Mesenchymal stem cells with rhBMP-2 inhibits the growth of canine osteosarcoma cells

Background: The bone morphogenetic proteins (BMPs) belong to a unique group of proteins that includes the growth factor TGF-beta. BMPs play important roles in cell differentiation, cell proliferation, and inhibition of cell growth. They also participate in the maturation of several cell types, depending on the microenvironment and interactions with other regulatory factors. Depending on their concentration gradient, the BMPs can attract various types of cells and act as chemotactic, mitogenic, or differentiation agents. BMPs can interfere with cell proliferation and the formation of cartilage and bone. In addition, BMPs can induce the differentiation of mesenchymal progenitor cells into various cell types, including chondroblasts and osteoblasts. The aim of this study was to analyze the effects of treatment with rhBMP-2 on the proliferation of canine mesenchymal stem cells (cMSCs) and the tumor suppression properties of rhBMP-2 in canine osteocarcoma (OST) cells. Osteosarcoma cell lines were isolated from biopsies and excisions of animals with osteosarcoma and were characterized by the Laboratory of Biochemistry and Biophysics, Butantan Institute. The mesenchymal stem cells were derived from the bone marrow of canine fetuses (cMSCs) and belong to the University of Sao Paulo, College of Veterinary Medicine (FMVZ-USP) stem cell bank. After expansion, the cells were cultured in a 12-well Transwell system; cells were treated with bone marrow mesenchymal stem cells associated with rhBMP2. Expression of the intracytoplasmic and nuclear markers such as Caspase-3, Bax, Bad, Bcl-2, Ki-67, p53, Oct3/4, Nanog, Stro-1 were performed by flow citometry. Results: We evaluated the regenerative potential of in vitro treatment with rhBMP-2 and found that both osteogenic induction and tumor regression occur in stem cells from canine bone marrow. rhBMP-2 inhibits the proliferation capacity of OST cells by mechanisms of apoptosis and tumor suppression mediated by p53. Conclusion: We propose that rhBMP-2 has great therapeutic potential in bone marrow cells by serving as a tumor suppressor to increase p53 and the pro-apoptotic proteins Bad and Bax, as well as by increasing the activity of phosphorylated caspase 3. Study design: Canine bone marrow mesenchymal stem cells associated with rhBMP2 in canine osteosarcoma treatment: "in vitro" study

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Adipose Tissue

Mesenchymal stem cells (MSCs) from human adipose tissue have a great potential for use in cell therapy due to their ease of isolation, expansion, and differentiation, besides the relative acceptance from the ethical point of view. Our intention was to isolate and promote in vitro expansion and differentiation of MSCs from human adipose tissue into cells with a pancreatic endocrine phenotype. Human adipose tissue obtained from patients undergoing abdominal dermolipectomy was digested with type I collagenase. MSCs isolated by plastic adherence and characterized by cytochemistry and FACS were expanded in vitro. MSC differentiation into an endocrine phenotype was induced over 2 to 4 months with high glucose (25 mmol/L) media containing nicotinamide, exendin-4, and 2-mercaptoethanol. Insulin and glucagon expressions were analyzed by immunofluorescence. Cells isolated from human adipose tissue and expanded in vitro expressed MSC markers as confirmed by FACS and cytochemistry. Insulin but not glucagon production by differentiated cells was demonstrated by irnmunofluorescence. MSCs isolated from human adipose tissue were induced to differentiate in vitro into an endocrine phenotype that expressed insulin

A new dic(7;12)(p12.21;p12.2) and i(12)(q10) during the lymphoid blast crisis of patient with Ph plus chronic myeloid leukemia

Chronic myelogenous leukemia (CML) is a common myeloproliferative disease that is characterized by the clonal expansion of marrow stem cells, and is associated with the Philadelphia chromosome. As the disease progresses, additional chromosome abnormalities may arise. The prognostic impact of secondary chromosomal abnormalities in CML is complex, heterogeneous, and sometimes related to previous treatment. Here, we describe a CML patient in lymphoid blast crisis associated with a new chromosomal abnormality identified, dic(7;12)(p12.21;p12.2) and i(12)(q10) using classical cytogenetics and spectral karyotype analysis. To the best of our knowledge, this is the first report of t(7;12)(p11.1;q11.1) and i(12)(q10) in a CML patient with lymphoid evolution.

Mesenchymal stem cells from umbilical cord blood: parameters for isolation, characterization and adipogenic differentiation

Isolation of mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) from full-term deliveries is a laborious, time-consuming process that results in a low yield of cells. In this study we identified parameters that can be helpful for a successful isolation of UCB-MSCs. According to our findings, chances for a well succeeded isolation of these cells are higher when MSCs were isolated from UCB collected from normal full-term pregnancies that did not last over 37 weeks. Besides the duration of pregnancy, blood volume and storage period of the UCB should also be considered for a successful isolation of these cells. Here, we found that the ideal blood volume collected should be above 80 mL and the period of storage should not exceed 6 h. We characterized UCB-MSCs by morphologic, immunophenotypic, protein/gene expression and by adipogenic differentiation potential. Isolated UCB-MSCs showed fibroblast-like morphology and the capacity of differentiating into adipocyte-like cells. Looking for markers of the undifferentiated status of UCB-MSCs, we analyzed the UCB-MSCs' protein expression profile along different time periods of the differentiation process into adipocyte-like cells. Our results showed that there is a decrease in the expression of the markers CD73, CD90, and CD105 that correlates to the degree of differentiation of UCB-MSCs We suggest that CD90 can be used as a mark to follow the differentiation commitment degree of MSCs. Microarray results showed an up-regulation of genes related to the adipogenesis process and to redox metabolism in the adipocyte-like differentiated MSCs. Our study provides information on a group of parameters that may help with successful isolation and consequently with characterization of the differentiated/undifferentiated status of UCB-MSCs, which will be useful to monitor the differentiation commitment of UCB-MSC and further facilitate the application of those cells in stem-cell therapy.

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic (TM) ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male: female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible "homogenous" subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country.

Molecular mapping of the regenerative niche in a murine model of myocardial infarction

Adult stem cells are distributed through the whole organism, and present a great potential for the therapy of different types of disease. For the design of efficient therapeutic strategies, it is important to have a more detailed understanding of their basic biological characteristics, as well as of the signals produced by damaged tissues and to which they respond. Myocardial infarction (MI), a disease caused by a lack of blood flow supply in the heart, represents the most common cause of morbidity and mortality in the Western world. Stem cell therapy arises as a promising alternative to conventional treatments, which are often ineffective in preventing loss of cardiomyocytes and fibrosis. Cell therapy protocols must take into account the molecular events that occur in the regenerative niche of MI. In the present study, we investigated the expression profile of ten genes coding for chemokines or cytokines in a murine model of MI, aiming at the characterization of the regenerative niche. MI was induced in adult C57BL/6 mice and heart samples were collected after 24 h and 30 days, as well as from control animals, for quantitative RT-PCR. Expression of the chemokine genes CCL2, CCL3, CCL4, CCL7, CXCL2 and CXCL10 was significantly increased 24 h after infarction, returning to baseline levels on day 30. Expression of the CCL8 gene significantly increased only on day 30, whereas gene expression of CXCL12 and CX3CL1 were not significantly increased in either ischemic period. Finally, expression of the IL-6 gene increased 24 h after infarction and was maintained at a significantly higher level than control samples 30 days later. These results contribute to the better knowledge of the regenerative niche in MI, allowing a more efficient selection or genetic manipulation of cells in therapeutic protocols.

Identificação do nicho de progenitores mesenquimais no fígado de embriões e fetos caninos: uma fonte de células-tronco para terapia celular

As células-tronco (CT) derivadas dos tecidos fetais (TF) foram as mais recentes descobertas entre as CT, e ultimamente tem demonstrado amplo potencial terapêutico, dentre os TF o fígado fetal (FF) apresenta grande potencial terapêutico. Este órgão durante o período fetal em mamíferos é um nicho hematopoético transitório, sendo o principal órgão responsável pela hematopoese no feto, além de contribuir com a formação do nicho definitivo na medula óssea adulta, portanto pode ser considerado um nicho de células-tronco mesenquimais (CTM) e progenitores. No entanto, pouco se sabe sobre a localização destas células no FF, desta forma o presente estudo visa identificar o nicho de CTM e progenitores em FF de cães, a fim de contribuir com as técnicas de isolamento e extração celular. Em conjunto foi realizada a verificação da expressão do fator de transcrição Oct-3/4 e da proteína delta polimerase do DNA (PCNA). Para a análise foram utilizados cinco embriões e 11 fetos caninos com idades gestacionais variando de 25-60 dias. Os resultados elucidaram a partir de 25 dias de gestação o FF apresentou-se volumoso e composto por todas as estruturas típicas, dentre elas a tríade portal, ductos biliares e ramos das artérias hepáticas. Com 30 dias de gestação foram identificados os primeiros sitos de progenitores mesenquimais (PM) enquanto que aos 60 dias os nichos estavam completamente formados com localização semelhante ao fígado adulto (FA). No entanto, células imunopositivas para Oct-3/4 não foram identificadas; sendo assim, destacamos que o FF é uma fonte de PM, apresentando-se como uma alternativa para a utilização terapêutica, bem como para os estudos da biologia do desenvolvimento das CTM e progenitores.

Thalidomide plus dexamethasone as a maintenance therapy after autologous hematopoietic stem cell transplantation improves progression-free survival in multiple myeloma

Despite the good response of stem cell transplant (SCT) in the treatment of multiple myeloma (MM), most patients relapse or do not achieve complete remission, suggesting that additional treatment is needed. We assessed the impact of thalidomide in maintenance after SCT in untreated patients with MM. A hundred and eight patients (<70 years old) were randomized to receive maintenance with dexamethasone (arm A; n = 52) or dexamethasone with thalidomide (arm B; n = 56; 200 mg daily) for 12 months or until disease progression. After a median follow-up of 27 months, an intention to treat analysis showed a 2-year progression-free survival (PFS) of 30% in arm A (95% CI 2238) and 64% in arm B (95% CI 5771; P = 0.002), with median PFS of 19 months and 36 months, respectively. In patients who did not achieve at least a very good partial response, the PFS at 2 years was significantly higher when in use of thalidomide (19 vs. 59%; P = 0.002). Overall survival at 2 years was not significantly improved (70 vs. 85% in arm A and arm B, respectively; P = 0.27). The addition of thalidomide to dexamethasone as maintenance improved the PFS mainly in patients who did not respond to treatment after SCT. Am. J. Hematol. (c) 2012 Wiley Periodicals, Inc.

Photodynamic therapy in periocular basal cell carcinoma: Case report

The authors report the use of photodynamic therapy with methyl aminolevulinate (Metvix (R)) in a patient with nodular and infiltrative basal cell carcinoma in the right lower eyelid. Side effects on the eye were evaluated weekly. After 12 weeks of treatment, to confirm cure the patient was submitted to a 2-mm punch biopsy, the anatomopathological findings of which were negative for neoplasia. Photodynamic therapy with methyl aminolevulinate was shown to be an attractive alternative to surgical excision(-)the current gold standard treatment worldwide.

Endostatin gene therapy stimulates upregulation of ICAM-1 and VCAM-1 in a metastatic renal cell carcinoma model

One of the greatest challenges in urological oncology is renal cell carcinoma (RCC), which is the third leading cause of death in genitourinary cancers. RCCs are highly vascularized and respond positively to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the potential of ES-based antiangiogenic therapy to activate tumor-associated endothelial cells in metastatic RCC (mRCC). Balb/c-bearing Renca cells were treated with NIH/3T3-LendSN or, as a control, with NIH/3T3-LXSN cells. The T-cell subsets and lymphocyte populations of tumors, mediastinal lymph nodes and the spleen were assessed by flow cytometry. The expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was assessed by real-time PCR, flow cytometry and immunohistochemistry analysis. ES gene therapy led to an increase in the percentage of infiltrating CD4-interferon (IFN)-gamma cells (P<0.05), CD8-IFN-gamma cells (P<0.01) and CD49b-tumor necrosis factor-alpha cells (P<0.01). In addition, ES therapy caused an increase at the mRNA level of ICAM-1 (1.4-fold; P<0.01) and VCAM-1 (1.5-fold) (control vs treated group; P<0.001). Through flow cytometry, we found a significant increase in the CD34/ICAM-1 cells (8.1-fold; P<0.001) and CD34/VCAM-1 cells (1.6-fold; P<0.05). ES gene therapy induced a significant increase in both T CD4 and CD8 cells in the lymph nodes and the spleen, suggesting that ES therapy may facilitate cell survival or clonal expansion. CD49b cells were also present in increased quantities in all of these organs. In this study, we demonstrate an antitumor inflammatory effect of ES in an mRCC model, and this effect is mediated by an increase in ICAM-1 and VCAM-1 expression in tumor-associated endothelial cells.

Low-Level Laser Therapy Influences Mouse Odontoblast-Like Cell Response in Vitro

Objective: The purpose of this study was to analyze the influence of two different irradiation times with 85mW/cm(2) 830nm laser on the behavior of mouse odontoblast-like cells. Background data: The use of low-level laser therapy (LLLT) to stimulate pulp tissue is a reality, but few reports relate odontoblastic responses to irradiation in in vitro models. Methods: Odontoblast-like cells (MDPC-23) were cultivated and divided into three groups: control/nonirradiated (group 1); or irradiated with 85mW/cm(2), 830nm laser for 10 sec (0.8 J/cm(2)) (group 2); or for 50 sec (4.2 J/cm(2)) (group 3) with a wavelength of 830 nm. After 3, 7, and 10 days, it was analyzed: growth curve and cell viability, total protein content, alkaline phosphatase (ALP) activity, calcified nodules detection and quantification, collagen immunolocalization, vascular endothelial growth factor (VEGF) expression, and real-time polymerase chain reaction (PCR) for DMP1 gene. Data were analyzed by Kruskall-Wallis test (alpha = 0.05). Results: Cell growth was smaller in group 2 (p < 0.01), whereas viability was similar in all groups and at all periods. Total protein content and ALP activity increased on the 10th day with 0.8 J/cm(2) (p < 0.01), as well as the detection and quantification of mineralization nodules (p < 0.05), collagen, and VEGF expression (p < 0.01). The expression of DMP1 increased in all groups (p < 0.05) compared with control at 3 days, except for 0.8 J/cm(2) at 3 days and control at 10 days. Conclusions: LLLT influenced the behavior of odontoblast-like cells; the shorter time/smallest energy density promoted the expression of odontoblastic phenotype in a more significant way.

Fibronectin expression is decreased in metastatic renal cell carcinoma following endostatin gene therapy

Tumor cells induce the disruption of homeostasis between cellular and extracellular compartments to favor tumor progression. The expression of fibronectin (FN), a matrix glycoprotein, is increased in several carcinoma cell types, including renal cell carcinoma (RCC). RCC are highly vascularized tumors and are often amenable to antiangiogenic therapy. Endostatin (ES) is a fragment of collagen XVIII that possesses antiangiogenic activity. In this study, we examined the modulation of FN gene expression by ES gene therapy in a murine metastatic renal cell carcinoma (mRCC) model. Balb/C mice bearing Renca cells were treated with NIH/3T3-LXSN cells or NIH/3T3-LendSN cells. At the end of the experiment, the ES serum levels were measured, and the FN gene expression was assessed using real-time PCR. The tissue FN was evaluated by western blotting and by immunofluorescence analysis. The ES serum levels in treated mice were higher than those in the control group (P < 0.05). ES treatment led to significant decreases at the FN mRNA (P < 0.001) and protein levels (P < 0.01). Here, we demonstrate the ES antitumor effect that is mediated by down-regulation of FN expression in mRCC. (C) 2012 Elsevier Masson SAS. All rights reserved.

Effect of Cholecalciferol as Adjunctive Therapy With Insulin on Protective Immunologic Profile and Decline of Residual beta-Cell Function in New-Onset Type 1 Diabetes Mellitus

Objective: To evaluate the effect of vitamin D-3 on cytokine levels, regulatory T cells, and residual beta-cell function decline when cholecalciferol (vitamin D-3 administered therapeutically) is given as adjunctive therapy with insulin in new-onset type 1 diabetes mellitus (T1DM). Design and Setting: An 18-month (March 10, 2006, to October 28, 2010) randomized, double-blind, placebo-controlled trial was conducted at the Diabetes Center of Sao Paulo Federal University, Sao Paulo, Brazil. Participants: Thirty-eight patients with new-onset T1DM with fasting serum C-peptide levels greater than or equal to 0.6 ng/mL were randomly assigned to receive daily oral therapy of cholecalciferol, 2000 IU, or placebo. Main Outcome Measure: Levels of proinflammatory and anti-inflammatory cytokines, chemokines, regulatory T cells, hemoglobin A(1c), and C-peptide; body mass index; and insulin daily dose. Results: Mean (SD) chemokine ligand 2 (monocyte chemoattractant protein 1) levels were significantly higher (184.6 [101.1] vs 121.4 [55.8] pg/mL) at 12 months, as well as the increase in regulatory T-cell percentage (4.55%[1.5%] vs 3.34%[1.8%]) with cholecalciferol vs placebo. The cumulative incidence of progression to undetectable (<= 0.1 ng/mL) fasting C-peptide reached 18.7% in the cholecalciferol group and 62.5% in the placebo group; stimulated C-peptide reached 6.2% in the cholecalciferol group and 37.5% in the placebo group at 18 months. Body mass index, hemoglobin A(1c) level, and insulin requirements were similar between the 2 groups. Conclusions: Cholecalciferol used as adjunctive therapy with insulin is safe and associated with a protective immunologic effect and slow decline of residual beta-cell function in patients with new-onset T1DM. Cholecalciferol may be an interesting adjuvant in T1DM prevention trials.

P16-39. T cell recognition of autologous and non-autologous HIV-1 protease peptides by HIV-1 infected patients undergoing PI therapy

NIH, ICGEB, FAPESP, CNPq

Periocular basal cell carcinoma: cost of topical immunotherapy versus estimated cost of surgical treatment

Purpose: The objective of this study was to compare the estimated cost of clinical and surgical treatment for basl cell carcinoma of the eyelid. Methods: This was a pilot study of 12 patients with basal cell carcinoma receiving treatment with 5% imiquimod cream at the ocular plastic surgery center, medical school University of Sao Paulo (HC-FMUSP, Brazil). The cost of clinical treatment was estimated based on the time of treatment and amount of medication consumed by patients in the home setting. The cost of surgical treatment was estimated by ophthalmologists with experience in reconstructive plastic surgery based on analysis of images of the same patients. Surgeons responded to a questionnaire with four questions about surgical technique, surgical materials required, estimated duration of surgery and type of anesthesia. Results: Immunotherapy lasted from 8 to 12 weeks. All patients reported each cold-stored sachet with 5% imiquimod cream lasted 3 days. According to the institution, a box with 12 sachets costs BRL 480.00. Patients required 1.58-3.11 boxes for complete treatment, corresponding to a total cost of BRL 758.40-1,492.80. Based on image analysis, surgeons evaluated surgery would require 1-3 hours. The estimated cost of surgery room and staff was BRL 263.00, to which the cost of supplies was added. Thus, the total cost of surgical treatment was BRL 272.61-864.82. On the average, immunotherapy was 57,64% more costly than surgical treatment. Conclusions: Malignant eyelid tumors are a common finding in clinical ophthalmology. Surgery is still the treatment of choice at our institution, but immunotherapy with 5% imiquimod cream may be indicated for patients with multiple lesions or high surgical risk and for patients declining surgery for reasons of fear or esthetic concerns. The ability to estimate costs related to the treatment of malignant eyelid tumors is an important aid in the financial planning of health care institutions. Further studies should evaluate the possibility of institutions equating the cost of immunotherapy and surgical treatment by acquiring similar but less expensive medications.