Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Humoral and T cell-mediated immune response against trichomoniasis.

Trichomonas vaginalis (T. vaginalis) infection leads to the synthesis of specific antibodies in the serum and local secretions. The profile of T. vaginalis-specific antibodies and T cell-mediated immune responses may influence the outcome of infection, towards parasite elimination, persistence or pathological reactions. Studies have indicated that Th1-, Th17- and Th22 cell-related cytokines may be protective or pathogenic, whereas Th2- and Treg cell-related cytokines can exert anti-inflammatory effects during T. vaginalis infection. A number of T. vaginalis-related components such as lipophosphoglycan (TvLPG), α-actinin, migration inhibitory factor (TvMIF), pyruvate:ferredoxin oxidoreductase (PFO), legumain-1 (TvLEGU-1), adhesins and cysteine proteases lead to the induction of specific antibodies. T. vaginalis has acquired several strategies to evade the humoral immune responses such as degradation of immunoglobulins by cysteine proteases, antigenic variation and killing of antibody-producing B cells. The characterization of the T. vaginalis-specific antibodies to significant immunogenic molecules and formulation of strategies to promote their induction in vaginal mucosa may reveal their potential protective effects against trichomoniasis. In this review, we discuss the current understanding of antibody and T cell-mediated immune responses to T. vaginalis and highlight novel insights into the possible role of immune responses in protection against parasite.

An ocular cysticercosis case: Caused by Asian genotype of Taenia solium.

An ocular cysticercosis case of a 42-year-old male, who presented with anterior uveitis is being reported. Microscopical examination of the cyst revealed presence of only one hooklet suggestive of T. solium cysticercus. Mitochondrial DNA analysis confirmed it to be T. solium cysticercus of Asian genotype. This is the first report on molecular typing of cysticercus isolate from ocular cysticercosis patient in India. The study suggests that the molecular analysis of cox1 gene may be a useful diagnostic tool in cases where microscopic examination is not confirmatory.

Immunity in urogenital protozoa.

Innate and adaptive immunity play a significant role in urogenital infections. Innate immunity is provided by the epithelial cells and mucus lining along with acidic pH, which forms a strong physical barrier against the pathogens in female reproductive tract. Cells of innate immune system, antimicrobial peptides, cytokines, chemokines and adaptive immunity in the reproductive tract are evolved during infection, and a pro-inflammatory response is generated to fight against the invading pathogen Trichomonas vaginalis, a primary urogenital protozoa, the etiological agent of human trichomoniasis, a curable sexually transmitted infection. The involvement of the urogenital tract by other protozoal infections such as P. falciparum, Trypanosoma, Leishmania, Toxoplasma, Entamoeba histolytica and Acanthamoeba infection is rarely reported. Trichomonas induce pro-inflammatory and immunosuppressive responses in infected subjects. Multifactorial pathogenic mechanisms including parasite adherence, cysteine proteases, lipophosphoglycan, free radical, cytokine generation and Toll-like receptors appear to interplay with the induction of local and systemic immune responses that ultimately determine the outcome of the infection. However, the involvement of urogenital pathogen-specific immune mechanisms and effect of normal local resident flora on the outcome (symptomatic vs. asymptomatic) of infection are poorly understood. Moreover, immune interactions in trichomoniasis subjects co-infected with bacterial and viral pathogens need to be elucidated.

Ophthalmomyiasis: three cases from North India.

Three cases of external ophthalmomyiasis are reported here. The larvae were identified to be Oestrus ovis in two cases and Cochliomyia hominivorax in one. Two of the patients were immunocompetent while one was undergoing treatment for squamous cell carcinoma of eyelid. In the latter myiasis led to complete destruction of the eye.

Serological screening for antenatal toxoplasma infection in India.

PURPOSE: Detection of infection caused by Toxoplasma gondii during pregnancy to prevent congenital infection. MATERIALS AND METHODS: This study was carried out from January 2005 to 2006 in 300 pregnant women. Antitoxoplasma IgG, IgM, IgA antibody and IgG avidity were assessed using ELISA. At least two samples were taken at least 3 weeks apart preferably one in each trimester. RESULT: Of these 300 pregnant women, anti toxoplasma IgG antibodies were detected in 46 (15.33%) cases, while 9 (3%) had positive anti toxoplasma IgM with IgA and /low IgG avidity antibodies suggestive of acute infection during or just before pregnancy. CONCLUSION: The results indicate that about 85% of female population of Chandigarh is susceptible to toxoplasma infection and thus should be specifically educated about prevention of this infection during pregnancy.

Evaluation of excretory secretory and 10-30 kDa antigens of Taenia solium Cysticerci by EITB assay for the diagnosis of neurocysticercosis.

Neurocysticercosis (NCC), caused by the presence of Taenia solium Cysticerci in the Central Nervous System is the most common neurological disease of parasite aetiology. The serodiagnostic methods available at present have variable sensitivity and specificity depending upon the antigen and technique used. The present study was aimed to assess the efficacy of T. solium Cysticerci excretory secretory (ES) and lower molecular mass (LMM) 10-30 kDa antigenic fractions for antibody detection in serum and urine samples by enzyme-linked immunoelectrotransfer blot (EITB) for the diagnosis of NCC. Serum and urine samples were collected from 125 clinically suspected and radiologically proven NCC children (111 patients with single lesion and 14 with multiple lesions) and 125 control subjects. With the use of ES and LMM antigenic fractions, the sensitivity of the EITB assay was 85.6% and 80.8% with serum and 76.8% and 50.4% with urine, respectively. The specificity was 64% and 61.6% with serum and 48% and 33.6% with urine samples, respectively. The study suggests that antibody detection to ES antigen in serum by EITB assay may serve better purpose for the serodiagnosis of human NCC.

Neurocysticercosis immunodiagnosis using Taenia solium cysticerci crude soluble extract, excretory secretory and lower molecular mass antigens in serum and urine samples of Indian children.

Neurocysticercosis (NCC), the most common neurological disorder of parasite etiology, results from lodgement of Taenia solium cysticerci in the central nervous system and is now increasingly being recognized in children. The confirmed diagnosis is based collectively on radiological findings and serodiagnostic techniques. The serodiagnostic techniques have variable sensitivity and specificity depending upon the technique, antigens used, location and number of cysts. Crude soluble extract (CSE), excretory secretory (ES) and lower molecular mass (LMM) (10-30 kDa) antigenic fraction of T. solium cysticerci were evaluated for antibody detection in serum and urine samples by ELISA. Serum and urine samples were collected each from 125 clinically suspected and radiologically proven NCC (111 with single Computed Tomography (CT) lesions and 14 with multiple CT lesions) and 125 control subjects (60 with neurological disorders other than NCC, 40 with other parasitic diseases and 25 apparently healthy subjects). The sensitivity of the ELISA with the use of CSE, ES and LMM antigenic fractions was 38.4%, 63.2% and 30.4% with serum (cut off dilution 400), 46.4%, 44% and 47.2% with neat urine and the specificity was 88%, 76.8% and 85.6% with serum (cut off dilution 400), 66.4%, 65.2% and 58.4% with neat urine samples, respectively. The study suggests that detection of antibody to ES antigen in serum samples may serve useful purpose for the serodiagnosis of human NCC.

Cysteine proteinase 30 (CP30) and antibody response to CP30 in serum and vaginal washes of symptomatic and asymptomatic Trichomonas vaginalis-infected women.

Infection with Trichomonas vaginalis may be asymptomatic or with symptoms suggestive of vaginitis. Because cysteine proteinase 30 (CP30) of T. vaginalis is known to be a virulence marker that plays a role in cytoadherence, the aim of this study was to analyse the presence of CP30 and antibody to CP30 in clinical samples of symptomatic and asymptomatic infected women. CP30 was detected in all the serum and vaginal washes (VWs) of symptomatic women and in 65% of the serum and 80% of the VWs of asymptomatic women. This suggested that the majority of asymptomatic women also exhibit CP30 in the serum and VWs. Antibody to CP30 was detected in all the serum samples of symptomatic and asymptomatic women and in the VWs of only 54.5% of the symptomatic and 35% of the asymptomatic women. Antibody to CP30 was also detected in 3/20 of the serum samples and in none of the VWs from uninfected women. Significantly higher amounts of antibody (mean OD values) were observed in serum and VWs of symptomatic as compared to asymptomatic and healthy women (P<0.001). These results indicate that besides CP30, other factors may also be playing a role in leading to symptomatic infection, because CP30 was detected in clinical samples from all the symptomatic and the majority of the asymptomatic women. Although anti-CP30 antibodies do not appear to be protective, detection of antibody to CP30 antigen in serum samples may be used as a diagnostic tool.

Evaluation of ELISA and dot blots for the serodiagnosis of neurocysticercosis, in children found to have single or multiple enhancing lesions in computerized tomographic scans of the brain.

Although human neurocysticercosis (NCC) is being increasingly recognized in children, diagnosis of the disease can be difficult, and the 'gold standard' criteria that indicate an unambiguous case have still to be established. In the present study, the performances of an ELISA and dot-blot assay, for the detection of antibodies against antigens from larval Taenia solium, were investigated and compared, using sera, from children aged 5-12 years, that were diluted to at least 1:400. Eighty of the subjects (20 aged 5-<8 years and 60 older children) each had the signs and symptoms of NCC, including one brain lesion (N=69) or multiple brain lesions (N=11) that were visible by computed tomography. Another 100 sera, from children who had tubercular meningitis (N=20) or a parasitic disease other than taeniasis/cysticercosis (N=20) or, apart from a minor respiratory-tract infection, appeared healthy (N=60), were also investigated. Most (86%) of the cases of NCC had presented with focal seizures. Analysis of antibody response indicated that the optimum threshold titres for seropositivity were 1:800 for the ELISA and 1:6400 for the dot-blot assays. When used with these thresholds, the ELISA gave a sensitivity, specificity, positive and negative predictive values and diagnostic efficacy of 89%, 81%, 79%, 90%, 85%, respectively. The corresponding values for the dot-blot assay were similar, at 89%, 73%, 72.5%, 89%, 82%, respectively. Both assays were more sensitive, in the detection of the specific antibody response, when used among the paediatric cases of NCC who had multiple brain lesions (100%) than when used among the single-lesion cases (87%). As the ELISA gave higher specificity and diagnostic efficacy than the dot-blot assay, it should be considered the better method for the serological confirmation of NCC in children.

Association of parasitic infections and cancers.

Recent advances in the fields of molecular biology, epidemiology and infectious diseases have led to significant revelations to clarify the relationship between cancer and infective agents. This article reviews the relationship between parasitic infections and carcinogenesis and the possible mechanisms involved. Few parasites, e.g., Schistosoma haematobium and Opisthorchis viverrini have been found to be strongly associated with bladder cancer and cholangiocarcinoma respectively. The evidence for the association of several other parasites and cancers has also been postulated.

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.

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.

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.