Vaccination with antioxidant enzymes confers protective immunity against challenge infection with Schistosoma mansoni

Schistosoma mansoni, an intravascular parasite, lives in a hostile environment in close contact with host humoral and cellular cytotoxic factors. To establish itself in the host, the parasite has evolved a number of immune evasion mechanisms, such as antioxidant enzymes. Our laboratory has demonstrated that the expression of antioxidant enzymes is developmentally regulated, with the highest levels present in the adult worm, the stage least susceptible to immune elimination, and the lowest levels in the larval stages, the most susceptible to immune elimination. Vaccination of mice with naked DNA constructs containing Cu/Zn cytosolic superoxide dismutase (CT-SOD), signal-peptide containing SOD or glutathione peroxidase (GPX) showed significant levels of protection compared to a control group. We have further shown that vaccination with SmCT-SOD but not SmGPX results in elimination of adult worms. Anti-oxidant enzyme vaccine candidates offer an advance over existing vaccine strategies that all seem to target the larval developmental stages in that they target adult worms and thus may have therapeutic as well as prophylactic value. To eliminate the potential for cross-reactivity of SmCT-SOD with human superoxide dismutase, we identified parasite-specific epitope-containing peptides. Our results serve as a basis for developing a subunit vaccine against schistosomiasis.

Sawfly larval poisoning in cattle: Report on new outbreaks and brief review of the literature

Sawfly larval poisoning (SLP) is an acute hepatotoxicosis documented in livestock in Australia, Denmark and in countries of South America. It is caused by the ingestion of the larval stage of insects of the suborder Symphyta, order Hymenoptera, commonly known as "sawfly". Three species of sawfly are reportedly involved in the toxicosis. The insect involved in Australian SLP is Lophyrotoma interrupta (Pergidae), in Denmark the cause of SLP is the ingestion of the larvae Arge pullata (Argidae), and in South American countries documented outbreaks of SLP were caused by the ingestion of yet another sawfly, Perreyia flavipes (Pergidae). In all geographical areas where it occurred, SLP causes important livestock losses. In cattle, as well as in other affected species, the disease has a short clinical course and in many outbreaks affected cattle can be found dead. When observed, clinical signs include apathy, recumbence, tremors, paddling movements and death in 24-48 hours. Neurological signs such aggressiveness attributable to hepatic encephalopathy are also observed. In cases with a more protracted course icterus and photodermatitis may develop. Gross findings included ascites, petechiae and ecchymosis over serosal surfaces of thoracic and abdominal cavities, and an enlarged liver that displays accentuation of the lobular pattern and edema of the gall bladder wall. Sawfly larval body fragments and heads are consistently found in the fore stomachs and occasionally abomasum of affected cattle. Main microscopic lesions are restricted to the liver and consist of centrolobular (periacinar) to massive hepatocellular necrosis. In most lobules necrotic areas extended up to the portal triads where only a few viable hepatocytes remain. Mild to moderate lymphocyte necrosis is seen in lymphatic tissues. Cases occur in the winter months when the larval stages of the sawfly are developing. D-amino acid-containing peptides have been found to be the toxic principle in each sawfly involved in SLP. The octapeptide lophyrotomin is the major toxin in the in the larvae of Australian and Danish sawflies and is present in small amounts in the larvae of South American sawfly. The heptadecapeptide pergidin is the main toxin in the South American sawfly while small amounts of pergidin have been found in the other two species of toxic sawfly. During the winter of 2011 (July-August) four outbreaks of SLP were diagnosed in the State of Rio Grande do Sul, Brazil. The findings in those outbreaks are reported here and a brief review of the literature regarding SLP around the world is provided.

Immunogenicity of multiple antigen peptides containing Plasmodium vivax CS epitopes in BALB/c mice

Multiple antigen peptide systems (MAPs) allow the incorporation of various epitopes in to a single synthetic peptide immunogen. We have characterized the immune response of BALB/c mice to a series of MAPs assembled with different B and T cell epitopes derived from the Plasmodium vivax circumsporozoite (CS) protein. A B-cell epitope from the central repeat domain and two T-cell epitopes from the amino and carboxyl flanking regions were used to assembled eight different MAPs. An additional universal T cell epitope (ptt-30) from tetanus toxin protein was included. Immunogenicity in terms of antibody responses and in vitro T lymphocyte proliferation was evaluated. MAPs containing B and T cell epitopes induced high titers of anti-peptides antibodies, which recognized the native protein on sporozoites as determined by IFAT. The antibody specificity was also determined by a competitive inhibition assay with different MAPs. A MAP containing the B cell epitope (p11) and the universal epitope ptt-30 together with another composed of p11 and the promiscuous T cell epitope (p25) proved to be the most immunogenic. The strong antibody response and specificity for the cognate protein indicates that further studies designed to assess the potential of these proteins as human malaria vaccine candidates are warranted.

Proline racemases: insights into Trypanosoma cruzi peptides containing D-proline

Trypanosoma cruzi proline racemases (TcPRAC) are homodimeric enzymes that interconvert the L and D-enantiomers of proline. At least two paralogous copies of proline racemase (PR) genes are present per parasite haploid genome and they are differentially expressed during T. cruzi development. Non-infective epimastigote forms that overexpress PR genes differentiate more readily into metacyclic infective forms that are more invasive to host cells, indicating that PR participates in mechanisms of virulence acquisition. Using a combination of biochemical and enzymatic methods, we show here that, in addition to free D-amino acids, non-infective epimastigote and infective metacyclic parasite extracts possess peptides composed notably of D-proline. The relative contribution of TcPRAC to D-proline availability and its further assembly into peptides was estimated through the use of wild-type parasites and parasites over-expressing TcPRAC genes. Our data suggest that D-proline-bearing peptides, similarly to the mucopeptide layer of bacterial cell walls, may be of benefit to T. cruzi by providing resistance against host proteolytic mechanisms.

Detection of the basement membrane-degrading proteolytic activity of Paracoccidioides brasiliensis after SDS-PAGE using agarose overlays containing Abz-MKALTLQ-EDDnp

We have characterized, in the Paracoccidioides brasiliensis yeast phase, an exocellular SH-dependent serine proteinase activity against Abz-MKRLTL-EDDnp and analogous fluorescent-quenched peptides, and showed that it is also active against constituents of the basement membrane in vitro. In the present study, we separated the components of P. brasiliensis culture filtrates by electrophoresis and demonstrated that the serine-thiol exocellular proteinase has a diffuse and heterogeneous migration by SDS-PAGE, localizing in a region between 69 and 43 kDa. The hydrolytic activity was demonstrable after SDS-PAGE using buffered agarose overlays of Abz-MKALTLQ-EDDnp, following incubation at 37oC, and detection of fluorescent bands with a UV transilluminator. Hydrolysis was more intense when incubation was carried out at basic pH, and was completely inhibited with 2.5 mM PMSF and partially with sodium 7-hydroxymercuribenzoate (2.5 mM p-HMB), suggesting its serine-thiol nature. A proteolytic band with similar characteristics was observed in conventional gelatin zymograms, but could not be correlated with a silver-stained component. Detection of the serine-thiol proteinase in substrate gels after SDS-PAGE provides a useful way of monitoring purification of the basement membrane degrading enzyme.

Production of cabbage grown in pots containing legumes' root and shoot

Roots effect is not generally considered in studies assessing the performance of crops in response to green manuring. However, such effect can contribute to a better understanding of crop rotation. The aim of this study was to assess the effect of root and shoot of two legumes on the production of cabbage. The experiment was conducted in pots of 10 liters containing substrate of 2:1 soil/sand. The experiment was arranged in a factorial scheme (2x3 + 2) in a randomized block design with five replicates using two legume species (Crotalaria juncea L. and Canavalia ensiformis L), three plant parts (root, shoot, or whole plant), and two additional treatments (mineral fertilization with 100% and 50% of the recommended dose of N for growing cabbage). Pots with legume treatments received mineral fertilizer with 50% of the recommended dose of N for growing cabbage. The experimental plot consisted of a pot containing one plant of cabbage. Legumes were grown in pots and harvested at 78 days. The root biomass was determined in extra pots. Production was assessed using head fresh and dry weight. The application of the whole plant of both legume species reduced cabbage production. However, root or shoot of both legume species was equivalent to 50% of mineral N fertilization required for the cultivation of cabbage.

Lethality of triatomines (Hemiptera: Reduviiae), vectors of Chagas' disease, feeding on blood baits containing synthetic insecticides, under laboratory conditions

A laboratory study was conducted to test the toxicity of synthetic insecticides added to defibrinated sheep blood kept at room temperature and offered as food to the following triatomine species: Triatoma infestans, Panstrongylus megistus, Triatoma vitticeps, Triatoma pseudomaculata, Triatoma brasiliensis and Rhodnius prolixus. The insecticides used, at a concentration of 1g/l, were: HCH, DDT, Malathion and Trichlorfon, and the lethalithy observed at the end of a 7-day period varied according to the active principle of each. HCH was the most effective by the oral route, killing 100% of the insects, except P. megistus (95.7%) and T. pseudomaculata (94.1%). Trichlorfon killed the insects at rates ranging from 71.8% (T. vitticeps) to 98% (R. prolixus). Malathion was slightly less efficient, killing the insects at rates from 56.8% (T. vitticeps) to 97% (T.brasiliensis). DDT was the least effective, with a killing rate of 10% (T. vitticeps) to 75% (T.brasiliensis). Since the tests were performed at room temperature, we suggest that baits of this type should be tried for the control of triatomines in the field.

Lethal effect of a bait for Rhodnius prolixus (Hemiptera: Reduviidae), the vector of Chagas' disease, containing hexachlorocyclohexane (HCH), under laboratory conditions

The lethal effect of a bait containing an aqueous hexachlorocyclohexane (HCH) suspension at the concentration of 1g/l and maintained at room temperature was studied in the laboratory over a period of 12 weeks. The suspension was placed in a latex bag hanging inside a 1000-ml beaker tightly covered with nylon netting, and left there with no changes for 85 days. Sixteen groups of R. prolixas bugs, consisting on average of 30 specimens each, were successively exposed to the bait and observed at different intervals for one week each. The mortality rate was 100% for all groups, except for the 16th, whose mortality rate was 96.7%. As the groups succeeded one another, mortality started to occur more rapidly and was more marked at the 6- and 24-h intervals. Later tests respectively started at 6:00 a.m. and 6:00 p.m. showed that diurnal and nocturnal periodicity in the offer of food had no effect on mortality. First- and 2nd- instar nymphs and adults male were more sensitive and 5th- instar nymphs were more resistant to the active principle of the bait.

Leishmania braziliensis: isolation of carbohydrate-containing antigen and possibility of its use in the immunodiagnosis of American Cutaneous Leishmaniasis

Leishmania braziliensis is a causative agent of American Cutaneous Leishmaniasis (ACL). The 034-JCG strain, isolated from a patient from the northern region of Paraná State, Brazil, was cultivated in Blood Agar Base medium, lyophilized and submitted to phenol-water extraction. The extract was treated with RNase I. The carbohydrate containing-antigen (Ag-CHO) was immunogenic to rabbits and showed at least a fraction with some negative charge at pH 8.2. This antigen showed cross-reactivity with the phenol-water extract of the growth medium used for the culture of promastigotes and with the surface antigens of promastigotes. Its composition is: 24.3% of total sugars, from which 11.2% of galactose, 7.5% of mannose and 5.6% of ribose. Protein content was 5.4% and phosphate 18.5%. The antigenic activity was maintained after: repeated freezing-thawing; lyophilization; heating at 100ºC for 30 minutes; treatment with RNase, trichloroacetic acid and sodium metaperiodate. The precipitin line obtained is Periodic Acid Schiff positive. The application of the Ag-CHO in counterimmunoelectrophoresis reaction for the immunodiagnosis of ACL showed 60% sensitivity, and no cross-reaction with the five sera of Chagas' disease patients tested. The use of this antigen in a more sensitive technique, with more samples of sera, may improve these results.

McCoy cell line as a possible model containing CD4+ receptors for the study of HIV-1 replication

Several studies have recently shown the use of recombinant rabies virus as potential vector-viral vaccine for HIV-1. The sequence homology between gp 120 and rabies virus glycoprotein has been reported. The McCoy cell line has therefore been used to show CD4+ or CD4+ like receptors. Samples of HIV-1 were isolated, when plasma of HIV-1 positive patients was inoculated in the McCoy cell line. The virus infection was then studied during successive virus passages. The proteins released in the extra cellular medium were checked for protein activity, by exposure to SDS Electrophoresis and blotting to nitro-cellulose filter, then reacting with sera of HIV positive and negative patients. Successive passages were performed, and showed viral replication, membrane permeabilization, the syncytium formation, and the cellular lysis (cytopathic effect). Flow cytometry analysis shows clear evidence that CD4+ receptors are present in this cell line, which enhances the likelihood of easy isolation and replication of HIV. The results observed allow the use of this cell line as a possible model for isolating HIV, as well as for carrying out studies of the dynamics of viral infection in several situations, including exposure to drugs in pharmacological studies, and possibly studies and analyses of the immune response in vaccine therapies.

Immunogenicity of three recombinant hepatitis B vaccines administered to students in three doses containing half the antigen amount routinely used for adult vaccination

We evaluated the immunogenicity of three recombinant hepatitis B vaccines, one Brazilian (Butang, Instituto Butantan) and two Korean vaccines (Euvax-B, LG Chemical Ltd. and Hepavax-Gene, Greencross Vaccine Corp.), administered intramuscularly to students aged 17 to 19 years in three 10-µg doses (corresponding to half the amount of antigen routinely used for adult vaccination) at intervals of one month between the first and second dose, and of four months between the second and third dose. A total of 316 students non-reactive for any serological marker of hepatitis B virus infection were vaccinated: 77 (24.4%) with the Butang vaccine, 71 (22.5%) with Euvax-B, 85 (26.9%) with Hepavax-Gene and, for comparison, 83 (26.2%) with Engerix-B (GlaxoSmithKline), whose efficacy in young adults at the dose used here has been confirmed in previous studies. Similar seroconversion rates (anti-HBs > 10 mIU/mL about one month after application of the third dose) were obtained for the Butang, Euvax-B, Hepavax-Gene and Engerix-B vaccines (96.2%, 98.6%, 96.5% and 97.6%, respectively). The frequency of good responders (anti-HBs > 100 mIU/mL) was also similar among students receiving the four vaccines (85.8%, 91.6%, 89.4% and 89.2%, respectively). The geometric mean titers (GMT) of anti-HBs about one month after the third dose obtained with these vaccines were 727.78 ± 6.46 mIU/mL, 2009.09 ± 7.16 mIU/mL, 1729.82 ± 8.85 mIU/mL and 2070.14 ± 11.69 mIU/mL, respectively. The GMT of anti-HBs induced by the Euvax-B and Engerix-B vaccines were higher than those obtained with the Butang vaccine (p < 0.05); this difference was not significant when comparing the other vaccines two-by-two. No spontaneous adverse effects attributable to the application of any dose of the four vaccines were reported.

High diagnostic efficiency of IgM-ELISA with the use of multiple antigen peptides (MAP1) from T. gondii ESA (SAG-1, GRA-1 and GRA-7), in acute toxoplasmosis

The main serological marker for the diagnosis of recent toxoplasmosis is the specific IgM antibody, along with IgG antibodies of low avidity. However, in some patients these antibodies may persist long after the acute/recent phase, contributing to misdiagnosis in suspected cases of toxoplasmosis. In the present study, the diagnostic efficiency of ELISA was evaluated, with the use of peptides derived from T. gondii ESA antigens, named SAG-1, GRA-1 and GRA-7. In the assay referred to, we studied each of these peptides individually, as well as in four different combinations, as Multiple Antigen Peptides (MAP), aiming to establish a reliable profile for the acute/recent toxoplasmosis with only one patient serum sample. The diagnostic performance of the assay using MAP1, with the combination of SAG-1, GRA-1 and GRA-7 peptides, demonstrated better discrimination of the acute/recent phase from non acute/recent phase of toxoplasmosis. Our results show that IgM antibodies to MAP1 may be useful as a serological marker, enhancing the diagnostic efficiency of the assay for acute/recent phase of toxoplasmosis.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

Different culture media containing methyldopa for melanin production by Cryptococcus species

INTRODUCTION: Melanin production by species of Cryptococcus is widely used to characterize C. neoformans complex in mycology laboratories. This study aims to test the efficacy of methyldopa from pharmaceutical tablet as a substrate for melanin production, to compare the production of melanin using different agar base added with methyldopa, and to compare the melanin produced in those media with that produced in Niger seed agar and sunflower seed agar by C. neoformans, C. laurentii, and C. albidus. Two isolates of each species, C. neoformans, C. laurentii, and C. albidus, and one of Candida albicans were used to experimentally detect conditions for melanin production. METHODS: The following media were tested: Mueller-Hinton agar (MHA), brain and heart infusion agar (BHIA), blood agar base (BAB), and minimal medium agar (MMA), all added with methyldopa, and the media Niger seed agar (NSA) and sunflower seed agar (SSA). RESULTS: All isolates grew in most of the culture media after 24h. Strains planted on media BAB and BHIA showed growth only after 48h. All isolates produced melanin in MMA, MHA, SSA, and NSA media. CONCLUSIONS: Methyldopa in the form pharmaceutical tablet can be used as a substrate for melanin production by Cryptococcus species; minimal medium plus methyldopa was more efficient than the BAB, MHA, and BHIA in the melanin production; and NSA and SSA, followed by MMA added with methyldopa, were more efficient than other media studied for melanin production by all strains studied.