This study aimed to characterise the pathomechanisms of diarrhoea and malabsorption in human small intestines chronically infected with G lamblia. In general, diarrhoea can be driven by changes in motility or by osmotic forces, and osmotic forces can result from active secretion, leak flux mechanisms and malabsorption.
As the first important finding of our study, impedance spectroscopy showed Re
that was considerably reduced in chronic giardiasis. However, Rsub
, and hence Rt
, was not altered considerably. This underlines the importance of applying impedance spectroscopy for investigating diseased mucosae, as conventional resistance analysis would have missed this epithelial barrier dysfunction. At first glance, the degree of this barrier disturbance seemed to be rather low. However, one has to keep in mind that mucosal surface area is markedly reduced. Taking this into account, epithelial barrier dysfunction has to be assumed to be significant. The first evidence for an epithelial change with increased intestinal permeability in giardiasis came from infants, using the lactulose–mannitol ratio, as well as from adults, using technetium‐labelled DTPA.26,27
Furthermore, mice infected with G muris
showed increased intestinal permeability from day 4 until day 21 after infection.12
However, these techniques cannot exclude area or blood flow changes as possible confounding factors. More direct evidence for epithelial barrier impairment in giardiasis resulted from in vitro studies. Here, the addition of G lamblia
sonicates on to the duodenal cell line SCBN showed decreased resistance and increased FITC‐dextran 3000 fluxes.11
After barrier dysfunction had been shown by rather indirect methods in human and animal models and by direct methods in the in vitro studies, our data from human biopsy specimens are the first that directly quantify this barrier dysfunction in human chronic giardiasis.
This G lamblia
‐induced epithelial barrier dysfunction contributes to diarrhoea by a leak flux mechanism. Normally, the epithelial barrier maintains the “milieu intérieur”. If it is defective, solutes and water will enter the lumen depending on the permeability of the paracellular pathway. Evidence that a leak flux mechanism is sufficient to elicit diarrhoea was already obtained in HIV enteropathy, where barrier dysfunction was primarily responsible for relevant diarrhoea in these patients.17
This form of diarrhoea was also found to be induced by Clostridium difficile
More direct evidence for this concept comes from Fasano et al
Attenuated Vibrio cholerae
strains depleted of the chloride secretion‐inducing cholera toxin gene were shown to still induce diarrhoea in human volunteers. This results from another toxin, zonula occludens toxin, which leads to a change in tight junction structure, and consequently ions and water are lost into the intestinal lumen.
When further investigating the nature of epithelial barrier dysfunction in patients with chronic lambliasis, we found that the epithelial apoptotic rate was increased by >50%. This is in accordance with previous in vitro studies showing that apoptotic events are stimulated in enterocytes after G lamblia
infection and that this is a caspase‐3‐dependent process.13
However, the total increase in apoptotic ratio was only moderate (a 0.5% increase) compared with the in vitro observations mentioned above or to other small intestinal diseases such as HIV enteropathy where the apoptotic ratio reaches a level as high as 4%.30
Nevertheless, it is well established that epithelial apoptoses can play an important role for epithelial barrier function, even if the increase is only moderate.22,31,32
Although it was originally thought that epithelial apoptosis at the villus tip is a regular and highly sealed process without much barrier importance, our own group has presented experimental data that apoptoses possess marked conductivity.33
In addition, apoptotic foci were directly demonstrated to contribute to the barrier dysfunction in ulcerative colitis using the conductance scanning technique.34
Thus, the relative contribution of apoptotic events may vary from minor to predominating, depending on the particular condition. The relatively weak increase in apoptotic rates in patients with giardiasis represents a relevant contribution to epithelial pathology but seems to play a rather moderate role for barrier dysfunction and diarrhoea. Presumably, apoptosis is triggered by direct contact of the parasites or their products with the epithelium as shown in in vitro studies using monolayers of duodenum‐derived cells.13
On the other hand, even if G lamblia
does not cause heavy local inflammation in the intestine, it is feasible that even low‐level release of pro‐inflammatory cytokines such as tumour necrosis factor α or interferon‐γ could lead to increased epithelial apoptosis in response to the chronic colonisation.
Besides epithelial apoptosis, tight junctions are one of the prominent features that determine epithelial barrier properties.35
The epithelial tight junction forms a barrier between neighbouring epithelial cells, which limits transepithelial movement of ions and solutes through the paracellular route. Thus, tight junctions are also a target of pathophysiological stimuli through pathogens.36,37
On first view, nearly all the examined tight junctional proteins seemed to be decreased in chronic giardiasis. However, this was mainly caused by the serious reduction in mucosal surface area, which in itself causes a reduction in the epithelial tight junction protein pool. Therefore, after mucosal surface correction, only claudin 1 expression was differentially downregulated in chronic giardiasis. Claudin 1 is known to have sealing properties as, for example, indicated by an increase in barrier function after overexpression in epithelial monolayers.38
Furthermore, claudin 1 knock‐out mice were shown to die shortly after birth owing to a loss of water through the leaky epidermis.39
Therefore, the decreased expression of claudin 1 has to be assumed to be another relevant structural correlate for the decreased epithelial resistance in chronic human giardiasis. Claudin 2, a protein with pore‐forming properties, which is primarily expressed in the crypt regions,23,40
was not detectable. This may reflect the rather low level of mucosal inflammation in giardiasis,41
as up regulation of claudin 2 had been shown under inflammatory conditions in ulcerative colitis and duodenal HIV enteropathy using the same technique.42,30
As another important observation, an increase in basal Isc
was found in chronic giardiasis. Such an increase in spontaneous Isc
in the presence of phlorizin usually reflects increased active anion (chloride/bicarbonate) secretion. This was supported by the blocking effect of bumetanide, which was considerably higher in giardiasis than in controls. In keeping with these findings in human biopsy specimens, previous reports have also shown chloride secretion to be activated in the small intestine of mice infected with G lamblia
Noteworthy, this anion secretion is activated in giardiasis despite the reduction in mucosal surface area. However, mucosal surface reduction in our study is predominantly due to villous surface reduction, whereas crypts as an important source of active anion secretion43
were not affected. Interestingly, active anion secretion was also observed in HIV enteropathy, and this was thought to result from an altered cytokine pattern released from the HIV‐infected subepithelial immune cells.44
Therefore, it may be reasonable to assume that the activation of active anion secretion in giardiasis could also be induced by an altered cytokine pattern.
Finally, we observed an impaired sodium‐coupled D
‐glucose absorption in the duodenum of patients chronically infected with G lamblia
. This was not surprising, as the mucosal surface area was considerably affected. Thus, the crucial factor for this glucose malabsorption seems to be the reduction in villous surface area, as the degree of the Na–glucose symport reduction did not substantially exceed the degree of mucosal surface decrease. However, it should be noted that overall loss of absorptive surface area may occur even in the absence of villous atrophy—namely, via a diffuse shortening of brush border microvilli, a mechanism that has been previously described in giardiasis.6,14
Furthermore, it was shown in a mouse model that IEL can induce brush border injury and malfunction.45
Interestingly, we observed an increased number of IEL in giardiasis, which may cause brush border changes and would be a further explanation for the Na–glucose malabsorption observed in our study.
A variable degree of mucosal architecture change is established in giardiasis in humans as well as from data obtained in animal models. This is generally assumed to account for the malabsorptive component of the diarrhoea in giardiasis.14,16
On the other hand, it is in sharp contrast with a large histological analysis that detected a change in villous architecture only in a minority of patients.8
In our own study, we were surprised to find a reduction in villous surface area in almost all specimens from individuals with chronic giardiasis. This may reflect the selected group of patients with pronounced chronic complaints, which were referred to our university centre for more extensive diagnostic procedures including upper gastrointestinal endoscopy. Furthermore and more important, it could also be due to the microdissection technique used in present protocol, which is a very sensitive method to analyse villous surface area changes. This is also supported by the fact that routine histological analysis of our specimens in the pathology department showed an altered mucosal architecture in only two thirds of the specimens (data not shown).
In conclusion, chronic infection of G lamblia in humans leads to a combination of epithelial transport and barrier dysfunction. We showed a dramatic reduction in villous surface, which in turn leads to reduced absorptive capacity as shown by reduced Na–glucose absorption. Moreover, duodenal mucosa exhibited increased anion secretion. Hence, malabsorption and active secretion as shown in animal models also seem extremely important in human disease. Furthermore, epithelial barrier dysfunction due to chronic G lamblia infection could be shown by impedance spectroscopy and, moreover, underlying molecular changes were identified—namely, a reduction in claudin 1 expression and increased epithelial apoptosis.