Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis

Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis. Soluble antigen from L. (V.) shawi promastigotes was submitted to reverse phase HPLC to purify F1, F2, F3, F4 and F5 antigens. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g protein. After 1 week, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 8 weeks, those same mice were sacrificed and parasite burden as well as the cellular and humoral immune responses were evaluated. F1 and F5-immunized mice restrained lesion progression and parasite load in the skin. However, only the F1 group was able to control the parasitism in lymph nodes, which was associated with low IL-4 and high IFN-gamma production; IgG2a isotype was increased in this group. Immunizations with F2, F3 and F4 antigens did not protect mice. The capability of antigens to restrain IL-4 levels and increase IFN-gamma was associated with protection, such as in immunization using F1 antigen.

Pharmacological analysis of cyclooxygenase-1 in inflammation

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 μM; COX-2 IC50 = 6.3 μM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.

Whole-body and intravital optical imaging of angiogenesis in orthotopically implanted tumors

The development of drugs for the control of tumor angiogenesis requires a simple, accurate, and economical assay for tumor-induced vascularization. We have adapted the orthotopic implantation model to angiogenesis measurement by using human tumors labeled with Aequorea victoria green fluorescent protein for grafting into nude mice. The nonluminous induced capillaries are clearly visible against the very bright tumor fluorescence examined either intravitally or by whole-body luminance in real time. The orthotopic implantation model of human cancer has been well characterized, and fluorescence shadowing replaces the laborious histological techniques for determining blood vessel density. Intravital images of orthotopically implanted human pancreatic tumors clearly show angiogenic capillaries at both primary and metastatic sites. A quantitative time course of angiogenesis was determined for an orthotopically growing human prostate tumor periodically imaged intravitally in a single nude mouse over a 19-day period. Whole-body optical imaging of tumor angiogenesis was demonstrated by injecting fluorescent Lewis lung carcinoma cells into the s.c. site of the footpad of nude mice. The footpad is relatively transparent, with comparatively few resident blood vessels, allowing quantitative imaging of tumor angiogenesis in the intact animal. Capillary density increased linearly over a 10-day period as determined by whole-body imaging. Similarly, the green fluorescent protein-expressing human breast tumor MDA-MB-435 was orthotopically transplanted to the mouse fat pad, where whole-body optical imaging showed that blood vessel density increased linearly over a 20-week period. These powerful and clinically relevant angiogenesis mouse models can be used for real-time in vivo evaluation of agents inhibiting or promoting tumor angiogenesis in physiological microenvironments.

Administration of interleukin 12 in combination with type II collagen induces severe arthritis in DBA/1 mice.

The induction of arthritis in DBA/1 mice usually requires immunization with the antigen type II collagen emulsified with Mycobacterium tuberculosis in oil. Here we describe that interleukin 12 (IL-12) can replace mycobacteria and cause severe arthritis of DBA/1 mice when administered in combination with type II collagen. Immunization of DBA/1 mice with type II collagen emulsified in oil alone resulted in a weak immune response, and only a few animals (10-30%) developed arthritis. Administration of IL-12 for 5 days simultaneously with each immunization strongly enhanced the anti-type II collagen immune response. Collagen-specific interferon gamma (IFN-gamma) synthesis by ex vivo activated spleen cells was enhanced 3- to 10-fold. IFN-gamma was almost completely produced by CD4+ T cells. Furthermore, the production of collagen-specific IgG2a and IgG2b antibodies was upregulated 10- to 100-fold. As a consequence, the incidence of arthritis in the group of mice immunized with collagen plus IL-12 was very high (80-100%). The developing arthritis was severe, involving approximately 50% of all limbs with strongly increased footpad thickness in most cases. Furthermore, histological examination revealed massive, mainly polymorphonuclear cell infiltration, synovial hyperplasia, cartilage and bone destruction, as well as new bone formation. In many cases, this resulted in the complete loss of joint structure. Neutralization of IFN-gamma in vivo prevented the development of arthritis in collagen-immunized and IL-12-treated mice. In conclusion, our data show that in vivo administered IL-12 can profoundly upregulate a T helper I-type autoimmune response, resulting in severe joint disease in DBA/1 mice.

Physical Health Problems and Environmental Challenges Influence Balancing Behaviour in Laying Hens

With rising public concern for animal welfare, many major food chains and restaurants are changing their policies, strictly buying their eggs from non-cage producers. However, with the additional space in these cage-free systems to perform natural behaviours and movements comes the risk of injury. We evaluated the ability to maintain balance in adult laying hens with health problems (footpad dermatitis, keel damage, poor wing feather cover; n = 15) using a series of environmental challenges and compared such abilities with those of healthy birds (n = 5). Environmental challenges consisted of visual and spatial constraints, created using a head mask, perch obstacles, and static and swaying perch states. We hypothesized that perch movement, environmental challenges, and diminished physical health would negatively impact perching performance demonstrated as balance (as measured by time spent on perch and by number of falls of the perch) and would require more exaggerated correctional movements.We measured perching stability whereby each bird underwent eight 30-second trials on a static and swaying perch: with and without disrupted vision (head mask), with and without space limitations (obstacles) and combinations thereof. Video recordings (600 Hz) and a three-axis accelerometer/gyroscope (100 Hz) were used to measure the number of jumps/falls, latencies to leave the perch, as well as magnitude and direction of both linear and rotational balance-correcting movements. Laying hens with and without physical health problems, in both challenged and unchallenged environments, managed to perch and remain off the ground. We attribute this capacity to our training of the birds. Environmental challenges and physical state had an effect on the use of accelerations and rotations to stabilize themselves on a perch. Birds with physical health problems performed a higher frequency of rotational corrections to keep the body centered over the perch, whereas, for both health categories, environmental challenges required more intense and variable movement corrections. Collectively, these results provide novel empirical support for the effectiveness of training, and highlight that overcrowding, visual constraints, and poor physical health all reduce perching performance.

Mechanisms of B and T lymphocyte accumulation in tumor-draining lymph nodes

Thesis (Master's)--University of Washington, 2016-06

iNOS-producing inflammatory dendritic cells constitute the major infected cell type during the chronic Leishmania major infection phase of C57BL/6 resistant mice.

Leishmania major parasites reside and multiply in late endosomal compartments of host phagocytic cells. Immune control of Leishmania growth absolutely requires expression of inducible Nitric Oxide Synthase (iNOS/NOS2) and subsequent production of NO. Here, we show that CD11b+ CD11c+ Ly-6C+ MHC-II+ cells are the main iNOS-producing cells in the footpad lesion and in the draining lymph node of Leishmania major-infected C57BL/6 mice. These cells are phenotypically similar to iNOS-producing inflammatory DC (iNOS-DC) observed in the mouse models of Listeria monocytogenes and Brucella melitensis infection. The use of DsRed-expressing parasites demonstrated that these iNOS-producing cells are the major infected population in the lesions and the draining lymph nodes. Analysis of various genetically deficient mouse strains revealed the requirement of CCR2 expression for the recruitment of iNOS-DC in the draining lymph nodes, whereas their activation is strongly dependent on CD40, IL-12, IFN-gamma and MyD88 molecules with a partial contribution of TNF-alpha and TLR9. In contrast, STAT-6 deficiency enhanced iNOS-DC recruitment and activation in susceptible BALB/c mice, demonstrating a key role for IL-4 and IL-13 as negative regulators. Taken together, our results suggest that iNOS-DC represent a major class of Th1-regulated effector cell population and constitute the most frequent infected cell type during chronic Leishmania major infection phase of C57BL/6 resistant mice.

Estudo da prevalência e da gravidade da dermatite de contacto em 'frango do campo'

O sector avícola enfrenta atualmente dois desafios muito estimulantes. O primeiro decorre do aumento, que se prevê continuar a crescer, nos níveis de procura de carne de aves no mercado interno e internacional; o segundo decorre do facto da criação avícola ter adotado métodos de produção mais intensivos (kg peso vivo/m2/ano) e em maior escala, i.e. com maior concentração animal na mesma exploração. Este carácter vincadamente “industrial” tem merecido uma natural atenção das sociedades e das autoridades pecuárias no sentido desta economia de escala passar a ter num conjunto de instrumentos legais e técnicos o devido contrapeso para a salvaguarda das aves enquanto ser vivo. O presente trabalho tem como ponto de partida a Directiva 2007/43/CE do Conselho de 28 de Junho, relativa ao estabelecimento de regras mínimas para a proteção de frangos de carne. Em virtude de não existir ainda informação suficiente sobre a forma como a qualidade do maneio animal pode ser monitorizada, ao nível do abate, por médicos veterinários e auxiliares oficiais, em frangos de criação especial segundo os modelos definidos no Regulamento (CE) n.º 543/2008, urge realizar estudos neste domínio. O principal objetivo da realização do presente trabalho de campo foi o estudo da ocorrência das dermatites de contacto plantar (pododermatites) e da bolsa sinovial préesternal em frangos produzidos em sistemas de produção considerados “protetores” do bem-estar animal, designadamente os seguintes: i) ar livre; e, ii) extensivo de interior. O estudo foi efetuado num centro de abate de frangos do campo, em Oliveira de Frades, entre Maio de 20012 e Março de 2013. Os animais abatidos foram criados em explorações com contratos de integração situadas no Distrito de Viseu. Os dados foram recolhidos em 39 bandos diferentes da espécie Gallus domesticus, dos quais 1021 carcaças foram avaliadas após evisceração, o que correspondeu ao exame de uma a cada quinze aves da linha de abate. Para a avaliação da pododermatite foi utilizado o método adaptado pela DGAV, enquanto para a avaliação da bursite esternal foi efetuada tendo em conta o modelo aplicado em perus por Berk em 2002. Apesar do modelo estatístico desenvolvido para a análise dos resultados obtidos no presente trabalho exigir um maior número de observações, foi possível identificar com grande precisão alguns fatores de risco que devem ser realçados pela sua relevância no contexto dos sistemas produtivos escrutinados ou no mecanismo fisiopatológico da dermatite de contacto, nomeadamente os seguintes: (i) a idade das aves que, apesar de não ter sido identificada uma relação directa com os scores de pododermatite e bursite, verificou-se que a idade elevada que os animais tipicamente atingem nos sistemas de produção extensivos está associada a uma taxa superior de rejeições pela inspecção sanitária; (ii) o peso pré-abate que, independentemente da inconsistência defendida por diversos autores em relação à influência do peso vivo do frango industrial sobre a dermatite de contacto, nos animais produzidos em regime extensivo, esta variável pode desempenhar um fator chave para a ocorrência desta lesão. De facto, há que realçar que o peso destes animais tem uma importância fulcral na modelação da biomecânica da ave, incluindo na pressão exercida sobre a superfície plantar; (iii) o tipo de sistema de abeberamento, tendo ficado demonstrado que a selecção do tipo de bebedouro tem uma importância peculiar sobre a ocorrência de pododermatite em “frango de campo”, algo que está provavelmente relacionado com a influência exercida sobre o teor de humidade da cama. Globalmente, as frequências de pododermatite e bursite apuradas neste trabalho devem ser consideradas inquietantes. Esta preocupação eleva-se quando se toma consciência que as aves provieram de regimes considerados “amigáveis” e “sustentáveis”, pelo que urge monitorizar adequadamente aqueles sistemas produtivos, melhorar as suas condições e reanalisar os benefícios ao nível do bem-estar animal.

Leishmania (Viannia) shawi purified antigens confer protection against murine cutaneous leishmaniasis

Leishmania (Viannia) shawi was characterized only recently, and few studies concerning the immunogenic and protective properties of its antigens have been performed. The present study aimed to evaluate the protective potential of the five antigenic fractions isolated from L. (V.) shawi promastigotes in experimental cutaneous leishmaniasis.Soluble antigen from L. (V.) shawi promastigotes was submitted to reverse phase HPLC to purify F1, F2, F3, F4 and F5 antigens. BALB/c mice were immunized once a week for two consecutive weeks by subcutaneous routes in the rump, using 25 mu g protein. After 1 week, groups were challenged in the footpad with L. (V.) shawi promastigotes. After 8 weeks, those same mice were sacrificed and parasite burden as well as the cellular and humoral immune responses were evaluated.F1 and F5-immunized mice restrained lesion progression and parasite load in the skin. However, only the F1 group was able to control the parasitism in lymph nodes, which was associated with low IL-4 and high IFN-gamma production; IgG2a isotype was increased in this group. Immunizations with F2, F3 and F4 antigens did not protect mice.The capability of antigens to restrain IL-4 levels and increase IFN-gamma was associated with protection, such as in immunization using F1 antigen.

Ameliorative effect and potential mechanism of Ermiao san on adjuvant-induced arthritis in rats

Purpose: To investigate the effect and mechanism of action of Ermiao san (EMS), a traditional Chinese herbal formula, on inflammation development and production of inflammatory mediators in adjuvantinduced arthritis (AIA). Methods: AIA was induced by injection of 0.1 ml Freund’s complete adjuvant (FCA, 10 mg/ml) in the left hind footpad of the rats. AIA rats were intragastricly treated with 0.5, 1, 2 g/kg EMS or 0.1 g/kg methotrexate from day 7 to 28 after FCA challenge. Foot volume and histological score were measured. Osteoclast number was calculated by tartrate-resistant acid phosphatase (TRAP) staining assay. Levels of prostaglandin (PG) E2, tumor necrosis factor (TNF) -α and interleukin (IL)-1β in serum were determined by enzyme-linked immunosorbent assay (ELISA) while the level of nitric oxide (NO) in serum was analyzed by Griess reaction method. Results: Foot volume, histological score, osteoclast number and serum levels of TNF-α, IL-1β, PGE2 and NO were all increased in AIA group rats on day 28 after FCA challenge (all p < 0.01) compared with control. EMS (1 and 2 g/kg) significantly decreased the foot volume of AIA rats by 10 % (p < 0.05) and 19 % (p < 0.01), respectively, compared with AIA group. Furthermore, 1 and 2 g/kg EMS significantly reduced histological score by about 28 % (p < 0.05) and 46 % (p < 0.01), respectively, as well as osteoclast number by 12 % (p < 0.05) and 15 % (p < 0.05), respectively, compared with AIA group. In addition, 1 and 2 g/kg EMS significantly decreased the serum levels of TNF-α about 23 % (p < 0.05) and 43 % (p < 0.01), IL-1β by15 % (p < 0.05) and 26 % (p < 0.01), NO 13 % (p < 0.05) and 26 % (p < 0.01) as well as PGE2 by 11 % (p < 0.05) and 15 % (p < 0.01), respectively, compared with AIA group. Conclusion: These results suggest that EMS probably alleviates arthritis development and joint destruction by decreasing the production of inflammatory mediators in AIA rats.