Prema Robinson

Interferon-γ Expression in Jejunal Biopsies in Experimental Human Cryptosporidiosis Correlates with Prior Sensitization and Control of Oocyst Excretion

To investigate the role of interferon (IFN)-gamma in human cryptosporidiosis, jejunal biopsies from experimentally infected volunteers and chronically infected AIDS patients were examined for IFN-gamma expression by in situ hybridization. IFN-gamma expression was compared with oocyst excretion, baseline serum anti-Cryptosporidium antibody, and symptoms. IFN-gamma mRNA was detected in biopsies from 13 of 26 volunteers after experimental infection but not in biopsies taken before C. parvum exposure or in biopsies from patients with AIDS-associated cryptosporidiosis. After challenge, 9 of 10 volunteers with baseline C. parvum antibody produced IFN-gamma, compared with 4 of 16 volunteers without baseline antibody (P<.01). Furthermore, IFN-gamma mRNA was detected in 9 of 13 volunteers who did not excrete oocysts, compared with 4 of 13 with organisms (P<.05). Thus, expression of IFN-gamma in the jejunum was associated with prior sensitization and absence of oocyst shedding. IFN-gamma production may explain the resistance to infection noted in sensitized persons but may not be involved in control of human primary infection.

Interleukin-15 Activates Human Natural Killer Cells to Clear the Intestinal Protozoan Cryptosporidium

Intracellular protozoans of the genus Cryptosporidium are a major cause of diarrheal illness worldwide, but little is known about the mechanisms that control intestinal infection. We have previously demonstrated interleukin (IL)-15 expression in the intestinal mucosa of seronegative symptomatic volunteers after oral challenge with C. parvum, which suggests a role for IL-15 in the control of acute infection. We hypothesize that IL-15 activates an innate cytolytic cell response that contributes to the clearance of initial C. parvum infection. We report here that IL-15 activates peripheral blood mononuclear cells to lyse Cryptosporidium-infected epithelial cells in a dose-dependent manner. Lysis was due to CD3(-)CD16(+)CD56+ cells (i.e., natural killer [NK] cells). Furthermore, flow cytometry revealed that IL-15 increased expression of the activation receptor NKG2D on NK cells, particularly among the CD16Hi cytolytically active cells. Major histocompatibility complex class I-related molecules A and B (MICA and MICB), ligands for NKG2D, were increased after infection of epithelial cell lines and human ileal tissue. These data suggest that IL-15 has an important role in activating an NK cell-mediated pathway that leads to the elimination of intracellular protozoans from the intestines, which is a previously unrecognized feature of NK cell function.

Intestinal Immune Response to Human Cryptosporidium sp. Infection

Cryptosporidium is an obligate intracellular protozoan parasite that is a major cause of diarrheal illness worldwide. Cryptosporidium primarily infects the distal small intestine. Immunocompetent hosts control and eliminate the infection, which typically causes acute, self-limited watery diarrhea lasting 5 to 10 days. However, in patients with defects in cellular immune responses (e.g., AIDS, malnutrition, or defects in the CD40-CD154 system), Cryptosporidium frequently causes persistent or chronic diarrhea and may also involve the biliary tract (40). In malnourished children, persistent diarrhea is associated with increased susceptibility to recurrent diarrheal episodes, which can lead to death or chronic nutritional and cognitive sequelae (1, 9, 33). Thus, the host immune response plays a critical role in the control of human cryptosporidiosis. Although extensive studies with various animal models have provided important insight into the host immune response towards Cryptosporidium parvum, the ability of these models to explain the human immune response is limited. The clinical picture in rodents differs from that in humans, as mice do not get diarrhea after infection. Nonhuman primates, although probably the best in vivo model to mimic human disease, are difficult to work with, expensive, and not widely available. Cryptosporidium hominis, the pathogen causing most human cryptosporidiosis, infects only humans and gnotobiotic pigs, thus limiting data from animal models. Most importantly, comparison of animal and human data has shown that the immune response towards Cryptosporidium in humans differs significantly from that in animals; for example, in mice gamma interferon (IFN-γ) production seems to be associated with the innate and primary immune responses (35, 47), whereas in humans it is most probably associated with the memory response towards the parasite (93). Conducting studies to elucidate human mucosal immune responses is difficult. Patients with a natural infection would be the ideal subjects to study, but it is difficult to identify cases. Healthy human volunteers can be studied, but they typically experience a milder illness than malnourished children and AIDS patients. Human intestinal tissue samples can be obtained only by invasive procedures, limiting the numbers of subjects and samples available. Some data can be obtained from in vitro infections, but most of the target cells are immortalized and may not be ideal for studying mechanisms involving apoptosis. Furthermore, the immune cells in the peripheral blood may exhibit properties different from the properties of cells found in the intestinal compartment. Thus, knowledge about the human immune response towards Cryptosporidium infection is far from complete. Still, important recent advances have been made. The goal of this paper is to review the current literature to provide an understanding of the human immune response towards the parasite. We include some relevant data from other models only when the data shed light on studies performed with human cells or tissues.

Substance P Expression Correlates with Severity of Diarrhea in Cryptosporidiosis

Cryptosporidiosis, caused by Cryptosporidium parvum, is self-limited in immunocompetent hosts but may cause chronic diarrhea in patients with acquired immunodeficiency syndrome (AIDS). Substance P (SP), a neuropeptide belonging to the tachykinin family, is expressed in gastrointestinal tract and can cause electrogenic chloride anion secretion. Therefore, we studied SP mRNA and protein expression in jejunal tissue samples of patients with AIDS with naturally occurring chronic cryptosporidiosis and healthy volunteers with mild cryptosporidiosis or asymptomatic infection after experimental C. parvum challenge. SP mRNA was associated with symptoms in cryptosporidiosis. SP protein levels were greater in symptomatic than asymptomatic volunteers. Similarly, greater expression of SP mRNA and protein were noted in patients with AIDS with chronic cryptosporidiosis versus immunocompetent volunteers with self-limited infection. This study demonstrates a direct correlation between SP levels and disease severity and may imply that SP plays a role in diarrhea mediation.

Epileptogenic activity of granulomas associated with murine cysticercosis.

Neurocysticercosis, caused by Taenia solium, is a common cause of neurologic disease in developing countries and among immigrants to the United States. Seizures are the most common clinical manifestation of neurocysticercosis. Imaging studies of patients with seizures from neurocysticercosis typically reveal evidence of an inflammatory reaction associated with the parasite or calcified granulomas. This study investigated whether a substance produced by the host granulomatous reaction to the dying parasite, in a mouse model of the infection, is sufficient to induce epileptiform activity. Granulomas associated with Taenia crassiceps cysticerci were removed from the peritoneal cavity of infected mice. One piece of the granuloma was used for blinded histological staging of the dying parasite. The second piece was used to generate extracts, which were injected into the hippocampus of an anesthetized Sprague-Dawley rat. Positive controls included animals injected with kainic acid, picrotoxin, or bicuculline. Seizures were recorded after injection of extracts from 6 out of 6 early stage granulomas, but only 1 out of 9 late stage granulomas. Injections of buffered saline, extracts from non-stimulated mouse spleen cells, and homogenates of viable parasite material caused no epileptiform activity. The data suggest that a substance in the granulomas early in the inflammatory response to the dying parasite is capable of inducing seizure activity. Further experiments are needed to dissect out the exact seizure mediator in the granuloma extracts.

Expression of tumor necrosis factor alpha and interleukin 1β in jejuna of volunteers after experimental challenge with Cryptosporidium parvum correlates with exposure but not with symptoms

ABSTRACT Jejunal biopsies from volunteers challenged withCryptosporidium parvum were examined for tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β mRNA. Postchallenge biopsies from 15 of 28 (54%) volunteers expressed TNF-α; 14% expressed IL-1β. Cytokine expression did not correlate with enteric symptoms, suggesting that TNF-α and IL-1β are not key mediators of diarrhea in human cryptosporidiosis.

Transforming growth factor β1 is expressed in the jejunum after experimental Cryptosporidium parvum infection in humans

ABSTRACT Biopsies from volunteers challenged with Cryptosporidium parvum were examined for transforming growth factor β1 (TGF-β1). None of the prechallenge biopsies exhibited TGF-β. Seven of 12 volunteers with oocyst shedding expressed TGF-β versus 2 of 13 volunteers without detected oocysts. The association of TGF-β expression with oocyst excretion and the timing of symptoms suggests that TGF-β mediates intestinal healing.

SUBSTANCE P IS ASSOCIATED WITH HEART ENLARGEMENT AND APOPTOSIS IN MURINE DILATED CARDIOMYOPATHY INDUCED BY TAENIA CRASSICEPS INFECTION

Dilated cardiomyopathy (degeneration of heart muscle and heart enlargement) is an important cause of heart failure among young adults. Dilated cardiomyopathy may be a complication during or after various viral, bacterial, or parasitic diseases. Substance P (SP) is a neurotransmitter that is involved in the pathogenesis of various diseases. To determine whether SP is associated with cardiac changes in murine cysticercosis, we compared heart-weight to body-weight ratio, cardiac pathology, cardiomyocyte size, and cardiac-apoptosis (TUNEL assay) in hearts from Taenia crassiceps–infected (wild-type vs. SP-knockout) mice. We noted that, as compared with control uninfected wild-type mice, elevated protein levels of SP and its receptor as studied by ELISA or immunohistochemistry, respectively, were elevated in the hearts of parasite-infected wild-type mice. The heart-weight to body-weight ratios were significantly higher in the parasite-infected wild-type mice versus those of the infected SP-knockout mice. Furthermore, wild-type infected mice developed dilated cardiomyopathy with increased chamber size of both ventricles, decreased ventricular wall thickness, compensatory cardiomyocyte hypertrophy, and increased cardiac apoptosis. This cardiac pathology did not develop in mice lacking SP activity (i.e., in infected SP knockout mice) or in uninfected mice. These data indicate that SP is associated with cardiac changes in an animal model of parasitic dilated cardiomyopathy.

Substance P is responsible for physiological alterations such as increased chloride ion secretion and glucose malabsorption in cryptosporidiosis

ABSTRACT Cryptosporidiosis, caused by the protozoan parasite Cryptosporidium, causes self-limited diarrhea in immunocompetent hosts and severe life-threatening diarrhea in AIDS patients. Highly active antiretroviral therapy has been used to effectively treat cryptosporiosis in some but not all AIDS patients. Therefore, there is an urgent need for innovative drugs to treat this disease. Cryptosporidium infection results in intestinal pathophysiological changes such as glucose malabsorption, increased chloride ion (Cl−) secretion, and epithelial barrier disruption, leading to disease pathogenesis. In order to develop tools to combat this opportunistic pathogen, it is vital to understand mediators involved in disease pathogenesis. Substance P (SP), a neuropeptide and pain transmitter, is located in the gastrointestinal tract. SP can cause Cl− secretion in human gastrointestinal explants. However, its role in cryptosporidiosis has not been fully studied. Jejunal samples from macaques before and after Cryptosporidium parvum infection were assayed for SP and SP receptor mRNA and protein levels by reverse transcription-PCR and by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The role of SP in pathophysiological alterations, such as Cl− secretion and glucose malabsorption, was studied using tissues derived from macaques infected with C. parvum by the Ussing chamber technique. SP and SP receptor mRNA and protein expression levels were increased in jejunal samples following C. parvum infection and were accompanied by increased basal ion secretion and glucose malabsorption. In vitro treatment of samples obtained from infected macaques with the SP receptor antagonist aprepitant (Emend; Merck, Whitehouse Station, NJ) completely reversed the increase in basal ion secretion and corrected the glucose malabsorption. Our findings raise the possibility of using SP receptor antagonists for the treatment of symptoms associated with cryptosporidiosis.

Sequential Expression of the Neuropeptides Substance P and Somatostatin in Granulomas Associated with Murine Cysticercosis

ABSTRACT Neurocysticercosis, a parasitic infection of the human central nervous system caused by Taenia solium, is a leading cause of seizures. Seizures associated with neurocysticercosis are caused mainly by the host inflammatory responses to dying parasites in the brain parenchyma. We previously demonstrated sequential expression of Th1 cytokines in early-stage granulomas, followed by expression of Th2 cytokines in later-stage granulomas in murine cysticercosis. However, the mechanism leading to this shift in cytokine response in the granulomas is unknown. Neuropeptides modulate cytokine responses and granuloma formation in murine schistosomiasis. Substance P (SP) induces Th1 cytokine expression and granuloma formation, whereas somatostatin inhibits the granulomatous response. We hypothesized that neuropeptides might play a role in regulation of the granulomatous response in cysticercosis. To test this hypothesis, we compared expression of SP and expression of somatostatin in murine cysticercal granulomas by using in situ hybridization and immunohistochemistry. We also compared expression with granuloma stage. Expression of SP mRNA was more frequent in the early-stage granulomas than in the late-stage granulomas (34 of 35 early-stage granulomas versus 1 of 13 late-stage granulomas). By contrast, somatostatin was expressed primarily in later-stage granulomas (13 of 14 late-stage granulomas versus 2 of 35 early-stage granulomas). The median light microscope grade of SP mRNA expression in the early-stage granulomas was significantly higher than that in the late-stage granulomas (P = 0.008, as determined by the Wilcoxon signed rank test). By contrast, somatostatin mRNA expression was higher at later stages (P = 0.008, as determined by the Wilcoxon signed rank test). SP and somatostatin are therefore temporally expressed in granulomas associated with murine cysticercosis, which may be related to differential expression of Th1 and Th2 cytokines.