Cytotoxicity of Reparative Endodontic Cements on Human Periodontal Ligament Stem Cells

ABSTRACT Objective To compare the cytotoxicity of commercial reparative endodontic cements on human periodontal ligament stem cells (hPDLSCs). Material and Methods The culture of hPDLSCs was established. Cell density was set at 2 × 104 cells/well in 96-well plates. Extracts of Biodentine, Bio-C Repair, Cimmo HD, MTA Repair HP and White MTA were prepared. Then, the extracts were diluted (pure, 1:4 and 1:16) and inserted into cell-seeded wells for 24, 48, and 72 h to assess cell viability through MTT assay. hPDLSCs incubated with culture medium alone served as a negative control group. Data were analyzed by Two-Way ANOVA and Tukey's test (α=0.05). Results At 24 h, pure extract of MTA Repair HP and Biodentine 1:16 presented higher cell viability compared to control. Lower cell viability was found for pure extract of Cimmo HD, MTA Repair HP 1:4 and 1:16, and White MTA 1:16. At 48 h, pure extract of Bio-C Repair and MTA Repair HP presented higher cell viability compared to control. At 72 h, only the pure extract of MTA Repair HP led to higher cell proliferation compared to control. Conclusion Biodentine, Bio-C Repair and MTA Repair HP were able to induce hPDLSCs proliferation. Cimmo HD and White MTA were found to be mostly cytotoxic in hPDLSCs.

CD147 as a Target for COVID-19 Treatment: Suggested Effects of Azithromycin and Stem Cell Engagement.

The expressive number of deaths and confirmed cases of SARS-CoV-2 call for an urgent demand of effective and available drugs for COVID-19 treatment. CD147, a receptor on host cells, is a novel route for SARS-CoV-2 invasion. Thus, drugs that interfere in the spike protein/CD147 interaction or CD147 expression may inhibit viral invasion and dissemination among other cells, including in progenitor/stem cells. Studies suggest beneficial effects of azithromycin in reducing viral load of hospitalized patients, possibly interfering with ligand/CD147 receptor interactions; however, its possible effects on SARS-CoV-2 invasion has not yet been evaluated. In addition to the possible effect in invasion, azithromycin decreases the expression of some metalloproteinases (downstream to CD147), induces anti-viral responses in primary human bronchial epithelial infected with rhinovirus, decreasing viral replication and release. Moreover, resident lung progenitor/stem are extensively differentiated into myofibroblasts during pulmonary fibrosis, a complication observed in COVID-19 patients. This process, and the possible direct viral invasion of progenitor/stem cells via CD147 or ACE2, could result in the decline of these cellular stocks and failing lung repair. Clinical tests with allogeneic MSCs from healthy individuals are underway to enhance endogenous lung repair and suppress inflammation.

The inhibition of adipogenesis via an in vitro assay can reduce animal use by more precisely estimating the starting dose for the acute toxic class method.

In the present work, we established an adipogenesis inhibition assay as an adequate and sensitive in vitro model for reducing animal use by estimating the starting dose for the acute toxic class (ATC) method. First, human adipose-derived stem cells (ADSCs) underwent adipogenic differentiation induction for 14 days. Then, by high-content imaging analysis, we determined the percentage and area of cell differentiation that we considered suitable for negative and positive internal control according to the quality control criteria strictly standardized mean difference (SSMD) and robust SSMD. Moreover, we established sodium dodecyl sulfate (SDS) as an external positive control in this assay. To measure reduction in animal use to estimate the starting dose for the ATC method, we evaluated 10 chemicals representing Globally Harmonized System of Classification and Labeling of Chemicals (GHS) toxicity categories 1-5 and unclassified toxicity and determined the dose-response curves for percentage and area of cell differentiation by using the Hill function with an R2 ≥ 0.85. The resulting IC50 values were used for LD50 prediction and for estimating the starting dose for the ATC method. Our results indicated that use of the inhibition of adipogenesis assay to estimate the starting dose for the ATC method would decrease animal use for 7 out of 10 tested substances, possibly all substances if we consider the more toxic test substances in GHS categories 1, 2, and 3. We can conclude that the present assay is a suitable alternative to reduce animal testing in the first steps of predicting highly toxic substances. Moreover, this method also presents internal and external controls as differentials, which guarantee the quality of the assay as well as the results. These features are important for suggesting a methodology for regulatory purposes.

Terapia com células-tronco mesenquimais geneticamente modificadas para superexpressão de G6GSF e IGF-1 na doença de chagas crônica experimental / Therapy with genetically modified mesenchymal stem cells for overexpression of G6GSF and IGF-1 in experimental chronic chagasic disease

INTRODUÇÃO: A doença de Chagas é uma doença parasitária causada pelo Trypanosoma cruzi, a qual representa uma das principais causas de morbimortalidade na América Latina. As intervenções terapêuticas existentes não são totalmente eficazes, sendo o transplante cardíaco a única alternativa para os pacientes com cardiopatia chagásica crônica (CCC) grave. Neste sentido, a ausência de terapias capazes de atuar diretamente sobre os determinantes fisiopatológicos da doença torna necessária a identificação de novas abordagens terapêuticas. Estudos previamente realizados pelo nosso grupo mostraram que a utilização de célulastronco obtidas da medula óssea e de outras fontes teve efeitos benéficos no tratamento da CCC experimental. A possibilidade de potencializar os efeitos parácrinos das células-tronco através de modificação genética tem sido alvo de investigações científicas. OBJETIVO: Avaliar os efeitos da terapia com células-tronco mesenquimais (CTMs) da medula óssea, modificadas geneticamente para superexpressar o fator estimulador de colônias de granulócitos (hG-CSF) ou o fator de crescimento semelhante à insulina 1 (hIGF-1) em um modelo experimental de CCC. MATERIAL E MÉTODOS: Camundongos C57BL/6 foram infectados com 1000 tripomastigotas da cepa Colombiana de T. cruzi e, após seis meses de infecção, foram tratados com CTM, CTM-G-CSF, ou CTM-IGF-1. Grupos de animais não infectados ou infectados tratados com salina (veículo) foram utilizados como controles. Todos os animais foram eutanasiados sob anestesia após dois meses de tratamento para análises histopatológicas e morfométricas do coração ou músculo esquelético, bem como para avaliação da expressão de citocinas inflamatórias. RESULTADOS: As secções de corações de camundongos dos grupos tratados com CTM, CTM-GCSF ou CTM-IGF-1 apresentaram redução significativa do número de células inflamatórias e do percentual de fibrose em comparação aos animais chagásicos tratados com salina, sendo esta diferença mais evidente no grupo que foi tratado com células-tronco que superexpressam o G-CSF. Além disto, a terapia com CTM-G-CSF induziu a mobilização de células imunomoduladoras para o coração, tais como células supressoras de origem mielóide (MDSC) e células T regulatórias Foxp3+ que expressam IL-10. A avaliação da expressão gênica das citocinas inflamatórias no tecido cardíaco mostrou um aumento das citocinas inflamatórias em animais chagásicos crônicos quando comparados aos controles não infectados, sendo a maioria delas moduladas de forma significativa nos grupos que foram tratados com CTM ou CTM-G-CSF. Apesar da terapia utilizando CTM-IGF-1 não ter apresentado benefício adicional ao tecido cardíaco comparado ao grupo que foi tratado com CTM não modificadas, foi observado um efeito regenerativo desta terapia no músculo esquelético dos animais, resultando em um aumento de fibras musculares esqueléticas 60 dias após o tratamento. CONCLUSÃO: Nossos resultados demonstram que o tratamento com CTM da medula óssea que superexpressam hG-CSF ou hIGF-1 potencializou o efeito terapêutico das CTMs através de ações imunomoduladoras e pró-regenerativas no coração e músculo esquelético de camundongos cronicamente infectados por T. cruzi. Desse modo, a modificação genética de CTMs para superexpressão de fatores com potencial terapêutico representa uma estratégia promissora para o desenvolvimento de novas terapias para a cardiomiopatia chagásica crônica

INTRODUCTION: Chagas' disease is a parasitic disease caused by Trypanosoma cruzi, which is one of the main causes of cardiovascular morbidity and mortality in Latin America. Existing therapeutic interventions are not fully effective, and heart transplantation is the only alternative for patients with severe chronic Chagas' heart disease. In this sense, the absence of therapies capable of acting directly on the pathophysiological determinants of the disease demonstrates the necessity of identifying new therapeutic approaches. Studies previously conducted by our group demonstrated that the use of stem cells obtained from bone marrow and other sources had beneficial effects in the treatment of experimental chagas disease. Additionally, the possibility of enhancing stem cell paracrine effects through genetic modification has been the subject of scientific investigations. OBJECTIVE: To evaluate the effects of genetically modified mesenchymal stem cell therapy to overexpress granulocyte colony stimulating factor (hG-CSF) or insulin-like growth factor 1 (hIGF-1) in an experimental model of Chagas disease. MATERIAL AND METHODS: C57BL/6 mice were infected with 1000 trypomastigotes from the Colombian strain of T. cruzi and, after six months of infection, were treated with CTM, CTM-G-CSF, or CTM-IGF-1. Groups of uninfected or infected animals treated with saline (vehicle) were used as controls. All animals were euthanized under anesthesia after two months of treatment for histopathological and morphometric analysis of the heart or skeletal muscle, as well as for evaluation of inflammatory cytokine expression. RESULTS: Mouse heart sections from groups treated with CTM, CTM-GCSF or CTM-IGF-1 showed a significant reduction in the number of inflammatory cells and the percentage of fibrosis when compared to chagasic animals treated with saline.This difference was more evident in the group that was treated with stem cells overexpressing G-CSF. In addition, CTM-G-CSF therapy induced mobilization of immunomodulatory cells to the heart, including myeloid suppressor cells (MDSC) and Foxp3 + regulatory T cells expressing IL-10. Expression of inflammatory cytokine genes in cardiac tissue revealed an increase in inflammatory cytokines in chronic chagasic animals when compared to uninfected controls, where most cytokines were significantly modulated in groups treated with CTM or CTM-G-CSF. Although CTM-IGF-1 therapy demonstrated no additional benefit to cardiac tissue when compared to the group treated with unmodified CTM, a regenerative effect of this therapy was observed in chagasic mice skeletal muscle, resulting in an increase in skeletal muscle fibers 60 days after treatment. CONCLUSION: Our results demonstrate that bone marrow derived CTM treatment overexpressing hG-CSF or hIGF-1 enhanced the therapeutic effects of MSCs through immunomodulation and proregenerative actions in the heart and skeletal muscle of mice chronically infected wthT. cruzi. Thus, the genetic modification of CTMs for overexpression of factors with therapeutic potential represents a promising strategy for the development of new therapies for chronic chagasic cardiomyopathy

Stem cells from human-exfoliated deciduous teeth reduce tissue-infiltrating inflammatory cells improving clinical signs in experimental autoimmune encephalomyelitis.

Stem cells from human exfoliated deciduous teeth (SHED) have great therapeutic potential and here, by the first time, we evaluated their immunomodulatory effect on experimental model of autoimmune encephalomyelitis (EAE). Specifically, we investigated the effect of SHED administration on clinical signs and cellular patterns in EAE model using Foxp3 GFP + transgenic mice (C57Bl/6-Foxp3GFP). The results showed that SHED infusion ameliorated EAE clinical score with reduced number of infiltrating IFN-γ+CD8+, IL-4+CD8+, IFN-γ+CD4+ and IL-4+CD4+ T cells into the central nervous system (CNS). In addition, we observed that SHED promoted a significant increase in CD4+FOXP3+ T cells population in the spleen of EAE-affected animals. Taken together, our results provide strong evidence that SHED can modulate peripherally the CD4+ T cell responses suggesting that SHED would be explored as part of cellular therapy in autoimmune diseases associated with CNS.

The influence of protein malnutrition on biological and immunomodulatory aspects of bone marrow mesenchymal stem cells.

Tissues that require a great supply of nutrients and possess high metabolic demands, such as lympho-hemopoietics tissues, are the first to be affected by protein malnutrition (PM). Thus, PM directly affects hemopoiesis and the production and function of immune cells. Consequently, malnourished individuals are more susceptible to infections. Mesenchymal stem cells (MSCs) have immunomodulatory properties and are important in the formation of lympho-hemopoietic stroma. Since an adequate supply of nutrients is essential to sustain stroma formation, which is mainly constituted of MSCs and differentiated cells originated from them, this study investigated whether PM would influence some biological and immunomodulatory aspects of MSCs. Two-month-old Balb/c mice were divided into control and malnourished groups receiving normoproteic or hypoproteic diets, respectively (12% and 2% of protein) for 28 days. MSCs obtained from control (MSCct) and malnourished (MSCmaln) animals were characterized. In addition, the proliferation rate and cell cycle protein expression were determined, but no differences in these parameters were observed. In order to evaluate whether PM affects the immunomodulatory properties of MSCs, the expression of NFκB and STAT-3, and the production of IL-1α, IL-1ß, IL-6, IL-10, TGF-ß and TNF-α by MSCs were assessed. MSCmaln expressed lower levels of NF-κB and the production of IL-1ß, IL-6 and TGF-ß was significantly influenced by PM. Furthermore, MSCct and MSCmaln culture supernatants affected lymphocyte and macrophage proliferation. However, MSCmaln did not reduce the production of IFN-γ nor stimulate the production of IL-10 in lymphocytes in the same manner as observed in MSCct. Overall, this study implied that PM modifies immunosuppressive properties of MSCs.

CD73-mediated adenosine production promotes stem cell-like properties in mouse Tc17 cells.

The CD73 ectonucleotidase catalyses the hydrolysis of AMP to adenosine, an immunosuppressive molecule. Recent evidence has demonstrated that this ectonucleotidase is up-regulated in T helper type 17 cells when generated in the presence of transforming growth factor-ß (TGF-ß), and hence CD73 expression is related to the acquisition of immunosuppressive potential by these cells. TGF-ß is also able to induce CD73 expression in CD8(+) T cells but the function of this ectonucleotidase in CD8(+) T cells is still unknown. Here, we show that Tc17 cells present high levels of the CD73 ectonucleotidase and produce adenosine; however, they do not suppress the proliferation of CD4(+) T cells. Interestingly, we report that adenosine signalling through A2A receptor favours interleukin-17 production and the expression of stem cell-associated transcription factors such as tcf-7 and lef-1 but restrains the acquisition of Tc1-related effector molecules such as interferon-γ and Granzyme B by Tc17 cells. Within the tumour microenvironment, CD73 is highly expressed in CD62L(+) CD127(+) CD8(+) T cells (memory T cells) and is down-regulated in GZMB(+) KLRG1(+) CD8(+) T cells (terminally differentiated T cells), demonstrating that CD73 is expressed in memory/naive cells and is down-regulated during differentiation. These data reveal a novel function of CD73 ectonucleotidase in arresting CD8(+) T-cell differentiation and support the idea that CD73-driven adenosine production by Tc17 cells may promote stem cell-like properties in Tc17 cells.

Geração e caracterização de linhagens de células-tronco mesenquimais de camundongo geneticamente modificadas para expressão / Generation and characterization of mousemesenchymal stem cells lines genetically modified for the ectopic expression of hG-CSFand hIGF-1ectópica de hIGF-1 ou hG-CSF

As células-tronco mesenquimais (CTM) constituem uma ferramenta promissora para o campo de terapia celular. Além de seu potencial de diferenciação em diferentes tipos celulares, as CTM apresentam a habilidade de secretar moléculas bioativas e, assim, exercer múltiplos efeitos biológicos, tais como indução da regeneração de tecidos lesionados, redução de fibrose e modulação do sistema imune. A superexpressão dos fatores de crescimento G-CSF e IGF-1, conhecidos por seus efeitos sobre os processos de imunomodulação, sobrevivência celular e reparo tecidual, pode ampliar as ações terapêuticas das CTM. O objetivo deste trabalho consiste em gerar e caracterizar linhagens de CTM de camundongo superexpressando hGCSF ou hIGF-1. Um sistema lentiviral de segunda geração foi utilizado para modificação de CTM para expressão ectópica dos genes de interesse. As sequências codificantes de hG-CSF e hIGF-1 foram amplificadas por PCR e subclonadas em um vetor lentiviral de transferência, contendo um promotor constitutivo. As partículas lentivirais foram produzidas a partir da cotransfecção de células da linhagem HEK293FT...

Mesenchymal stem cells (MSCs) are a promising tool for the cell therapy field. In addition to their potential for differentiation into different cell types, MSCs have the ability to secrete bioactive molecules and thus exert multiple biological effects such as induction of the injured tissue regeneration, fibrosis reduction and modulation of the immune system. The overexpression of the growth factors G-CSF and IGF-1, known for their effects on immune modulation processes, cell survival and tissue repair, can result in a magnification of MSCs' therapeutic actions. The objective of this work is to generate and characterize mouse MSCs lines overexpressing hG-CSF or hIGF-1. A second generation lentiviral system was used to modify MSCs derived from mice for the ectopic expression of the genes of interest. The coding sequences of hG-CSF and hIGF-1 were amplified by PCR and subcloned into a lentiviral transfer vector containing a constitutive promoter. The lentiviral particles were produced from the co-transfection of HEK293FT...

The CD8⁺ memory stem T cell (T(SCM)) subset is associated with improved prognosis in chronic HIV-1 infection.

UNLABELLED: Memory stem T cells (T(SCM)) constitute a long-lived, self-renewing lymphocyte population essential for the maintenance of functional immunity. The hallmarks of HIV-1 pathogenesis are CD4(+) T cell depletion and abnormal cellular activation. We investigated the impact of HIV-1 infection on the T(SCM) compartment, as well as any protective role these cells may have in disease progression, by characterizing this subset in a cohort of 113 subjects with various degrees of viral control on and off highly active antiretroviral therapy (HAART). We observed that the frequency of CD8(+) T(SCM) was decreased in all individuals with chronic, untreated HIV-1 infection and that HAART had a restorative effect on this subset. In contrast, natural controllers of HIV-1 had the highest absolute number of CD4(+) T(SCM) cells among all of the infected groups. The frequency of CD4(+) T(SCM) predicted higher CD8(+) T(SCM) frequencies, consistent with a role for the CD4(+) subset in helping to maintain CD8(+) memory T cells. In addition, T(SCM) appeared to be progenitors for effector T cells (TEM), as these two compartments were inversely correlated. Increased frequencies of CD8(+) T(SCM) predicted lower viral loads, higher CD4(+) counts, and less CD8(+) T cell activation. Finally, we found that T(SCM) express the mucosal homing integrin α4ß7 and can be identified in gut-associated lymphoid tissue (GALT). The frequency of mucosal CD4(+) T(SCM) was inversely correlated with that in the blood, potentially reflecting the ability of these self-renewing cells to migrate to a crucial site of ongoing viral replication and CD4(+) T cell depletion. IMPORTANCE: HIV-1 infection leads to profound impairment of the immune system. T(SCM) constitute a recently identified lymphocyte subset with stem cell-like qualities, including the ability to generate other memory T cell subtypes, and are therefore likely to play an important role in controlling viral infection. We investigated the relationship between the size of the CD8(+) T(SCM) compartment and HIV-1 disease progression in a cohort of chronically infected individuals. Our results suggest that HAART restores a normal frequency of CD8(+) T(SCM) and that the natural preservation of this subset in the setting of untreated HIV-1 infection is associated with improved viral control and immunity. Therefore, the CD8(+) T(SCM) population may represent a correlate of protection in chronic HIV-1 infection that is directly relevant to the design of T cell-based vaccines, adoptive immunotherapy approaches, or the pharmacologic induction of T(SCM).

Avaliação da atividade biológica de células-tronco da polpa dental humana submetida à irradiação laser de baixa intensidade / Evaluation of biological activity of stem cells from human dental pulp subjected to low-intensity laser irradiation

O laser de baixa intensidade (LBI) tem sido utilizado com a finalidade de promover cicatrização e regeneração dos tecidos. A literatura mostra um efeito positivo do LBI na proliferação celular, porém pouco se sabe sobre a sua eficácia na proliferação de célulastronco dentais. O objetivo deste estudo foi avaliar o efeito da irradiação do LBI na atividade biológica de células-tronco da polpa de dente permanente (DPSCs). Extratos de polpa dental foram isolados de cinco terceiros molares hígidos removidos por indicação cirúrgica e/ou ortodôntica. Após a digestão enzimática, as células foram caracterizadas e cultivadas em meio de cultura αMEM suplementado com antibióticos e 15% de soro fetal bovino. No terceiro subcultivo, as células foram irradiadas com um laser diodo InGaAlP, utilizando-se duas diferentes densidades de energia (0,5 J/cm2 - 16 segundos e 1,0 J/cm² - 33 segundos), comprimento de onda de 660nm e potência de 30mW. Uma nova irradiação, utilizando os mesmos parâmetros, foi realizada 48 h após a primeira. Um grupo controle (não irradiado) foi mantido nas mesmas condições experimentais de cultivo. A fim de avaliar a proliferação celular, foram utilizados o método exclusão por Azul de Tripan e a viabilidade celular medida através do ensaio de MTT, nos intervalos de 24, 48, 72 e 96 h após a primeira aplicação do laser. Nos mesmos intervalos foram avaliados os eventos do ciclo celular. Eventos relacionados à morte celular foram analisados por citometria de fluxo nos intervalos de 24 e 72 horas. Os dados das contagens celulares foram submetidos a testes estatísticos não paramétricos de Kruskal-Wallis e Mann-Whitney, considerando um intervalo de confiança de 95%. Os resultados mostraram que os dois grupos irradiados exibiram uma curva de proliferação mais ascendente, com diferença estatisticamente significante (p<0,05) em relação ao grupo controle nos intervalos de 72 e 96h, sem alterações consideráveis na viabilidade celular ao longo do experimento. A distribuição das células nas fases do ciclo celular foi coerente com células em proliferação nos três grupos. Os resultados da curva de crescimento, MTT, Anexina/PI e Ciclo celular foram concordantes, dessa forma é possível concluir que o LLLI, principalmente com dose de 1,0J/cm 2 , seja uma terapia de grande importância para o futuro da engenharia tecidual e medicina regenerativa envolvendo DPSCs, uma vez que neste estudo o LLLI contribuiu com o crescimento e viabilidade celular. (AU)

The low-level laser therapy (LLLT) has been used in order to improve wound healing and tissue regeneration. The literature shows a positive effect of LLLT on cell proliferation, but little is known about its effectiveness on the proliferation of dental pulp stem cells. The aim of this study was to evaluate the effect of irradiation LLLT on biological activity of dental pulp stem cells from permanent teeth (DPSCs). Dental pulp extracts were isolated from healthy five third molars removed by surgical and/or orthodontic indication. After enzymatic digestion, the cells were examined and cultured in αMEM supplemented with antibiotics and 15% fetal bovine serum. On the third subculture, the cells were either irradiated with a laser diode InGaAlP, using two different energy densities (0.5 J/cm2 - 16 seconds and 1.0 J / cm ² - 33 seconds), wavelength of 660nm and power of 30mW. A new irradiation using the same parameters was performed 48 hours after the first. A control group (non-irradiated) was kept under the same experimental conditions of culture. Cell proliferation was evaluated by Trypan blue exclusion method and by measuring mitochondrial activity using the MTT-based cytotoxicity assay, at intervals of 24, 48, 72 and 96 h after the first laser application. Events of the cell cycle were evaluated in the same intervals, and the events related to cell death were analyzed by flow cytometry in the ranges of 24 to 72 hours. Data from cell counts were submitted to non-parametric Kruskal-Wallis and Mann-Whitney test, considering a confidence interval of 95%. The results showed that the both groups irradiated exhibited an upward cell proliferation curve, with statistically significant difference (p < 0.05) compared to the control group in intervals of 72 and 96 hours. No significant changes were observed in cell viability throughout the experiment. The distribution of the cells in the cell cycle phases was consistent with proliferating cells in all three groups. The results of growth curve, MTT, Anexin/PI and cell cycle were concordant, then it is possible conclude that the LLLI, especially with the dose of 1,0J/cm2 , it is a therapy of great importance for the future of tissue engineering and regenerative medicine involving stem cells, once in this study it contributed to the growth and viability of DPSCs. (AU)

CD34 affinity pheresis attenuates a surge among circulating progenitor cells following vascular injury.

BACKGROUND: Intimal hyperplasia (restenosis) is an exaggerated healing response leading to failure of half of vascular interventions. Increasing evidence suggests that circulating progenitor cells contribute to intimal pathology, and clinical studies have demonstrated a correlation between progenitor cells and the incidence of restenosis after cardiovascular interventions. The aims of this study were to characterize the temporal response of CD34+ progenitors following vascular injury in an ovine model and to evaluate an affinity pheresis approach to attenuate this response. METHODS: An ovine model underwent either operative vascular injury or a nonvascular surgery (n = 3 per group). Blood was examined perioperatively over 2 weeks by flow cytometry. Next, an affinity pheresis approach to mediate systemic depletion of CD34 progenitors was designed. Custom agarose pheresis matrix with antibody affinity toward CD34 or an isotype control was evaluated in vitro. Next, following vascular injury, sheep underwent perioperative whole blood volume pheresis toward either the progenitor cell marker CD34 (n = 3) or an isotype control (n = 4) for 14 days. Animals were monitored by physical exam as well as complete blood counts. Cells recovered by pheresis were eluted and examined by flow cytometry. RESULTS: Flow cytometry revealed a focal surge of circulating CD34 cells after vascular injury but not among surgical controls (P = .05). Toward the goal of an approach to attenuate the surge of CD34 progenitors, an evaluation of high-flow affinity matrix revealed efficacy in removal of progenitors from ovine blood in vitro. Next, a separate group of animals undergoing affinity pheresis after vascular injury was evaluated to mediate systemic depletion of CD34+ cells. Again, a surge of CD34+ cells was observed among isotype pheresis animals following vascular intervention but was attenuated over 20-fold by a CD34 pheresis approach (P = .029). Furthermore, an average of 77 million CD34-positive cells were eluted from the CD34 pheresis matrix. Despite multiple sessions of pheresis, complete blood counts remained essentially unchanged over 2 weeks. CONCLUSIONS: Despite evidence suggesting a role for CD34+ circulating progenitor cells in restenotic pathology, the temporal pattern of CD34 progenitors after vascular injury has not been previously defined. We have demonstrated a surge among circulating CD34+ cells that appears confined to procedures involving vascular injury and that this event seems to occur early after vascular injury. We further conclude that CD34 affinity pheresis attenuates the surge. This approach for direct depletion of progenitors may have important implications for the study of progenitors in vascular restenosis.

B-cell lymphopoiesis is regulated by cathepsin L.

Cathepsin L (CTSL) is a ubiquitously expressed lysosomal cysteine peptidase with diverse and highly specific functions. The involvement of CTSL in thymic CD4+ T-cell positive selection has been well documented. Using CTSL(nkt/nkt) mice that lack CTSL activity, we have previously demonstrated that the absence of CTSL activity affects the homeostasis of the T-cell pool by decreasing CD4+ cell thymic production and increasing CD8+ thymocyte production. Herein we investigated the influence of CTSL activity on the homeostasis of peripheral B-cell populations and bone marrow (BM) B-cell maturation. B-cell numbers were increased in lymph nodes (LN), spleen and blood from CTSL (nkt/nkt) mice. Increases in splenic B-cell numbers were restricted to transitional T1 and T2 cells and to the marginal zone (MZ) cell subpopulation. No alterations in the proliferative or apoptosis levels were detected in peripheral B-cell populations from CTSL (nkt/nkt) mice. In the BM, the percentage and the absolute number of pre-pro-B, pro-B, pre-B, immature and mature B cells were not altered. However, in vitro and in vivo experiments showed that BM B-cell production was markedly increased in CTSL (nkt/nkt) mice. Besides, BM B-cell emigration to the spleen was increased in CTSL (nkt/nkt) mice. Colony-forming unit pre-B (CFU pre-B) assays in the presence of BM stromal cells (SC) and reciprocal BM chimeras revealed that both BM B-cell precursors and SC would contribute to sustain the increased B-cell hematopoiesis in CTSL (nkt/nkt) mice. Overall, our data clearly demonstrate that CTSL negatively regulates BM B-cell production and output therefore influencing the homeostasis of peripheral B cells.

The REMEDEE trial: a randomized comparison of a combination sirolimus-eluting endothelial progenitor cell capture stent with a paclitaxel-eluting stent.

OBJECTIVES: This study sought to compare the efficacy and safety results after coronary implantation of a combined sirolimus-eluting CD34 antibody coated Combo stent (OrbusNeich Medical, Ft. Lauderdale, Florida) with the paclitaxel-eluting Taxus Liberté stent (PES) (Boston Scientific, Natick, Massachusetts). This report summarizes the first-in-man randomized, controlled multicenter REMEDEE trial (Randomized study to Evaluate the safety and effectiveness of an abluMinal sirolimus coatED bio-Engineered StEnt) angiographic, intravascular ultrasound, and clinical results up to 12 months. BACKGROUND: Drug-eluting stents have limited restenosis and reintervention but are complicated by especially late and very late stent thrombosis and accelerated neoatherosclerosis. Alternative or adjunct technologies should address these limitations. METHODS: One hundred eighty-three patients with de novo native coronary artery stenoses were randomized 2:1 to Combo stent or PES implantation. The primary endpoint is the angiographic in-stent late lumen loss at 9 months, which was tested for noninferiority between the 2 stent groups. Secondary endpoints include the occurrence of major adverse cardiac events. RESULTS: The Combo stent was found to be noninferior to the PES in 9-month angiographic in-stent late lumen loss with 0.39 ± 0.45 mm versus 0.44 ± 0.56 mm (pnoninferiority = 0.0012). At 12 months, the occurrence of major adverse cardiac events was 8.9% in the Combo group and 10.2% in the PES group (p = 0.80) with no difference in mortality, occurrence of myocardial infarction, or target lesion revascularization. No stent thrombosis was reported in either group. CONCLUSIONS: In the REMEDEE trial the Combo stent has shown to be effective by meeting the primary noninferiority angiographic endpoint and safe, with an overall low rate of clinical events in both stent groups, including no stent thrombosis up to 12 months.

Toward personalized cell therapies by using stem cells: seven relevant topics for safety and success in stem cell therapy.

Stem cells, both embryonic and adult, due to the potential for application in tissue regeneration have been the target of interest to the world scientific community. In fact, stem cells can be considered revolutionary in the field of medicine, especially in the treatment of a wide range of human diseases. However, caution is needed in the clinical application of such cells and this is an issue that demands more studies. This paper will discuss some controversial issues of importance for achieving cell therapy safety and success. Particularly, the following aspects of stem cell biology will be presented: methods for stem cells culture, teratogenic or tumorigenic potential, cellular dose, proliferation, senescence, karyotyping, and immunosuppressive activity.

Derivation and characterization of progenitor stem cells from canine allantois and amniotic fluids at the third trimester of gestation.

Fetal tissues are frequently discarded before (amniocentesis) or after birth, which both facilitates stem cell access and helps to overcome ethical concerns. In the present study, we aimed to isolate and characterize stem cells from the allantoic and amniotic fluids (ALF; AMF) of third trimester canine fetuses. This gestation age has not been previously explored for stem cells isolation. The gestational age, cell culture conditions and method of isolation used in this study allowed for the establishment and efficient expansion of ALF and AMF cells. We showed that the majority of ALF and ALF cells express the stem cell markers, such as vimentin, nestin and cytokeratin 18 (CK18). Under appropriate culture conditions AMF derived cells can undergo differentiation into osteogenic, adipogenic, chondrogenic and neuron-like lineages. ALF derived cells showed adipogenic, and chondrogenic potential. Therefore, ALF and AMF cells derived at the third gestation trimester can be qualified as progenitor stem cells, accordingly referred as (alantoic fluid progenitor/stem) ALF PS cells and (amniotic fluid progenitor/stem) AMF PS cells.

In vitro biology of human hematopoietic stem and progenitor cells.

Hematopoietic stem and progenitor cells from human umbilical cord blood have been the focus of both basic and clinical research during the last 20 years. It has been clearly demonstrated that such sells possess higher proliferation and expansion potentials, as compared to their adult counterparts, and their capacity to reconstitute the hematopoietic system of mammals has also been shown. Different In vitro systems have been used to characterize the biology of these hematopoietic cells and some culture methods are being currently used to expand the numbers of such cells for clinical purposes.

Stem Cell Therapy to Restore Pancreatic Function in Dogs and Cats

Diabetes mellitus is a common disease in dogs and cats. It consists of a group of metabolic diseases characterized by hyperglycemia resulting from defects in secretion and/or insulin activity. The islets of Langerhans from donor pancreas may be an alternative for the cure of diabetes, however, this approach is limited because the donation is scarce and complications occur due to the concurrent use of immunosuppressive drugs. For many decades researchers have sought ways to replace pancreatic islets in diabetic individuals. Current studies in progress with stem cell culture for production of pancreatic islet cells are promising, despite the difficulties in their production. This review reports several aspects concerning the use of stem cells in diabetes cell therapy. Recent studies in mice have shown that embryonic stem cells can be induced to differentiate into insulin-producing ß-cells. In parallel with this study, a new class of stem cells has emerged, i.e. induced pluripotent stem cells (iPSCs) aimed at clinical and therapeutic use. Adult stem cells may circumvent the ethical issues surrounding embryonic stem cells and allow auto-transplantation.(AU)

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.

Myeloid-derived suppressor cells help protective immunity to Leishmania major infection despite suppressed T cell responses.

Th1/Th2 cytokines play a key role in immune responses to Leishmania major by controlling macrophage activation for NO production and parasite killing. MDSCs, including myeloid precursors and immature monocytes, produce NO and suppress T cell responses in tumor immunity. We hypothesized that NO-producing MDSCs could help immunity to L. major infection. Gr1(hi)(Ly6C(hi)) CD11b(hi) MDSCs elicited by L. major infection suppressed polyclonal and antigen-specific T cell proliferation. Moreover, L. major-induced MDSCs killed intracellular parasites in a NO-dependent manner and reduced parasite burden in vivo. By contrast, treatment with ATRA, which induces MDSCs to differentiate into macrophages, increased development of lesions, parasite load, and T cell proliferation in draining LNs. Altogether, these results indicate that NO-producing MDSCs help protective immunity to L. major infection, despite suppressed T cell proliferation.

Stem cells and androgen receptor in human periapical periodontitis / Células madre y receptor de andrógenos en periodontitis periapical humana

Activation of macrophages in periapical granulomas occurs through the presence of cytokines, endotoxin and other genetic and epigenetic factors, allowing the initiation of inflammation and bone resorption. The present study aims to analyze the presence of CD133 protein (marker of stem cells) and the AR (androgen receptor) protein in biopsies of human odontogenic periapical granuloma. Biopsies from 14 adult male patients with diagnosis of periapical granuloma included in paraffin blocks were processed histologically to obtain 5-um thick sections. Protein presence was detected and analyzed by immunohistochemistry of CD133 and AR. The quantification considered the number of positive cells in 0.17 mm2 random areas under the microscope using a 1000X objective. Both CD133 and AR proteins are expressed abundantly in cells in pathological periapical granulomas tissue. The number of cells expressing CD133 and AR shows a wide variation coefficient, so its variation is a particular feature for each individual. We concluded that in human odontogenic periapical granuloma there are abundant stem cells and cells expressing AR that may be important for the pathogenic inflammatory process.

La activación de los macrófagos en los granulomas periapicales humanos se producen a través de la presencia de citoquinas, endotoxinas y otros factores genéticos y epigenéticos que permiten la iniciación de la inflamación y la reabsorción ósea. El presente estudio pretende analizar la presencia de proteína CD133 (marcador de células madre) y de la proteína RA (receptor de andrógenos) en las biopsias de granulomas periapicales odontogénicos humanos. Las biopsias de 14 pacientes varones adultos con diagnóstico de granuloma periapical fueron incluidos en bloques de parafina y se procesaron histológicamente para obtener secciones de 5 micras de espesor. La presencia de CD133 y RA fueron detectadas y analizadas por inmunohistoquímica. La cuantificación se realizó considerando el número de células positivas en áreas al azar de 0,17mm2, utilizando microscopio con objetivo de 1000X. Ambas proteínas, CD133 y RA se expresan en abundancia en las células del tejido patológico con granuloma periapical. El número de células que expresan CD133 y RA presentan un amplio coeficiente de variación, por lo que su variación es una característica particular de cada individuo. Se concluye que en granuloma periapical odontogénico humano se expresan abundantes células madre y proteínas receptoras de andrógenos, antecedentes que pueden sermuy importantes en la expresión y diagnosis de los procesos patológicos inflamatorios.