusetts 02111. Fax: (617) 636-8590. Supported by National Institutes of Health and National institute of Allergy and Infectious Disease grant U01 AI33384.