Microbes and microbial toxins: paradigms for microbial-mucosal interactions I. Pathophysiological aspects of enteric infections with the lumen-dwelling protozoan pathogen Giardia lamblia

Giardia lamblia is one of the most important causes of waterborne diarrheal disease worldwide, and giardiasis is the most common protozoan infection of the human small intestine. Symptomatic infection is characterized by diarrhea, abdominal pain, and malabsorption, leading to malnutrition and weight loss, particularly in children. The pathogen resides strictly in the lumen of the small intestine, and infection is typically not accompanied by significant mucosal inflammation. Clinical and experimental studies indicate that B cell-dependent host defenses, particularly IgA, are important for controlling and clearing Giardia infection, although B cell-independent mechanisms also contribute to this outcome. In contrast to antigiardial host defenses, much less is known about the pathophysiological mechanisms underlying the clinical symptoms of giardiasis, partly because of the current lack of suitable model systems. In addition to being an important human enteric pathogen, Giardia is an interesting model organism for gaining basic insights into genetic innovations that led to evolution of eukaryotic cells, since it belongs to the earliest diverging eukaryotic lineage known. The completion of the giardial genome project will increase understanding of the basic biology of the protozoan and will help us to better understand host pathogen-interactions as a basis for developing new vaccination and therapeutic strategies.     

Recent insights into the mucosal reactions associated with Giardia lamblia infections

Giardia lamblia is an intestinal protozoan parasite infecting humans and various other mammalian hosts. The most important clinical signs of giardiasis are diarrhoea and malabsorption. Giardia lamblia is able to undergo continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). While intestinal antibodies, and more specifically anti-VSP IgA antibodies, were proven to be involved in modulating antigenic variation of the parasite the participation of the local antibody response in control of the parasite infection is still controversial. Conversely, previous studies based on experimental infections in mice showed that cellular immune mechanisms are essential for elimination of the parasite from its intestinal habitat. Furthermore, recent data indicated that inflammatory mast cells have a potential to directly, or indirectly, interfere in duodenal growth of G. lamblia trophozoites. However, this finding was challenged by other reports, which did not find a correlation between intestinal inflammation and resistance to infection. Since intestinal infiltration of inflammatory cells and/or CD8+T-cells were demonstrated to coincide with villus-shortening and crypt hyperplasia immunological reactions were considered to be a potential factor of pathogenesis in giardiasis. The contribution of physiological factors to pathogenesis was essentially assessed in vitro by co-cultivation of G. lamblia trophozoites with epithelial cell lines. By using this in vitro model, molecular (through surface lectins) and mechanical (through ventral disk) adhesion of trophozoites to the epithelium was shown to be crucial for increased epithelial permeability. This phenomenon as well as other Giardia-induced intestinal abnormalities such as loss of intestinal brush border surface area, villus flattening, inhibition of disaccharidase activities, and eventually also overgrowth of the enteric bacterial flora seem to be involved in the pathophysiology of giardiasis. However, it remains to be elucidated whether at least part of these pathological effects are causatively linked to the clinical manifestation of the disease.     

Pathophysiology of enteric infections with Giardia duodenalius

Giardia is the most prevalent human intestinal parasitic protist in the world, and one of the most common parasite of companion animals and young livestock. Giardia is a major cause of diarrhea in children and in travelers. The host-microbial interactions that govern the outcome of infection remain incompletely understood. Findings available to date indicate that the infection causes diarrhea via a combination of intestinal malabsorption and hypersecretion. Malabsorption and maldigestion mainly result from a diffuse shortening of epithelial microvilli. This enterocytic injury is mediated by activated host T lymphocytes. Pathophysiological activation of lymphocytes is secondary to Giardia-induced disruption of epithelial tight junctions, which in turn increases intestinal permeability. Loss of epithelial barrier function is a result of Giardia-induced enterocyte apoptosis. Recent findings suggest that these effects may facilitate the development of chronic enteric disorders, including inflammatory bowel disease, irritable bowel syndrome, and allergies, via mechanisms that remain poorly understood. A newly discovered SGLT-1 glucose uptake-mediated host cytoprotective mechanism may represent an effective modulator of the epithelial apoptosis induced by this parasite, and, possibly, by other enteropathogens. A better understanding of the pathogenesis of giardiasis will shed light on new potential therapeutic targets.     

Substance P receptor antagonism for treatment of cryptosporidiosis in immunosuppressed mice

Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium parvum, causes self-limited diarrhea in normal hosts but can cause life-threatening diarrhea for immunosuppressed patients. There is an urgent need for new drugs to treat this chronic disease. Cryptosporidium parvum infection is associated with intestinal structural and pathophysiologic changes, including villi blunting and glucose malabsorption. Substance P (SP), a neuropeptide and pain transmitter, is associated with the gastrointestinal tract and is elevated in humans and macaques after experimental C. parvum challenge. To examine the relevance of SP in the pathogenesis of cryptosporidiosis, and to determine if SP receptor antagonism can be employed for treatment of cryptosporidiosis in immunosuppressed hosts, we used an immunosuppressed murine model (dexamethasone-immunosuppressed mice) that is frequently utilized for examining chemotherapeutic potential of drugs. Quantitative ELISA was used to measure intestinal SP levels in immunosuppressed mice with, and without, C. parvum infection. Intestinal physiological alterations, as studied by the Ussing chamber technique, plus weight change, fecal oocyst shedding, and villi measurements, were compared in infected mice with, and without, SP receptor antagonist (aprepitant) treatment. Immunosuppressed mice infected with C. parvum demonstrated increased SP levels as well as physiological alterations (glucose malabsorption), weight loss, fecal oocyst shedding, and structural alterations (increased intestinal villi blunting) compared to uninfected mice. Each of these defects was significantly inhibited by aprepitant treatment. These studies demonstrate the potential of SP receptor antagonism for treatment of pathogenesis of cryptosporidiosis in immunosuppressed hosts.     

Weight loss through ileal transposition is accompanied by increased ileal hormone secretion and synthesis in rats

Bariatric surgeries, such as gastric bypass, result in dramatic and sustained weight loss that is usually attributed to a combination of gastric volume restriction and intestinal malabsorption. However, studies parceling out the contribution of enhanced intestinal stimulation in the absence of these two mechanisms have received little attention. Previous studies have demonstrated that patients who received intestinal bypass or Roux-en-Y surgery have increased release of gastrointestinal hormones. One possible mechanism for this increase is the rapid transit of nutrients into the intestine after eating. To determine whether there is increased secretion of anorectic peptides produced in the distal small intestine when this portion of the gut is given greater exposure to nutrients, we preformed ileal transpositions (IT) in rats. In this procedure, an isolated segment of ileum is transposed to the jejunum, resulting in an intestinal tract of normal length but an alteration in the normal distribution of endocrine cells along the gut. Rats with IT lost more weight (P < 0.05) and consumed less food (P < 0.05) than control rats with intestinal transections and reanastomosis without transposition. Weight loss in the IT rats was not due to malabsorption of nutrients. However, transposition of distal gut to a proximal location caused increased synthesis and release of the anorectic ileal hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY; P < 0.01). The association of weight loss with increased release of GLP-1 and PYY suggests that procedures that promote gastrointestinal endocrine function can reduce energy intake. These findings support the importance of evaluating the contribution of gastrointestinal hormones to the weight loss seen with bariatric surgery.     

Recent advances in human protozoan parasites of the gastrointestinal tract

An attempt was made in this report to present an update on the recent development on intestinal protozoan infections in humans. Except for a few historical references the review covers the period from 1980 to the time of writing, mid-1985. The emphasis was on the more important parasites and an effort made to cover the latest developments in their biology, epidemiology and pathogenesis. During preparation of this paper I was impressed with the plethora of papers published on some parasites and the paucity of reports on others. There are an increasing number of papers on Cryptosporidium sp. and the interest in the organisms should continue. Furthermore, it will be of interest to follow the association between Blastocystis hominis and disease. These are essentially new protozoan parasites of man, and one wonders how many more intestinal protozoan parasitosis are still waiting to be found. Like the Cryptosporidium sp., it may be a matter of finding the right diagnostic technique to detect the unknown organism.Giardiasis continues to be a cause of diarrhea among various groups especially campers who are drinking untreated water and G. lamblia as well as E. histolytica are being found more frequently in homosexuals with and without AIDS. The ability to predict virulence in strains of E. histolytica by enzyme patterns is intriguing but some skeptics still prefer the older test for virulence by cecal scoring in animals. New animal models are being evaluated and new techniques applied to the study of pathogenic protozoa. In the future the use of new biotechnological methods will most certainly lead to a better understanding of intestinal protozoa as well as of other parasitic organisms.     

Prevalence and Risk Factors for Intestinal Protozoan Infections with Cryptosporidium,Giardia, Blastocystis and Dientamoeba among Schoolchildren in Tripoli,Lebanon

BackgroundIntestinal protozoan infections are confirmed as major causes of diarrhea, particularly in children, and represent a significant, but often neglected, threat to public health. No recent data were available in Lebanon concerning the molecular epidemiology of protozoan infections in children, a vulnerable population at high risk of infection.ConclusionsThis is the first study performed in Lebanon reporting the prevalence and the clinical and molecular epidemiological data associated with intestinal protozoan infections among schoolchildren in Tripoli. A high prevalence of protozoan parasites was found, with Blastocystis spp. being the most predominant protozoans. Although only 50% of children reported digestive symptoms, asymptomatic infection was observed, and these children may act as unidentified carriers. This survey provides necessary information for designing prevention and control strategies to reduce the burden of these protozoan infections, especially in children.     

Interactions between rotavirus and gastrointestinal cells

Rotaviruses are the leading cause of life-threatening diarrheal disease in infants and in young animals worldwide. The outcome of rotavirus infection of intestinal epithelial cells is more complex and involves induction of more diverse cellular responses than initially appreciated. Similar to bacteria, the pathogenesis of rotavirus-induced disease involves an enterotoxin, activation of the enteric nervous system and malabsorption, suggesting that common mechanisms of pathogenesis may exist between viral and bacterial pathogens.     

Cellular changes in the enteric nervous system during ageing

The intrinsic neurons of the gut, enteric neurons, have an essential role in gastrointestinal functions. The enteric nervous system is plastic and continues to undergo changes throughout life, as the gut grows and responds to dietary and other environmental changes. Detailed analysis of changes in the ENS during ageing suggests that enteric neurons are more vulnerable to age-related degeneration and cell death than neurons in other parts of the nervous system, although there is considerable variation in the extent and time course of age-related enteric neuronal loss reported in different studies. Specific neuronal subpopulations, particularly cholinergic myenteric neurons, may be more vulnerable than others to age-associated loss or damage. Enteric degeneration and other age-related neuronal changes may contribute to gastrointestinal dysfunction that is common in the elderly population. Evidence suggests that caloric restriction protects against age-associated loss of enteric neurons, but recent advances in the understanding of the effects of the microbiota and the complex interactions between enteric ganglion cells, mucosal immune system and intestinal epithelium indicate that other factors may well influence ageing of enteric neurons. Much remains to be understood about the mechanisms of neuronal loss and damage in the gut, although there is evidence that reactive oxygen species, neurotrophic factor dysregulation and/or activation of a senescence associated phenotype may be involved. To date, there is no evidence for ongoing neurogenesis that might replace dying neurons in the ageing gut, although small local sites of neurogenesis would be difficult to detect. Finally, despite the considerable evidence for enteric neurodegeneration during ageing, and evidence for some physiological changes in animal models, the ageing gut appears to maintain its function remarkably well in animals that exhibit major neuronal loss, indicating that the ENS has considerable functional reserve.     

Cell death and human intestinal protozoa: a brief overview

Protozoan programmed cell death or apoptosis is an important factor in the survival of the parasite and its pathogenicity. The most amazing aspect of protozoan cell death is in its molecular architecture. To date, protozoa lack most of the components of the highly complex cell death machinery studied in multicellular organisms. Hence the unique apoptotic machinery in protozoa can be exploited for the development of therapeutic drugs and diagnostic markers. This review focuses on human intestinal protozoa undergoing cell death and inducing or inhibiting host cell apoptosis. The first part of this review focuses on intestinal protozoa that undergo PCD under various stress conditions. The second part focuses on protozoa that induce or inhibit PCD in their host cell. Although these intestinal parasites differ in their mechanism of infection and intracellular localization, they may activate conserved cell death pathways within themselves and in the host cell. Understanding conserved cell death pathways in the intestinal protozoa and their host-parasite PCD relationship may lead to drug targets which can be used for a broad range of parasitic diseases.     

Caspase-dependent apoptosis of the HCT-8 epithelial cell line induced by the parasite Giardia intestinalis

Giardia intestinalis is a flagellated protozoan which causes enteric disease worldwide. Giardia trophozoites infect epithelial cells of the proximal small intestine and can cause acute or chronic diarrhea. The mechanism of epithelial injury in giardiasis remains unknown. A number of enteric pathogens, including protozoan parasites, are able to induce enterocyte apoptosis. The aim of this work was to assess whether G. intestinalis strain WB clone C6 is able to induce apoptosis in the human intestinal epithelial cell line HCT-8, and to investigate the role of caspases in this process. Results demonstrated that the parasite induces cell apoptosis, as confirmed by DNA fragmentation analysis, detection of active caspase-3 and degradation of the caspase-3 substrate PARP [poly(ADP-ribose) polymerase]. Furthermore, G. intestinalis infection induces activation of both the intrinsic and the extrinsic apoptotic pathways, down-regulation of the antiapoptotic protein Bcl-2 and up-regulation of the proapoptotic Bax, suggesting a possible role for caspase-dependent apoptosis in the pathogenesis of giardiasis.     

Colony-dependent sex differences in protozoan communities of the lower termite <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> (Isoptera: Rhinotermitidae)

In many animals, sex differences in hormones, behavior, and immunity lead to differences in their gut microbial communities. One of the best-known examples of mutualistic symbiosis is that between lower termites and their intestinal protozoa. Although differences in the protozoan communities of different castes have been studied in lower termites, nothing is known about the sex differences in protozoan communities in neuter castes. Here, we show that termite workers have different protozoan communities according to sex depending on the colony. We investigated the communities of symbiotic protozoa living in lower termites, Reticulitermes speratus, and how they are affected by sex and caste. Workers had the largest numbers of protozoa, followed by soldiers, whereas reproductives (primary kings and secondary queens) had no protozoa. Workers showed colony-dependent sex differences in the total abundance of protozoa, whereas soldiers showed no such sex differences. There were significant sex effect and/or interaction effect between colony and sex in abundances of five species of protozoa in workers. Workers also showed significant sex differences and/or colony-dependent sex differences in proportion of six species of protozoa. These may result in sex differences in the host–symbiont interaction due to physiological or behavioral sex differences in workers that have not been recognized previously. This study has an important implication: although workers are not engaged in reproduction, their potential sex difference may affect various aspects of social interactions.     

Giardia duodenalis: The double-edged sword of immune responses in giardiasis

Giardiasis is one of the most common intestinal protozoan infections worldwide. The etiological agent, Giardia duodenalis (syn. Giardia lamblia, Giardia intestinalis), is a flagellated, binucleated protozoan parasite which infects a wide array of mammalian hosts (Adam, 2001). The symptoms of giardiasis include abdominal cramps, nausea, and acute or chronic diarrhea, with malabsorption and failure of children to thrive occurring in both sub-clinical and symptomatic disease (Thompson et al., 1993). Infections are transmitted by cysts which are excreted in the feces of infected humans and animals. Human giardiasis is distributed worldwide, with rates of detection between 2-5% in the developed world and 20-30% in the developing nations (Farthing, 1994). There is significant variation in the outcome of Giardia infections. Most infections are self-limiting, although re-infection is common in endemic areas and chronic infections also occur. Moreover, some individuals suffer from severe cramps, nausea and diarrhea while others escape these overt symptoms. This review will describe recent advances in parasite genetics and host immunity that are helping to shed light on this variability.     

Clinical and parasitologic aspects of cryptosporidiosis in nonhuman primates

Eighty-one cases of acute cryptosporidiosis were diagnosed among 157 (52%) infant primates, predominantly Macaca nemestrina, housed in the nursery unit of the Washington Regional Primate Research Center. The mean age at onset of oocyst passage was 38 +/- 25 days. The outbreak was confined to the nursery and no cases were detected among juvenile or adult primates housed in other rooms within the colony. All but one animal manifested symptoms of enteric infection, including severe diarrhea and dehydration. Infected animals excreted oocysts for a mean of 36 days (range 7-78 days). No reinfections occurred. Cryptosporidium was the second most common enteric pathogen detected in the population, after Campylobacter jejuni. The risk of infection was related to the length of time the animal was housed in the nursery and to social interaction with other monkeys. These findings are relevant to the understanding of the epidemiology of cryptosporidiosis among human infants and children in environments with close social interactions and minimal learned personal hygiene practices.     

The pathogenesis of cryptosporidiosis

Human infection with the protozoan parasite Cryptosporidium parvum has recently emerged as a global public health problem. Although infection is unrelenting in patients classically regarded as immunocompromised, a tantalizing observation is that infection with this parasite results in both acute self-limited as well as chronic diarrhea in young children. Recent data have begun to elucidate multiple potential mechanisms by which parasitism of the intestinal epithelium may yield an intestinal secretory response. However, a central issue for future studies is to understand how Cryptosporidium infection in young children results in such a broad spectrum of clinical presentation. An answer to this question is likely to result through a dual understanding of how systemic or enteric immunity impacts on intestinal secretory responses and how intra-cellular parasitism alters intestinal epithelial cell function and signals the submucosal intestinal compartment. The virulence factors of Cryptosporidium mediating these events need to be identified. Douglas Clark and Cynthia Sears here review the current understanding of the pathogenesis of intestinal secretion in response to Cryptosporidium infection, and discuss key questions requiring additional study.     

微囊藻毒素-LR对小鼠肠道消化酶的影响

微囊藻毒素-LR（MCLR）是一类对动物和人类健康影响很大的蓝藻毒素。已有调查认为MCLR能导致某些胃肠道疾病,相关实验室资料也证实MCLR能在肠道积累并引起肠道损伤。研究对小鼠连续腹腔染毒MCLR 28d,观察肠道病理水平及超微结构的变化,并测定肠黏膜刷状缘膜酶活性。结果显示,小肠绒毛受损较严重,绒毛数量减少、部分脱落至肠腔,固有层及黏膜下层水肿、充血;电镜观察发现肠细胞细胞质电子密度降低,伴有线粒体肿胀、细胞核变形现象;肠黏膜二糖酶（蔗糖酶、麦芽糖酶、乳糖酶）、碱性磷酸酶及-谷氨酰转移酶活性均呈下降趋势。在亚慢性MCLR染毒条件下,肠道的消化功能可能受到抑制,进而导致机体对营养物质吸收不良。研究对理解蓝藻毒素引起的胃肠道不适（如恶心、呕吐、腹痛、腹泻）提供了新的证据。       

Effect of giardiasis combined with low-protein diet on intestinal absorption of glucose and electrolytes in gerbils

Studies have shown that symptomatic infection by Giardia lamblia causes acute or chronic diarrhea, dehydration, abdominal pain and malabsorption, leading to undernutrition and weight loss. The aim of the present study was to evaluate the effects of giardiasis and its combination with a low-protein diet on the intestinal absorption of glucose and electrolytes in gerbils. The intestinal absorption of glucose, sodium and potassium was investigated in male gerbils weighing 46-64 g (n≥5). A Tyrode solution containing twice the glucose, sodium and potassium concentration (pH 7.4) was infused through the intestinal loops for 40 min. Glucose absorption was not significantly affected by diet and infection. However, there was a significant increase in sodium absorption in the Giardia-infected group (57.2±6.1, p<0.05) in comparison to the control, low-protein diet and low-protein diet+Giardia-infected groups (8.9±6.5, 2.8±11.1 and 0.8±7.9, respectively; p<0.05). Moreover, potassium was absorbed in the Giardia-infected group (0.45±0.30), while the other groups exhibited potassium secretion. A low-protein diet and Giardia infection had no influence over glucose absorption. However, Giardia infection increased sodium and potassium uptake, suggesting a compensatory mechanism for maintaining homeostasis after likely hypernatremia and hypokalemia caused by the diarrhea that accompanies giardiasis.     

Adaptive physiological processes in the host during gastrointestinal parasitism

Parasite infection of the gastrointestinal tract with helminths or protozoa induces detrimental effects on host tissues and host physiology, which have been extensively studied and reviewed. However, parasitism of the digestive system is also associated with adaptive, compensatory phenomena based on changes in host physiology or structures and which tend to counterbalance the negative consequences. The objective of this review is to describe these adaptive processes and their possible underlying mechanisms. Different processes which tend to attenuate the effect of either the loss of appetite, the intestinal malabsorption or the increased tissue losses have been assessed. These processes have been reported both for helminth and protozoan infections, where they present similar characteristics. The mechanisms involved in the adaptation to parasitism remain largely unidentified. The role of feedback mechanisms based on host regulation, possibly through gastrointestinal hormones, has been raised. On the other hand, some data support the proposal that parasites themselves may initiate some of the adaptive processes and consequently favour their own survival. These adaptive phenomena appear to be an essential component in the dynamic balance between host and parasites. Also, parasite infections represent unique models to study the adaptation of the gastrointestinal tract to aggressors.     

A case of fatal malabsorption syndrome caused by strongyloidiasis complicated with isosporiasis and human cytomegalovirus infection]

This 54-year-old Korean coal miner suffered from continuous watery diarrhea and weight loss after corticosteroid treatment (beta-methasone, 4 mg daily for 1 week) due to hip-bone fracture in January 1991. Except for the short therapy of steroid, no other histories were contributory. The malabsorption syndrome was aggravated while the case was treated under the impression of amebiasis or intestinal tuberculosis. AIDS antibody test by EIA was negative and quantitative analysis of serum immunoglobulins was in normal ranges. Nine months after the onset of symptoms, the case was diagnosed as malabsorption syndrome caused by complexed and aggravated infection by Strongyloides stercoralis, Isospora and cytomegalovirus in the small intestine, which were proved by stool examination and duodenal biopsy. His clinical course became worse even after high-dosed and prolonged albendazole treatment for strongyloidiasis with supportive fluid therapy. The patient was discharged in hopeless status in November, 1991 and died after one week at home.