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Autologous hematopoietic stem cell transplantation (AHSCT) increases C-peptide levels and induces insulin independence in patients with type 1 diabetes. This study aimed to investigate how clinical outcomes may associate with the immunological status, especially concerning the balance between immunoregulation and autoreactivity. Twenty-one type 1 diabetes patients were monitored after AHSCT and assessed every 6 months for duration of insulin independence, C-peptide levels, frequencies of islet-specific autoreactive CD8 + T cells (CTL), regulatory lymphocyte subsets, thymic function, and T-cell repertoire diversity. In median follow-up of 78 (range 15-106) months, all patients became insulin-independent, resuming insulin after median of 43 (range 6-100) months. Patients were retrospectively divided into short- or prolonged-remission groups, according to duration of insulin independence. For the entire follow-up, CD3 + CD4 + T-cell numbers remained lower than baseline in both groups, whereas CD3 + CD8 + T-cell levels did not change, resulting in a CD4/CD8 ratio inversion. Memory CTL comprehended most of T cells detected on long-term follow-up of patients after AHSCT. B cells reconstituted to baseline levels at 2-3 months post-AHSCT in both patient groups. In the prolonged-remission-group, baseline islet-specific T-cell autoreactivity persisted after transplantation, but regulatory T cell counts increased. Patients with lower frequencies of autoreactive islet-specific T cells remained insulin-free longer and presented greater C-peptide levels than those with lower frequencies of these cells. Therefore, immune monitoring identified a subgroup of patients with superior clinical outcome of AHSCT. Our study shows that improved immunoregulation may balance autoreactivity endorsing better metabolic outcomes in patients with lower frequencies of islet-specific T cells. Development of new strategies of AHSCT is necessary to increase frequency and function of T and B regulatory cells and decrease efficiently autoreactive islet-specific T and B memory cells in type 1 diabetes patients undergoing transplantation.

Pericytes (PCs) are a subset of perivascular cells that can give rise to mesenchymal stromal cells (MSCs) when culture-expanded, and are postulated to give rise to MSC-like cells during tissue repair in vivo. PCs have been suggested to behave as stem cells (SCs) in situ in animal models, although evidence for this role in humans is lacking. Here, we analyzed the transcriptomes of highly purified, non-cultured adipose tissue (AT)-derived PCs (ATPCs) to detect gene expression changes that occur as they acquire MSC characteristics in vitro, and evaluated the hypothesis that human ATPCs exhibit a gene expression profile compatible with an AT SC phenotype. The results showed ATPCs are non-proliferative and express genes characteristic not only of PCs, but also of AT stem/progenitor cells. Additional analyses defined a gene expression signature for ATPCs, and revealed putative novel ATPC markers. Almost all AT stem/progenitor cell genes differentially expressed by ATPCs were not expressed by ATMSCs or culture-expanded ATPCs. Genes expressed by ATMSCs but not by ATPCs were also identified. These findings strengthen the hypothesis that PCs are SCs in vascularized tissues, highlight gene expression changes they undergo as they assume an MSC phenotype, and provide new insights into PC biology., (Copyright © 2016 Elsevier Inc. All rights reserved.)

During the early thymus colonization, Notch signaling activation on hematopoietic progenitor cells (HPCs) drives proliferation and T cell commitment. Although these processes are driven by transcription factors such as HOXB4 and GATA3, there is no evidence that Notch directly regulates their transcription. To evaluate the role of NOTCH and TNF signaling in this process, human CD34 + HPCs were cocultured with OP9-DL1 cells, in the presence or absence of TNF. The use of a Notch signaling inhibitor and a protein synthesis inhibitor allowed us to distinguish primary effects, mediated by direct signaling downstream Notch and TNF, from secondary effects, mediated by de novo synthesized proteins. A low and physiologically relevant concentration of TNF promoted T lymphopoiesis in OP9-DL1 cocultures. TNF positively modulated the expression of both transcripts in a Notch-dependent manner; however, GATA3 induction was mediated by a direct mechanism, while HOXB4 induction was indirect. Induction of both transcripts was repressed by a GSK3β inhibitor, indicating that activation of canonical Wnt signaling inhibits rather than induces their expression. Our study provides novel evidences of the mechanisms integrating Notch and TNF-alpha signaling in the transcriptional induction of GATA3 and HOXB4. This mechanism has direct implications in the control of self-renewal, proliferation, commitment, and T cell differentiation.

Background: Type 1 diabetes mellitus (T1D) is characterized by autoimmune responses resulting in destruction of insulin-producing pancreatic beta cells. Multipotent mesenchymal stromal cells (MSCs) exhibit immunomodulatory potential, migratory capacity to injured areas and may contribute to tissue regeneration by the secretion of bioactive factors. Therefore, MSCs are considered as a promising approach to treat patients with different autoimmune diseases (AID), including T1D patients. Phenotypical and functional alterations have been reported in MSCs derived from patients with different AID. However, little is known about the properties of MSCs derived from patients with T1D. Since autoimmunity and the diabetic microenvironment may affect the biology of MSCs, it becomes important to investigate whether these cells are suitable for autologous transplantation. Thus, the aim of the present study was to evaluate the in vitro properties and the in vivo therapeutic efficacy of MSCs isolated from bone marrow of newly diagnosed T1D patients (T1D-MSCs) and to compare them with MSCs from healthy individuals (C-MSCs)., Methods: T1D-MSCs and C-MSCs were isolated and cultured until third passage. Then, morphology, cell diameter, expression of surface markers, differentiation potential, global microarray analyses and immunosuppressive capacity were in vitro analyzed. T1D-MSCs and C-MSCs therapeutic potential were evaluated using a murine experimental model of streptozotocin (STZ)-induced diabetes., Results: T1D-MSCs and C-MSCs presented similar morphology, immunophenotype, differentiation potential, gene expression of immunomodulatory molecules and in vitro immunosuppressive capacity. When administered into diabetic mice, both T1D-MSCs and C-MSCs were able to reverse hyperglycemia, improve beta cell function and modulate pancreatic cytokine levels., Conclusions: Thus, bone marrow MSCs isolated from T1D patients recently after diagnosis are not phenotypically or functionally impaired by harmful inflammatory and metabolic diabetic conditions. Our results provide support for the use of autologous MSCs for treatment of newly diagnosed T1D patients.

Methods: this was a cross-sectional study using the Oral Health Services Quality Assessment Questionnaire, with a probabilistic sample of 256 patients, using multiple linear regression to identify variables associated with their satisfaction., Results: most patients stated they were satisfied (86.7%), followed by barely satisfied (10.2%) and very satisfied (3.1%); there were differences in satisfaction among patients attending the different SDCs; independent variables associated with patient satisfaction were 'improved self-perceived oral health' (p=0.001) and less 'waiting time at the clinic' (p<0.001)., Conclusion: he majority of patients were satisfied with the service provided; variables not analyzed in this study, including infrastructure, human resources and management, may have influenced the differences in patient satisfaction found between the different SDCs.

Major skin burns are difficult to treat. Patients often require special care and long-term hospitalization. Besides specific complications associated with the wounds themselves, there may be impairment of the immune system and of other organs. Mesenchymal stromal cells (MSCs) are a recent therapeutic alternative to treat burns, mainly aiming to accelerate the healing process. Several MSC properties favor their use as therapeutic approach, as they promote angiogenesis, stimulate regeneration, and enhance the immunoregulatory function. Moreover, since patients with extensive burns require urgent treatment and because the expansion of autologous MSCs is a time-consuming process, in this present study we chose to evaluate the therapeutic potential of xenogeneic MSCs in the treatment of severe burns in rats. MSCs were isolated from mouse bone marrow, expanded in vitro, and intradermally injected in the periphery of burn wounds. MSC-treated rats presented higher survival rates (76.19%) than control animals treated with PBS (60.86%, p < 0.05). In addition, 60 days after the thermal injury, the MSC-treated group showed larger proportion of healed areas within the burn wounds (90.81 ± 5.05%) than the PBS-treated group (76.11 ± 3.46%, p = 0.03). We also observed that CD4(+) and CD8(+) T cells in spleens and in damaged skin, as well as the percentage of neutrophils in the burned area, were modulated by MSC treatment. Plasma cytokine (TGF-β, IL-10, IL-6, and CINC-1) levels were also altered in the MSC-treated rats, when compared to controls. Number of injected GFP(+) MSCs progressively decreased over time, and 60 days after injection, few MSCs were still detected in the skin of treated animals. This study demonstrates the therapeutic effectiveness of intradermal application of MSCs in a rat model of deep burns, providing basis for future regenerative therapies in patients suffering from deep burn injuries.

Mesenchymal stromal cells (MSCs) are cultured cells that can give rise to mature mesenchymal cells under appropriate conditions and secrete a number of biologically relevant molecules that may play an important role in regenerative medicine. Evidence indicates that pericytes (PCs) correspond to mesenchymal stem cells in vivo and can give rise to MSCs when cultured, but a comparison between the gene expression profiles of cultured PCs (cPCs) and MSCs is lacking. We have devised a novel methodology to isolate PCs from human adipose tissue and compared cPCs to MSCs obtained through traditional methods. Freshly isolated PCs expressed CD34, CD140b, and CD271 on their surface, but not CD146. Both MSCs and cPCs were able to differentiate along mesenchymal pathways in vitro, displayed an essentially identical surface immunophenotype, and exhibited the ability to suppress CD3(+) lymphocyte proliferation in vitro. Microarray expression data of cPCs and MSCs formed a single cluster among other cell types. Further analyses showed that the gene expression profiles of cPCs and MSCs are extremely similar, although MSCs differentially expressed endothelial cell (EC)-specific transcripts. These results confirm, using the power of transcriptomic analysis, that PCs give rise to MSCs and suggest that low levels of ECs may persist in MSC cultures established using traditional protocols.

Background: The therapeutic use of human embryonic stem cells (hESCs) is dependent on an efficient cryopreservation protocol for long-term storage. The aim of this study was to determine whether the combination of three cryoprotecting reagents using two freezing systems might improve hESC recovery rates with maintenance of hESC pluripotency properties for potential cell therapy application., Methods: Recovery rates of hESC colonies which were frozen in three cryoprotective solutions: Me2SO/HES/SR medium, Defined-medium® and Me2SO/SFB in medium solution were evaluated in ultra-slow programmable freezing system (USPF) and a slow-rate freezing system (SRF). The hESC pluripotency properties after freezing-thawing were evaluated., Results: We estimated the distribution frequency of survival colonies and observed that independent of the freezing system used (USPF or SRF) the best results were obtained with Me2SO/HES/SR as cryopreservation medium. We showed a significant hESC recovery colonies rate after thawing in Me2SO/HES/SR medium were 3.88 and 2.9 in USPF and SRF, respectively. The recovery colonies rate with Defined-medium® were 1.05 and 1.07 however in classical Me2SO medium were 0.5 and 0.86 in USPF and SRF, respectively. We showed significant difference between Me2SO/HES/SR medium×Defined-medium® and between Me2SO/HES/SR medium×Me2SO medium, for two cryopreservation systems (P<0.05)., Conclusion: We developed an in house protocol using the combination of Me2SO/HES/SR medium and ultra-slow programmable freezing system which resulted in hESC colonies that remain undifferentiated, maintain their in vitro and in vivo pluripotency properties and genetic stability. This approach may be suitable for cell therapy studies., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Statins are widely used hypocholesterolemic drugs that block the mevalonate pathway, responsible for the biosysnthesis of cholesterol. However, statins also have pleiotropic effects that interfere with several signaling pathways. Mesenchymal stromal cells (MSC) are a heterogeneous mixture of cells that can be isolated from a variety of tissues and are identified by the expression of a panel of surface markers and by their ability to differentiate in vitro into osteocytes, adipocytes and chondrocytes. MSC were isolated from amniotic membranes and bone marrows and characterized based on ISCT (International Society for Cell Therapy) minimal criteria. Simvastatin-treated cells and controls were directly assayed by CFSE (Carboxyfluorescein diacetate succinimidyl ester) staining to assess their cell proliferation and their RNA was used for microarray analyses and quantitative PCR (qPCR). These MSC were also evaluated for their ability to inhibit PBMC (peripheral blood mononuclear cells) proliferation. We show here that simvastatin negatively modulates MSC proliferation in a dose-dependent way and regulates the expression of proliferation-related genes. Importantly, we observed that simvastatin increased the percentage of a subset of smaller MSC, which also were actively proliferating. The association of MSC decreased size with increased pluripotency and the accumulating evidence that statins may prevent cellular senescence led us to hypothesize that simvastatin induces a smaller subpopulation that may have increased ability to maintain the entire pool of MSC and also to protect them from cellular senescence induced by long-term cultures/passages in vitro. These results may be important to better understand the pleiotropic effects of statins and its effects on the biology of cells with regenerative potential.

Introduction: Mesenchymal stromal/stem cells (MSCs) are multipotent cells that have the ability to express and secrete a wide range of immunomodulatory molecules, cytokines, growth factors and antiapoptotic proteins. MSCs modulate both innate and adaptive immune responses making them potential candidates for the treatment of patients with type 1 diabetes mellitus (T1D). However, one problem frequently associated with the systemic MSCs administration is the entrapment of the cells mainly in the lungs. In this sense, trying to avoid the lung barrier, the purpose of this study was to evaluate the long-term therapeutic efficacy and biodistribution of allogeneic adipose tissue-derived MSCs (ADMSCs) injected via two different delivery routes (intrasplenic/I.Sp and intrapancreatic/I.Pc) in a murine model of diabetes induced by streptozotocin (STZ)., Methods: Experimental diabetes was induced in C57BL/6 male mice by multiple low-doses of STZ. MSCs were isolated from adipose tissue (ADMSCs) of Balb/c mice. A single dose of 1x10(6) ADMSCs was microinjected into the spleen or into the pancreas of diabetic mice. Control group received injection of PBS by I.Sp or I.Pc delivery routes. Glycemia, peripheral glucose response, insulin-producing β cell mass, regulatory T cell population, cytokine profile and cell biodistribution were evaluated after ADMSCs/PBS administration., Results: ADMSCs injected by both delivery routes were able to decrease blood glucose levels and improve glucose tolerance in diabetic mice. ADMSCs injected by I.Sp route reverted hyperglycemia in 70% of diabetic treated mice, stimulating insulin production by pancreatic β cells. Using the I.Pc delivery route, 42% of ADMSCs-treated mice responded to the therapy. Regulatory T cell population remained unchanged after ADMSCs administration but pancreatic TGF-β levels were increased in ADMSCs/I.Sp-treated mice. ADMSCs administrated by I.Sp route were retained in the spleen and in the liver and ADMSCs injected by I.Pc route remained in the pancreas. However, ADMSCs injected by these delivery routes remained only few days in the recipients., Conclusion: Considering the potential role of MSCs in the treatment of several disorders, this study reports alternative delivery routes that circumvent cell entrapment into the lungs promoting beneficial therapeutic responses in ADMSCs-treated diabetic mice.

Autologous hematopoietic SCT (AHSCT) has been investigated in the past as a therapeutic alternative for multiple sclerosis (MS). Despite advances in clinical management, knowledge about mechanisms involved with clinical remission post transplantation is still limited. Abnormal microRNA and gene expression patterns were described in MS and have been suggested as disease biomarkers and potential therapeutic targets. Here we assessed T- and B-cell reconstitution, microRNAs and immunoregulatory gene expression after AHSCT. Early immune reconstitution was mainly driven by peripheral homeostatic proliferation. AHSCT increased CD4(+)CD25(hi)FoxP3(+) regulatory T-cell counts and expression of CTLA-4 and GITR (glucocorticoid-induced TNFR) on CD4(+)CD25(hi) T cells. We found transient increase in exhausted PD-1(+) T cells and of suppressive CD8(+)CD28(-)CD57(+) T cells. At baseline, CD4(+) and CD8(+) T cells from MS patients presented upregulated miR-16, miR-155 and miR-142-3p and downregulated FOXP3, FOXO1, PDCD1 and IRF2BP2. After transplantation, the expression of FOXP3, FOXO1, PDCD1 and IRF2BP2 increased, reaching control levels at 2 years. Expression of miR-16, miR-155 and miR-142-3p decreased towards normal levels at 6 months post therapy, remaining downregulated until the end of follow-up. These data strongly suggest that AHSCT normalizes microRNA and gene expression, thereby improving the immunoregulatory network. These mechanisms may be important for disease control in the early periods after AHSCT.

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system, due to an immune reaction against myelin proteins. Multipotent mesenchymal stromal cells (MSCs) present immunosuppressive effects and have been used for the treatment of autoimmune diseases. In our study, gene expression profile and in vitro immunomodulatory function tests were used to compare bone marrow-derived MSCs obtained from MS patients, at pre- and postautologous hematopoietic stem cell transplantation (AHSCT) with those from healthy donors. Patient MSCs comparatively exhibited i) senescence in culture; ii) similar osteogenic and adipogenic differentiation potential; iii) decreased expression of CD105, CD73, CD44, and HLA-A/B/C molecules; iv) distinct transcription at pre-AHSCT compared with control MSCs, yielding 618 differentially expressed genes, including the downregulation of TGFB1 and HGF genes and modulation of the FGF and HGF signaling pathways; v) reduced antiproliferative effects when pre-AHSCT MSCs were cocultured with allogeneic T-lymphocytes; vi) decreased secretion of IL-10 and TGF-β in supernatants of both cocultures (pre- and post-AHSCT MSCs); and vii) similar percentages of regulatory cells recovered after MSC cocultures. The transcriptional profile of patient MSCs isolated 6 months posttransplantation was closer to pre-AHSCT samples than from healthy MSCs. Considering that patient MSCs exhibited phenotypic changes, distinct transcriptional profile and functional defects implicated in MSC immunomodulatory and immunosuppressive activity, we suggest that further MS clinical studies should be conducted using allogeneic bone marrow MSCs derived from healthy donors. We also demonstrated that treatment of MS patients with AHSCT does not reverse the transcriptional and functional alterations observed in patient MSCs.

Recent studies have demonstrated the role of adenosine (ADO) in sickle-cell anemia (SCA). ADO is produced by CD39 and CD73 and converted to inosine by adenosine deaminase (ADA). We evaluated the effects of hydroxycarbamide (HU) treatment on the modulation of adenosine levels in SCA patients. The expressions of CD39, CD73, and CD26 were evaluated by flow cytometry on blood cells in 15 HU-treated and 17 untreated patients and 10 healthy individuals. RNA was extracted from monocytes, and ADA gene expression was quantified by real-time PCR. ADA activity was also evaluated. We found that ADA transcripts were two times higher in monocytes of HU-treated patients, compared with untreated (P = 0.039). Monocytes of HU-treated patients expressed CD26, while monocytes of controls and untreated patients did not (P = 0.023). In treated patients, a lower percentage of T lymphocytes expressed CD39 compared with untreated (P = 0.003), and the percentage of T regulatory (Treg) cells was reduced in the treated group compared with untreated (P = 0.017) and controls (P = 0.0009). Besides, HU-treated patients displayed increased ADA activity, compared with untreated. Our results indicate a novel mechanism of action of HU mediated by the reduction of adenosine levels and its effects on pathophysiological processes in SCA.

Objective: Asthma is the most common chronic disease among adolescents. This study assessed the quality of life (QOL) related to health in adolescents with asthma and its determining factors (demographic, socioeconomic, and clinical). We also separately evaluated each of the parameters that comprised the asthma control classification., Methods: This was an observational, cross-sectional study of 114 adolescents who had doctor-diagnosed asthma. QOL was assessed using a version of the Pediatric Asthma Quality of Life Questionnaire (PAQLQ) that was adapted and validated for Brazil, and higher scores indicated a better QOL. The level of asthma control was assessed using the rating system proposed by the Global Initiative for Asthma, and sociodemographic factors were evaluated., Results: When the averages of the PAQLQ domains and overall scores were compared to the potentially explanatory variables, significantly lower average PAQLQ scores were obtained for individuals with an inadequate level of asthma control (p < 0.001). Of the control components, daytime symptoms, nighttime symptoms, and limited physical activity were related to QOL. However, the use of the β2 agonist and the peak flow functional parameter were not related to QOL., Conclusions: The level of asthma control was related to QOL, but this association manifested mainly in the subjective control domains, such as nighttime and daytime symptoms and physical activity limitations. The objective domain for control classification, represented by pulmonary function, was not an independent predictor or determinant of the QOL of adolescent asthma patients.

In Brazil, the existing reference values for T-lymphocytes subsets are based on data originated in other countries. There is no local information on normal variation for these parameters in Brazilian adults and children. We evaluated the normal variation found in blood donors from five large Brazilian cities, in different regions, and in children living in Salvador, and Rio de Janeiro. All samples were processed by flow cytometry. The results were analyzed according to region, gender, and lifestyle of blood donors. A total of 641 adults (63% males), and 280 children (58% males) were involved in the study. The absolute CD3+, and CD4+ cells count were significantly higher for females (adults and children). Higher CD4+ cell count in adults was associated with smoking, while higher CD8+ count was found among female children. Higher counts, for all T-cells subsets, were detected in blood donors from southeast / south regions while those living in the northern region had the lowest values. Individuals from midwestern and northeastern regions had an intermediate count for all these cells subsets. However, these differences did not reach statistical significance. In Brazil, gender and smoking, were the main determinants of differences in T-lymphocytes reference values.

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Hek-293 cell line presents good production platform for recombinant therapeutic proteins, however little is known about the components that contribute to the cellular control of recombinant protein production. In this study, we generated a Hek-293 producing recombinant factor VIII (FVIII) and we evaluated the immunoglobulin-binding protein (BiP) and phytanoil-CoA α-hydroxylase (PAHX) expression levels which are known for diminishing FVIII production. Our analyses showed that the recombinant cell population expresses 3.1 ± 1.4 fold of BIP mRNA (P = 0.0054) and 97.8 ± 0.5 fold of PAHX mRNA (P = 0.0016) compared to nontransduced cells. The amount of these proteins was inversely correlated to the secreted FVIII. In conclusion, BIP and PAHX expression are augmented in human cells producing FVIII and they antagonize the amount of therapeutic factor VIII in the cell culture.

Objective: The aim of this study is to analyze subjectively, using the SWAL-QOL questionnaire, swallowing dysfunction and associated factors after treatment with radiotherapy and chemotherapy in patients treated for head and neck cancer., Material and Methods: This is a cross-sectional study, based on the selection of patients with tumors of the head and neck area, treated with radiotherapy with or without chemotherapy during the years 2000 to 2006 at the Oncology Institute of Juiz de Fora. The data were analyzed using SPSS 15.0 software, and were evaluated using the chi-square test to compare differences in proportions between groups. The statistical significance level was set at 5%., Results: It was observed that with respect to foods of solid consistency, there was a statistically significant difference for mouth tumors (p<0.01), with a tendency in this group to use softer foods, easier to chew (stews, boiled vegetables, creamy soups, canned fruit). With reference to the domains of the SWAL-QOL, the location of the tumor in the mouth was statistically associated with the lowest quality of life in the symptoms domain (p<0.05). The female gender variable was associated with the lowest perceived quality of life in several domains, namely swallowing (p=0.02); fatigue (p=0.008); symptoms (p=0.009). Age (split below and above 60 years) was not associated with differences in perceived quality of life in any domain., Conclusion: Tumor in the mouth and the total dose of radiation in the superior fossa were associated with the lowest quality of life in the symptoms domain. The female gender variable was associated with the lowest perceived quality of life in several domains This study shows that speech therapy should maintain a presence in the teams, to then guide the rehabilitation of organic dysphonia and mechanical dysphagia possibly afflicting patients after cancer treatment with radiation therapy and chemotherapy. Key words:Quality of life, dysphagia, head and neck neoplasms, rehabilitation.

A balance between proinflammatory (Th17 and Tc17) and anti-inflammatory (regulatory T cells) subsets of T cells is essential to maintain immunological tolerance and prevent the onset of several autoimmune diseases, including type 1 diabetes. However, the kinetics of these subsets and disease severity during the streptozotocin (STZ)-induced diabetes course has not been determined. Thus, susceptible C57BL/6 mice were administrated with multiple low doses of STZ and we evaluated the frequency/absolute number of these T cell subsets in the pancreatic lymph nodes (PLNs) and spleen and Th1, Th17, Treg cytokine production in the pancreatic tissue. At different time points of the disease progression (6, 11, 18 and 25 days after the last STZ administration), the histopathological alterations were also evaluated by H&E and immunohistochemistry staining. During the initial phase of diabetes development (day 6), we noted increased numbers of CD4(+) and CD8(+) T cells in spleen and PLNs. At the same time, the frequencies of Th17 and Tc17 cells in PLNs were also enhanced. In addition, the early augment of interferon gamma (IFN-γ), tumoral necrosis factor (TNF-α), IL-6 and IL-17 levels in pancreatic tissue correlated with pancreatic islet inflammation and mild β-cell damage. Notably, the absolute number of Treg cells increased in PLNs during over time when compared to control group. Interestingly, increased IL-10 levels were associated with control of the inflammatory process during the late phase of the type 1 diabetes (day 25). In agreement, mice lacking the expression of IL-17 receptor (Il17r) showed impairment in STZ-induced diabetes progression, reduced peri-insulitis and beta cells preservation when compared with wild-type mice. Our findings suggest that dynamic changes of pathogenic Th17/Tc17 and regulatory T cell subsets numbers is associated with early strong inflammation in the pancreatic islets followed by late regulatory profile during the experimental STZ-induced diabetes course., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Background: Lymphedema is a highly prevalent condition in women who have undergone treatment for breast cancer. Lymphedema negatively affects the quality of life. The objective of this study was to estimate the prevalence of lymphedema and associated factors in women treated for breast cancer in the municipality of Juiz de Fora., Methods: We performed a cross-sectional study that evaluated 250 women who were being treated for breast cancer. Pre-screening of the sample by analysis of medical records was performed to select women who met the inclusion criteria as follows: women who had an operation more than 6 months ago; absence of active disease, locoregional or distant; the absence of functional change in the affected limb before surgery, which could lead to swelling of the limb; and simulating or masking symptoms of lymphedema, such as bursitis, tendonitis, and work-related musculoskeletal disorders. Women with bilateral breast cancer, absence of axillary intervention (partial or complete axillary dissection and/or SLN biopsy), active disease in the region, or lympho-venous alteration of the limb before surgery were excluded. Data were collected from the medical records of the selected cases, and they subsequently underwent an interview and a physical assessment., Results: The prevalence of lymphedema was 44.8%. There were medical records on the presence of this condition in 5.4% of cases. With regard to shoulder joint mobility, restrictions on abduction movements, internal and external rotation, and anterior shoulder adduction were significantly associated with lymphedema. Variables, including the presence of seroma, vascular changes, time elapsed after surgery, episodes of redness in the extremities, and cuticle removal from the hand with pliers were considered as major associated factors for lymphedema (p<0.05)., Conclusions: The prevalence of 44.8% for lymphedema found in this study is considered to be relevant because it is a morbidity that produces psychological, physical, and functional damage in patients with this condition. The planning of health programs and services appropriate for the immediate postoperative treatment of women with breast cancer, and increasing the awareness of health professionals regarding the early diagnosis of lymphedema, can help minimize the morbidity of this disease.

Dendritic cells (DCs) are potent antigen-presenting cells that initiate the primary immune response and whose functional properties in vivo depend on the maturation stimulus. We describe the functional properties of human monocyte-derived DCs after the maturation of immature DCs (iDCs) for 2 days with LPS (100 ng/ml), PGE2 (1 μg/ml), CD40L (1 μg/ml) or IL-18 (200 ng/ml) and with CD40L+PGE2 and IL-18+PGE2 mixtures at the same concentrations as above. Neither IL-18 nor PGE2 alone stimulated IL-12 or IFN-γ secretion. When administered simultaneously to 1×10(6)iDCs/ml, IL-18+PGE2 induced the secretion of 131.4±6.7 pg IL-12/ml and 355±87 pg IFN-γ/ml but there was no detectable IL-10 secretion. However, PGE2 alone stimulated the secretion of 208±89 pg IL-10/ml whereas IL-18 alone did not stimulate the secretion of IL-10, IL-12, TNF-α or INF-γ. When the mixture of CD40L+PGE2 was used, only migration toward CCL19 and CCL21 was induced. CD40L did not stimulate the secretion of IL-10, IL-12, TNF-α or IFN-γ and did not stimulate migration toward CCL19 or CCL21. The extent of stimulation of T cell proliferation was essentially the same for all stimuli at the concentrations given above. New properties such as IL-12 and INF-γ secretion and migration toward CCL21 emerged when a mixture of IL-18+PGE2 was employed. These data show that when the pairs of stimuli reported here were used simultaneously their effect was not additive. This system can be used to prepare mDCs with properties useful for cell therapy and also as a model to investigate the mechanisms of cytokine secretion and cell migration., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

It has been demonstrated that genomic alterations of cells in the hematopoietic microenvironment could induce myelodysplastic syndromes (MDS) with ineffective hematopoiesis and dysmorphic hematopoietic cells, and subsequent transformation to acute myeloid leukemia. This investigation is the first attempt to correlate the gene expression profile of AURKA and AURKB in a cytogenetically stratified population of mesenchymal stem cells (MSCs) from MDS patients. We found that AURKA messenger RNA was expressed at significantly higher levels in MSCs even with normal/altered karyotype when compared with hematopoietic cells and healthy donors. In addition, we found that the presence of chromosomal abnormalities (mainly aneuploidy) in hematopoietic cells/MSCs was also associated with higher levels of AURKA. Different from previous investigations, our findings, regarding AURKA expression support the hypothesis that the presence of chromosomal abnormalities in MSCs from MDS is not a consequence of the method used for chromosome preparation. They may reflect the genomic instability present in the bone marrow microenvironment of MDS patients. This information is also supported by differences observed in the growth kinetics between MSCs from healthy donors (normal karyotype) and from MDS patients with abnormal karyotype. In summary, our results may not be considered evidence that MDS and MSCs are originated from a single neoplastic clone. In fact, both cells (hematopoietic and MSCs) may probably be altered in response to damage-inducing factors, and the presence of genomic abnormalities in MSCs suggests that an unstable bone marrow microenvironment may facilitate the expansion of MDS/leukemic cells., (Copyright © 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc. All rights reserved.)

Introduction: This study evaluated the intracellular profile of interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-10 (IL-10) and interferon-γ (IFN-γ) in peripheral blood mononuclear cells (PBMCs) from leprosy patients based on oral infections presence to determine whether these coinfections could be associated with pro-inflammatory activity in leprosy., Methods: Leprosy patients regardless of clinical form and specific leprosy treatment (n=38) were divided into two groups: Group I - leprosy patients with oral infections (n=19), and Group II - leprosy patients without oral infections (n=19). Non-leprosy patients presenting oral infections were assigned to the control Group (n=10). Intracellular IL-2, IL-4, IL-10 and IFN-γ production was evaluated by flow cytometry (FACS) before and 7 days after controlling the oral infection in the Group I, before and 7 days after dental prophylaxis in the Group II, and during oral infection process in control Group., Results: Low percentages of CD3+ lymphocytes bearing IL-2, IL-10 and IFN-γ were observed in the Group I and Group II at baseline and 7 days after therapy or prophylaxis compared to controls. Group I showed reduced percentages of IL-4 at baseline and 7 days after therapy compared to controls, or at baseline of Group II, and the Group II showed reduced percentages of CD3+ cells bearing IL-4 compared to control. An increase of the percentages of CD3+cells bearing IL-4 was observed in the Group I after the oral infections treatment., Conclusions: The occurrence of oral infections favors the intracellular cytokines expression and, probably, the inflammatory reaction operating as a stimulatory signal triggering the leprosy reactions.

Objective. This study assessed the impact of periodontal diseases on health-related quality of life of adult users of the Brazilian Unified Health System. Study Design. A cross-sectional study was conducted on an outpatient basis. The sample included 151 adults treated in the Periodontics section at Dental Specialty Centres of Juiz de Fora (Minas Gerais, Brazil). The Oral Health Impact Profile (OHIP-14) measured the impact of periodontal disease on quality of life. Participants were interviewed to obtain self-perception of general and oral health and socioeconomic data, and dental records were consulted to obtain periodontal status data. The values of central tendency of the OHIP-14 were compared with socioeconomic, demographic, and self-reported health predictors using nonparametric tests. The final analysis was performed using multiple linear regressions. Results. The results showed that psychological discomfort and physical disability exhibited a negative impact. The following variables can explain approximately 27% of the impact of oral health conditions on health-related quality of life in this group: periodontal disease, self-perceived oral health, and the need to use or replace dental prosthesis. Conclusion. The need for prosthetic rehabilitation and worse periodontal status are associated with health-related quality of life, which can be predicted by the self-perception of health.

Background Aims: Mesenchymal stromal cells (MSC) are being used to treat and prevent a variety of clinical conditions. To be readily available, MSC must be cryopreserved until infusion. However, the optimal cryopreservation methods, cryoprotector solutions and MSC sensitivity to dimethyl sulfoxide (DMSO) exposure are unknown. This study investigated these issues., Methods: MSC samples were obtained from human umbilical cord (n = 15), expanded with Minimal Essential Medium-alpha (α-MEM) 10% human serum (HS), resuspended in 25 mL solution (HS, 10% DMSO, 20% hydroxyethyl starch) and cryopreserved using the BioArchive® system. After a mean of 18 ± 7 days, cell suspensions were thawed and diluted until a DMSO concentration of 2.5% was reached. Samples were tested for cell quantification and viability, immunophenotype and functional assays., Results: Post-thaw cell recovery: 114 ± 2.90% (mean ± SEM). Recovery of viable cells: 93.46 ± 4.41%, 90.17 ± 4.55% and 81.03 ± 4.30% at 30 min, 120 min and 24 h post-thaw, respectively. Cell viability: 89.26 ± 1.56%, 72.71 ± 2.12%, 70.20 ± 2.39% and 63.02 ± 2.33% (P < 0.0001) pre-cryopreservation and 30 min, 120 min and 24 h post-thaw, respectively. All post-thaw samples had cells that adhered to culture bottles. Post-thaw cell expansion was 4.18 ± 0.17 ×, with a doubling time of 38 ± 1.69 h, and their capacity to inhibit peripheral blood mononuclear cells (PBMC) proliferation was similar to that observed before cryopreservation. Differentiation capacity, cell-surface marker profile and cytogenetics were not changed by the cryopreservation procedure., Conclusions: A method for cryopreservation of MSC in bags, in xenofree conditions, is described that facilitates their clinical use. The MSC functional and cytogenetic status and morphologic characteristics were not changed by cryopreservation. It was also demonstrated that MSC are relatively resistant to exposure to DMSO, but we recommend cell infusion as soon as possible.

There is much discussion about effective public policies that allow the proper treatment of asthma, providing care that is comprehensive and centered on asthma patients within their social context. Educating health professionals and asthma patients provides better recognition of symptoms and the triggers of asthma exacerbations, as well as disseminating strategies for avoiding such triggers, thereby ensuring better treatment and quality of life for asthma patients. Asthma imposes an ever-increasing burden on society, in terms of impaired quality of life, morbidity, and health care costs, making this a very important discussion in the field of public policy.

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: Essential Thrombocythemia (ET) and Primary Myelofibrosis (PMF) are Chronic Myeloproliferative Neoplasms (MPN) characterized by clonal myeloproliferation/myeloaccumulation without cell maturation impairment. The JAK2 V617F mutation and PRV1 gene overexpression may contribute to MPN physiopathology. We hypothesized that deregulation of the apoptotic machinery may also play a role in the pathogenesis of ET and PMF. In this study we evaluated the apoptosis-related gene and protein expression of BCL2 family members in bone marrow CD34+ hematopoietic stem cells (HSC) and peripheral blood leukocytes from ET and PMF patients. We also tested whether the gene expression results were correlated with JAK2 V617F allele burden percentage, PRV1 overexpression, and clinical and laboratory parameters., Results: By real time PCR assay, we observed that A1, MCL1, BIK and BID, as well as A1, BCLW and BAK gene expression were increased in ET and PMF CD34+ cells respectively, while pro-apoptotic BAX and anti-apoptotic BCL2 mRNA levels were found to be lower in ET and PMF CD34+ cells respectively, in relation to controls. In patients' leukocytes, we detected an upregulation of anti-apoptotic genes A1, BCL2, BCL-XL and BCLW. In contrast, pro-apoptotic BID and BIMEL expression were downregulated in ET leukocytes. Increased BCL-XL protein expression in PMF leukocytes and decreased BID protein expression in ET leukocytes were observed by Western Blot. In ET leukocytes, we found a correlation between JAK2 V617F allele burden and BAX, BIK and BAD gene expression and between A1, BAX and BIK and PRV1 gene expression. A negative correlation between PRV1 gene expression and platelet count was observed, as well as a positive correlation between PRV1 gene expression and splenomegaly., Conclusions: Our results suggest the participation of intrinsic apoptosis pathway in the MPN physiopathology. In addition, PRV1 and JAK2 V617F allele burden were linked to deregulation of the apoptotic machinery.

The LX-2 cell line has characteristics of hepatic stellate cells (HSCs), which are considered pericytes of the hepatic microcirculatory system. Recent studies have suggested that HSCs might have mesenchymal origin. We have performed an extensive characterization of the LX-2 cells and have compared their features with those of mesenchymal cells. Our data show that LX-2 cells have a phenotype resembling activated HSCs as well as bone marrow-derived mesenchymal stem cells (BM-MSCs). Our immunophenotypic analysis showed that LX-2 cells are positive for activated HSC markers (αSMA, GFAP, nestin and CD271) and classical mesenchymal makers (CD105, CD44, CD29, CD13, CD90, HLA class-I, CD73, CD49e, CD166 and CD146) but negative for the endothelial marker CD31 and endothelial progenitor cell marker CD133 as well as hematopoietic markers (CD45 and CD34). LX-2 cells also express the same transcripts found in immortalized and primary BM-MSCs (vimentin, annexin 5, collagen 1A, NG2 and CD140b), although at different levels. We show that LX-2 cells are capable to differentiate into multilineage mesenchymal cells in vitro and can stimulate new blood vessel formation in vivo. LX-2 cells appear not to possess tumorigenic potential. Thus, the LX-2 cell line behaves as a multipotent cell line with similarity to BM-MSCs. This line should be useful for further studies to elucidate liver regeneration mechanisms and be the foundation for development of hepatic cell-based therapies., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Mesenchymal stromal cells (MSCs) suppress T cell responses through mechanisms not completely understood. Adenosine is a strong immunosuppressant that acts mainly through its receptor A(2a) (ADORA2A). Extracellular adenosine levels are a net result of its production (mediated by CD39 and CD73), and of its conversion into inosine by Adenosine Deaminase (ADA). Here we investigated the involvement of ADO in the immunomodulation promoted by MSCs. Human T lymphocytes were activated and cultured with or without MSCs. Compared to lymphocytes cultured without MSCs, co-cultured lymphocytes were suppressed and expressed higher levels of ADORA2A and lower levels of ADA. In co-cultures, the percentage of MSCs expressing CD39, and of T lymphocytes expressing CD73, increased significantly and adenosine levels were higher. Incubation of MSCs with media conditioned by activated T lymphocytes induced the production of adenosine to levels similar to those observed in co-cultures, indicating that adenosine production was mainly derived from MSCs. Finally, blocking ADORA2A signaling raised lymphocyte proliferation significantly. Our results suggest that some of the immunomodulatory properties of MSCs may, in part, be mediated through the modulation of components related to adenosine signaling. These findings may open new avenues for the development of new treatments for GVHD and other inflammatory diseases., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Human T-lymphotropic virus I (HTLV-1) is associated with the T-cell malignancy known as adult T-cell leukemia/ lymphoma (ATLL) and with a disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, the treatment of these diseases is based on symptom relief. RNA interference (RNAi) technology has been described as an efficient mechanism for development of new therapeutic methods. Thus, the aim of this study was to evaluate the inhibition of HTLV-1 structural proteins using short hairpin RNAs (shRNAs) expressed by non-viral vectors., Materials and Methods: Reporter plasmids that express enhanced green fluorescent protein-Gag (EGFP-Gag) and EGFP-Env fusion proteins and vectors that express shRNAs corresponding to the HTLV-1 gag and env genes were constructed. shRNA vectors and reporter plasmids were simultaneously transfected into HEK 293 cells., Results: Fluorescence microscopy, flow cytometry and real-time PCR showed that shRNAs were effective in inhibiting the fusion proteins., Conclusion: These shRNAs are effective against the expression of structural genes and may provide an approach to the development of new therapeutic agents.

Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection., (Copyright © 2011 Elsevier B.V. All rights reserved.)

A DNA vaccine expressing dengue-4 virus premembrane (prM) and envelope (E) genes was produced by inserting these genes into a mammalian expression plasmid (pCI). Following a thorough screening, including confirmation of protein expression in vitro, a recombinant clone expressing these genes was selected and used to immunize BALB/c mice. After 3 immunizations all the animals produced detectable levels of neutralizing antibodies against dengue-4 virus. The cytokines levels and T cell proliferation, detected ex vivo from the spleen of the immunized mice, showed that our construction induced substantial immune stimulation after three doses. Even though the antibody levels, induced by our DNA vaccine, were lower than those obtained in mice immunized with dengue-4 virus the levels of protection were high with this vaccine. This observation is further supported by the fact that 80% of the vaccine immunized group was protected against lethal challenge. In conclusion, we developed a DNA vaccine employing the genes of the prM and E proteins from dengue-4 virus that protects mice against this virus., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objective: High-dose chemotherapy (HDCT) followed by autologous stem cell transplantation is a widely applied treatment for hematological and autoimmune diseases. Little is known about the effects of this therapy on multipotent mesenchymal stromal cells (MSCs). We aimed to characterize, morphologically and functionally, MSCs isolated from bone marrow aspirates of patients after HDCT., Materials and Methods: We studied 12 consecutive lymphoma patients submitted to BEAM conditioning regimen followed by autologous stem cell transplantation 28 to 1836 days before the sample collection. Thirteen normal donors were used as control. MSCs were isolated by adherence to plastic and expanded ex vivo by culture in flasks containing alpha-minimum essential medium plus 15% fetal bovine serum., Results: The cell population isolated showed a typical MSC morphology, immunophenotype, and differentiation capacity into adipogenic, osteogenic, and chondrogenic lineages. The MSCs obtained from patients with Hodgkin's disease and non-Hodgkin's lymphoma showed decreased fibroblastoid colony-forming unit count (p = 0.023) and increased doubling time (p = 0.031) related to the control group. The total cell expansion of MSCs from normal subjects was marginally superior to the patient group (p = 0.064). There were no differences in gene expression profile, MSCs plasticity, or hematopoiesis support capability between control and patient group., Conclusions: Results suggest that HDCT applied to lymphoma patients damaged MSCs, which was demonstrated by their reduced clonogenic potential, doubling time, and cell expansion rates when compared to controls.

As previously shown, higher levels of NOTCH1 and increased NF-kappaB signaling is a distinctive feature of the more primitive umbilical cord blood (UCB) CD34+ hematopoietic stem cells (HSCs), as compared to bone marrow (BM). Differences between BM and UCB cell composition also account for this finding. The CD133 marker defines a more primitive cell subset among CD34+ HSC with a proposed hemangioblast potential. To further evaluate the molecular basis related to the more primitive characteristics of UCB and CD133+ HSC, immunomagnetically purified human CD34+ and CD133+ cells from BM and UCB were used on gene expression microarrays studies. UCB CD34+ cells contained a significantly higher proportion of CD133+ cells than BM (70% and 40%, respectively). Cluster analysis showed that BM CD133+ cells grouped with the UCB cells (CD133+ and CD34+) rather than to BM CD34+ cells. Compared with CD34+ cells, CD133+ had a higher expression of many transcription factors (TFs). Promoter analysis on all these TF genes revealed a significantly higher frequency (than expected by chance) of NF-kappaB-binding sites (BS), including potentially novel NF-kappaB targets such as RUNX1, GATA3, and USF1. Selected transcripts of TF related to primitive hematopoiesis and self-renewal, such as RUNX1, GATA3, USF1, TAL1, HOXA9, HOXB4, NOTCH1, RELB, and NFKB2 were evaluated by real-time PCR and were all significantly positively correlated. Taken together, our data indicate the existence of an interconnected transcriptional network characterized by higher levels of NOTCH1, NF-kappaB, and other important TFs on more primitive HSC sets.

Development of hypoxic areas occurs during infectious and inflammatory processes and dendritic cells (DCs) are involved in both innate and adaptive immunity in diseased tissues. Our group previously reported that macrophages exposed to hypoxia were infected with the intracellular parasite Leishmania amazonensis, but showed reduced susceptibility to the parasite. This study shows that although hypoxia did not alter human DC viability, it significantly altered phenotypic and functional characteristics. The expression of CD1a, CD80, and CD86 was significantly reduced in DCs exposed to hypoxia, whereas CD11c, CD14, CD123, CD49 and HLA-DR expression remained unaltered in DCs cultured in hypoxia or normoxia. DC secretion of IL-12p70, the bioactive interleukin-12 (IL-12), a cytokine produced in response to inflammatory mediators, was enhanced under hypoxia. In addition, phagocytic activity (Leishmania uptake) was not impaired under hypoxia, although this microenviroment induced infected DCs to reduce parasite survival, consequently controlling the infection rate. All these data support the notion that a hypoxic microenvironment promotes selective pressure on DCs to assume a phenotype characterized by pro-inflammatory and microbial activities in injured or inflamed tissues and contribute to the innate immune response.

Urethane is a chemical carcinogen which causes lung tumorigenesis in mice with similarities to human adenocarcinoma (AC). The sphingosine 1-phosphate agonist FTY720 administered to mice in doses above 5 mg/kg/day has been able to prevent hepatocellular carcinoma and bladder cancer. We used BALB/c mice in urethane-induced lung cancer model to investigate the effects of a lower dose of FTY720 (1 mg/kg/day). The benefits of FTY720 were associated with the time point of the compound administration. FTY720 30 Group presented lower incidence and smaller area of lung nodules, decreased PCNA and increased Caspase-3 expressions. The findings in FTY720 0 Group (nodule multiplicity and area, PCNA expression) were similar to Urethane Group suggesting that the administration of the compound at early time point did not affect lung tumor development. FTY720 90 Group presented the biggest nodule area which was associated with increased PCNA and decreased Caspase-3 expressions. FTY720 (30 days and 90 days) administration decreased CD4 + splenocytes and blood lymphocytes which caused opposite effects in lung tumor development - impairment and improvement respectively.In conclusion, FTY720 in low dose did not provide lung tumor inhibition in mice but its administration 30 days after the chemical carcinogen (Urethane) injection was associated with impaired tumor development.

Calcineurin inhibitors such as cyclosporine (CsA) and tacrolimus (FK506) show similar efficacy to prevent rejection within the first year after organ transplantation. However, their use is limited by side effects, such as kidney damage, hypertension, new-onset diabetes, and hyperlipidemia. The consensus opinion suggests that compared with CsA, FK506 has fewer negative effects on blood pressure, serum lipids, and renal function. Nevertheless, FK506 use is associated with a higher incidence of posttransplantation diabetes mellitus. FTY720 is a new compound that has shown beneficial effects in animal models of rejection in transplantation, ischemia/reperfusion injury, autoimmune diseases, and tumor development. Our aim was to investigate whether FTY720 + tacrolimus association could provide additional immunosuppression without causing renal toxicity. FTY720 as a monotherapy or in association with FK506 was administered to C57BL/6 mice for 21 days to prevent skin graft rejection and to evaluate renal function and structure. Increased skin allograft survival in the FTY720 + FK506 group was associated with decreased cell numbers in the spleen, blood, and axillary lymph nodes. Changes in major histocompatibility complex (MHC) class II and intercellular adhesion molecule-1 (ICAM-1) expressions in splenocytes were also found in this group. The major effects already described for FK506 (diabetes) or FTY720 (lymphopenia) were observed after 21 days administration even when the drugs were associated. FTY720 associated with FK506 caused fewer changes in kidney structure, and blood glucose levels were lower than in FK506 monotherapy.

The innate immune response facilitates the quality of the adaptive immune response and is critical to an individual's susceptibility to infection and disease. Mannose-binding lectin (MBL) is a plasma protein with anti-microbial properties that binds a wide range of pathogens to flag them for immune destruction independent of antibodies. In this study, serum MBL levels were measured in 81 children <5 years old experiencing acute respiratory syncytial virus infection and in 40 control children to determine the association with disease severity. Almost 70% of all RSV-infected children had low to intermediate MBL levels (<500 ng/ml) compared to controls, and most of the <6 months old RSV interned patients had low to intermediate levels. No differences were detected in MBL levels between case and control children <1 month old. Analysis of the T-cell compartment in peripheral blood mononuclear cells (PBMC) from acute RSV-infected and control children showed that the percent CD4+ T cells was statistically lower in RSV-infected children > or =6 months old compared to controls, while the percent CD8+ T cells in RSV-infected and control PBMC was generally similar. These results suggest that low serum MBL levels may be a marker of RSV disease severity in children and that MBL may be important in limiting RSV disease pathogenesis.

Background: Previous publications have documented the damage caused to red blood cells (RBCs) irradiated with X-rays produced by a linear accelerator and with gamma rays derived from a 137Cs source. The biologic effects on RBCs of gamma rays from a 60Co source, however, have not been characterized., Study Design and Methods: This study investigated the effect of 3000 and 4000 cGy on the in vitro properties of RBCs preserved with preservative solution and irradiated with a cobalt teletherapy unit. A thermal device equipped with a data acquisition system was used to maintain and monitor the blood temperature during irradiation. The device was rotated at 2 r.p.m. in the irradiation beam by means of an automated system. The spatial distribution of the absorbed dose over the irradiated volume was obtained with phantom and thermoluminescent dosimeters (TLDs). Levels of Hb, K+, and Cl(-) were assessed by spectrophotometric techniques over a period of 45 days. The change in the topology of the RBC membrane was investigated by flow cytometry., Results: Irradiation caused significant changes in the extracellular levels of K+ and Hb and in the organizational structure of the phospholipid bilayer of the RBC membrane. Blood temperature ranged from 2 to 4 degrees C during irradiation. Rotation at 2 r.p.m. distributed the dose homogeneously (92%-104%) and did not damage the RBCs., Conclusions: The method used to store the blood bags during irradiation guaranteed that all damage caused to the cells was exclusively due to the action of radiation at the doses applied. It was demonstrated that prolonged storage of 60Co-irradiated RBCs results in loss of membrane phospholipids asymmetry, exposing phosphatidylserine (PS) on the cells' surface with a time and dose dependence, which can reduce the in vivo recovery of these cells. A time- and dose-dependence effect on the extracellular K+ and plasma-free Hb levels was also observed. The magnitude of all these effects, however, seems not to be clinically important and can support the storage of irradiated RBC units for at last 28 days.

Ischemia-reperfusion injury is a common early event in kidney transplantation and contributes to a delay in organ function. Acute tubular necrosis, impaired kidney function and organ leukocyte infiltration are the major findings. The therapeutic potential of stem cells has been the focus of recent research as these cells possess capabilities such as self-renewal, multipotent differentiation and aid in regeneration after organ injury. FTY720 is a new synthetic compound that has been associated with preferential migration of blood lymphocytes to peripheral lymph nodes instead of inflammatory sites. Bone marrow stem cells (BMSC) and/or FTY720 were used as therapy to promote recovery of tubule cells and avoid inflammation at the renal site, respectively. Mice were submitted to renal ischemia-reperfusion injury and were either treated with two doses of FTY720, 10x10(6) BMSC, or both in order to compare the therapeutic effect with non-treated and control animals. Renal function and structure were investigated as were cell numbers in peripheral blood and spleen. Activation and apoptosis markers were also evaluated in splenocytes using flow cytometry. We found that the combined therapy (FTY720+BMSC) was associated with more significant changes in renal function and structure after ischemia-reperfusion injury when compared with the other groups. Also a decrease at cell numbers and prevention of spleen cells activation and apoptosis was observed. In conclusion, in our model it was not possible to demonstrate the potential of stem cells alone or in combination with FTY720 to promote early kidney recovery after ischemia-reperfusion injury.

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34%) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

Background: To identify the most appropriate dose for the prevention of transfusion-associated graft-versus-host disease, the radiosensitivity of T cells has been determined in blood bags irradiated with X-rays produced by a linear accelerator and gamma rays derived from the cesium-137 source of a specific irradiator. In this study, the influence of doses ranging from 500 to 2500 cGy was investigated on T cells isolated from red blood cell (RBC) units preserved with ADSOL and irradiated with a cobalt teletherapy unit., Study Design and Methods: A thermal device consisting of acrylic and foam was constructed to store the blood bags during irradiation. Blood temperature was monitored with an automated data acquisition system. Dose distribution in the blood bags was analyzed based on isodose curves obtained with a polystyrene phantom constructed for this purpose. The influence of cobalt-60 gamma radiation on T cells was determined by limiting-dilution analysis, which measures clonable T cells. T-cell content of the mononuclear cell population plated was assessed by flow cytometry with a monoclonal antibody specific for CD3., Results: Blood temperature ranged from 2 to 4.5 degrees C during irradiation. Dosimetry performed on the phantom showed a homogenous dose distribution when the phantom was irradiated with a parallel-opposite field. A radiation dose of 1500 cGy led to the inactivation of T cells by 4 log, but T-cell growth was observed in all experiments. At 2500 cGy, no T-cell growth was detected in any of the experiments and a greater than 5 log reduction in functional T cells was noted., Conclusion: The results showed that a dose of 2500 cGy completely inactivates T cells in RBC units irradiated with cobalt-60 source.

Allogeneic bone marrow transplantation (alloBMT) is the only curative therapy for chronic myelogenous leukemia (CML). This success is explained by the delivery of high doses of antineoplastic agents followed by the rescue of marrow function and the induction of graft-versus-leukemia reaction mediated by allogeneic lymphocytes against host tumor cells. This reaction can also be induced by donor lymphocyte infusion (DLI) producing remission in most patients with CML who relapse after alloBMT. The immunological mechanisms involved in DLI therapy are poorly understood. We studied five CML patients in the chronic phase, who received DLI after relapsing from an HLA-identical BMT. Using flow cytometry we evaluated cellular activation and apoptosis, NK cytotoxicity, lymphocytes producing cytokines (IL-2, IL-4 and IFN-gamma), and unstimulated (in vivo) lymphocyte proliferation. In three CML patients who achieved hematological and/or cytogenetic remission after DLI we observed an increase of the percent of activation markers on T and NK cells (CD3/DR, CD3/CD25 and CD56/DR), of lymphocytes producing IL-2 and IFN-gamma, of NK activity, and of in vivo lymphocyte proliferation. These changes were not observed consistently in two of the five patients who did not achieve complete remission with DLI. The percent of apoptotic markers (Fas, FasL and Bcl-2) on lymphocytes and CD34-positive cells did not change after DLI throughout the different study periods. Taken together, these preliminary results suggest that the therapeutic effect of DLI in the chronic phase of CML is mediated by classic cytotoxic and proliferative events involving T and NK cells but not by the Fas pathway of apoptosis.

Treatment with interferon-alpha is effective for chronic myelogenous leukemia in the chronic phase (CML-CP), but the immunological mechanisms of the antileukemic effect of this substance are still unclear. The objective of this study was to investigate the immunological effects of interferon-alpha in CML patients. Markers of cellular activation and apoptosis, natural killer (NK) cell cytotoxicity and production of intracellular cytokines (IFN-gamma, IL-2 and IL-4) were determined by flow cytometry in the peripheral blood mononuclear cells (PBMC) of 26 CML-CP patients before and 3, 6 and 9 months after IFN-alpha treatment. The results were correlated with the hematological response. In the whole group of patients, INF-alpha use was followed by a significant increase of lymphocytes producing IL-2 and IFN-gamma, an increase in NK activity and a decrease in the number of CD34+ cells. Out of 26 CML patients, 15 achieved hematological remission and 7 achieved partial cytogenetic remission after 9 months of IFN-alpha treatment. There was an increase in the percentage of CD8/FasL+, DR/CD3+, DQ/CD3+, CD34/Fas+, DR/CD56+, CD56/FasL+ cells and of IFN-gamma- and IL-2-producing lymphocytes and an increase in NK cytotoxicity only in the group of patients who achieved complete hematological remission. Our results indicate that IFN-alpha use in CML-CP reduces the number of CD34+ cells, activates T cells, enhances stem cell apoptotic markers and increases the production of intracellular IFN-gamma and IL-2 by lymphocytes. Taken together, these results indicate that the therapeutic effect of IFN-alpha in CML-CP is mediated at least in part by immunological mechanisms.

Myosin-V is involved in organelle and vesicle trafficking in Saccharomyces cerevisiae and in other eukaryotic cells from yeast to human. In the present study, we determined by FACS that the major subpopulations of the peripheral blood mononuclear cells express myosin-V with similar fluorescence intensity. Confocal microscopy showed intense labeling for myosin-V at the centrosomal region and a punctate staining throughout the cytoplasm, frequently associated with the central microtubule arrays and the actin-rich cortex. Some degree of overlap with an endolysosomal marker and dynein light-chain 8 k was found at the cell center. Striking colocalization was observed with the major histocompatibility complex (MHC) class II molecules near the cell surface. Treatment with phytohemagglutinin, which induces T-lymphocyte activation, associated with MHC class II expression, increased the levels of myosin-V protein and mRNA for the three members of class V myosins. These data suggest that class V myosins might be involved in relevant functions in the immune response.

Natural killer (NK) cells are CD3- CD56+ and/or CD16+ cytotoxic lymphocytes that mediate first-line defense against various types of target cells without prior immunization. To assess the effect of the menstrual cycle and gender on NK activity we evaluated 30 healthy women (mean age 28.1 years, range 21-39) in follicular and luteal phases, 29 postmenopausal women (mean age 58.8 years, range 42-72) and 48 healthy men (mean age 31.6 years, range 21-40). In a flow cytometric test of NK activity, peripheral blood mononuclear effector cells were mixed with K562 targets cells labeled with DiO (3,3'-dioctadecyloxacarbocyanine perchlorate) at effector:target cell ratios of 40, 20, 10 and 5:1. Dead cells were stained with propidium iodide and results were expressed as lytic units per 10(7) cells. In addition, progesterone levels were determined in the luteal phase of the menstrual cycle of healthy women by a chemiluminescence assay. Our results showed that (1) NK cytotoxicity was higher in the follicular than in the luteal phase of the menstrual cycle (P < 0.0001); (2) postmenopausal women and men showed NK activity similar to women in the follicular phase but higher than women in the luteal phase of the menstrual cycle (P < 0.05); and (3) there was no correlation between NK activity and levels of progesterone. The data suggest that progesterone does not influence NK activity directly and that other factors may explain the reduction of NK activity in the luteal phase of the menstrual cycle.