Morphological characterization of the hemocytes of the pulmonate snail Biomphalaria tenagophila

The blood cells of the pulmonate snail Biomphalaria tenagophila, an important transmiter of the trematode Schistosoma mansoni in Brazil, were examined by ligth and transmission electron microscopy (TEM). Two hemocyte types were identified: hyalinocytes and granulocytes. Hyalinocytes are small young (immature), poorly spreading cells, which have a high nucleocytoplasmic ratio and are especially rich in free ribosomes. They do not appear to contain lysosome-like bodies and represent less than 10% of the circulating hemocytes. Granulocytes are larger hemocytes which readily spread on glass surface and which strongly react to the Gomori substrate, indicating the enzyme acid phosphatase usually found in lysosomes. Ultra-structurally, they contain a well-developed rough endoplasmic reticulum, dictyosomes and some some lysosome-like dense bodies. Granulocytes do not exhibit a characteristic granular aspect and the few granules observed in the cytoplasm should correspond to a lysosome system. They were named granulocytes instead of amoebocytes to use the same terminology adopted for Biomphalaria glabrata in order to make easier comparative studies. This is a preface study for more specific investigations on the functional activities of the blood cells of B. tenagophila and their interactions with the trematode parasite.

Morphologic and morphometric aspects of oenocytes of Apis mellifera queens and workers in different phases of life

Oenocytes of adult workers and queens of Apis mellifera L. were studied in different ages or life stages, by means of morphometric and histologic techniques. In workers, the oenocytes were found in the head, near the mandibles and in the abdomen, immersed in the parietal fat body mainly below the sterna, close to the wax gland. In queens, two populations of oenocytes different in size and localization were found within the parietal and visceral fat body, respectively. The oenocytes of workers and queens show the presence of acid lipids and acid phosphatase. The role of these cells in the castes differences is discussed.

Standardization and evaluation of ELISA for the serodiagnosis of amoebic liver abscess

An ELISA test for the serological diagnosisof amoebic liver abscess (ALA) was standardized and evaluated in sera from three groups of patients: (1) three patients with diagnosis confirmed by isolation of the parasite,(2) thirty seven patients with diagnosis established by clinical findings and ultrasound studies and (3) seven patients whose diagnosis were established by clinical findings and a positive double immunodifusion test. Ninety one serum samples from healthy subjects and 22 from patients with other liver or parasitic diseases were also included in the study. the optimum concentration of Entamoeba histolytica antigen was 1.25 µg/ml and optimum dilutions of serum and anti-human IgG-alkaline phosphatase conjugate were 1:400 and 1:4000 respectively. The cut-off point of the ELISA test in this study was an absorbance value of 0.34. The test parameters were: sensitivity = 95.7 per cent, specificty = 100 per cent, positive predictive value = 100 per cent and negative predictive value = 98.2 per cent.The ELISA test was found to be of great use as a diagnostic tool for the establishment of amoebic etiology in patients with clinical supposition of ALA. The test could also be used for seroepidemiological surveys of the prevalence of invasive amoebiasis in a given population, since it allows the processing of a greater number of samples at a lower cost tahn other serological tests.

Role of the cyclosporin-sensitive transcription factor NFAT1 in the allergic response

Proteins belonging to the NFAT (nuclear factor of activated T cells) family of transcription factors are expressed in most immune cell types, and play a central role in the transcription of cytokine genes, such as IL-2, IL-4, IL-5, IL-13, IFN-gamma, TNF-alpha, and GM-CSF. The activity of NFAT proteins is regulated by the calcium/calmodulin-dependent phosphatase calcineurin, a target for inhibition by CsA and FK506. Recently, two different groups have described that mice lacking the NFAT1 transcription factor show an enhanced immune response, with tendency towards the development of a late Th2-like response. This review evaluates the possible role of NFAT proteins in the Th2 immune response and in the eosinophil-mediated allergic response.

Adenosine Deaminase and Guanosine Deaminase Activities in Sera of Patients with Viral Hepatitis

In order to investigate purin and primidin metabolism pathways in hepatitis, adenosine deaminase (ADA) and guanosine deaminase (GDA) activities in sera of patients with different types and manifestations of viral hepatitis disease (A, B, C, D, E, chronic, acute) were investigated and compared with the control group of healthy individuals. Hepatitis cases were classified with respect to their serological findings and clinics. When compared all the hepatitis cases with the controls, levels of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase enzymes, as well as ADA and GDA, were significantly higher than the control group (p<0.01). Levels of ADA and GDA in hepatitis cases were determined as 26.07±11.98 IU/l and 2.37±1.91 IU/l, respectively. When compared their ADA and GDA levels amongst the classified hepatitis groups, there was no difference in ADA levels amongst cases (p>0.05). However, GDA levels in hepatitis A group were closed to the controls. Increase in serum ADA activities in hepatitis forms may be dependent on and reflect the increase in phagocytic activity of macrophages and maturation of T-lymphocytes, and may be valuable in monitoring in viral hepatitis cases.

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 µg/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.

Laboratory diagnosis of Schistosomiasis in areas of low transmission: a review of a line of research

After 57 years of successful control of schistosomiasis in Venezuela, the prevalence and intensity of infection have declined. Approximately 80% of the individuals eliminate less than 100 eggs/g of stools, therefore morbidity is mild and the majority are asymptomatic. The sensitivity of Kato-Katz decreases to approximately 60%. Available serological methods for the detection of circulating antigens only reach a 70% of sensitivity. Tests based on the detection of antibodies by immunoenzymatic assays have been improved. The circumoval precipitine test has shown a high sensitivity (97%), specificity (100%), and correlation with oviposition, being considered the best confirmatory diagnostic test. Additionally to the classical immunoenzymatic assays, the development of the alkaline phosphatase immunoassay, allowed to reach a 100% specificity with an 89% sensitivity. Recently, we have developed a modified ELISA in which the soluble egg antigen is treated with sodium metaperiodate (SMP-ELISA) in order to eliminate the glycosilated epitopes responsible for the false positive reactions. The specificity and sensitivity reaches 97% and 99%, respectively. Synthetic peptides from the excretory-secretory enzymes, cathepsin B (Sm31) legumain (Sm32) and cathepsin D (Sm45), have been synthesized. The combination of two peptides derived from the Sm31 have been evaluated, reaching a sensitivity of 96% when analyzed independently and with a 100% specificity. Antibodies raised in rabbits against peptides derived from the Sm31 and Sm32 are currently evaluated in two different antigen-capture-based assays. The development of a simple, cheap and reliable test that correlates with parasite activity is a major goal.

The association of genetic markers and malaria infection in the Brazilian Western Amazonian region

Almost all individuals (182) belonging to an Amazonian riverine population (Portuchuelo, RO, Brazil) were investigated for ascertaining data on epidemiological aspects of malaria. Thirteen genetic blood polymorphisms were investigated (ABO, MNSs, Rh, Kell, and Duffy systems, haptoglobins, hemoglobins, and the enzymes glucose-6-phosphate dehydrogenase, glyoxalase, phosphoglucomutase, carbonic anhydrase, red cell acid phosphatase, and esterase D). The results indicated that the Duffy system is associated with susceptibility to malaria, as observed in other endemic areas. Moreover, suggestions also arose indicating that the EsD and Rh loci may be significantly associated with resistance to malaria. If statistical type II errors and sample stratification could be ruled out, hypotheses on the existence of a causal mechanism or an unknown closely linked locus involved in susceptibility to malaria infection may explain the present findings.

Efficacy of Citrus reticulata and Mirazid in treatment of Schistosoma mansoni

This work has been carried out to investigate the effect of Schistosoma mansoni infection on mice livers after treatment with the ethanolic extract of Citrus reticulata root or the oleo-resin extract from Myrrh of Commiphora molmol tree (Mirazid), as a new antishistosomal drug. Marker enzymes for different cell organelles were measured; succinate dehydrogenase (SDH); lactate dehydrogenase (LDH) and its isoenzymes; glucose-6-phosphatase (G-6-Pase); acid phosphatase (AP) and 5'- nucleotidase. Liver function enzymes; aspartate aminotransferase (AST); alanine aminotransferase (ALT), and alkaline phosphatase (ALP) were also estimated. Parasitological studies through ova count and worm burden will also be taken into consideration. The results showed a marked reduction in SDH, LDH, AST, and ALT enzyme activities and a significant increase in G-6-Pase, AP, 5'- nucleotidase, and ALP after S. mansoni infection. A noticeable alteration in LDH subunits were also noticed. Treatment with C. reticulata or Mirazid improved all the previous enzyme activities with a noticeable reduction in ova count and worm burden.

Low transmission areas of schistosomiasis in Venezuela: consequences on the diagnosis, treatment, and control

Schistosomiasis low transmission areas as Venezuela, can be defined as those where the vector exists, the prevalence of active cases is under 25%, individuals with mild intensity of infection predominate and are mostly asymptomatic. These areas are the consequence of effective control programs, however, "silent" epidemiological places are difficult to trace, avoiding the opportune diagnosis and treatment of infected persons. Clinic and abdominal ultrasound have not shown to discriminate infected from uninfected persons in areas where besides Schistosoma mansoni, intestinal parasites are the rule. Under these conditions, serology remains as a very valuable diagnostic tool, since it gives a closer approximation to the true prevalence. In this sense, circumoval precipitin test, ELISA-SEA with sodium metaperiodate, and alkaline phosphatase immunoassay joined to coprology allow the identification of the "schistosomiasis cases". In relation to public health, schistosomiasis has been underestimated by the sanitary authorities and the investment on its control is being transferred to other diseases of major social and political relevance neglecting sanitary efforts and allowing growth of snail population. Some strategies of diagnosis and control should be done before schistosomiasis reemergence occurs in low transmission areas.

A contribution to the pathobiology of Biomphalaria glabrata hemocytes

This study attempts to investigate the relationship between the hemocytes in the two compartments: circulating peripheral lymph and the connective tissues. The hemocytes are compared with the vertebrate macrophages and constitute the principal line of defense against external aggression. The hemocytes were counted in circulating hemolymph and their phagocytic capability was evaluated in Schistosoma mansoni-infected Biomphalaria glabrata and the results were compared with those obtained from normal intact control snails. Although the number of circulating hemocytes revealed a mild increase in snails at the 6th week of infection, the overall findings were similar and pointed out that the cells in the two compartments are not functionally connected. However, the hemocytes found within the connective tissues of infected snails showed definite ultrastructural differences in the number and disposition of cytoplasmic prolongations and organelles in comparison with the hemocytes from non-infected snails. Histochemically, the staining for acid phosphatase activity served as a marker to hemocytes, sometimes being found in extracellular material at the foci of parasite-hemocyte interactions.

Correlation of biological serum markers with the degree of hepatic fibrosis and necroinflammatory activity in hepatitis C and schistosomiasis patients

Liver biopsy is the gold-standard method to stage fibrosis; however, it is an invasive procedure and is potentially dangerous. The main objective of this study was to evaluate biological markers, such as cytokines IL-13, IFN-γ, TNF-α and TGF-β, platelets, bilirubins (Bil), alanine aminotransferase (ALT) and aspartate aminotransferase (AST), total proteins, γ-glutamil transferase (γ-GT) and alkaline phosphatase (AP), that could be used to predict the severity of hepatic fibrosis in schistosomiasis and hepatitis C (HC) as isolated diseases or co-infections. The following patient groups were selected: HC (n = 39), HC/hepatosplenic schistosomiasis (HSS) (n = 19), HSS (n = 22) and a control group (n = 13). ANOVA and ROC curves were used for statistical analysis. P < 0.05 was considered significant. With HC patients we showed that TNF-α (p = 0.020) and AP (p = 0.005) could differentiate mild and severe fibrosis. With regard to necroinflammatory activity, AST (p = 0.002), γ-GT (p = 0.034) and AP (p = 0.001) were the best markers to differentiate mild and severe activity. In HC + HSS patients, total Bil (p = 0.008) was capable of differentiating between mild and severe fibrosis. In conclusion, our study was able to suggest biological markers that are non-invasive candidates to evaluate fibrosis and necroinflammatory activity in HC and HC + HSS.

Biomphalaria alexandrina snails as immunogens against Schistosoma mansoni infection in mice

Despite effective chemotherapy, schistosomiasis remains the second largest public health problem in the developing world. Currently, vaccination is the new strategy for schistosomiasis control. The presence of common antigenic fractions between Schistosoma mansoni and its intermediate host provides a source for the preparation of a proper vaccine. The objective of this paper is to evaluate the nucleoprotein extracted from either susceptible or resistant snails to protect against schistosomiasis. The vaccination schedule consisted of a subcutaneous injection of 50 µg protein of each antigen followed by another inoculation 15 days later. Analyses of marker enzymes for different cell organelles [succinate dehydrogenase, lactate dehydrogenase (LDH), glucose-6-phosphatase, acid phosphatase and 5'-nucleotidase] were carried out. Energetic parameters (ATP, ADP, AMP, phosphate potentials, inorganic phosphate, amino acids and LDH isoenzymes) were also investigated. The work was extended to record worm and ova counts, oogram determination in the liver and intestine and the histopathological pattern of the liver. The nucleoprotein of susceptible snails showed reduction in worm and ova counts by 70.96% and 51.31%, respectively, whereas the nucleoprotein of resistant snails showed reductions of 9.67% and 16.77%, respectively. In conclusion, we found that the nucleoprotein of susceptible snails was more effective in protecting against schistosomiasis.

A transient increase in total head phosphotyrosine levels is observed upon the emergence of Aedes aegypti from the pupal stage

Phosphorylation and dephosphorylation of protein tyrosine residues constitutes a major biochemical regulatory mechanism for the cell. We report a transient increase in the total tyrosine phosphorylation of the Aedes aegypti head during the first days after emergence from the pupal stage. This correlates with an initial reduction in total head protein tyrosine phosphatase (PTP) activity. Similarly, phosphotyrosine (pTyr)-containing bands are seen in extracts prepared from both male and female heads and are spread among a variety of structures including the antennae, proboscis and the maxillary palps combined with the proboscis. Also, mosquitoes treated with sodium orthovanadate, a classical PTP inhibitor, show reduced blood-feeding activity and higher head tyrosine phosphorylation levels. These results suggest that pTyr-mediated signalling pathways may play a role in the initial days following the emergence of the adult mosquito from the pupal stage.

Interferon-γ inhibits group B Streptococcus survival within human endothelial cells

Endothelial dysfunction is a major component of the pathophysiology of septicaemic group B Streptococcus (GBS) infections. Although cytokines have been shown to activate human umbilical vein endothelial cells (HUVECs), the capacity of interferon (IFN)-γ to enhance the microbicidal activity of HUVECs against GBS has not been studied. We report that the viability of intracellular bacteria was reduced in HUVECs activated by IFN-γ. Enhanced fusion of lysosomes with bacteria-containing vacuoles was observed by acid phosphatase and the colocalisation of Rab-5, Rab-7 and lysosomal-associated membrane protein-1 with GBS in IFN-γ-activated HUVECs. IFN-γ resulted in an enhancement of the phagosome maturation process in HUVECs, improving the capacity to control the intracellular survival of GBS.