Amanda dos Santos Cavalcanti

Parasite Load Induces Progressive Spleen Architecture Breakage and Impairs Cytokine mRNA Expression in Leishmania infantum- Naturally Infected Dogs

Canine Visceral Leishmaniasis (CVL) shares many aspects with the human disease and dogs are considered the main urban reservoir of L. infantum in zoonotic VL. Infected dogs develop progressive disease with a large clinical spectrum. A complex balance between the parasite and the genetic/immunological background of the host are decisive for infection evolution and clinical outcome. This study comprised 92 Leishmania infected mongrel dogs of various ages from Mato Grosso, Brazil. Spleen samples were collected for determining parasite load, humoral response, cytokine mRNA expression and histopathology alterations. By real-time PCR for the ssrRNA Leishmania gene, two groups were defined; a low (lowP, n = 46) and a high parasite load groups (highP, n = 42). When comparing these groups, results show variable individual humoral immune response with higher specific IgG production in infected animals but with a notable difference in CVL rapid test optical densities (DPP) between highP and lowP groups. Splenic architecture disruption was characterized by disorganization of white pulp, more evident in animals with high parasitism. All cytokine transcripts in spleen were less expressed in highP than lowP groups with a large heterogeneous variation in response. Individual correlation analysis between cytokine expression and parasite load revealed a negative correlation for both pro-inflammatory cytokines: IFNγ, IL-12, IL-6; and anti-inflammatory cytokines: IL-10 and TGFβ. TNF showed the best negative correlation (r2 = 0.231; p<0.001). Herein we describe impairment on mRNA cytokine expression in leishmania infected dogs with high parasite load associated with a structural modification in the splenic lymphoid micro-architecture. We also discuss the possible mechanism responsible for the uncontrolled parasite growth and clinical outcome.

Canine cutaneous leishmaniasis caused by neotropical Leishmania infantum despite of systemic disease: A case report

Visceral leishmaniasis is an anthropozoonosis caused by a protozoan Leishmania infantum (syn. Leishmania chagasi). Here, we report a typical case of canine cutaneous leishmaniasis due to L. infantum infection without any other systemic symptom in one dog in the city of Rio de Janeiro, Brazil. A mongrel female dog was admitted in a veterinary clinic with reports of chronic wounds in the body. Physical examination revealed erosive lesions in the limbs, nasal ulcers, presence of ectoparasites and seborrheic dermatitis. Blood samples and fragments of healthy and injured skin were collected. The complete hemogram revealed aregenerative normocytic normochromic anemia and erythrocyte rouleaux, and biochemical analysis revealed normal renal and hepatic functions. Cytology of the muzzle and skin lesions suggested pyogranulomatous inflammatory process. The histopathology of a skin fragment was performed and revealed suspicion of protozoa accompanied by necrotizing dermatitis. The diagnosis of leishmaniasis was accomplished by positive serology, isolation of Leishmania from the skin lesion, and also by molecular test (PCR targeting the conserved region of Leishmania kDNA). Culture was positive for damaged skin samples. PCR targeting a fragment of Leishmania hsp70 gene was performed employing DNA extracted from damaged skin. RFLP of the amplified hsp70 fragment identified the parasite as L. infantum, instead of Leishmania braziliensis, the main agent of cutaneous leishmaniasis in Rio de Janeiro. Characterization of isolated promastigotes by five different enzymatic systems confirmed the species identification of the etiological agent. Serology was positive by ELISA and rapid test. This case warns to the suspicion of viscerotropic Leishmania in cases of chronic skin lesions and brings the discussion of the mechanisms involved in the parasite tissue tropism.

Detection and quantification of Leishmania infantum in naturally and experimentally infected animal samples.

Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL). VL is the most severe form of leishmaniasis and can be fatal if it is not properly treated. Although several PCR works are intended to detect L. infantum, in silico analysis of available primers and/or primer-probes reveals potential cross species amplification. Here, a TaqMan-based quantitative real time PCR (qPCR) assay was developed for specific detection and quantitation of L. infantum in tissue samples from experimentally or naturally infected animals, mice or dogs, respectively. For this assay, primers and probes were designed for the kinetoplast minicircle DNA of L. infantum. The qPCR assay achieved a detection limit of 0.01pg of parasite DNA, and allowed specific amplification of L. infantum in both asymptomatic and symptomatic naturally infected dogs with inter-assay variation coefficients between 0.05-0.11. There was no cross amplification with dog DNA or with L. braziliensis, L. donovani, L. major, L. tropica or Trypanosoma cruzi. In addition, our assay detected a significantly higher parasite load in symptomatic than in the asymptomatic animals (p<0.0001). We believe this approach will be a valuable tool for the specific detection of L. infantum in regions of sympatric transmission of VL-causing parasites.

Characterization of Leishmania infantum species in dogs from the urban area of Cuiabá, State of Mato Grosso, Brazil

INTRODUCTIONnVisceral leishmaniasis presents urban behavior in some Brazilian cities, with domestic dogs as the main infection source. In Cuiabá, MT, canine visceral leishmaniasis was diagnosed and characterized as recommended by the Ministry of Health.nnnMETHODSnBiological samples from suspected canine carriers were analyzed by the isoenzyme electrophoresis technique. The 6PGDH enzyme and reference strain IOC/L0566 (MHOM/BR/1975/M2903) of Leishmania (Leishmania) infantum was used as one of the controls.nnnRESULTSnElectrophoresis analysis revealed that the canine isolates belonged to the species L. (L.) infantum.nnnCONCLUSIONSnThe authors emphasize the importance of species characterization, particularly in areas of mixed infection like Cuiabá.

The obturator oblique and iliac oblique/outlet views predict most accurately the adequate position of an anterior column acetabular screw

ObjectivePercutaneous fixation of the acetabulum is a treatment option for select acetabular fractures. Intra-operative fluoroscopy is required, and despite various described imaging strategies, it is debatable as to which combination of fluoroscopic views provides the most accurate and reliable assessment of screw position.Materials and methodsUsing five synthetic pelvic models, an experimental setup was created in which the anterior acetabular columns were instrumented with screws in five distinct trajectories. Five fluoroscopic images were obtained of each model (Pelvic Inlet, Obturator Oblique, Iliac Oblique, Obturator Oblique/Outlet, and Iliac Oblique/Outlet). The images were presented to 32 pelvic and acetabular orthopaedic surgeons, who were asked to draw two conclusions regarding screw position: (1) whether the screw was intra-articular and (2) whether the screw was intraosseous in its distal course through the bony corridor.ResultsIn the assessment of screw position relative to the hip joint, accuracy of surgeon’s response ranged from 52% (iliac oblique/outlet) to 88% (obturator oblique), with surgeon confidence in the interpretation ranging from 60% (pelvic inlet) to 93% (obturator oblique) (Pu2009<u20090.0001). In the assessment of intraosseous position of the screw, accuracy of surgeon’s response ranged from 40% (obturator oblique/outlet) to 79% (iliac oblique/outlet), with surgeon confidence in the interpretation ranging from 66% (iliac oblique) to 88% (pelvic inlet) (Pu2009<u20090.0001).ConclusionsThe obturator oblique and obturator oblique/outlet views afforded the most accurate and reliable assessment of penetration into the hip joint, and intraosseous position of the screw was most accurately assessed with pelvic inlet and iliac oblique/outlet views.EvidenceClinical Question

Mesenchymal stromal cells from bone marrow treated with bovine tendon extract acquire the phenotype of mature tenocytes

Objective This study evaluated in vitro differentiation of mesenchymal stromal cells isolated from bone marrow, in tenocytes after treatment with bovine tendon extract. Methods Bovine tendons were used for preparation of the extract and were stored at −80 °C. Mesenchymal stromal cells from the bone marrow of three donors were used for cytotoxicity tests by means of MTT and cell differentiation by means of qPCR. Results The data showed that mesenchymal stromal cells from bone marrow treated for up to 21 days in the presence of bovine tendon extract diluted at diminishing concentrations (1:10, 1:50 and 1:250) promoted activation of biglycan, collagen type I and fibromodulin expression. Conclusion Our results show that bovine tendon extract is capable of promoting differentiation of bone marrow stromal cells in tenocytes.

Epidemiological study on giant cell tumor recurrence at the Brazilian National Institute of Traumatology and Orthopedics

Objective Giant cell tumors are benign bone neoplasms that are relatively rare in adults and their biological behavior is still unpredictable. The incidence of local recurrence has presented variation between 0% and 65% in studies conducted worldwide, but few data are available on this complication in the Brazilian population. Methods Information on 155 patients with confirmed histological diagnoses of giant cell tumor who were treated in our institutions orthopedic oncology service between January 2000 and July 2014 was gathered. Demographic characteristics were evaluated and compared between patients who presented local recurrence during the clinical follow-up. Results Local recurrence was observed in 26 patients (16.7%), of whom 22 were female (84.6%). The most common site of local recurrence was the distal femur (38.4%). Eleven patients presented early recurrence, while 15 cases were diagnosed after 15 months, representing 42.3% and 57.7%, respectively. Metastases were identified in five patients (3.2%). Conclusion Tumor-related factors did not show any increased incidence of local recurrence of giant cell tumors. Surgical treatment with an intralesional margin is a valid option for treating local recurrences and does not show any difference in disease-free survival in relation to other types of procedures. Clinical treatment is reserved for cases of unresectable tumors or when surgical treatment is impossible.

Late Adherent Human Bone Marrow Stromal Cells Form Bone and Restore the Hematopoietic Microenvironment In Vivo

Bone marrow stromal cells (BMSCs) are a valuable resource for skeletal regenerative medicine because of their osteogenic potential. In spite of the very general term “stem cell,” this population of cells is far from homogeneous, and different BMSCs clones have greatly different phenotypic properties and, therefore, potentially different therapeutic potential. Adherence to a culture flask surface is a primary defining characteristic of BMSCs. We hypothesized that based on the adherence time we could obtain an enriched population of cells with a greater therapeutic potential. We characterized two populations of bone marrow-derived cells, those that adhered by three days (R-cells) and those that did not adhere by three days but did by six days (L-cells). Clones derived from L-cells could be induced into adipogenic, chondrogenic, and osteogenic differentiation in vitro. L-cells appeared to have greater proliferative capacity, as manifested by larger colony diameter and clones with higher CD146 expression. Only clones from L-cells developed bone marrow stroma in vivo. We conclude that the use of late adherence of BMSCs is one parameter that can be used to enrich for cells that will constitute a superior final product for cell therapy in orthopedics.

Fatores que influenciam o resultado da osteossíntese na fratura do colo do fêmur em paciente adulto jovem

Objectives u2003To evaluate the factors that influence the outcome of osteosynthesis after closed reduction of the fracture of the femoral neck in young adult patients. Methods u2003A retrospective study was conducted, reviewing the data of patients operated in a large orthopedic hospital from 2003 to 2011; a total of 81 patients met the inclusion criteria. The time interval between the fracture and the surgery, the initial fracture deviation, the quality of the reduction, and the placement of the implant were evaluated. Results u2003The present study observed a strong relationship between the quality of the reduction and therapeutic success. The degree of the initial deviation and the time elapsed between the initial trauma and the osteosynthesis did not influence the surgical outcome regarding bone consolidation. The correct positioning of the implants was associated with a satisfactory evolution in the postoperative period. Conclusion u2003The quality of the reduction and the positioning of the implants are factors that influence the results of osteosynthesis in fractures of the femoral neck in young adult patients.

Patient-derived osteosarcoma cells are resistant to methotrexate

Osteosarcoma is the most common primary bone tumor in children and young adults. The median survival of osteosarcoma patients has not significantly improved since 1990, despite administration of different classes of chemotherapy agents, such as methotrexate, cisplatin and doxorubicin. Cancer stem cells (CSCs) are responsible for the resistance of osteosarcoma to chemotherapy and OCT4, SOX2 and SSEA4 have been used to identify CSCs in osteosarcoma. Here, we used low-passage patient-derived osteosarcoma cells and osteosarcoma cells directly isolated from patients before and after chemotherapy treatments to evaluate the effects of chemotherapy on stem cell markers expression. We demonstrate that primary osteosarcoma cells are resistant to methotrexate treatment and sensitive to cisplatin and doxorubicin in vitro. We also verified that cisplatin and doxorubicin reduce the expression of SOX2 and OCT4 in primary osteosarcoma cells whereas methotrexate does not alter SOX2 and OCT4 expression, however it increases SSEA4 expression in primary osteosarcoma cells. Finally, we found that, although the combination treatment cisplatin plus doxorubicin inhibited the in vivo growth of osteosarcoma cells in NOD-SCID gamma mice subcutaneously injected with SaOs2, the combination treatment cisplatin plus doxorubicin plus methotrexate did not inhibit the in vivo growth of these cells. These observations may provide an explanation for the poor response of osteosarcomas to chemotherapy and point to the need of reevaluating the therapeutic strategies for human osteosarcomas.