Therapeutic Efficacy of a Mixed Formulation of Conventional and PEGylated Liposomes Containing Meglumine Antimoniate, Combined with Allopurinol, in Dogs Naturally Infected with Leishmania infantum.

The treatment of dogs naturally infected with Leishmania infantum using meglumine antimoniate (MA) encapsulated in conventional liposomes (LC) in association with allopurinol has been previously reported to promote a marked reduction in the parasite burden in the main infection sites. Here, a new assay in naturally infected dogs was performed using a novel liposome formulation of MA consisting of a mixture of conventional and long-circulating (PEGylated) liposomes (LCP), with expected broader distribution among affected tissues of the mononuclear phagocyte system. Experimental groups of naturally infected dogs were as follows: LCP plus Allop, receiving LCP intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg of body weight/dose) at 4-day intervals plus allopurinol at 30 mg/kg/12 h per os (p.o.) during 130 days (LCP+Allop); LC plus Allop, receiving LC intravenously as 2 cycles of 6 doses (6.5 mg Sb/kg/dose) plus allopurinol during 130 days (LC+Allop); Allop, treated with allopurinol only; and a nontreated control. Parasite loads were evaluated by quantitative PCR in liver, spleen, and bone marrow tissue and by immunohistochemistry in the ear skin, before treatment, just after treatment, and 4 months later. The LCP+Allop and LC+Allop groups, but not the Allop group, showed significant suppression of the parasites in the liver, spleen, and bone marrow 4 months after treatment compared to the pretreatment period or the control group. Only LCP+Allop group showed significantly lower parasite burden in the skin in comparison to the control group. On the basis of clinical staging and parasitological evaluations, the LCP formulation exhibited a more favorable therapeutic profile than the LC one, being therefore promising for the treatment of canine visceral leishmaniasis.

Assessment of histological liver alterations in dogs naturally infected with Leishmania infantum.

BACKGROUND: The liver plays a central role in the development of canine visceral leishmaniasis. Studies of natural infection in animals and humans indicate a direct relationship between resolution of infection and the formation and maturation of granulomas in the liver. However, in contrast to other reports in the literature, the present study found no differences in the characteristics of hepatic granulomas that could be related to resistance or susceptibility to Leishmania. Here, we describe the hepatic alterations observed in dogs with differing clinical manifestations of visceral leishmaniasis in an endemic area in the state of Bahia, Brazil. METHODS: We examined 148 animals in an endemic area. The animals were clinically examined, and the infection was determined by ELISA, spleen aspirate culture and quantitative PCR. The animals were grouped into asymptomatic or symptomatic based on the number of signs of LV. The histological liver evaluation was performed in a blinded way. RESULTS: Our results indicated no association between the characteristics of granulomas and clinical presentation. We found an association between the intensity of this inflammatory response and parasite load in the animals' spleens. It is important to note that while hepatic alterations, such as portal and perivascular inflammation and the presence of larger amounts of granulomas, were linked with higher parasite loads, we found the inverse to be true with respect to intrasinusoidal lymphocytosis, the formation of intrasinusoidal inflammatory cell aggregates and Kupffer cell hypertrophy. CONCLUSIONS: Our findings suggest that the presence of mononuclear inflammatory cells inside the sinusoids is more important than that of organized granulomas in terms of the containment of parasitism by the host. We suggest that the presence of granulomas indicates the failure of a first line of defense mechanism in the control of parasite infection, which could be related to the presence of inflammatory cells and Kupffer cell hypertrophy inside the sinusoids. We further demonstrated that dogs with active Leishmania spp. infection present a higher frequency of inflammatory changes in the liver. In addition to being correlated with the severity of clinical manifestation, these hepatic alterations were also associated with changes in hematological and biochemical parameters.

Host and parasite responses in human diffuse cutaneous leishmaniasis caused by L. amazonensis.

Diffuse cutaneous leishmaniasis (DCL) is a rare form of leishmaniasis where parasites grow uncontrolled in diffuse lesions across the skin. Meta-transcriptomic analysis of biopsies from DCL patients infected with Leishmania amazonensis demonstrated an infiltration of atypical B cells producing a surprising preponderance of the IgG4 isotype. DCL lesions contained minimal CD8+ T cell transcripts and no evidence of persistent TH2 responses. Whereas localized disease exhibited activated (so-called M1) macrophage presence, transcripts in DCL suggested a regulatory macrophage (R-Mϕ) phenotype with higher levels of ABCB5, DCSTAMP, SPP1, SLAMF9, PPARG, MMPs, and TM4SF19. The high levels of parasite transcripts in DCL and the remarkable uniformity among patients afforded a unique opportunity to study parasite gene expression in this disease. Patterns of parasite gene expression in DCL more closely resembled in vitro parasite growth in resting macrophages, in the absence of T cells. In contrast, parasite gene expression in LCL revealed 336 parasite genes that were differently upregulated, relative to DCL and in vitro macrophage growth, and these transcripts may represent transcripts that are produced by the parasite in response to host immune pressure.

Hepatic fibropoiesis in dogs naturally infected with Leishmania (Leishmania) infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol.

Hepatic fibropoiesis in canine visceral leishmaniasis (CVL) were evaluated by histological (morphometrical collagen deposition) and immunohistochemical assays characterizing alpha-actin (α-SMA), vimentin, calprotectin (L1 antigen), and TGF-ß in 46 naturally infected dogs with Leishmania infantum treated with liposome-encapsulated meglumine antimoniate and allopurinol separately and in combination. Six treatment groups were defined: meglumine antimoniate encapsulated in nanometric liposomes (LMA), allopurinol (ALLOP); liposome-encapsulated meglumine antomoniate combined with allopurinol (LMA+ALLOP); empty liposomes (LEMP); empty liposomes combined with allopurinol (LEMP+ALLOP) and saline. Relative liver weight was lower in LMA, LMA+ALLOP, and ALLOP groups compared to the LEMP control. Significantly lower granulomatous chronic inflammatory reaction was seen in the ALLOP group compared to a control group. Calprotectin was lowest in liver of those dogs showing lower numbers of intralobular hepatic granulomas. Collagen deposits were significantly higher in LMA compared to ALLOP, LEMP+ALLOP, and Saline groups. LMA+ALLOP group collagen deposition was higher than dogs treated only with allopurinol. Immunohistochemical analysis showed significant higher α-SMA in hepatic stellate cells (HSCs), hepatic perisinusoidal cells, in control groups than LMA+ALLOP and LEMP+ALLOP. Alpha-actin and Vimentin positive cells were diffusely distributed throughout the liver parenchyma in the hepatic lobule, mainly in HSCs. Vimentin expression was significantly higher in the saline group than in the ALLOP group. Our data suggest that allopurinol inhibits HSC and results in lower collagen deposits in liver during CVL progression, as supported by the significantly lower expression of TGF-ß in the ALLOP group compared to other groups. Results demonstrated that treatment with allopurinol inhibited chronic granulomatous inflammatory reaction and hepatic fibrosis in CVL.

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis.

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

Immunohistochemical study of hepatic fibropoiesis associated with canine visceral leishmaniasis.

Hepatic fibropoiesis has been confirmed in canine visceral leishmaniasis. In fibrotic disease, hepatic stellate cells (HSC) play an important role in fibropoiesis, undergoing activation by TGF-ß to acquire characteristics of myofibroblasts. These cells show extensive capacity for proliferation, motility, contractility, collagen synthesis and extracellular matrix component synthesis. The aim of this work was to identify markers of HSC activation in 10 symptomatic and 10 asymptomatic dogs naturally infected with Leishmania (Leishmania) infantum. Eight uninfected dogs were used as controls. Alpha-actin (α-SMA), vimentin and cytokeratin were investigated by immunohistochemistry as HSC markers. The cytokine TGF-ß in tissue was also evaluated by immunohistochemistry. All infected dogs showed higher numbers of reticular fibres than controls. Fibropoiesis found in infected dogs was always associated with the presence of parasites and chronic granulomatous hepatitis. Positive correlation was found among fibropoiesis, parasite tissue load and expression of α-SMA. There was no correlation between fibropoiesis, vimentin and cytokeratin markers. The expression of cytokine TGF-ß was higher in infected dogs than in controls, but not significantly different between symptomatic and asymptomatic dogs. These results confirm previous work describing the intense hepatic fibropoiesis in dogs naturally infected with Leishmania infantum, but now associated them with overexpression of TGF-ß, where α-SMA may be a superior marker for activated HSC cells in CVL.

Expression of regulatory T cells in jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum.

Using flow cytometry, we evaluated the frequencies of CD4(+) and CD8(+) T cells and Foxp3(+) regulatory T cells (Tregs) in mononuclear cells in the jejunum, colon, and cervical and mesenteric lymph nodes of dogs naturally infected with Leishmania infantum and in uninfected controls. All infected dogs showed chronic lymphadenitis and enteritis. Despite persistent parasite loads, no erosion or ulcers were evident in the epithelial mucosa. The colon harbored more parasites than the jejunum. Frequencies of total CD4(+), total Foxp3, and CD4(+) Foxp3(+) cells were higher in the jejunum than in the colon. Despite negative enzyme-linked immunosorbent assay (ELISA) serum results for cytokines, levels of interleukin-10 (IL-10), gamma interferon (IFN-γ), transforming growth factor beta (TGF-ß), and tumor necrosis factor alpha (TNF-α) were higher in the jejunum than in the colon for infected dogs. However, IL-4 levels were higher in the colon than in the jejunum for infected dogs. There was no observed correlation between clinical signs and histopathological changes or immunological and parasitological findings in the gastrointestinal tract (GIT) of canines with visceral leishmaniasis. However, distinct segments of the GIT presented different immunological and parasitological responses. The jejunum showed a lower parasite load, with increased frequencies and expression of CD4, Foxp3, and CD8 receptors and IL-10, TGF-ß, IFN-γ, and TNF-α cytokines. The colon showed a higher parasite load, with increasing expression of IL-4. Leishmania infantum infection increased expression of CD4, Foxp3, IL-10, TGF-ß, IFN-γ, and TNF-α and reduced CD8 and IL-4 expression in both the jejunum and the colon.

Complement activation-related pseudoallergy in dogs following intravenous administration of a liposomal formulation of meglumine antimoniate / Pseudoalergia relacionada à ativação do complemento em cães após administração intravenosa de uma formulação lipossomal de antimoniato de meglumina

The increasing use of nanotechnologies in advanced therapies has allowed the observation of specific adverse reactions related to nanostructures. The toxicity of a novel liposome formulation of meglumine antimoniate in dogs with visceral leishmaniasis after single dose has been investigated. Groups of 12 animals received by the intravenous route a single dose of liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg), empty liposomes (GII) or isotonic saline (GIII). Evaluation of hematological and biochemical parameters showed no significant changes 4 days after administration. No undesired effects were registered in the GIII. However, adverse reactions were observed in 67.7% of dogs from both groups that received liposomal formulations. The side effects began moments after bolus administration and disappeared during the first 15 minutes after treatment. Prostation, sialorrhea and defecation were the most frequent clinical signs, registered in 33.3% and 41.6 % of animals from the groups GI and GII, respectively. Tachypnea, mydriasis, miosis, vomiting and cyanosis were also registered in both groups. The adverse reactions observed in this study were attributed to the activation of the complement system by lipid vesicles in a phenomenon known as Complement Activation-Related Pseudoallergy (CARPA). The influence of the physical-chemical characteristics of liposomal formulation in the triggering of CARPA is discussed.

O crescente uso das nanotecnologias nas terapias avançadas tem permitido a observação de reações adversas específicas relacionadas às nanoestruturas. A toxicidade de uma nova formulação lipossomal de antimoniato de meglumina após dose única foi avaliada em cães com leishmaniose visceral. Grupos de 12 animais receberam por via intravenosa uma dose única de antimoniato de meglumina lipossomal (grupo I [GI], 6,5 mg Sb/kg), lipossomas vazios (GII) ou solução salina isotônica (GIII). A avaliação de parâmetros hematológicos e bioquímicos não revelou alterações significativas quatro dias após a administração. Nenhum efeito indesejável foi registrado no GIII. No entanto, reações adversas foram observadas em 67,7% dos cães de ambos os grupos que receberam formulações lipossomais. Os efeitos colaterais iniciaram momentos após a administração em "bolus" e desapareceram no decurso dos primeiros 15 minutos após o tratamento. Prostração, sialorréia e defecação foram os sinais clínicos mais frequentes, registrados em 33,3% e 41,6% dos animais dos grupos GI e GII, respectivamente. Taquipnéia, midríase, miose, vômitos e cianose também foram registrados em ambos os grupos. As reações adversas observadas neste trabalho foram atribuídas à ativação do sistema complemento pelas vesículas lipídicas em fenômeno conhecido como Pseudoalergia Relacionada à Ativação do Complemento (PARAC). A influência das características físico-químicas da formulação lipossomal no desencadeamento de PARAC é abordada.

Ecto-nucleotidase activities of promastigotes from Leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome.

BACKGROUND: Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. METHODOLOGY/PRINCIPAL FINDINGS: Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. CONCLUSIONS/SIGNIFICANCE: Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

Efficacy of combined therapy with liposome-encapsulated meglumine antimoniate and allopurinol in treatment of canine visceral leishmaniasis.

An innovative liposomal formulation of meglumine antimoniate (LMA) was recently reported to promote both long-term parasite suppression and reduction of infectivity to sand flies in dogs with visceral leishmaniasis. However, 5 months after treatment, parasites were still found in the bone marrow of all treated dogs. In order to improve treatment with LMA, the present study aimed to evaluate its efficacy in combination with allopurinol. Mongrel dogs naturally infected with Leishmania infantum were treated with six doses of LMA (6.5 mg Sb/kg of body weight/dose) given at 4-day intervals, plus allopurinol (20 mg/kg/24 h per os) for 140 days. Comparison was made with groups treated with LMA, allopurinol, empty liposomes plus allopurinol, empty liposomes, and saline. Dogs remained without treatment from day 140 to 200 after the start of treatment. The drug combination promoted both clinical improvement of dogs and significant reduction in the parasitic load in bone marrow and spleen on days 140 and 200 compared to these parameters in the pretreatment period. This is in contrast with the other protocols, which did not result in significant reduction of the bone marrow parasite load on day 200. Strikingly, the combined treatment, in contrast to the other regimens, induced negative quantitative PCR (qPCR) results in the liver of 100% of the dogs. Both xenodiagnosis and skin parasite determination by qPCR indicated that the drug combination was effective in blocking the transmission of skin parasites to sand flies. Based on all of the parasitological tests performed on day 200, 50% of the animals that received the combined treatment were considered cured.

NADPH phagocyte oxidase knockout mice control Trypanosoma cruzi proliferation, but develop circulatory collapse and succumb to infection.

(•)NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91(phox) (-/-) or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with (•)NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi.

Reduced tissue parasitic load and infectivity to sand flies in dogs naturally infected by Leishmania (Leishmania) chagasi following treatment with a liposome formulation of meglumine antimoniate.

The toxicity and antileishmanial effectiveness of a novel liposome formulation of meglumine antimoniate in mongrel dogs with visceral leishmaniasis (VL) obtained from a region where VL is endemic in Brazil have been investigated. Groups of 12 animals received by the intravenous route four doses (with 4-day intervals) of either liposomal meglumine antimoniate (group I [GI], 6.5 mg Sb/kg of body weight/dose), empty liposomes (GII), or isotonic saline (GIII). Evaluation of markers of hematopoietic, hepatic, and renal functions before and just after treatment showed no significant change. On the other hand, transitory adverse reactions, including prostration, defecation, tachypnea, and sialorrhea, were observed during the first 15 min after injections in GI and GII. Parasitological evaluation of sternal bone marrow 4 days after the last dose showed a significant reduction of parasite burden in GI, compared to the other groups. Immunocytochemical evaluations of the skin, bone marrow, cervical lymph nodes, livers, and spleens of dogs for parasites, 150 days after treatment, indicated significant parasite suppression (higher than 95.7%) in the lymph nodes, livers, and spleens of GI, compared to control groups. Feeding of Lutzomyia longipalpis phlebotomines on dogs from GI, 150 days after treatment, resulted in a significant reduction of sand fly infection efficiency, compared to feeding on animals from GII and GIII. This is the first report of both long-term parasite suppression and reduction of infectivity to sand flies in naturally infected dogs following treatment with a liposome-encapsulated drug. Importantly, this was achieved using a 20-fold-lower cumulative dose of Sb than is used for conventional antimonial treatment.

In vitro binding and survival assays of Leishmania parasites to peripherical blood monocytes and monocyte-derived macrophages isolated from dogs naturally and experimentally infected with Leishmania (Leishmania) chagasi.

BACKGROUND: There are a few works considering the characterization of canine monocyte-derived macrophages as well as a standardized procedure for isolation, culture, and infection of these cells with Leishmania. We have performed several modifications in order to improve the canine monocyte-derived macrophage cultures. In addition, we have done a comparative study between monocytes and monocyte-derived macrophages from dogs naturally and experimentally infected with L. chagasi. RESULTS: In the presence of exogenous serum, opsonized Leishmania promastigotes binds better to monocytes/macrophages than without serum. Otherwise, this binding occurs due to the strict correlation between the opsonized biologic particles with the third receptor of the complement (CR3-CD11b/CD18). In fact, our assays with CD11b confirmed the importance of this receptor for canine cells and the L. chagasi experimental system. Moreover, monocytes obtained from naturally infected dogs have shown a higher number of monocytes bounded to promastigotes. The experimental results regarding survival have shown that promastigote forms of opsonized L. chagasi were more infective, because we found higher numbers of promastigotes bound to the different cells. As a consequence, after forty-eight hours of binding, higher numbers of amastigotes appeared inside monocyte-macrophages. CONCLUSION: These studies have given support to continue comparative studies involving canine monocytes, monocyte-derived macrophages and peritoneal macrophages. Since we have standardized the canine cell culture, we are looking forward to determining the phenotypic properties of these cells before and after L. chagasi infection using flow cytometry.

Platelet activating factor receptor-deficient mice present delayed interferon-gamma upregulation and high susceptibility to Leishmania amazonensis infection.

We investigated the role of the platelet activation factor (PAF) receptor (PAFR) in the outcome of infection with Leishmania amazonensis. PAFR deficient (PAFR(-/-)) mice were infected with L. amazonensis and the course of infection was followed. We found that PAFR(-/-) mice in the C57BL/6 background were more susceptible to infection with L. amazonensis than the wild-type controls, as seen both by lesion size and parasite number at the site of infection. Interferon (IFN)-gamma production was delayed in PAFR(-/-) mice, and lower levels of Ccl5 were found in lesions. Expression of nitric oxide synthase-2 mRNA was found impaired in PAFR(-/-) associated with higher levels of arginase-1 mRNA. Moreover, higher levels of antibodies were produced in response to L. amazonensis by PAFR(-/-) mice. We conclude that signaling through the PAFR is essential for the ability of the murine host to control L. amazonensis infection by driving an adequate immune response.

Toxoplasma gondii: in vitro and in vivo activities of the hydroxynaphthoquinone 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone alone or combined with sulfadiazine.

The compound 2-hydroxy-3-(1'-propen-3-phenyl)-1,4-naphthoquinone (PHNQ6) was evaluated for activity against Toxoplasma gondii, alone or combined with sulfadiazine. Treatment with PHNQ6 combined with sulfadiazine protected at least 70 and 90% of mice infected with RH and EGS strains, respectively. Mice were treated with PHNQ6 (50 mg/kg/day) alone or combined with sulfadiazine (40 mg/L) 30 days after infection with P strain. The number of brain cysts was lower in mice treated with PHNQ6 alone or combined with sulfadiazine compared to that in control mice. Degenerated bradyzoites were observed in animals treated with PHNQ6. Infectivity of bradyzoites treated with PHNQ6 alone or combined with sulfadiazine was inhibited after in vitro incubation.

Anti-inflammatory and analgesic effects of atorvastatin in a rat model of adjuvant-induced arthritis.

Statins exert favorable effects on lipoprotein metabolism but may also possess anti-inflammatory effects. Here, we explored the effects of atorvastatin in a model of adjuvant-induced arthritis in rat. Oral treatment with atorvastatin (1-10 mg/kg) from days 10 to 15 after arthritis induction caused inhibition of the increase in paw volume. Maximal inhibition occurred at a dose of 10 mg/kg. At this dose, atorvastatin markedly ameliorated the histopathological findings of joints obtained from day 16 of arthritic animals. This was mirrored by an effective blockade of neutrophil influx, as assessed by the tissue myeloperoxidase levels. The concentrations of the cytokines interleukin-1beta, interleukin-6 and tumor necrosis factor-alpha and the chemokines CCL5 and CCL2 were significantly decreased in arthritic rats treated with atorvastatin. In contrast, the levels of interleukin-10 were enhanced by the drug treatment. The drug also prevented the hypernociception observed in the inflamed joints. These data clearly illustrate the therapeutic potential of a statin-sensitive pathway in inflammatory arthritis.

A sensitive flow cytometric methodology for studying the binding of L. chagasi to canine peritoneal macrophages.

BACKGROUND: The Leishmania promastigote-macrophage interaction occurs through the association of multiple receptors on the biological membrane surfaces. The success of the parasite infection is dramatically dependent on this early interaction in the vertebrate host, which permits or not the development of the disease. In this study we propose a novel methodology using flow cytometry to study this interaction, and compare it with a previously described "in vitro" binding assay. METHODS: To study parasite-macrophage interaction, peritoneal macrophages were obtained from 4 dogs and adjusted to 3 x 10(6) cells/mL. Leishmania (Leishmania) chagasi parasites (stationary-phase) were adjusted to 5 x 10(7) cells/mL. The interaction between CFSE-stained Leishmania chagasi and canine peritoneal macrophages was performed in polypropylene tubes to avoid macrophage adhesion. We carried out assays in the presence or absence of normal serum or in the presence of a final concentration of 5% of C5 deficient (serum from AKR/J mice) mouse serum. Then, the number of infected macrophages was counted in an optical microscope, as well as by flow citometry. Macrophages obtained were stained with anti-CR3 (CD11b/CD18) antibodies and analyzed by flow citometry. RESULTS: Our results have shown that the interaction between Leishmania and macrophages can be measured by flow cytometry using the fluorescent dye CFSE to identify the Leishmania, and measuring simultaneously the expression of an important integrin involved in this interaction: the CD11b/CD18 (CR3 or Mac-1) beta2 integrin. CONCLUSION: Flow cytometry offers rapid, reliable and sensitive measurements of single cell interactions with Leishmania in unstained or phenotypically defined cell populations following staining with one or more fluorochromes.

Toxoplasma gondii: in vivo expression of BAG-5 and cyst formation is independent of TNF p55 receptor and inducible nitric oxide synthase functions.

Wild type, TNFRp55(-/-), iNOS(-/-) and IFN-gamma(-/-) mice were infected with Toxoplasma gondii strain ME-49, and the central nervous system (CNS), lungs, liver, spleen, heart and kidneys were examined for the presence of parasites expressing tachyzoite-specific (SAG-1) and bradyzoite-specific (BAG-5) antigens. During the acute phase of infection, the peripheral organs, but not the CNS, of the IFN-gamma(-/-) mice are heavily parasitized by tachyzoites and there are no signs of parasites expressing BAG-5. In contrast, the tissues from TNFRp55(-/-) and inducible nitric oxide synthase (iNOS)(-/-) mice, mainly the CNS, presented high numbers of parasites expressing SAG-1 and/or BAG-5. Tachyzoite transformation into bradyzoite and cyst development was shown to be normal in the tissues from TNFRp55(-/-) and iNOS(-/-) mice, as indicated by the high numbers of BAG-5/PAS positive cysts. Consistently, reactivation of infection in IFN-gamma(-/-) mice was rapid and characterized by a dramatic increase in SAG-1, contrasting with slow course in the TNFRp55(-/-) or iNOS(-/-) mice associated with a relatively small increase in SAG-1- and/or BAG-5-positive parasites. In conclusion, our results suggest that the control of multiplication of tachyzoites is largely dependent on endogenous IFN-gamma with partial involvement of TNFRp55 and iNOS. In contrast, induction of BAG-5 expression and cyst formation during toxoplasmosis seems to be dependent on IFN-gamma, but independent of TNFRp55 and iNOS functions.

Leishmania (Leishmania) chagasi is not vertically transmitted in dogs.

The most frequent and most important mode of human or canine visceral leishmaniasis (CVL) transmission is through the bite of infected sand flies. This study investigates Leishmania (Leishmania) chagasi vertical transmission in offspring of naturally infected dogs. Thus 63 puppies from 18 female dogs with CVL were used. Parasite presence was evaluated through parasitologic and histopathologic examination of lymphatic organs, as well as polymerase chain reaction (PCR) on samples from adults (milk, uterus, placenta, spleen, liver and bone marrow) and offspring (spleen, liver, lymph nodes and bone marrow). PCR sensitivity and specificity were calculated using a microscope as the gold standard on samples of bone marrow, spleen and liver. Specificity was 100% for all organs and sensitivity was 100% for bone marrow, 71.4% for spleen and 66.6% for liver. Bone marrow smears (n = 63), histopathology and imprint of spleen (n = 25), liver (n = 25) and lymph nodes (n = 25) were performed to evaluate congenital transmission in the 63 offspring. PCR was done on 92 samples collected from 56 of the offspring. No test performed on the offspring was positive. It was not possible to confirm vertical transmission of CVL (95% confidence interval for the observed prevalence), despite positive PCR in the placenta of seropositive adults.