Pathophysiology of visceral leishmaniasis - some recent concepts.

Visceral leishmaniasis is characterized by diversity and complexity of clinical manifestations ranging from asymptomatic infection to life threatening illness. Experimental evidence and clinical studies indicate multifaceted role of various factors leading to parasite survival and multiplication. In early stage of infection, generation of reactive oxygen and nitrogen intermediates play significant role in curtailing the parasite multiplication while in later phase on one hand, hepatic resistance is expressed by the dominant role played by nitric oxide synthase (NOS)-2 gene regulation and on the other hand, production of inhibitors of NOS-2 gene expression, interleukin 10 (IL-10) and transforming growth factor beta (TGFbeta) correlate well with reduced parasite killing. The hepatic infection is usually self-limiting due to production of multiple cytokine responses including moderate level of tumour necrosis factor (TNF) while in spleen excess TNF mediates destructive pathology. CD8+ T cells appear to play multiple roles comprising both cytotoxic activity and secretion of cytokines and chemokines. Capacity to produce ThI cytokines is associated with asymptomatic or subclinical self-healing infection. However, in symptomatic patients, Th I cytokine production is not depressed but there appears to be unresponsiveness to the stimuli of these cytokines. Experimental evidences indicate genetic basis for such a phenomenon.

Specific IgA response, T-cell subtype and cytokine profile in experimental intravaginal trichomoniasis.

Trichomoniasis caused by Trichomonas vaginalis may lead to either a complete absence of symptoms or to severe inflammatory manifestations in infected women. Studies of the role of immune responses in the pathogenesis and varied symptomatology of this disease are lacking. Mice may prove useful as an experimental model for intravaginal trichomoniasis in developing an understanding of the role of local immune responses in the pathogenesis and varied symptomatology of this disease. The present study reports the levels of anti-Trichomonas IgA antibodies in serum and vaginal washes, and T-cell subtype and cytokine profile in vaginal cervical tissues of mice infected intravaginally with T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. It also correlates the responses with symptomatology of the patients. Successful intravaginal infection was established by inoculating T. vaginalis in BALB/c mice preinoculated with Lactobacillus acidophilus and pretreated with oestradiol. A significant increase in specific IgA antibody levels was detected with enzyme-linked immunosorbent assay in vaginal secretions and serum samples collected on the 7th post-infection day from animals infected with isolates from asymptomatic women when compared with mice infected with isolates from symptomatic women. T-cell subset analysis showed significant differences, with increased CD4+ T-cell count in animals infected with isolates from asymptomatic women compared with animals infected using isolates from symptomatic women. No difference in CD8+ T cells was observed between the two groups. Cytokine profile revealed significantly higher (P < 0.001) production of gamma-IFN and IL-2 in mice infected with asymptomatic isolates compared with animals infected with symptomatic isolates, using T. vaginalis crude antigen extract and nonspecific mitogen (ConA) as stimulants for vaginal cervical lymphocytes. However, no difference in IL-4 levels was observed in the two groups of animals. In contrast, significant increase in tumour necrosis factor (TNF-alpha) levels was observed in animals infected with asymptomatic isolates compared with those infected with isolates from symptomatic women and controls, thereby indicating that TNF-alpha may play an important role in the inflammatory response to trichomoniasis. The study further suggests that specific IgA antibodies might help to protect asymptomatic individuals from severe infection and T-lymphocytes may play an important function in the eradication of the parasite. The cytokine profile indicated the involvement of Th-1 like responses in mice infected with asymptomatic isolates, compared with those infected with symptomatic isolates.

Detection of antigen B of Cysticercus cellulosae in cerebrospinal fluid for the diagnosis of human neurocysticercosis.

Neurocysticercosis (NCC) is a major cause of morbidity and mortality in developed and developing countries. The diagnosis of this disease remains a problem. We report the detection of specific antigenic fraction (antigen B) of Cysticercus cellulosae by enzyme-linked immunosorbent assay (ELISA) in various fractions of cerebrospinal fluid (CSF) obtained by high performance liquid chromatographic (HPLC) separation, for the diagnosis of human NCC. Forty patients attending or admitted to Nehru Hospital, Chandigarh were included in the study: 10 with suspected NCC, 20 with other neurological diseases and 10 undergoing surgery under spinal anaesthesia for non-neurological conditions, who served as controls. CSF samples collected from all patients and controls were subjected to chromatographic separation on an HPLC system. Antigen B (AgB) was detected in separated fractions by an ELISA test and compared with the detection of antibody response in CSF samples by indirect haemagglutination (IHA) technique. Antigen B was detected in 9 out of 10 patients with suspected NCC based on clinical symptoms and radioimaging reports, but in none of the control subjects. However, antigen B was also detected in 9 out of 20 patients with other neurological disorders, mostly tubercular meningitis. Antibody response by IHA was found positive in only 2 of 10 cases clinically suspected of NCC. In conclusion, antigen B detection in CSF samples may be a useful adjunct to clinical suspicion and radiological reports for the diagnosis of NCC as there is no gold standard criteria to confirm this disease. However, the test needs to be evaluated on more patients in countries where tuberculosis and cysticercosis are endemic due to the high cross reactivity with samples from tubercular meningitis patients.

Ca2+ signaling in the transformation of promastigotes to axenic amastigotes of Leishmania donovani.

The present study is an attempt to look into the role of Ca2+ in signaling the transformation of promastigotes to axenic amastigotes. An estimation of intracellular free calcium concentration at 6 h intervals during the conversion of promastigotes to axenic amastigotes (72 h) revealed a 10 fold increase in [Ca2+]i at the initial 6-12 h during the conversion. This was followed by declining levels till 60 h and the concentration thereafter remained constant. Axenic amastigotes (72 h) had a 5 fold higher [Ca+]i as compared to the promastigotes. A 30-40% decrease in [Ca2+]i after pretreatment of cells with dentrolene and a gradual rise of intracellular Ca2+ in [Ca2+] free medium indicates the role of intracellular calcium pools in the elevation of [Ca2+]i. A sudden increase in [Ca2+]i on addition of NH4Cl (20 mM) in the cells grown in Ca2+ free medium indicates the presence of acidocalcisomes, as intracellular Ca2+ storing pool, in L. donovani. To study the role of Ca2+ influx from the external medium in the morphogenetic transformation and in the elevation of [Ca2+]i a 45Ca2+ uptake study was performed. Maximum uptake of 45Ca2+ was observed in the initial 24 h of transformation and maximum Ca2+ ATPase activity was also observed between 24-42 h. So the presence of low Ca2+ in the cytosol, existence of intracellular Ca2+ pools and presence of mechanisms to maintain the Ca2+ homeostasis in the cells suggests that Ca2+ can be an appropriate candidate for a second messenger during the morphogenetic transformation of L. donovani.

Entamoeba histolytica: rapid detection of indian isolates by cysteine proteinase gene-specific polymerase chain reaction.

Amoebiasis, caused by Entamoeba histolytica, is still one of the major problems for developing countries like India. Early detection of the parasite is a must for its prevention and control. In this study, PCR analysis of the cysteine proteinase gene from clinical isolates of symptomatic intestinal and amoebic liver abscess (ALA) cases has been compared with the stool microscopy, serology, and ultrasonography methods. The clinical isolates negative for E. histolytica by stool microscopy demonstrated the presence of the cysteine proteinase gene by PCR amplification. Also the gene copy number was increased in ALA samples compared with intestinal cases. Hence an accurate, early, and easier detection was possible by cysteine proteinase gene amplification directly from the clinical samples.

Cellular immune responses in human neurocysticercosis.

Studies on the role of cell-mediated immune responses in human neurocysticercosis (NCC) are lacking. Various cell-mediated immune responses such as lymphocyte subpopulation, lymphocyte transformation to cysticercus antigens and cytokine profile were carried out in NCC patients. Lymphocyte transformation assays using larval antigens showed significantly higher (3)H-thymidine uptake. Immunophenotyping analysis showed an insignificant increase in B cells and a decrease in total T cells. However, there was a significant decrease (P < 0.05) in CD8+ T cells whereas there was no change in other cells like CD4+, HLA-DR+ and CD16+/CD56+. Cytokine profile revealed significantly higher (P < 0.01) production of Th1 cytokines (gamma-IFN and IL-2) using cysticercal antigens as stimulants for peripheral blood mononuclear cells, while there was no difference in IL-4 levels between NCC patients and healthy controls. The cytokine profile indicated the involvement of Th-1-like responses in NCC patients.

Kinetics of humoral & cellular immune responses in experimental cysticercosis in pigs infected with Taenia solium.

Studies were undertaken to assess the kinetics of antibody responses, lymphocyte transformation to Taenia solium larval antigens (crude soluble extract antigen and antigen B), and T cell subpopulation in piglets following experimental infection. Cysticercosis was established in 1-2 month old piglets after feeding 5,00,000 T. solium eggs per pig. The anti-CD4 and anti-CD8 monoclonal antibodies against swine T cells were raised indigenously. It was observed that at 60 days post infection (PI) there was a significant increase (P < 0.01) in CD4+ T cells without any change in CD8+ T cells. Increased 3H-thymidine uptake was found in infected piglets at 45 days PI using both CSE and antigen B. Kinetics of antibody responses indicated significant increase (P < 0.01) at 15 days PI (with CSE antigen) and 30 days PI (with antigen B) by ELISA. This increase persisted till 90 days PI (the time up to which the animals were followed). It was also observed that the cellular mechanisms were triggered in late stage (60 days PI) as compared to humoral responses (15-30 days PI) and may persist longer as seen by both lymphocyte transformation and T cell subpopulation studies. The study suggests that in cysticercosis, both humoral and cellular mechanisms may play a role in the host defences.

Diagnostic accuracy of rapid enzyme linked immunosorbent assay for the diagnosis of human hydatidosis.

Rapid enzyme linked immunosorbent assay (ELISA) was compared with the standard ELISA and indirect haemagglutination (IHA) techniques for the diagnosis of human hydatidosis. Eighty nine serum samples including 17 from hydatidosis patients (10 surgically confirmed and 7 clinically suspected), 50 from patients with other parasitic diseases and 22 samples from normal healthy individuals were analysed for anti-hydatid IgG antibodies using sheep hydatid cyst fluid antigen. The sensitivity and specificity respectively was found to be 82.3 and 100 per cent by rapid ELISA; 88.23 and 90.27 per cent by standard ELISA and 70.58 and 100 per cent by IHA technique. No cross reactions were observed with rapid ELISA technique using samples from cysticercosis and amoebiasis patients and normal healthy controls. The present study indicates that rapid ELISA can easily be performed in place of the standard ELISA for the serodiagnosis of human hydatidosis with the advantage of minimising reporting time and manpower hours.

Role of calcium influx & energy metabolism in histamine release by filarial antigen in Mastomys natalensis experimentally infected with Brugia malayi.

The present work was undertaken to study the biochemical pathways involved in terms of role of calcium influx and status of energy metabolism in the activation of mast cells obtained from Mastomys natalensis infected with Brugia malayi when challenged in vitro with a potentially allergenic antigen (60 kDa) of Brugia malayi. It was observed that histamine release from sensitized lung and peritoneal mast cells was associated with intracellular influx of radioactive Ca2+, thus establishing the role of calcium in histamine release. The process of activation of mast cells by 60 kDA antigen was an energy requiring process as it utilized the energy phosphates in the form of ATP and the cells followed the aerobic respiratory pathway for the generation of energy molecules.

Microbicidal mechanisms of liver macrophages in experimental visceral leishmaniasis.

To study the differential microbicidal potentials of liver macrophages, the oxygen-dependent and oxygen-independent pathways in Kupffer cells and immigrant macrophages of Leishmania donovani-infected BALB/c mice were investigated. Hydrogen peroxide assay was performed using horse radish peroxidase-dependent oxidation of phenol red to quantitate the reactive oxygen species produced. To examine the oxygen-independent pathway, the enzymes N-acetyl-beta-glucosaminidase (NAG) and beta-glucuronidase (beta G) were investigated after exposure of cells to lipopolysaccharide. Hydrogen peroxide release by Kupffer cells was significantly decreased only at 21 days postinfection, whereas hydrogen peroxide release by immigrant macrophages was significantly increased on all postinfection days with a maximum at 21 days postinfection. The pattern of release of NAG and beta G was similar in both cell populations with a peak at 21 days postinfection. The present study therefore suggests that Kupffer cells and immigrant macrophages adopt different pathways to cope with this infection.

Evaluation of the efficacy of albendazole against the larvae of Taenia solium in experimentally infected pigs, and kinetics of the immune response.

Cysticercosis, a disease of economic and public health importance, is caused by Cysticercus cellulosae, the metacestode stage of Taenia solium. Experimental induction of cysticercosis was achieved in young pigs by feeding an optimum dose of 20,000 T. solium (Indian strain) eggs after immunosuppression, to assess the effect of albendazole and development of the immune response to cysticercus antigens before and after treatment. Histopathological studies revealed the presence of cysticerei in liver, lungs and muscles. Treatment with albendazole at 15 mg kg-1 body weight daily for 30 days starting from day 0 or 15 days post-infection resulted in 100% cure rates. Increases in antibody titre to crude soluble extract and a Sephadek G-200 purified antigenic fraction of Cysticercus cellulosae were found on days 25, 40 and 55 post-infection in untreated pigs and those in which treatment started on day 15 post-infection, whereas no increase in antibody response was observed in pigs in which treatment started on day 0.

Increased glutathione cycling and vitamin E of P. falciparum infected erythrocytes fail to prevent spontaneous haemolysis.

In an attempt to understand the pathogenesis of anaemia in Plasmodium falciparum infection, the status of erythrocyte glutathione and vitamin E content in relation to the susceptibility of infected red cells to peroxide haemolysis was examined. Synchronized cultures of the parasite with either ring-, trophozoite or schizont-infected red cells showed a gradual increase in the reduced glutathione content which was significantly higher (p < 0.05) in schizont-infected cells. Trophozoite-infected cells revealed significant increase in oxidized glutathione (p < 0.01) suggesting an increase in glutathione utilization during active erythrocytic schizogony of the parasites. The membrane antioxidant vitamin E also showed an increased accumulation in trophozoite- and schizont-infected red cells (p < 0.05) but not in the uninfected or ring-infected erythrocytes. Despite a favourable change in these antioxidants, the infected as well as uninfected red cells from parasite cultures showed enhanced peroxide haemolysis (uninfected, p < 0.05; ring-rich, p < 0.05, trophozoite- and schizont-rich, p < 0.001). The study provided direct evidence for enhanced susceptibility of red cells to lysis, including those of uninfected cells exposed to parasite products. This might explain the cause for much higher red cell loss and anaemia during P. falciparum infection than all the infected cells put together.

Plasmodium falciparum and blood monocyte induced abnormalities in human erythrocyte cation homeostasis.

The role of Plasmodium falciparum and activated blood monocytes in bringing about erythrocyte membrane lipid peroxidation and in altering the enzyme activity associated with Ca2+ and K+ efflux was studied. An attempt was made to investigate the role of parasite and monocyte-mediated reactive oxygen species (ROS) in inhibiting Ca(2+)-Mg2+ ATPase and Na(+)-K+ ATPase in order to find out the cause of reported high intra-erythrocytic calcium and depleted potassium levels in parasitized erythrocytes (PRBC). The PRBC showed enhanced lipid peroxidation as indicated by increased malonyldialdehyde (MDA) formation which coincided with the maturity of the parasite. This was further enhanced following exposure of PRBC to activated blood monocytes. The Ca(2+)-Mg2+ ATPase activity was decreased as the parasite matured and was further hampered significantly in mature parasite-infected red cells exposed to activated blood monocytes. There was a good negative correlation between MDA formation and Ca(2+)-efflux from red blood cells suggesting the negative influence of ROS on Ca(2+)-efflux. The Na(+)-K+ ATPase activity did not reveal any significant change, both during parasite maturation as well as upon exposure to ROS from activated monocytes. We therefore suggest that inhibition of Ca(2+)-efflux and the resulting increased cytosolic Ca2+ in PRBC might have a role in structural and functional abnormalities of red blood cell, thus enhancing the red cell loss during P. falciparum infection.

Leishmania donovani: in vitro evidence of hepatocyte damage by Kupffer cells and immigrant macrophages in a murine model.

Infection with Leishmania donovani leads to activation of liver macrophages. The role of different macrophage populations of liver in this infection is not clearly defined. Thus, the mechanism involved in hepatocyte damage was studied by coculturing hepatocytes with two populations of liver macrophages, the kupffer cells and immigrant macrophages. The results indicated maximum tissue damage at peak infection in both the macrophage populations cocultured with hepatocytes (P < 0.001). Kupffer cell-hepatocyte coculture treated with scavengers of reactive oxygen intermediates failed to inhibit the hepatocyte damage (P > 0.05). But with heparin and phenylmethylsulfonyl fluoride, a sharp decrease in the damage was noticed (P < 0.001). In contrast, immigrant macrophage-hepatocyte coculture showed a significant reduction in tissue damage when treated with both the scavengers of reactive oxygen intermediates and enzyme inhibitors (P < 0.001). Therefore the murine infection with L. donovani is speculated to involve two distinct subpopulations of liver macrophages with marked differences in morphology and functional capabilities.

Effect of nifedipine on oxidative damage of erythrocytes in Plasmodium berghei-infected mice.

It is known that the calcium channel blocker (CCB), nifedipine, can inhibit phagocyte oxidative burst in Plasmodium berghei-infected mice. The extent of immunopathological changes as seen by the course of infection and membrane lipid peroxidation in nifedipine-treated mice was examined in comparison with untreated mice at different parasite loads. The glutathione antioxidant system was also studied in these animals to assess its capacity to neutralize reactive oxygen species (ROS) in infected erythrocytes. The survival period of nifedipine-treated, infected mice decreased significantly. It was observed that the accumulation of reduced glutathione was greater and the decrease in glutathione peroxidase activity was less marked in drug-treated animals, suggesting better protection of the parasites against oxidative injury. The accumulation of the lipid peroxidation product, malonyldialdehyde was significantly lower in nifedipine-treated animals at all parasitemia levels studied, indicating decreased ROS generation and parasite damage. These observations reveal the shortcomings of using CCB to reverse the chloroquine resistance in malaria as this would minimize oxidative damage of parasitized red cells and phagocyte-mediated parasite killing.

Macrophage-mediated enterocyte damage in BALB/c mice infected with different strains of Giardia lamblia.

The mechanism of mucosal injury in Giardia lamblia-infected animals and humans is not well understood, although the role of gut macrophages in killing the trophozoites is well known. It is speculated, however, that macrophage products have a role in tissue injury and inflammatory response during infection, as in other inflammatory diseases. Therefore, in the present study an attempt was made to examine the mechanism involved in enterocyte damage during giardiasis. This was achieved using co-culture of enterocytes and gut macrophages obtained from infected BALB/c mice. The extent of tissue damage was assessed by measuring the marker enzyme of enterocyte damage, lactate dehydrogenase. To investigate the role of the various proteases and free oxygen radicals released by activated macrophages on enterocyte damage, inhibitors of various proteases and free oxygen radicals were used. Superoxide radical and certain proteases were found to have important roles in bringing about enterocyte damage during this infection in mice. Parasite load, lactate dehydrogenase release, and extent of lipid peroxidation were more pronounced in mice infected with symptomatic strains than in asymptomatic ones. The theory of inflammatory cell-mediated enterocyte damage in Giardia lamblia infection is proposed.

Effect of nifedipine on calcium status and chemiluminescence response of phagocytes during Plasmodium berghei infection in mice.

The macrophages and neutrophils from nifedipine-treated mice, both Plasmodium berghei-infected and uninfected, showed suppressed capacity to generate oxygen free radicals as compared with untreated controls. Nifedipine treatment did not affect resting state free calcium levels in these cells. But the rise in intracellular calcium levels of macrophages and neutrophils following P. berghei infection was significantly less (P < 0.05) in nifedipine-treated mice as compared with untreated groups at various parasitaemia levels. Probably this reflects a more potent effect of nifedipine on these cells in the depolarized state. Similarly, the rise in intracellular calcium levels of these cells following formyl-Met-Leu-Phe (fMLP) stimulation was also significantly less in nifedipine-treated groups than in untreated controls at different parasitaemia levels. A positive correlation between this fMLP-stimulated rise in calcium levels and the chemiluminescence response of macrophages and neutrophils was observed in nifedipine-treated and untreated groups at various parasitaemia levels. Thus the respiratory-burst responses of these cells during P. berghei infection depend on the calcium homeostasis in the cells. The disturbances of the calcium-regulating mechanisms by nifedipine treatment resulted in subnormal phagocytic cell responses which lead to more severe and rapidly fatal P. berghei infection in these animals.

Oxidative damage of erythrocytes infected with Plasmodium falciparum. An in vitro study.

The extent of reduced glutathione, activity of glutathione peroxidase, amount of membrane lipid peroxidation products, and the extent of hemoglobin release from host erythrocytes during in vitro Plasmodium falciparum growth was studied. Highly synchronized parasite cultures were studied to examine the alterations caused by different growth stages of the parasite. There was a moderate increase in the reduced glutathione content as the parasite matured, which was significant only in schizont-rich erythrocyte lysates (p < 0.05) whereas the activity of glutathione peroxidase was significantly low in all the parasitized red blood cells (ring-infected RBC, p < 0.005; trophozoite- and schizont-infected RBC, p < 0.001). The lipid peroxidation product, malonyldialdehyde, of the host red cells increased gradually to more than fourfold in schizont-rich cells as compared with normal erythrocytes (p < 0.001). The hemoglobin release from cultured cells was significantly higher in all parasitized red cell cultures as well as in uninfected cells kept in in vitro, as compared with normal erythrocytes. The consequence of such changes induced by the malarial parasites in the host red cells in the pathogenesis of erythrocyte destruction and anemia of P. falciparum malaria is discussed.

Plasmodium falciparum induced perturbations of the erythrocyte antioxidant system.

Erythrocyte antioxidants catalase, superoxide dismutase, reduced glutathione and glutathione peroxidase were studied in cells harbouring different growth stages of Plasmodium falciparum. Catalase and superoxide dismutase showed significant decrease during parasite maturation indicating hampered metabolism of hydrogen peroxide and superoxide anions. Glutathione peroxidase also exhibited a downward trend during the growth of P. falciparum, while there was a moderate accumulation of reduced glutathione. These findings suggest decreased utilization of the reduction potential in detoxification of reactive oxygen species. The fall in all three antioxidant enzymes studied was highly significant (P less than 0.001) in erythrocytes with mature stages of the parasite (trophozoites, schizonts). The increased vulnerability of erythrocytes to damage, which parallels the growth phases of the parasite emphasizes the need for early treatment of P. falciparum malaria to minimise red cell destruction and the resulting anaemia.

An experimental model of ameboma in guinea pig.

Among the wide variety of clinicopathological manifestations of intestinal amebiasis, amebomas occur rarely and their pathogenesis is not well understood. When cholesterol-fed, 2- to 4-week-old guinea pigs were infected intracecally with a virulent, monoaxenic strain of Entamoeba histolytica, gross and histologically characteristic amebomas developed in 85% of the animals by the 3rd day, in 94% by the 9th day, and in 96% by the 12th day postinfection, by which time most of them had died. Amebomas were confirmed by histopathology. Thus, a model of consistent production of amebomas was documented.