Meniscal transection rather than excision increases pain behavior and structural damage in experimental osteoarthritis in mice.

OBJECTIVE: To evaluate pain behavior and structural damage in mice subjected to either meniscal transection or removal. METHODS: Mice (10/group) were subjected to transection of the medial collateral and anterior cruciate ligaments (ACLT/MCLT) followed by either transection (meniscotomy) or removal (meniscectomy) of the medial meniscus. A control group was subjected only to transection of the ligaments. Pain was assessed using the electronic pressure-meter paw test. Cell influx, measured in joint exudates, and joint histopathology were assessed after 49 days. Four other groups subjected to meniscotomy received indomethacin, the inducible nitric oxide synthase (iNOS) inhibitor 1400W, morphine or the vehicles. RESULTS: Both meniscotomy and meniscectomy groups displayed persistent and significant increase in pain behavior as compared to controls, being significantly more severe in the former. Cell influx was more intense in the meniscotomy as compared to the meniscectomy group. Structural damage at the tibia, but not at the femur, was also more severe in the meniscotomy group. Indomethacin and 1400W partially but significantly reduced pain whereas morphine abrogated pain behavior in meniscotomized mice. CONCLUSION: Meniscal transection rather than resection promotes more severe pain and structural damage in mice. Administration of opioids, cyclooxygenase and nitric oxide (NO) synthase inhibitors provide analgesia in this model. Careful description of the structures damaged is crucial when reporting experimental osteoarthritis (OA).

The role of the SLAM-SAP signaling pathway in the modulation of CD4+ T cell responses

The signaling lymphocytic activation molecule (SLAM), present on the surface of hematopoietic cells, can regulate some events of the immune responses. This modulatory action is associated with the capacity of SLAM to interact with an intracytoplasmic adapter, such as SLAM-associated protein (SAP). SLAM is constitutively expressed in most of these cells, is rapidly induced after antigenic or inflammatory stimuli, and participates in the immunological synapse. Defects in the function of the SLAM-SAP pathway contribute to immunological abnormalities, resulting in autoimmune diseases, tumors of the lymphoid tissues and inadequate responses to infectious agents. Initially, the role of SLAM was investigated using an anti-SLAM monoclonal antibody (α-SLAM mAb) identified as an agonist of the SLAM-SAP pathway, which could induce the production of interferon-γ and could redirect the immune response to a T helper 1 (Th1) cell profile. However, in this review we postulate that the SLAM-SAP pathway primarily induces a Th2 response and secondarily suppresses the Th1 response.

The role of the SLAM-SAP signaling pathway in the modulation of CD4+ T cell responses.

The signaling lymphocytic activation molecule (SLAM), present on the surface of hematopoietic cells, can regulate some events of the immune responses. This modulatory action is associated with the capacity of SLAM to interact with an intracytoplasmic adapter, such as SLAM-associated protein (SAP). SLAM is constitutively expressed in most of these cells, is rapidly induced after antigenic or inflammatory stimuli, and participates in the immunological synapse. Defects in the function of the SLAM-SAP pathway contribute to immunological abnormalities, resulting in autoimmune diseases, tumors of the lymphoid tissues and inadequate responses to infectious agents. Initially, the role of SLAM was investigated using an anti-SLAM monoclonal antibody (α-SLAM mAb) identified as an agonist of the SLAM-SAP pathway, which could induce the production of interferon-γ and could redirect the immune response to a T helper 1 (Th1) cell profile. However, in this review we postulate that the SLAM-SAP pathway primarily induces a Th2 response and secondarily suppresses the Th1 response.

The susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis.

Over the last few years, several cases of feline leishmaniasis (FL) with cutaneous and visceral forms have been reported around the world. Nonetheless, the real susceptibility of cats to infection with Leishmania spp. and the outcome of leishmaniasis in these animals are poorly understood. Experimental studies on feline models will contribute to the knowledge of natural FL. Thus, in order to determine the susceptibility of domestic cats (Felis catus) to experimental infection with Leishmania braziliensis, 13 stray cats were infected with 10(7) promastigotes by the intradermal route in the ear and nose simultaneously and followed up for 72 weeks. Soon after infection, the earliest indication of a lesion was a papule on the ear at 2 weeks post-infection (w.p.i.). The emergence of satellite papules around the primary lesion was observed about 4 w.p.i. Two weeks later these papules coalesced and formed a huge and irregular nodule. Thereafter, there was lesion dissemination to the external and marginal surface of the ipsilateral ear, and later to the contralateral ear. At 10 w.p.i., some nodules became ulcerated. Nose lesions presented a similar evolution. At both sites, the largest lesion sizes occurred at 10 w.p.i. and started to decrease 15 days later. Ear and nose nodules healed at 32 and 40 w.p.i., respectively. Specific L. braziliensis IgG antibody titers (optical density> or = 0.01 as positive result) were detected as early as 2 w.p.i. (0.09 +/- 0.02) in only three animals (23%), and all cats had positive titers at 20 w.p.i. (0.34 +/- 0.06). Only three animals (38%) continued to show positive serology at 72 w.p.i. (0.08 +/- 0.02). Up to that time, none of the cats had lesion recurrence. In a feline model of cutaneous leishmaniasis, it seems that there is no correlation between active lesions and positive serology. The implications of these data are discussed.

Differences in gamma interferon production in vitro predict the pace of the in vivo response to Leishmania amazonensis in healthy volunteers.

The initial encounter of Leishmania cells and cells from the immune system is fundamentally important in the outcome of infection and determines disease development or resistance. We evaluated the anti-Leishmania amazonensis response of naive volunteers by using an in vitro priming (IVP) system and comparing the responses following in vivo vaccination against the same parasite. In vitro stimulation allowed us to distinguish two groups of individuals, those who produced small amounts of gamma interferon (IFN-gamma) (n = 16) (low producers) and those who produced large amounts of this cytokine (n = 16) (high producers). IFN-gamma production was proportional to tumor necrosis factor alpha and interleukin 10 (IL-10) levels but did not correlate with IL-5 production. Volunteers who produced small amounts of IFN-gamma in vitro remained low producers 40 days after vaccination, whereas high producers exhibited increased IFN-gamma production. However, 6 months after vaccination, all individuals tested produced similarly high levels of IFN-gamma upon stimulation of their peripheral blood mononuclear cells with Leishmania promastigotes, indicating that low in vitro producers respond slowly in vivo to vaccination. In high IFN-gamma producers there was an increased frequency of activated CD8(+) T cells both in vitro and in vivo compared to the frequency in low producers, and such cells were positive for IFN-gamma as determined by intracellular staining. Such findings suggest that IVP responses can be used to predict the pace of postvaccination responses of test volunteers. Although all vaccinated individuals eventually have a potent anti-Leishmania cell-mediated immunity (CMI) response, a delay in mounting the CMI response may influence resistance against leishmaniasis.

1,8-cineole protects against liver failure in an in-vivo murine model of endotoxemic shock.

The effects of 1,8-cineole on D-galactosamine/lipopolysaccharide (GalN/LPS)-induced shock model of liver injury was investigated in mice. The co-administration of GalN (700 mg kg(-1), i.p.) and LPS (5 microg kg(-1), i.p.) greatly elevated serum concentrations of tumour necrosis factor-alpha (TNF-alpha), alanine aminotransferase and aspartate aminotransferase, and induced massive hepatic necrosis and lethality in 100% of control mice. Pretreatment with 1,8-cineole (400 mg kg(-1), p.o.) and dexamethasone (1 mg kg(-1), s.c.), 60 min before GalN/LPS, offered complete protection (100%) against the lethal shock and acute elevation in serum TNF-alpha and serum transaminases. Hepatic necrosis induced by GalN/LPS was also greatly reduced by both 1,8-cineole and dexamethasone treatment. The results indicate that 1,8-cineole protects mice against GalN/LPS-induced liver injury through the inhibition of TNF-alpha production, and suggest that 1,8-cineole may be a promising agent to combat septic-shock-associated pathologies.

In vitro and in vivo Leishmanicidal activity of 2-hydroxy-3-(3-methyl-2-butenyl)-1,4-naphthoquinone (lapachol).

This study aims to evaluate the in vitro and in vivo leishmanicidal activity of lapachol, a naphthoquinone found in the seeds and heartwood of certain tropical plants, and to compare its efficacy with a reference drug, sodium stibogluconate (Pentostam(R)). These compounds (0.0125-4.0 mg/mL) were evaluated in vitro against intracellular amastigotes of Leishmania (Viannia) braziliensis (LVb), then tested in an animal model (hamster) to try to reproduce the leishmanicidal activity. In vitro, lapachol exhibited an anti-amastigote effect, whereas in vivo it did not prevent the development of LVb-induced lesions at an oral dose of 300 mg/kg/day for 42 days. Pentostam(R) demonstrated a significant anti-amastigote effect in vitro for LVb and apparent clinical cure in vivo (60 mg/kg/day). However, it could not completely eradicate parasites from the tissues of infected animals. The observation that lapachol exerts leishmanicidal activity in vitro without offering significant protection against LVb-infected lesions in hamsters suggests that lapachol in vivo might possibly inhibit the microbicidal functioning of macrophages. Alternatively, it might be transformed into an inactive metabolite(s) or neutralized, losing its leishmanicidal activity. It is also possible that an optimal and sustained plasma level of the drug could not be achieved at the dose used in this study.

Periarthritis promotes gait disturbance in zymosan-induced arthritis in rats.

OBJECTIVE AND DESIGN: We studied the contribution of periarthritis and synovitis to gait disturbance in zymosan (Zy)-induced arthritis. METHODS: Sixty Wistar rats were subjected to injection of Zy (1 mg) into their right knee joints. A first group of animals (GI) had Zy injected through the intact skin. A second group (GII) had Zy injected directly into the articular cavity after excision of the skin and subcutaneous tissue surrounding the joint. Gait disturbance was evaluated using the rat-knee joint incapacitation test. Increase in vascular permeability and cell influx were assessed in joint fluids and joint histology was performed. RESULTS: Zy injection induced a dose-dependent gait disturbance which was maximal at the third/fourth hour of arthritis, being significantly greater in GI rats, whereas cell influx (neutrophils > or = 80%) was maximal at the sixth hour. Cell influx and increase in vasopermeability did not differ between both groups. Histology revealed no significant difference between GI and GII. A third group (GIII), subjected to immune-complex arthritis, that received anti-bovine serum albumin (BSA) antibodies intra-articularly and BSA i.v., did not present gait disturbance, despite the increase in cell counts. CONCLUSIONS: Vascular permeability increase and cell influx are phenomena independent of gait disturbance. Neutrophils do not seem to contribute to development of gait disturbance in Zy arthritis. Sensitization of specific pain receptors in periarticular rather than in synovial tissue is responsible for gait disturbance in Zy-induced arthritis.

[Control of canine visceral leishmaniasis: comparison of results from a rapid elimination program of serum-reactive dogs using an immunoenzyme assay and slower elimination of serum-reactive dogs using filter paper elution indirect immunofluorescence]. / Controle do calazar canino: comparação dos resultados de um programa de eliminação rápida de cães sororreagentes por ensaio imuno-enzimático com outro de eliminação tardia de cães sororreagentes por teste de imunofluorescência indireta de eluato de papel filtro.

The kala-azar control program, adopted by the Fundação Nacional de Saúde-FNS (National Health Foundation) has not been able to reduce to an acceptable level the incidence of human cases. The diagnostic method utilized is a blood eluate immunofluorescence. A dogs diagnosed as infected is eliminated a mean of eighty days after the blood collection. The low sensitivity of the test used and the continuing residence of the infected dog in the region due to the elimination delay may be critical in the lack of success of this program. In this study, the FNS standard canine control method is compared to a strategy based on ELISA identification of infected dog and elimination within 7 days. In both study areas the canine seroprevalence was noted ten months before and ten months after the intervention. In the routine FNS area a 9% decrease in seroprevalence was noted, compared to statistically significant greater 27%, reduction (p = 0.0015) in the ELISA intervention area.

Controle do calazar canino: comparação dos resultados de um programa de eliminação rápida de cães sororreagentes por ensaio imuno-enzimático com outro de eliminação tardia de cães sororreagentes por teste de imunofluorescência indireta de eluato de papel filtro / Control of canine visceral leishmaniasis: comparison of results from a rapid elimination program of serum-reactive dogs using an immunoenzyme assay and slower elimination of serum-reactive dogs using filter paper elution indirect immunofluorescence

O programa de controle do calazar, adotado pela Fundação Nacional de Saúde (FNS), não tem conseguido reduzir a níveis aceitáveis a incidência de calazar humano. Utiliza como método diagnóstico a imunofluorescência de eluato de sangue e sacrifíca os cães infectados com uma média de 80 dias após a coleta. A longa permanência do cão infectado na área e a baixa sensibilidade do teste utilizado podem ser importantes para esta falha. Neste trabalho, compara-se o programa de rotina da FNS, com outra estratégia baseada na identificação de cães infectados pelo enzyme-linked immunosorbent assay (ELISA) e eliminação dos cães infectados dentro de um prazo máximo de 7 dias. A prevalência do calazar canino foi medida nas duas áreas, antes e 10 meses após a medição inicial. Na área submetida ao controle de rotina da FNS observou-se um decréscimo de 9% na prevalência, enquanto na submetida ao método proposto a redução observada foi de 27%, sendo esta diferença sinificativamente maior (p = 0,0015).

The kala-azar control program, adopted by the Fundação Nacional de Saúde-FNS (National Health Foundation) has not been able to reduce to an acceptable level the incidence of human cases. The diagnostic method utilized is a blood eluate immunofluorescence. A dogs diagnosed as infected is eliminated a mean of eighty days after the blood collection. The low sensitivity of the test used and the continuing residence of the infected dog in the region due to the elimination delay may be critical in the lack of success of this program. In this study, the FNS standard canine control method is compared to a strategy based on ELISA identification of infected dog and elimination within 7 days. In both study areas the canine seroprevalence was noted ten months before and ten months after the intervention. In the routine FNS area a 9% decrease in seroprevalence was noted, compared to statistically significant greater 27%, reduction (p = 0.0015) in the ELISA intervention area.

Involvement of nitric oxide in the pathogenesis of cyclophosphamide-induced hemorrhagic cystitis.

The involvement of nitric oxide (NO) and the potential modulation of NO synthase (NOS) activity by platelet-activating factor were investigated in a rat model of cyclophosphamide-induced hemorrhagic cystitis. Male Wistar rats received a single intraperitoneal injection of cyclophosphamide, and cystitis was evaluated 6, 12, 24, 48, and 72 hours later by determining the changes in bladder wet weight and plasma protein extravasation and the macro- and microscopic morphological alterations. In addition, NOS activity and NADPH-diaphorase histochemistry were studied in bladder tissues. Normal bladders showed extensive NADPH-diaphorase staining and a high level of constitutive NOS whereas the activity of inducible NOS was almost undetectable. Cyclophosphamide dose- and time-dependently increased the bladder wet weight and bladder plasma protein extravasation. These events were accompanied at a microscopic level by urothelial necrosis, sloughing, ulceration, hemorrhage, and leukocyte infiltration. Cyclophosphamide also increased the levels of inducible NOS but reduced those of constitutive NOS. The NOS inhibitors L-NG-nitroarginine methyl ester and L-NG-nitroarginine significantly reduced the cyclophosphamide-induced plasma protein extravasation and urothelial damage. This reduction was completely reversed by L-arginine but not by D-arginine. The administration of the platelet-activating factor antagonist BN 52021 decreased the cyclophosphamide-induced plasma protein extravasation as well as the rise in inducible NOS activity but had no effect on the fall in constitutive NOS activity. These results suggest that endogenous NO participates in the urothelial damage and in the inflammatory events leading to cyclophosphamide-induced hemorrhagic cystitis. Platelet-activating factor also seems to be involved in the pathogenesis of this condition, possibly by inducing NOS.

Bubonic leishmaniasis: a common manifestation of Leishmania (Viannia) braziliensis infection in Ceara, Brazil.

Enlarged regional lymph nodes have been reported to accompany the cutaneous lesions of Leishmania (Viannia) braziliensis (= L. braziliensis). A survey in Ceara State, Brazil indicated that 77% of persons (456 of 595) with parasitologically confirmed cutaneous leishmaniasis reported lymphadenopathy in addition to skin lesions. A group of 169 persons with recently diagnosed leishmaniasis and lymph nodes measuring > or = 2 cm in diameter (mean = 3.6 cm, maximum = 10.5 cm) underwent detailed clinical examination. Lymphadenopathy preceded the skin lesions in more than two-thirds of these, on the average by two weeks. Cultures of lymph node aspirates yielded Leishmania more frequently (86%) than cultures of aspirates of skin (53%) or biopsies of skin (74%). Parasites were isolated from the peripheral blood of one patient. Persons with lymphadenopathy gave a history of fever and had enlarged livers or spleens more often than a comparison group of 50 persons with cutaneous lesions but no lymphadenopathy. Persons with lymphadenopathy had more intense leishmanin skin reactions and lymphocyte proliferation following stimulation with specific antigens, whereas persons without lymphadenopathy had a higher frequency of previous infection. Isolates of parasites from both groups were identified as L. braziliensis. These data demonstrate the early spread of L. braziliensis beyond the skin and suggest differences in host immunity between persons with and without lymphadenopathy. Leishmaniasis braziliensis should be considered in cases of unexplained lymphadenopathy in endemic areas.

Potential role for interleukin-10 in the immunosuppression associated with kala azar.

Patients with acute kala azar are generally nonreactive in a number of immunologic assays, including T cell proliferation and generation of macrophage-activating cytokines, principally IFN-gamma, in response to leishmania antigens in vitro. To test for potential immunosuppressive factors, a series of T cell lines and clones were established from patients with acute kala azar, from patients after chemotherapy for kala azar, and from skin test-positive adults from the same endemic region. Although CD4+ T cell lines and clones could be readily established from the skin test-positive adults, lines and clones from acute or treated patients were heavily biased in expression of CD8+. The CD8+ cells from acute patients did not themselves release cytokines in response to leishmania antigens in vitro, but markedly affected the cytokine profile of peripheral blood mononuclear cells isolated 1 yr later after recovery. Addition of the CD8+ cells caused inhibition of lymphoproliferation and IFN-gamma release, with augmentation of IL-6 and IL-10 release. The inhibitory effects of the CD8+ cells could be partially abrogated by antibodies to IL-10 but not by antibodies to IL-4. Analysis of four patients with acute kala azar demonstrated release of IL-10 that could not be demonstrated in supernatants from asymptomatic skin test-positive individuals. Generation of IL-10 may contribute to the profound suppression of IFN-gamma release that occurs during kala azar due to Leishmania chagasi.

Correlates of Leishmania-specific immunity in the clinical spectrum of infection with Leishmania chagasi.

Patients from across the spectrum of clinical manifestations of Leishmania chagasi infection were evaluated for in vitro correlates of immunity. Peripheral blood mononuclear cells were assayed for parasite-specific lymphoproliferation, cytokine generation, and the capacity to activate autologous macrophages to kill intracellular amastigotes. Patients with acute kala-azar were generally unreactive in each of these assays. Children with subclinical infection demonstrated relatively low levels of proliferation and interferon-gamma production, but none went on to develop overt kala-azar during the study. Patients evaluated after therapy for kala-azar demonstrated yet higher levels of lymphoproliferation and cytokine generation and produced low but significant levels of cytokines in vitro in response to parasite antigens, but not during the activation of infected macrophages. Finally, peripheral blood mononuclear cells from adults with positive delayed-type hypersensitivity responses and no history of kala-azar showed the broadest reactivity in vitro. These patients' cells generated the largest amounts of activating cytokines in vitro during the activation of autologous macrophages to a leishmanicidal state.