The Comorbidity of Bipolar Disorder and Migraine: The Role of Inflammation and Oxidative and Nitrosative Stress.

Comorbid migraine in the course of bipolar disorder has been reported as highly prevalent and associated with increased morbidity. Patients with bipolar disorder and comorbid migraine tend to present with higher rates of rapid cycling, increased number of depressive episodes, more severe depression, and increased suicidality when compared to subjects with bipolar disorder alone. Both conditions display similar clinical features, such as relapsing-recovering presentation, and vulnerability to psychological and physical stress. Clinical implications of this association have been well established, however the biological underpinnings involved in both conditions remain poorly understood. Inflammation and oxidative and nitrosative stress seem to play a role as mediators in the cross-sensitization between bipolar disorder and migraine. Therefore, the present study aims to review the role of inflammation, oxidative and nitrosative stress as underlying mechanisms in the natural history of bipolar disorder comorbid with migraine.

An antigenic domain of the Leishmania amazonensis nucleoside triphosphate diphosphohydrolase (NTPDase 1) is associated with disease progression in susceptible infected mice.

An antigenic conserved B domain was previously identified within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, the r-potDomain B, a 6× His-tag polypeptide belonging to the conserved B domain from the potato apyrase, and synthetic peptides LbB1LJ and LbB2LJ derived from the B domain from Leishmania NTPDase 1 were used as molecular tools for studies of the Leishmania amazonensis NTPDase 1. Widespread subcellular location of the specific NTPDase 1 was detected by Western blots of promastigote fractions and ultrastructural immunocytochemical microscopy using immune sera raised against these biomolecules. In addition, the L. amazonensis-infected BALB/c mice were evaluated at 12 to 120 days after infection, which progresses showing typical nodular lesion. High antibody reactivity with either r-potDomain B, LbB1LJ, or LbB2LJ was found in L. amazonensis-infected BALB/c mice indicating the antigenicity of the B domain from NTPDase 1 isoform. The IgG1 antibody reactivity significantly increased at 90-120 days postinfection, 18- to 24-fold when compared to the 12th day, and remained elevated even at 120th after infection, coinciding with the most active stage of the disease. In contrast, significantly higher IgG2a antibody reactivity with each biomolecule was observed at 40th day, about two- to fourfold higher than those found at 12th or 20th day, and decreased along 120-day period. Apparently, the conserved B domain is capable to induce IgG2a production in early disease stages. All together, these results suggest that r-potDomain B or synthetic peptides could be molecular starting points in experimental protocols of immunotherapy and/or vaccination for leishmaniasis.

Isolation and in vitro culture of trypanosomes from Leptodactylus ocellatus from the Atlantic Forest in a new experimental culture medium.

The purpose of this study was to verify the in vitro development of Trypanosoma sp. isolated from Leptodactylus ocellatus frogs under a new protocol using a biphasic medium composed of Novy, McNeal, and Nicolle (NNN) blood agar medium as a solid phase and liver infusion, brain heart infusion, and tryptose (LIBHIT) medium as a liquid phase. Blood forms, collected by cardiac puncture or after the maceration of different organs, were inoculated in culture tubes containing the biphasic medium composed by NNN and LIBHIT. Trypanosomes were observed 4 days postinoculation; most bloodstream trypomastigotes had differentiated into epimastigotes and amastigotes by this time. Trypomastigotes were again observed in older cultures (7 days). Parasites were successfully subcultured for 8 mo in this medium and successfully cryopreserved. The present study provides a new protocol medium for the isolation and culture of anuran trypanosomes.

Leishmania (Leishmania) infantum/chagasi: Histopathological aspects of the skin in naturally infected dogs in two endemic areas.

In the New World, visceral leishmaniasis (VL), which is a progressive disease and frequently fatal, is caused by Leishmania (Leishmania) infantum/chagasi. It is endemic in many regions of Brazil and occasionally occurs in non-endemic regions when dogs from an endemic area are introduced. The aim of the present study is to compare different skin infection patterns of dogs from two leishmaniasis endemic areas. A histological analysis of dogs from Campo Grande, Mato Grosso do Sul state, a region where epidemic episodes are currently taking place, showed dermic inflammatory infiltrates, composed of numerous vacuolated parasitized macrophages, few lymphocytes, plasma cells and many degranulated mast cells. In the other region of the study, São Luís, Maranhão state, the skin of dogs presented a remarkable inflammatory reaction composed mainly of plasma cells, lymphocytes and very few parasites. We concluded that there is a difference in the skin lesion patterns of dogs with leishmaniasis that is directly related to the endemic area where the animals live.

A Leishmania (L.) amazonensis ATP diphosphohydrolase isoform and potato apyrase share epitopes: antigenicity and correlation with disease progression.

A Leishmania (Leishmania) amazonensis ATP diphosphohydrolase isoform was partially purified from plasma membrane of promastigotes by preparative non-denaturing polyacrylamide gel electrophoresis. SDS-PAGE followed by Western blots developed with polyclonal anti-potato apyrase antibodies identified diffuse bands of about 58-63 kDa, possibly glycosylated forms of this protein. By ELISA technique, a significantly higher total IgG antibody level against potato apyrase was found in serum from promastigote-infected mice, as compared to the uninfected mice, confirming both the existence of shared epitopes between the parasite and vegetable proteins, and the parasite ATP diphosphohydrolase antigenicity. By Western blotting, serum from amastigote-infected BALB/c mice recognizes both potato apyrase and this antigenic ATP diphosphohydrolase isoform isolated from promastigotes, suggesting that it is also expressed in the amastigote stage. The infection monitored along a 90-day period in amastigote-infected mice showed reactivity of IgG2a antibody in early steps of infection, while the disappearance of the IgG2a response and elevation of IgG1 antibody serum levels against that shared epitopes were associated with the progression of experimental leishmaniasis. This is the first observation of the antigenicity of a L. (L.) amazonensis ATP diphosphohydrolase isoform, and of the ability of cross-immunoreactivity with potato apyrase to differentiate serologically stages of leishmaniasis in infected mice.

Experimental Leishmania (L.) amazonensis leishmaniasis: characterization and immunogenicity of subcellular fractions.

A technique developed in Trypanosoma cruzi biochemical studies was successfully used to fractionate Leishmania (Leishmania) amazonensis promastigotes. Ultrastructural analyses revealed a membrane fraction (MF) associated to subpellicular microtubules, a ribosomal-rich microsomal fraction (MicF), and a flagellar fraction (FF) free of associated membrane. All fractions proved to be immunogenic through delayed type hypersensitivity reaction assays. Therefore, a protocol was designed to test whether these fractions could elicit a protective response in mice infected by L. (L), amazonensis. The protocol consisted of a BCG injection (as cellular immunity inducer), followed by cyclophosphamide (once its cytotoxic effect is over, this immunosuppressor can increase the number of circulating leukocytes), then an injection with one of the fractions followed by a challenge. When compared to infected control animals, mice injected with any of the fractions presented a smaller footpad swelling, especially those injected with MicF or FF. Macroscopically, immunized mice under modulation by BCG presented no swelling. Histopathological studies performed on day 120 revealed fewer amastigotes and more intense inflammation in lesions of MicF and FF injected mice. Animals injected with MF presented an intermediate pattern. Parasite quantification corroborated these results. The results show that all fractions are potent immunostimulators, but MicF and FF have the strongest protective ability.

Canine visceral leishmaniasis in São José de Ribamar, Maranhão State, Brazil.

Here, we describe the situation of canine visceral leishmaniasis in two villages of São José de Ribamar in Maranhão State/Brazil, where human cases have been registered. Blood samples of 36 household crossbred dogs from Sergio Tamer village and 43 dogs from Quinta village were collected and the serum used for serological diagnosis. An Indirect Fluorescent Antibody Test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were used to detect antibodies against Leishmania. The clinical examination showed that 25% of the canine population of Quinta presented a poor body condition and in 39%, ectoparasites (ticks and fleas) were detected. In both tests, serology revealed that 21% (9 out of 43) of the dogs presented antibodies against Leishmania (55% were asymptomatic and 45% were symptomatic). In the Vila Sérgio Tamer, 25% (9 out of 36) of the dogs were seropositive for Leishmania (66.67% were asymptomatic and 33.33% were symptomatic), 33% presented poor body condition, and 22% have ectoparasites. The clinical signs more frequent were skin lesions. The statistical analysis showed that there was no statistical difference (p>0.05) between the seropositivity of the dogs from the two villages. The same was observed when the clinical signs were compared (p>0.05). Both villages have favorable conditions to maintain the cycle of leishmaniasis.

Canine visceral leishmaniosis in Anastácio, Mato Grosso do Sul state, Brazil.

Canine visceral leishmaniosis (CVL) may be an important factor preceding human outbreaks of the disease. We report that the prevalence of canine visceral leishmaniosis infection has been increasing in recent years in Anastácio town, located in the central western region of Brazil. Serological investigations showed that 75.3% of dogs presented antibody titres ranging from 1/40 to 1/160 in the indirect immunofluorescence antibody test (IFAT). Bone marrow and lymph node aspirates provided positive cultures and furnished parasites for enzymological and serological typing in 42.5% and 41.1% of the cases, respectively. All the strains were typed as Leishmania (L.) chagasi. This is primarily a canine disease that spills over into the human population as a zoonosis. The study showed the epidemiological features of the infection in a region in which the problem of visceral leishmaniosis has been underestimated.

Histopathological studies of visceralized Leishmania (Leishmania) amazonensis in mice experimentally infected.

BALB/c, C57BL/6, and DBA/2 mice were subcutaneously infected in the left footpad by injecting 10(4) Leishmania (Leishmania) amazonensis amastigotes. Mice were sacrificed 20, 30, 40, 60 and 90 days post-infection. Fragments of liver, kidney, spleen, skin, and draining lymph node were collected for histological examination. Light microscopy showed that at 20 days after infection BALB/c mice presented discrete inflammatory infiltrates in the skin made up of eosinophils, lymphocytes, and rare parasitized macrophages. Ninety days post-infection, the dermis showed necrotic tissue, large numbers of mononuclear cells and vacuolated macrophages filled with amastigotes. Forty days post-infection, the draining lymph nodes showed hyperplastic germinal centers, increase of high endothelial venules and apoptosis in germinal center cells. After the first 3 months post-infection, the involvement of spleen, kidney and liver was discreet, being characterized by multifocal inflammatory infiltrates. Eight months after infection, the animals presented metastatic lesions in the contralateral footpad and nose. In deep dermis, there was remarkable proliferation of fibroblasts associated with collagen fibers. The liver showed multifocal granulomas and mononuclear infiltrate around the blood vessels, but no parasites were observed, except in one animal. In some mice there were immature cells of the hematopoietic lineage. Both BALB/c and C57BL/6 mice presented osteonecrosis, which is characterized by pycnotic osteocytes and empty lacunae at the point of inoculation and subsequently, replacement of this tissue by fibrous connective tissue and colonization of the bone marrow. A diffuse inflammatory process composed of mononuclear cells and rare parasites were seen in the kidneys. In one mouse, bone marrow cells were observed in the renal medulla along with where free amastigotes. DBA/2 mice developed a mild infection and they did not visceralize. In conclusion, our data demonstrates that in susceptible mice L. (L.) amazonensis, a causative agent of tegumentary leishmaniasis, causes pathological changes similar to those produced by Leishmania (L.) infantum in both humans and canids.

Extracellular matrix alterations in experimental murine Leishmania (L.) amazonensis infection.

Here we describe extracellular matrix alterations in footpad lesions and draining lymph nodes caused by Leishmania (L.) amazonensis in mouse strains with distinct susceptibilities to this parasite: BALB/c (susceptible), C57BL/6 (intermediate), and DBA/2 (resistant). Changes in ECM were observed mainly in BALB/c mice that, in general, presented tissue damage associated with high parasite burden. Under polarized light, Sirius Red revealed type I collagen that was predominant in the primary lesion in all strains studied at the early phase of infection, but gradually decreased and was replaced by abundant type III collagen fibres in chronic phase lesions. The presence of type III collagen seemed to provide support to inflammatory cells, mainly vacuolated and parasitized macrophages. Laminin expression was not altered during infection by L. (L.) amazonensis in any of the mouse strains studied. Furthermore, the decreased fibronectin expression, in all strains, in areas where amastigotes have been found, indicated that this decline was also not related to the genetic background.

Interaction of Leishmania (L.) chagasi with the Vero cell line.

The Vero cell line, a non-phagocytic cell, has supported the intracellular mechanism of Leishmania (L.) chagasi. This strain (MHOM/BR/501/MS00) was isolated from a human case of visceral leishmaniasis in Mato Grosso do Sul, Brazil and cultivated in Schneider's Drosophila medium with 20% of heat inactivated fetal calf serum. It was allowed to infect the Vero cells at a ratio of 10 to 20 promastigotes per cell. Within six hours of incubation, promastigote forms were found attached to Vero cells without any particular orientation. The number of amastigotes per cell increased during the incubation period. Results showed that promastigotes of L. (L.) chagasi could interact, transform to amastigote forms and multiply in non-phagocytic cells, demonstrating a new model to study the intracellular cycle of this protozoan.

Biological behavior of Leishmania (L.) amazonensis isolated from a human diffuse cutaneous leishmaniasis in inbred strains of mice.

After a subcutaneous injection of 100000 purified amastigotes of an isolate from a diffuse case of cutaneous leishmaniasis caused by the MHOM/BR/76/Ma-5 strain of Leishmania amazonensis, three inbred mouse strains developed a progressive nodular lesion, which evolved to an ulcerated lesion. Based on these data, mice of BALB/c, C57BL/6 or C57BL/10 could be classified as susceptible. The majority of mice developed metastases in the footpads, ear, tail, nose and oral mucosa. Amputation of the members related to the primary lesion was frequent. Experiments using the limiting dilution analysis showed that there was no correlation between lesion and parasite load. It has been demonstrated that these mouse strains could be considered excellent models for mucocutaneous leishmaniasis when infected with L. amazonensis. Metastatic lesions caused destruction of the nasal region with many parasitized macrophages under the epithelial surface of the nasal mucosa. Bone destruction occurred with an extensive inflammatory reaction presenting macrophages heavily parasitized by amastigotes. The parasites also spread to the periodontal ligament and other structures of the oral cavity, which could induce a severe inflammatory process. This study indicates that both nasal and oral lesions in mice infected by L. amazonensis were characterized by an inflammatory reaction with the presence of a high parasite load within macrophages.

Central nervous system involvement in experimental infection with Leishmania (Leishmania) amazonensis.

We describe the pathologic alterations of the central nervous system (CNS) observed in experimental tegumentary leishmaniasis in BALB/c and Swiss mice. The mice were subcutaneously infected with 10(4) amastigotes of Leishmania (Leishmania) amazonensis. Animals were killed and brains were removed for histologic and immunocytochemical studies. Histologic examination showed that 66.6% of infected mice had a discrete hyperemia and inflammatory infiltrate in the meninges, composed of mononuclear cells and neutrophils with no detectable parasites. However, parasitized macrophages were detected in the cerebral parenchyma, as well as mast cells, lymphocytes, and polymorphonuclear cells. Necrosis in the cerebral parenchyma was also observed. Confocal fluorescence microscopy showed that CD8+ T lymphocytes are the major component of the inflammatory infiltrate in the CNS. In addition to these cells, CD4+, CD11b, and dendritic cells are present, in small numbers, in the inflammatory processes of the CNS. Thus, L. amazonensis is able to cross the blood-brain barrier and cause significant pathologic changes in the CNS.

T cell subpopulations in myocardial inflammatory infiltrates detected by confocal microscopy: dose dependence in mice treated with cyclophosphamide during acute Trypanosoma cruzi infection.

In this article, we have characterized cell subpopulations found in the hearts of mice presenting acute Chagas' disease by immunocytochemistry and subjected to different schedules of an immunosuppressive therapy with cyclophosphamide (CY). In this comparative study, CY treatment with different doses was carried out before or after infection with Trypanosoma cruzi Y strain trypomastigotes, enabling us to discriminate the parasitemic kinetics and inflammatory processes in the heart, 12 d after infection. Animals treated with 200 mg/kg of CY 2 d before infection presented high parasitaemia as well as heavy inflammation and low parasite loads in the heart. Mice treated 5 d after infection with the same dose, developed the same parasitaemic peak but were not able to control it. Their heart did not present inflammation, but a high number of parasites could be seen. Animals treated with five 3 mg/kg doses of CY every other day presented heavy inflammatory reaction and low parasitaemia. In this group, as well as the one treated before infection, immunocytochemistry studies have shown predominance of CD8(+) T cells in the myocardium. On the other hand, mice treated with 200 mg/kg of CY 5 d after infection, presented small amounts of CD4(+) T cells while no CD8(+) could be found. These results have confirmed the dose dependence influence of this drug on the T cell populations in the inflammatory infiltrates as well as the importance of the schedule employed.

Trypanosoma cruzi: infection patterns in intact and athymic mice of susceptible and resistant genotypes.

Inbred strains of mice inoculated with the T cruzi Y strain behaved as susceptible (A/J, C3H/HeN), intermediate (BALB/c) or relatively resistant (C57BL/6) with respect to the magnitude of parasitaemia and mortality rate. C57BL/10 mice were susceptible in relation to parasitaemia but resistant when mortality was analyzed. Infection with T cruzi CL strain presented the same results, except for C57BL/6 which behaved as susceptible mice. Athymic mice of various backgrounds revealed no differences in susceptibility, presenting the same dramatic parasitaemia, tissue colonization pattern and no inflammatory reaction in any of the tissues studied. Infection of euthymic and athymic BALB/c mice elicited the production of parasite-specific antibodies, which reached similar levels on the first 9 days but differed after day 13. Serum transfer experiments in BALB/c mice did not show great differences in parasitaemia but altered T. cruzi polymorphism reducing the slender forms in athymic mice. Histopathology of athymic BALB/c mice showed the same tissue tropism when infected either with T cruzi Y or CL strain.

Characterization and cytochemical localization of an ATP diphosphohydrolase from Leishmania amazonensis promastigotes.

An ATP diphosphohydrolase was identified in the plasma membranes isolated from promastigote forms of Leishmania amazonensis. Both ATP and ADP were hydrolysed at similar rates by the enzyme. Other nucleotides such as UTP, GTP and CTP were also degraded, revealing a broad substrate specificity. Adding ATP and ADP simultaneously, the amount of hydrolysis achieved was compatible with the presence of a single enzyme. ATPase activity was not affected by addition of vanadate, ouabain, thapsigargin, dicyclohexylcarbodiimide, oligomycin and bafilomycin A, thus excluding involvement of P-, F- and V-type ATPases. The effects of pH in the range 6.5-8.5 were examined using ATP or p-NPP as substrate. At pH 7.4, the phosphatase activity decreased, and did not show a significant contribution to ATP hydrolysis. In addition, the enzyme was not inhibited by levamisole and ammonium molybdate, excluding alkaline phosphatase and nucleotidase activities, respectively. Sodium azide (5-10 mM) caused inhibition of the ATP and ADP hydrolysis in a dose-dependent manner. Calcium was the best activating metal ion for both ATPase and ADPase activities. Ultrastructural cytochemical microscopy showed ATP diphosphohydrolase on the surface and flagellar pocket of the parasite. We have proposed that L. amazonensis ATP diphosphohydrolase may participate in the salvage pathway of nucleosides.

Leishmanial antigens in the diagnosis of active lesions and ancient scars of American tegumentary leishmaniasis patients.

Cutaneous biopsies (n = 94) obtained from 88 patients with American tegumentary leishmaniasis were studied by conventional and immunohistochemical techniques. Specimens were distributed as active lesions of cutaneous leishmaniasis (n = 53) (Group I), cicatricial lesions of cutaneous leishmaniasis (n = 35) (Group II) and suggestive scars of healed mucosal leishmaniasis patients (n = 6) (Group III). In addition, active cutaneous lesions of other etiology (n = 24) (Group C1) and cutaneous scars not related to leishmaniasis (n = 10) (Group C2) were also included in the protocol. Amastigotes in Group I biopsies were detected by routine histopathological exam (30.2%), imprint (28.2%), culture (43.4%), immunofluorescence (41.4%) and immunoperoxidase (58.5%) techniques; and by the five methods together (79.3%). In Group II, 5.7% of cultures were positive. Leishmanial antigen was also seen in the cytoplasm of macrophages and giant cells (cellular pattern), vessel walls (vascular pattern) and dermal nerves (neural pattern). Positive reaction was detected in 49 (92.5%), 20 (57%) and 4 (67%) biopsies of Groups I, II and III, respectively. Antigen persistency in cicatricial tissue may be related to immunoprotection or, on the contrary, to the development of late lesions. We suggest that the cellular, vascular and neural patterns could be applied in the immunodiagnosis of active and cicatricial lesions in which leishmaniasis is suspected.

Independent genetic control of B- and T-cell tolerance in strains of mouse selected for extreme phenotypes of oral tolerance.

Two strains of mice were genetically selected for susceptibility (TS-Ab/HetS strain) or resistance (TR-Ab/HetS strain) to oral tolerance of the humoral response by using ovalbumin (OVA). The progressive interstrain divergence produced by bi-directional selective breeding during 15 generations demonstrated the polygenic nature of oral tolerance. This paper shows the humoral and delayed-type hypersensitivity (DTH) responses, after intragastric administration of OVA and subsequent immunization with that immunogen in complete Freund adjuvant (CFA). Only the TS-Ab/HetS mice were tolerant for immunoglobulin (Ig)G production with its tolerance degree being the same as that obtained when Al ((OH)(3)) was employed. The DTH reactivity was not correlated to the antibody responsiveness, because both the TS-Ab/HetS and TR-Ab/HetS strains had their DTH reactions suppressed. The cyclophosphamide (Cy) pretreatment prevented DTH suppression on TR-Ab/HetS but do not in TS-Ab/HetS mice. Interstrain difference was also observed for the splenic index in the Cy-treated groups, although the number of splenocytes was the same. Flux cytometry cell analysis showed the recovery of CD3(+) cell numbers in both strains, but only the TR-Ab/HetS mice had their CD4/CD8 pattern restored. These results suggest: firstly, the independent control of DTH and humoral tolerance responsiveness; secondly no support for the clonal anergy concept; and thirdly the matrix proteins neo-synthesis after Cy treatment may facilitate the tolerance abrogation.

Chitinolytic activity in viable spores of encephalitozoon species

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80§C for 10 min or at 55§C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.

Chitinolytic activity in viable spores of Encephalitozoon species.

By employing 4-methylumbelliferyl-beta-D-NN',N"-triacetylchitotriose substrate in a semi quantitative assay, chitinolytic activity in viable spores of Encephalitozoon cuniculi and E. intestinalis was detected and dependence on reaction time, spore concentration, concentration of substrate and temperature were demonstrated. It was possible to block the chitinolytic activity by chitin hydrolysate. By incubation at 80 degrees C for 10 min or at 55 degrees C for 20 min the spores were loosing the chitinolytic activity. Incubation of the spores in trypsin reduced the chitinolytic activity. Cellulase activity could not be detected.