Anti-Rickettsia spp. antibodies in free-ranging and captive capybaras from southern Brazil

Capybaras (Hydrochaeris hydrochaeris) are among the main hosts of Amblyomma spp. ticks, which is able to transmit Rickettsia species to human beings and animals. Since they are often infested with potential vector ticks, capybaras may be used as sentinels for rickettsiosis, such as the Brazilian Spotted Fever. The aim of the present study was to determine the prevalence of antibodies against Rickettsia spp. using the indirect immunofluorescence assay (IFA) in 21 free-ranging and 10 captive animals from the Zoological Park of the 'Bela Vista Biological Sanctuary' (BVBS), Itaipu Binational, Foz do Iguaçu, Southern Brazil. Antigens of six rickettsial species already identified in Brazil (Rickettsia rickettsii, R. parkeri, R. bellii, R. rhipicephali, R. amblyommii and R. felis) were used for IFA. Ticks from each capybara were collected for posterior taxonomic identification. A total of 19 (61.3%) samples reacted to at least one of tested species. Seropositivity was found in 14 (45.2%), 12 (38.7%), 5 (16.1%), 4 (12.9%), 3 (9.7%) and 3 (9.7%) animals for R. rickettsii, R. bellii, R. parkeri, R. amblyommii, R. felis and R. rhipicephali, respectively. Two captive capybaras presented suggestive titers of R. rickettsii infection and one sample showed homologous reaction to R. parkeri. Only one free-ranging capybara presented evidence R. bellii infection. Ticks collected on capybaras were identified as Amblyomma dubitatum e Amblyomma sp. Results evidenced the rickettsial circulation in the area, suggesting a potential role of capybaras on bacterial life cycle.

Infection by Toxoplasma gondii in Neotropical non-human primates

Toxoplasma gondii (Nicolle et Manceaux, 1909) is an obligatory intracellular protozoan parasite of warm animals, including human and non-human primates. Domestic and wild felids are considered definitive hosts. Several authors have already identified lesions in New World primates caused by T. gondii. Nevertheless, little is known about serological studies on those animals. With this reason, New World non-human primates of the genera Cebus and Callithrix that were apprehended by governmental authorities and sent to the Wildlife Screening Center (Cetas)/IBAMA, at the municipality of Seropédica, state of Rio Janeiro, were bled and sera were submitted to the indirect hemagglutination test for detection of anti-T. gondii antibodies. From 21 sera of Cebus primates, 76.19% (16/21) had anti-T. gondii antibodies. Titles varied from 16 to 2048. In samples from 21 Callithrix, only 4.5% (1/22) had anti-T. gondii antibodies. Only one animal had a title of 32. During all the time those animals were clinical evaluated until sample was collected; none of them had any clinical sign or sequel related to infection by T. gondii. The fact that the origin of these primates is unknown and that there is no information about their feeding habits before captivity makes it difficult to determine the source of T. gondii infection.

Testing pigs of non-technified rearing farms for serum antibodies against Taenia solium in a region of the state of São Paulo, Brazil

Abstract: Taenia solium is a zoonotic tapeworm of great importance in developing countries, due to the occurrence of human taeniasis and cysticercosis. Pigs have an important role in the biological cycle of the parasite as intermediate hosts. The scientific literature has been describing risk factors associated with the occurrence of this disease that must be avoided in countries with poor sanitation, in order to reduce the exposure of swine to the parasite eggs. This research focused on testing pigs of non-technified rearing farms for serum antibodies against Taenia solium in the region of Jaboticabal municipality, in the state of São Paulo, Brazil. The found prevalence was 6.82% (CI 95% 4.18 - 9.45) at animal level and 28.87% (CI 95% 16.74 - 40.40) at herd level. These figures are probably associated with low technification adoption during animal rearing in the studied area, which increased the exposure of the animals to risk factors associated with the occurrence of Taenia solium complex. The results found based on serological evidences of swine cysticercosis in the studied region serves as a warning to public sanitary authorities to improve public health and control T. solium.

Cytokines as determinants of resistance and pathology in human Schistosoma mansoni infection

The role of different cytokines in the peripheral blood mononuclear cell (PBMC) proliferative response and in in vitro granuloma formation was evaluated in a cross-sectional study with patients with the different clinical forms and phases of Schistosoma mansoni infection, as well as a group of individuals "naturally" resistant to infection named normal endemic (NE). The blockage of IL-4 and IL-5 using anti-IL-4 and anti-IL-5 antibodies significantly reduced the PBMC proliferative response to soluble egg (SEA) and adult worm (SWAP) antigens in acute (ACT), chronic intestinal (INT) and hepatosplenic (HS) patients. Similar results were obtained in the in vitro granuloma formation. Blockage of IL-10 had no significant effect on either assay using PBMC from ACT or HS. In contrast, the addition of anti-IL-10 antibodies to PBMC cultures from INT patients significantly increased the proliferative response to SEA and SWAP as well as the in vitro granuloma formation. Interestingly, association of anti-IL-4 and anti-IL-10 antibodies did not increase the PBMC proliferative response of these patients, suggesting that IL-10 may act by modulating IL-4 and IL-5 secretion. Addition of recombinant IL-10 decreased the proliferative response to undetectable levels when PBMC from patients with the different clinical forms were used. Analysis of IFN-g in the supernatants showed that PBMC from INT patients secreted low levels of IFN-g upon antigenic stimulation. In contrast, PBMC from NE secreted high levels of IFN-g. These data suggest that IL-10 is an important cytokine in regulating the immune response and possibly controlling morbidity in human schistosomiasis mansoni, and that the production of IFN-g may be associated with resistance to infection.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

The pathogenesis of tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy

Tropical spastic paraparesis/human T-cell leukemia type I-associated myelopathy (TSP/HAM) is caused by a human T-cell leukemia virus type I (HTLV-I) after a long incubation period. TSP/HAM is characterized by a chronic progressive paraparesis with sphincter disturbances, no/mild sensory loss, the absence of spinal cord compression and seropositivity for HTLV-I antibodies. The pathogenesis of this entity is not completely known and involves a multivariable phenomenon of immune system activation against the presence of HTLV-I antigens, leading to an inflammatory process and demyelination, mainly in the thoracic spinal cord. The current hypothesis about the pathogenesis of TSP/HAM is: 1) presence of HTLV-I antigens in the lumbar spinal cord, noted by an increased DNA HTLV-I load; 2) CTL either with their lytic functions or release/production of soluble factors, such as CC-chemokines, cytokines, and adhesion molecules; 3) the presence of Tax gene expression that activates T-cell proliferation or induces an inflammatory process in the spinal cord; 4) the presence of B cells with neutralizing antibody production, or complement activation by an immune complex phenomenon, and 5) lower IL-2 and IFN-gamma production and increased IL-10, indicating drive to a cytokine type 2 pattern in the TSP/HAM subjects and the existence of a genetic background such as some HLA haplotypes. All of these factors should be implicated in TSP/HAM and further studies are necessary to investigate their role in the development of TSP/HAM.

Detection of human parvovirus B19 in a patient with hepatitis

Parvovirus B19 has been associated by some investigators with cases of severe hepatitis. The aim of the present study was to determine the presence of active parvovirus B19 infection among 129 Brazilian patients with non-A-E hepatitis. The patients were assayed for antibodies against parvovirus B19, IgM class, by ELISA. In IgM-positive cases, parvovirus B19 DNA was assayed by PCR in serum and liver tissue and parvovirus VP1 antigen in liver tissue was assayed by immunohistochemistry. Antibodies against parvovirus B19, IgM class, were detected in 3 (2.3%) of 129 patients with non-A-E hepatitis. Previous surgery and blood transfusions were reported by these 3 patients. One patient was a 56-year-old female with severe hepatitis, with antimitochondrial antibody seropositivity and submassive necrosis at liver biopsy, who responded to corticosteroid therapy. Strong evidence for active parvovirus B19 infection was found in this patient, with parvovirus B19 DNA being detected by PCR in liver tissue. Furthermore, parvovirus VP1 antigen was also detected in liver tissue by immunohistochemistry. The other two IgM-positive patients were chronic hepatitis cases, but active infection was not proven, since neither viral DNA nor antigen were detected in their liver tissues. This and other reports suggest a possible relation between parvovirus B19 infection and some cases of hepatitis.

Estrogen and progesterone receptors in human papilloma virus-related cervical neoplasia

Estrogen (ER) and progesterone (PR) receptors in the normal uterine cervix, cervical intraepithelial neoplasia and invasive carcinoma were studied in consecutive samples from Hospital do Câncer, São Paulo, between 1996 and 1997. Tissue was collected by removing a fragment of the tumoral area using a 5-mm diameter biopsy punch, followed by removal of a macroscopically normal area as close as possible from the tumor. Histopathological confirmation was obtained for all specimens analyzed. A total of 24 normal tissues, 17 cases of cervical intraepithelial neoplasia and 7 of invasive carcinomas were studied. The ER/PR ratio was determined by immunohistochemistry using monoclonal antibodies specific for each receptor. Adjacent tissue slides were submitted to generic PCR for human papillomavirus (HPV) DNA detection followed by typing by dot blot hybridization. About half (45.8%) of the tumors were HPV DNA positive while 29.1% of the patients were also HPV positive in their respective normal tissue. ER was negative in the tumoral epithelium of 11 HPV-positive patients (P = 0.04). There was a trend in the ER distribution in normal tissue that was opposite to that from lesions, but it was not statistically significant (P = 0.069). No difference in ER distribution in stromal tissues was observed between HPV-positive and HPV-negative tissues. PR staining was negative in the epithelium of all cases studied. The results obtained from this small number of cases cannot be considered to be conclusive but do suggest that factors related to viral infection affect the expression of these ER/PR cervix receptors.

The role of calcium, calcium-activated K+ channels, and tyrosine/kinase in psoralen-evoked responses in human melanoma cells

8-Methoxy psoralen (8-MOP) exerts a short-term (24 h) mitogenic action, and a long-term (48-72 h) anti-proliferative and melanogenic action on two human melanoma cell lines, SK-Mel 28 and C32TG. An increase of intracellular calcium concentration was observed by spectrofluorometry immediately after the addition of 0.1 mM 8-MOP to both cell lines, previously incubated with calcium probe fluo-3 AM (5 µM). The intracellular Ca2+ chelator BAPTA/AM (1 µM) blocked both early (mitogenic) and late (anti-proliferative and melanogenic) 8-MOP effects on both cell lines, thus revealing the importance of the calcium signal in both short- and long-term 8-MOP-evoked responses. Long-term biological assays with 5 and 10 mM tetraethylammonium chloride (TEA, an inhibitor of Ca2+-dependent K+ channels) did not affect the responses to psoralen; however, in 24-h assays 10 mM TEA blocked the proliferative peak, indicating a modulation of Ca2+-dependent K+ channels by 8-MOP. No alteration of cAMP basal levels or forskolin-stimulated cAMP levels was promoted by 8-MOP in SK-Mel 28 cells, as determined by radioimmunoassay. However, in C32TG cells forskolin-stimulated cAMP levels were further increased in the presence of 8-MOP. In addition, assays with 1 µM protein kinase C and calcium/calmodulin-dependent kinase inhibitors, Ro 31-8220 and KN-93, respectively, excluded the participation of these kinases in the responses evoked by 8-MOP. Western blot with antibodies anti-phosphotyrosine indicated a 92% increase of the phosphorylated state of a 43-kDa band, suggesting that the phosphorylation of this protein is a component of the cascade that leads to the increase of tyrosinase activity.

Seroprevalence of human herpesvirus 8 infection in children born to HIV-1- infected women in São Paulo, Brazil

Human herpesvirus-8 (HHV-8) appears to be transmitted mainly by sexual contact. However, several studies suggest that in developing countries the infection may be acquired early in life by routes other than sexual transmission. The present study estimated the seroprevalence of HHV-8 in Brazilian children born to HIV-1-infected mothers. The serum samples were collected in a cross-sectional cohort study from 99 children born to HIV-infected mothers (median age 3.27 years; range 1.5-13.8 years) attending the outpatient clinic of the Federal University of São Paulo. IgG antibodies to HHV-8 latency-associated nuclear antigen and lytic phase antigens were detected by immunofluorescence assays. The samples tested were collected from children aged 12 months or older to exclude the possibility of cross-placental antibody transport. The total prevalence of anti-lytic antibodies in this population (5/99; 5%) reveals that HHV-8 infection can occur during childhood. Children aged 1.5 to 2 years had a seroprevalence of 2% (1/50) and children aged 3.25 to 13.8 years had a seroprevalence of 8% (4/49). This difference was not statistically significant, probably because of the small size of the sample, but it suggests that HHV-8 infection occurs more commonly late in infancy. Further prospective studies are necessary to evaluate the timing and risk factors for primary HHV-8 infection in the pediatric population.

Autoimmunity and molecular mimicry in tropical spastic paraparesis/human T-lymphotropic virus-associated myelopathy

Viruses share antigenic sites with normal host cell components, a phenomenon known as molecular mimicry. It has long been suggested that viral infections might trigger an autoimmune response by several mechanisms including molecular mimicry. More than 600 antiviral monoclonal antibodies generated against 11 different viruses have been reported to react with 3.5% of cells specific for uninfected mouse organs. The main pathological feature of tropical spastic paraparesis/human T-lymphotropic virus type I (HTLV-I)-associated myelopathy (TSP/HAM) is a chronic inflammation of the spinal cord characterized by perivascular cuffing of mononuclear cells accompanied by parenchymal lymphocytic infiltration. We detected the presence of autoantibodies against a 98- to 100-kDa protein of in vitro cultured human astrocytes and a 33- to 35-kDa protein from normal human brain in the serum of HTLV-I-seropositive individuals. The two cell proteins exhibited molecular mimicry with HTLV-I gag and tax proteins in TSP/HAM patients, respectively. Furthermore, the location of 33- to 35-kDa protein cross-reaction correlated with the anatomical spinal cord areas (in the rat model) in which axonal damage has been reported in several cases of TSP/HAM patients. Our experimental evidence strongly suggests that the demyelinating process occurring in TSP/HAM may be mediated by molecular mimicry between domains of some viral proteins and normal cellular targets of the spinal cord sections involved in the neurodegeneration.

High expression of human carboxypeptidase M in Pichia pastoris: Purification and partial characterization

Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes

Epidemiological and functional implications of molecular variants of human papillomavirus

Human papillomavirus genomes are classified into molecular variants when they present more than 98% of similarity to the prototype sequence within the L1 gene. Comparative nucleotide sequence analyses of these viruses have elucidated some features of their phylogenetic relationship. In addition, human papillomavirus intratype variability has also been used as an important tool in epidemiological studies of viral transmission, persistence and progression to clinically relevant cervical lesions. Until the present, little has been published concerning the functional significance of molecular variants. It has been shown that nucleotide variability within the long control region leads to differences in the binding affinity of some cellular transcriptional factors and to the enhancement of the expression of E6 and E7 oncogenes. Furthermore, in vivo and in vitro studies revealed differences in E6 and E7 biochemical and biological properties among molecular variants. Nevertheless, further correlation with additional functional information is needed to evaluate the significance of genome intratypic variability. These results are also important for the development of vaccines and to determine the extent to which immunization with L1 virus-like particles of one variant could induce antibodies that cross-neutralize other variants.

Effect of 30 mCi radioiodine on multinodular goiter previously treated with recombinant human thyroid-stimulating hormone

Recombinant human thyroid-stimulating hormone (rhTSH) enhances 131I uptake, permitting a decrease in radiation for the treatment of multinodular goiter (MNG). Our objective was to evaluate the safety and efficacy of a single 0.1-mg dose of rhTSH, followed by 30 mCi 131I, in patients with MNG. Seventeen patients (15 females, 59.0 ± 13.1 years), who had never been submitted to 131I therapy, received a single 0.1-mg injection of rhTSH followed by 30 mCi 131I on the next day. Mean basal thyroid volume measured by computed tomography was 106.1 ± 64.4 mL. 131I 24-h uptake, TSH, free-T4, T3, thyroglobulin, anti-thyroid antibodies, and thyroid volume were evaluated at regular intervals of 12 months. Mean 131I 24-h uptake increased from 18.1 ± 9.7 to 49.6 ± 13.4% (P < 0.001), a median 2.6-fold increase (1.2 to 9.2). Peak hormonal levels were 10.86 ± 5.44 mU/L for TSH (a median 15.5-fold increase), 1.80 ± 0.48 ng/dL for free-T4, 204.61 ± 58.37 ng/dL for T3, and a median of 557.0 ng/mL for thyroglobulin. The adverse effects observed were hyperthyroidism (17.6%), painful thyroiditis (29.4%) and hypothyroidism (52.9%). Thyroid volume was reduced by 34.3 ± 14.3% after 6 months (P < 0.001) and by 46.0 ± 14.6% after 1 year (P < 0.001). Treatment of MNG with a single 0.1-mg dose of rhTSH, followed by a fixed amount of radioactivity of 131I, leads to an efficacious decrease in thyroid volume for the majority of the patients, with a moderate incidence of non-serious and readily treatable adverse effects.

Prokaryotic expression of a novel mouse pro-apoptosis protein PNAS-4 and application of its polyclonal antibodies

Mouse PNAS-4 (mPNAS-4) has 96% identity with human PNAS-4 (hPNAS-4) in primary sequence and has been reported to be involved in the apoptotic response to DNA damage. However, there have been no studies reported of the biological functions of mPNAS-4. In studies conducted by our group (unpublished data), it was interesting to note that overexpression of mPNAS-4 promoted apoptotic death in Lewis lung carcinoma cells (LL2) and colon carcinoma cells (CT26) of mice both in vitro and in vivo. In our studies, mPNAS-4 was cloned into the pGEX-6P-1 vector with GST tag at N-terminal in Escherichia coli strain BL21(DE3). The soluble and insoluble expression of recombinant protein mPNAS-4 (rmPNAS-4) was temperature-dependent. The majority of rmPNAS-4 was insoluble at 37°C, while it was almost exclusively expressed in soluble form at 20°C. The soluble rmPNAS-4 was purified by one-step affinity purification, using a glutathione Sepharose 4B column. The rmPNAS-4 protein was further identified by electrospray ionization-mass spectrometry analysis. The search parameters of the parent and fragment mass error tolerance were set at 0.1 and 0.05 kDa, respectively, and the sequence coverage of search result was 28%. The purified rmPNAS-4 was further used as immunogen to raise polyclonal antibodies in New Zealand white rabbit, which were suitable to detect both the recombinant and the endogenous mPNAS-4 in mouse brain tissue and LL2 cells after immunoblotting and/or immunostaining. The purified rmPNAS-4 and our prepared anti-mPNAS-4 polyclonal antibodies may provide useful tools for future biological function studies for mPNAS.