Multidisciplinary treatment program for improvement in pain and disability associated with nonspecific chronic low back pain / Programa de tratamento multidisciplinar para melhora da dor e incapacidade associada a dor crônica lombar inespecífica

ABSTRACT BACKGROUND AND OBJECTIVES: Lumbar disorders, which contribute to significant workplace absenteeism and chronic disability, are associated with a considerable financial and social burden. Although a conservative approach provides satisfactory pain relief, biomechanical improvement and is associated with a low risk of adverse effects, there is lack of consensus in the literature regarding the best therapeutic strategy in such cases. METHODS: This retrospective longitudinal study used secondary data from the institutional medical records of patients who completed a multidisciplinary program for the treatment of low back pain between 2019 and 2021. Data regarding pain levels and motor skills were obtained from patients who completed the care program at a private hospital in Bento Gonçalves, RS. The following step-wise treatment algorithm was used: evaluation by a specialist physician for the etiological diagnosis of pain, pharmacological management and dry needling, followed by standard rehabilitation intervention performed by the physiotherapy team and exercises by the physical education team. The visual analogue scale (VAS) was used to measure pain at the start and at the completion of the intervention, and the Oswestry Disability Index (ODI) was used to measure motor skills at the start and at 6 and 12 months following the multiprofessional intervention for rehabilitation. RESULTS: A reduction in pain and motor disability in patients who completed all stages of the treatment program was observed. Pain by the VAS presented the following scores: baseline 7 [5-8] and after treatment 2 [0-4]; and the scores of the ODI were: at baseline 0.34 [0.26 - 0.40], at 6 months 0.16 [0.08 - 0.26] and after treatment 0.12 [0.04 - 0.21]. CONCLUSION: The treatment program reduced the pain and disability associated with low back pain and can serve as the basis for further studies carried out to confirm the effectiveness of this intervention.

RESUMO JUSTIFICATIVA E OBJETIVOS: As doenças lombares, que contribuem para um absenteísmo significativo no local de trabalho e para a incapacidade crônica, estão associadas a um encargo financeiro e social considerável. Embora a abordagem conservadora proporcione alívio satisfatório da dor, melhore a biomecânica e esteja associada a baixo risco de efeitos adversos, não há consenso na literatura sobre a melhor estratégia terapêutica nesses casos. MÉTODOS: Neste estudo longitudinal retrospectivo, foram utilizados dados secundários dos prontuários médicos institucionais de pacientes que completaram um programa multidisciplinar para tratamento de dor lombar entre 2019 e 2021. Dados sobre níveis de dor e habilidades motoras foram obtidos de pacientes que completaram o programa assistencial de um hospital privado de Bento Gonçalves, RS. Foi utilizado o seguinte tratamento passo a passo: avaliação por médico especialista para diagnóstico etiológico da dor, manejo farmacológico e agulhamento a seco, seguido de intervenção de reabilitação padrão realizada pela equipe de fisioterapia e exercícios pela equipe de educação física. A escala analógica visual (EAV) foi utilizada para medir a dor no início e após a conclusão da intervenção, e o Índice de Incapacidade de Oswestry (ODI) foi usado para medir as habilidades motoras no início e aos 6 e 12 meses após a intervenção multiprofissional para reabilitação. RESULTADOS: Observou-se redução na dor e na incapacidade motora em pacientes que completaram todas as etapas do programa de tratamento. A intensidade da dor medida pela EAV apresentou as seguintes pontuações: basal 7 [5-8] e após tratamento 2 [0-4]; enquanto o ODI apresentou as pontuações: basal 0,34 [0,26 - 0,40], até 6 meses 0,16 [0,08 - 0,26] e após o tratamento 0,12 [0,04 - 0,21]. CONCLUSÃO: O programa de tratamento reduziu a dor e a incapacidade associadas à dor lombar e pode servir de base para novos estudos realizados para confirmar a eficácia desta intervenção.

Melatonin for prevention of acute kidney injury in patients treated with intravenous polymyxin B: a double-blind, placebo-controlled randomized clinical trial.

OBJECTIVES: To evaluate the effect of melatonin versus placebo on the incidence of acute kidney injury (AKI) in patients treated with polymyxin B. METHODS: We performed a single-centre, double-blind, randomized clinical trial (NCT03725267) of 30-mg oral melatonin versus placebo for patients treated with intravenous polymyxin B. Patients aged ≥18 years receiving polymyxin B for ≤48 hours were eligible. Melatonin or placebo pills were administered until the end of polymyxin B treatment or for a maximum of 14 days. The main outcome was any level of AKI. RESULTS: Eighty-eight patients were randomized: 44 in the melatonin group and 44 in the placebo group. The study ended prematurely because of polymyxin B shortage during the COVID-19 pandemic. The patients' mean age was 63.6 ± 17.3 years, and 60.2% of the patients were men. Forty-six (52.3%, 23 in each group) patients developed AKI during the follow-up period. The incidence rate of AKI was 81.9/1000 and 77.4/1000 patients per day in melatonin and placebo groups, respectively (hazard ratio, 1.09; 95% CI, 0.61-1.94; p 0.78). Renal failure and 30-day mortality were similar between the groups. Moreover, the incidence of AKI was not different in pre-specified sub-groups. DISCUSSION: Melatonin initiated in the first 48 hours of therapy did not reduce the incidence of AKI in patients treated with polymyxin B.

RvD1 treatment during primary infection modulates memory response increasing viral load during respiratory viral reinfection.

Resolvin D1 (RvD1), which is biosynthesized from essential long-chain fatty acids, is involved in anti-inflammatory activity and modulation of T cell response. Memory CD8+ T cells are important for controlling tumor growth and viral infections. Exacerbated inflammation has been described as impairing memory CD8+ T cell differentiation. This study aimed to verify the effects of RvD1 on memory CD8+ T cells in vitro and in vivo in a respiratory virus infection model. Peripheral blood mononuclear cells were treated at different time points with RvD1 and stimulated with anti-CD3/anti-CD28 antibodies. Pre-treatment with RvD1 increases the expansion of memory CD8+ T cells. The IL-12 level, a cytokine described to control memory CD8+ T cells, was reduced with RvD1 pre-treatment. When the mTOR axis was inhibited, the IL-12 levels were restored. In a respiratory virus infection model, Balb/c mice were treated with RvD1 before infection or after 7 days after infection. RvD1 treatment after infection increased the frequency of memory CD8+ T cells in the lung expressing II4, II10, and Ifng. During reinfection, RvD1-treated and RSV-infected mice present a high viral load in the lung and lower antibody response in the serum. Our results show that RvD1 modulates the expansion and phenotype of memory CD8+ T cells but contributed to a non-protective response after RSV reinfection.

DNA Methylation and Immune Memory Response.

The generation of memory is a cardinal feature of the adaptive immune response, involving different factors in a complex process of cellular differentiation. This process is essential for protecting the second encounter with pathogens and is the mechanism by which vaccines work. Epigenetic changes play important roles in the regulation of cell differentiation events. There are three types of epigenetic regulation: DNA methylation, histone modification, and microRNA expression. One of these epigenetic changes, DNA methylation, occurs in cytosine residues, mainly in CpG dinucleotides. This brief review aimed to analyse the literature to verify the involvement of DNA methylation during memory T and B cell development. Several studies have highlighted the importance of the DNA methyltransferases, enzymes that catalyse the methylation of DNA, during memory differentiation, maintenance, and function. The methylation profile within different subsets of naïve activated and memory cells could be an interesting tool to help monitor immune memory response.

Circulating Type I Interferon Levels and COVID-19 Severity: A Systematic Review and Meta-Analysis.

Introduction: Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, resulting in a range of clinical manifestations and outcomes. Laboratory and immunological alterations have been considered as potential markers of disease severity and clinical evolution. Type I interferons (IFN-I), mainly represented by IFN-α and ß, are a group of cytokines with an important function in antiviral responses and have played a complex role in COVID-19. Some studies have demonstrated that IFN-I levels and interferon response is elevated in mild cases, while other studies have noted this in severe cases. The involvement of IFN-I on the pathogenesis and outcomes of SARS-CoV-2 infection remains unclear. In this study, we summarize the available evidence of the association of plasma protein levels of type I IFN with the severity of COVID-19. Methods: The PRISMA checklist guided the reporting of the data. A systematic search of the MEDLINE (PubMed), EMBASE, and Web of Science databases was performed up to March of 2021, looking for articles that evaluated plasma protein levels of IFN-I in mild, severe, or critical COVID-19 patients. Comparative meta-analyses with random effects were performed to compare the standardized mean differences in plasma protein levels of IFN-I of mild versus severe and mild versus critical patients. Meta-regressions were performed to test the moderating role of age, sex, time that the IFN-I was measured, and limit of detection of the assay used in the difference between the means. Results: There was no significant difference in plasma levels of IFN-α when comparing between mild and severe patients (SMD = -0.236, 95% CI -0.645 to 0.173, p = 0.258, I2 = 82.11), nor when comparing between patients mild and critical (SMD = 0.203, 95% CI -0.363 to 0.770, p = 0.481, I2 = 64.06). However, there was a significant difference between healthy individuals and patients with mild disease (SMD = 0.447, 95% CI 0.085 to 0.810, p = 0.016, I2 = 62.89). Conclusions: Peripheral IFN-α cannot be used as a severity marker as it does not determine the clinical status presented by COVID-19 patients.

Cytotoxic Effects of Diclofenac and Ibuprofen Zinc (II)-Nicotinamide Ternary Complexes in Breast Cancer Cell Lines

Abstract Breast cancer is one of the leading types of cancer worldwide, and the search for new treatment options are crucial. Nonsteroidal anti-inflammatory drugs (NSAIDs) -specially ibuprofen and diclofenac-, have shown antitumoral effect against several types of cancer. The synthesis of organometallic compounds has shown significant improvements in pharmacological properties and efficacy of organic molecules. Two zinc II ternary complexes containing the NSAIDs diclofenac and ibuprofen and nicotinamide neutral linker (Nic) were obtained by the two-step solvent metalligand complexation method. The compounds Zn2(Diclof)4(Nic)2 (complex 1) and Zn2(Ibup)4(Nic)2 (complex 2) were tested in breast cancer cell lines (4T1, MCF-7 and MDA-MB-231) to evaluate their cytotoxicity, comparing to ibuprofen and diclofenac as controls. We found that both complex 1 and 2 exerted more than 60% reduction in 4T1 viability at 250µM, and complex 2 decreased cell viability at 250 µM and 137.5 µM in MCF-7 (34.35% and 26.42% reduction, respectively) and in MDA-MB-231 (57.2% and 22.88% reduction, respectively), all compared to controls. Complex 1 was selective only in MCF-7, and complex 2 was selective in both MCF-7 and MDA-MB-231. In summary, our data showed that the cytotoxic effect of complex 1 and 2 is increased comparing to their original NSAID in different breast cancer cell lines, highlighting their potential anti-tumoral activity.

March1-dependent modulation of donor MHC II on CD103+ dendritic cells mitigates alloimmunity.

In transplantation, donor dendritic cells (do-DCs) initiate the alloimmune response either by direct interaction with host T cells or by transferring intact donor MHC to host DCs. However, how do-DCs can be targeted for improving allograft survival is still unclear. Here we show CD103+ DCs are the major do-DC subset involved in the acute rejection of murine skin transplants. In the absence of CD103+ do-DCs, less donor MHC-II is carried to host lymph nodes, fewer allogenic T cells are primed and allograft survival is prolonged. Incubation of skin grafts with the anti-inflammatory mycobacterial protein DnaK reduces donor MHC-II on CD103+DCs and prolongs graft survival. This effect is mediated through IL-10-induced March1, which ubiquitinates and decreases MHC-II levels. Importantly, in vitro pre-treatment of human DCs with DnaK reduces their ability to prime alloreactive T cells. Our findings demonstrate a novel therapeutic approach to dampen alloimmunity by targeting donor MHC-II on CD103+DCs.

High IL-1R8 expression in breast tumors promotes tumor growth and contributes to impaired antitumor immunity.

Tumors develop numerous strategies to fine-tune inflammation and avoid detection and eradication by the immune system. The identification of mechanisms leading to local immune dysregulation is critical to improve cancer therapy. We here demonstrate that Interleukin-1 receptor 8 (IL-1R8 - previously known as SIGIRR/TIR8), a negative regulator of Toll-Like and Interleukin-1 Receptor family signaling, is up-regulated during breast epithelial cell transformation and in primary breast tumors. IL-1R8 expression in transformed breast epithelial cells reduced IL-1-dependent NF-κB activation and production of pro-inflammatory cytokines, inhibited NK cell activation and favored M2-like macrophage polarization. In a murine breast cancer model (MMTV-neu), IL-1R8-deficiency reduced tumor growth and metastasis and was associated with increased mobilization and activation of immune cells, such as NK cells and CD8+ T cells. Finally, immune-gene signature analysis in clinical specimens revealed that high IL-1R8 expression is associated with impaired innate immune sensing and T-cell exclusion from the tumor microenvironment. Our results indicate that high IL-1R8 expression acts as a novel immunomodulatory mechanism leading to dysregulated immunity with important implications for breast cancer immunotherapy.

Pre-clinical evaluation of voltage-gated calcium channel blockers derived from the spider P. nigriventer in glioma progression.

This study investigated the effects of P/Q- and N-type voltage-gated calcium channel (VGCC) blockers derived from P. nigriventer in glioma progression, by means of in vitro and in vivo experiments. Glioma cells M059J, U-138MG and U-251MG were used to evaluate the antiproliferative effects of P/Q- and N-type VGCC inhibitors PhTx3-3 and Phα1ß from P. nigriventer (0.3-100 pM), in comparison to MVIIC and MVIIA from C. magus (0.3-100 pM), respectively. The toxins were also analyzed in a glioma model induced by implantation of GL261 mouse cells. PhTx3-3, Phα1ß and MVIIA displayed significant inhibitory effects on the proliferation and viability of all tested glioma cell lines, and evoked cell death mainly with apoptosis characteristics, as indicated by Annexin V/propidium iodide (PI) positivity. The antiproliferative effects of toxins were confirmed by flow cytometry using Ki67 staining. None of the tested toxins altered the proliferation rates of the N9 non-tumor glial cell line. Noteworthy, the administration of the preferential N-type VGCC inhibitors, Phα1ß (50 pmol/site; i.c.v.), its recombinant form CTK 01512-2 (50 pmol/site; i.c.v. and i.t.), or MVIIA (10 pmol/site; i.c.v.) caused significant reductions of tumor areas in vivo. N-type VGCC inhibition by Phα1ß, CTK 01512-2, and MVIIA led to a marked increase of GFAP-activated astrocytes, and Iba-1-positive microglia, in the peritumoral region, which might explain, at least in part, the inhibitory effects of the toxins in tumor development. This study provides novel evidence on the potential effects of P. nigriventer-derived P/Q-, and mainly, N-type VGCC inhibitors, in glioma progression.

IL-10 is required for polarization of macrophages to M2-like phenotype by mycobacterial DnaK (heat shock protein 70).

Macrophages are key cells in the innate immune system. They phagocytose pathogens and cellular debris, promote inflammation, and have important roles in tumor immunity. Depending on the microenvironment, macrophages can polarize to M1 (inflammatory) or M2 (anti-inflammatory) phenotypes. Extracellular DnaK (the bacterial ortholog of the mammalian Hsp70) from Mycobacterium tuberculosis (Mtb) was described to exert immune modulatory roles in an IL-10 dependent manner. We have previously observed that endotoxin-free DnaK can polarize macrophages to an M2-like phenotype. However, the mechanisms that underlie this polarization need to be further investigated. IL-10 has been described to promote macrophage polarization, so we investigated the involvement of this cytokine in macrophages stimulated with extracellular DnaK. IL-10 was required to induce the expression of M2 markers - Ym1 and Fizz, when macrophages were treated with DnaK. Blockade of IL-10R also impaired DnaK induced polarization, demonstrating the requirement of the IL-10/IL-10R signaling pathway in this polarization. DnaK was able to induce TGF-ß mRNA in treated macrophages in an IL-10 dependent manner. However, protein TGF-ß could not be detected in culture supernatants. Finally, using an in vivo allogeneic melanoma model, we observed that DnaK-treated macrophages can promote tumor growth in an IL-10-dependent manner. Our results indicate that the IL-10/IL-10R axis is required for DnaK-induced M2-like polarization in murine macrophages.

P2X7 receptor as predictor gene for glioma radiosensitivity and median survival.

Glioblastoma multiforme (GBM) is considered the most lethal intracranial tumor and the median survival time is approximately 14 months. Although some glioma cells present radioresistance, radiotherapy has been the mainstay of therapy for patients with malignant glioma. The activation of P2X7 receptor (P2X7R) is responsible for ATP-induced death in various cell types. In this study, we analyzed the importance of ATP-P2X7R pathway in the radiotherapy response P2X7R silenced cell lines, in vivo and human tumor samples. Both glioma cell lines used in this study present a functional P2X7R and the P2X7R silencing reduced P2X7R pore activity by ethidium bromide uptake. Gamma radiation (2Gy) treatment reduced cell number in a P2X7R-dependent way, since both P2X7R antagonist and P2X7R silencing blocked the cell cytotoxicity caused by irradiation after 24h. The activation of P2X7R is time-dependent, as EtBr uptake significantly increased after 24h of irradiation. The radiotherapy plus ATP incubation significantly increased annexin V incorporation, compared with radiotherapy alone, suggesting that ATP acts synergistically with radiotherapy. Of note, GL261 P2X7R silenced-bearing mice failed in respond to radiotherapy (8Gy) and GL261 WT-bearing mice, that constitutively express P2X7R, presented a significant reduction in tumor volume after radiotherapy, showing in vivo that functional P2X7R expression is essential for an efficient radiotherapy response in gliomas. We also showed that a high P2X7R expression is a good prognostic factor for glioma radiosensitivity and survival probability in humans. Our data revealed the relevance of P2X7R expression in glioma cells to a successful radiotherapy response, and shed new light on this receptor as a useful predictor of the sensitivity of cancer patients to radiotherapy and median survival.

Pathological concentrations of homocysteine increases IL-1ß production in macrophages in a P2X7, NF-ĸB, and erk-dependent manner.

Elevated plasma levels of homocysteine (Hcy) are associated with the development of coronary artery disease (CAD), peripheral vascular disease, and atherosclerosis. Hyperhomocysteinemia is likely related to the enhanced production of pro-inflammatory cytokines including IL-1ß. However, the mechanisms underlying the effects of Hcy in immune cells are not completely understood. Recent studies have established a link between macrophage accumulation, cytokine IL-1ß, and the advance of vascular diseases. The purpose of the present study is to investigate the effects of Hcy on IL-1ß secretion by murine macrophages. Hcy (100 µM) increases IL-1ß synthesis via enhancement of P2X7 expression and NF-ĸB and ERK activation in murine macrophages. In addition, the antioxidant agent N-acetylcysteine (NAC) reduces NF-κB activation, ERK phosphorylation, and IL-1ß production in Hcy-exposed macrophages, indicating the importance of ROS in this pro-inflammatory process. In summary, our results show that Hcy may be involved in the synthesis and secretion of IL-1ß via NF-ĸB, ERK, and P2X7 stimulation in murine macrophages.

Involvement of purinergic system in the release of cytokines by macrophages exposed to glioma-conditioned medium.

Macrophages are involved in cancer progression. M1 macrophages have an antitumor effect, whereas M2 phenotype are associated with tumor growth. The progression of gliomas involves the participation of an inflammatory microenvironment. Adenosine triphosphate (ATP) can act as pro-inflammatory signal, whereas adenosine has opposite properties. The biological effects of extracellular nucleotides/nucleosides mediated by purinergic receptors are controlled by ectonucleotidases. In the present work, we evaluated whether glioma-conditioned medium (GL-CM) modulates macrophage differentiation and the participation of ATP and adenosine in the release of pro-and anti-inflammatory cytokines by these cells. The results show that macrophages exposed to GL-CM were modulated to an M2-like phenotype. HPLC analysis of GL-CM demonstrated the presence of significant amounts of ATP and its metabolites. Macrophages exposed to GL-CM presented decreased ATP and AMP hydrolysis and increased IL-10 and MCP-1 secretion, effects that were diminished by P1 or P2 antagonists. GL-CM did not alter the release of IL-6 by macrophages, although treatment with ATP promoted an increase in the release of IL-6, which was prevented by a P2X7 antagonist. In summary, we found that A2A and P2X7 activation is necessary for IL-10, MCP-1, and IL-6 release by macrophages exposed to GL-CM, which, in turn, modulates the macrophages to M2-phenotype. The present study establishes a relationship between M2-like polarization, cytokine release and purinergic receptor activation in macrophages exposed to GL-CM. Therefore, the data presented herein contributes to advancing in the field of cancer-related inflammation and point specific purinergic receptors as targets for modulation of the phenotype of glioma-associated macrophages.

Extracellular mycobacterial DnaK polarizes macrophages to the M2-like phenotype.

Macrophages are myeloid cells that play an essential role in inflammation and host defense, regulating immune responses and maintaining tissue homeostasis. Depending on the microenvironment, macrophages can polarize to two distinct phenotypes. The M1 phenotype is activated by IFN-γ and bacterial products, and displays an inflammatory profile, while M2 macrophages are activated by IL-4 and tend to be anti-inflammatory or immunosupressive. It was observed that DnaK from Mycobacterium tuberculosis has immunosuppressive properties, inducing a tolerogenic phenotype in dendritic cells and MDSCs, contributing to graft acceptance and tumor growth. However, its role in macrophage polarization remains to be elucidated. We asked whether DnaK was able to modulate macrophage phenotype. Murine macrophages, derived from bone marrow, or from the peritoneum, were incubated with DnaK and their phenotype compared to M1 or M2 polarized macrophages. Treatment with DnaK leads macrophages to present higher arginase I activity, IL-10 production and FIZZ1 and Ym1 expression. Furthermore, DnaK increased surface levels of CD206. Importantly, DnaK-treated macrophages were able to promote tumor growth in an allogeneic melanoma model. Our results suggest that DnaK polarizes macrophages to the M2-like phenotype and could constitute a virulence factor and is an important immunomodulator of macrophage responses.

Mechanisms involved in kinin-induced glioma cells proliferation: the role of ERK1/2 and PI3K/Akt pathways.

Gliomas are the most common malignant brain tumors in adults. Bradykinin (BK) displays an important role in cancer, although the exact role of kinin receptors in the glioma biology remains unclear. This study investigated the role of kinin B1 and B2 receptors (B1R and B2R) on cell proliferation in human glioblastoma cell lineages. The mRNA expression of B1R and B2R was verified by RT-qPCR, whereas the effects of kinin agonists (des-Arg(9)-BK and BK) were analyzed by cell counting, MTT assay and annexin-V/PI determination. The PI3K/Akt and ERK1/2 signaling activation was assessed by flow cytometry. Our results demonstrated that both human glioblastoma cell lines U-138MG and U-251MG express functional B1R and B2R. The proliferative effects induced by the incubation of des-Arg(9)-BK and BK are likely related to the activation of PI3K/Akt and ERK 1/2 pathways. Moreover, the pre-incubation of the selective PI3Kγ blocker AS252424 markedly prevented kinin-induced AKT phosphorylation. Noteworthy, the selective B1R and B2R antagonists SSR240612 and HOE-140 were able to induce cell death of either lineages, with mixed apoptosis/necrosis characteristics. Taken together, the present results show that activation of B1R and B2R might contribute to glioblastoma progression in vitro. Furthermore, PI3K/Akt and ERK 1/2 signaling may be a target for adjuvant treatment of glioblastoma with a possible impact on tumor proliferation.

Mesenchymal stem cells from different murine tissues have differential capacity to metabolize extracellular nucleotides.

Mesenchymal stem cells (MSCs) have shown a great potential for cell-based therapy and many different therapeutic purposes. Despite the recent advances in the knowledge of MSCs biology, their biochemical and molecular properties are still poorly defined. Ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases) and ecto-5'-nucleotidase (eNT/CD73) are widely expressed enzymes that hydrolyze extracellular nucleotides, generating an important cellular signaling cascade. Currently, studies have evidenced the relationship between the purinergic system and the development, maintenance, and differentiation of stem cells. The objective of this study is to identify the NTPDases and eNT/CD73 and compare the levels of nucleotide hydrolysis on MSCs isolated from different murine tissues (bone marrow, lung, vena cava, kidney, pancreas, spleen, skin, and adipose tissue). MSCs from all tissues investigated expressed the ectoenzymes at different levels. In MSCs from pancreas and adipose tissue, the hydrolysis of triphosphonucleosides was significantly higher when compared to the other cells. The diphosphonucleosides were hydrolyzed at a higher rate by MSC from pancreas when compared to MSC from other tissues. The differential nucleotide hydrolysis activity and enzyme expression in these cells suggests that MSCs play different roles in regulating the purinergic system in these tissues. Overall MSCs are an attractive adult-derived cell population for therapies, however, the fact that ecto-nucleotide metabolism can affect the microenvironment, modulating important events, such as immune response, makes the assessment of this metabolism an important part of the characterization of MSCs to be applied therapeutically.

Homocysteine modifies extracellular ATP availability in macrophages.

Increased levels of plasma homocysteine (hyperhomocysteinemia-HHcy) are associated to the development of coronary artery disease (CAD), peripheral vascular disease and thrombosis. In addition, recent studies have shown that inflammation, probably mediated by macrophages, mediates the pathogenesis associated to high levels of homocysteine (Hcy). In the present study, we evaluated the Hcy effects in the ATP hydrolysis and its breakdown products in murine macrophages. The results showed that micromolar concentrations of Hcy increased the ATP, ADP and AMP hydrolysis. Additionally, our results show decreased inosine levels in the extracellular milieu of Hcy-exposed macrophages. The increasing in ATP, ADP and AMP hydrolysis are not explained by increased transcription or protein expression of NTPDases and ecto-5'-nucleotidase (ecto-5'-NT/CD73) enzymes. Moreover, the formation of reactive oxygen species did not interfere in the Hcy effects, which suggest that Hcy or Hcy metabolites act directly on the modulation of NTPDases and ecto-5'-NT/CD73 activities. In conclusion, Hcy induces the rapid breakdown of ATP, ADP and AMP to adenosine (ADO), which is classically known as an anti-inflammatory response in immune cells. However, by the action of these enzymes, the extracellular adenosine generated during Hcy treatment probably is uptaken into the cells, as evidenced by the decreased in inosine formation, and thus collaborating to the inflammatory complications associates to HHcy.

Implication of purinergic P2X7 receptor in M. tuberculosis infection and host interaction mechanisms: a mouse model study.

In the present study, we analyzed the role of purinergic P2X7 receptor in Mycobacterium tuberculosis infection and host interaction mechanisms in vitro and in vivo. For experimental procedures, a macrophage murine cell line RAW 264.7, and male Swiss, wild-type C57BL/6 and P2X7 receptor knockout (P2X7R−/−) mice were used throughout this study. We have demonstrated that treatment of RAW 264.7 cells with ATP (3 and 5 mM) resulted in a statistically significant reduction of M. tuberculosis-colony-forming units. The purinergic P2X7 receptor expression was found significantly augmented in the lungs of mice infected with M. tuberculosis H37Rv. Infected wild-type mice showed a marked increase in the spleen weight, in comparison to non-infected animals. Furthermore, M. tuberculosis-infected P2X7R−/− mice showed an increase of M. tuberculosis burden in lung tissue, when compared to infected wild-type mice. In P2X7R−/− spleens, we observed a significant decrease in the populations of Treg (CD4+Foxp3+), T cells (CD4+, CD8+CD25+ and CD4+CD25+), dendritic cells (CD11c+) and B220+ cells. However, a significant increase in CD11b+ cells was observed in P2X7R−/− mice, when compared to wild-type animals. In the lungs, P2X7R−/− M. tuberculosisinfected mice exhibited pulmonary infiltrates containing an increase of Treg cells (CD4+Foxp3+), T cells (CD4+ and CD8+) and a decrease in the B220+ cells, when compared with wild-type M. tuberculosis-infected mice. The findings observed in the present study provide novel evidence on the role of P2X7 receptors in the pathogenesis of tuberculosis.

P2X7 receptor is required for neutrophil accumulation in a mouse model of irritant contact dermatitis.

Irritant contact dermatitis (ICD) is an inflammatory reaction caused by chemical toxicity on the skin. The P2X7 receptor (P2X7R) is a key mediator of cytokine release, which recruits immune cells to sites of inflammation. We investigated the role of P2X7R in croton oil (CrO)-induced ICD using in vitro and in vivo approaches. ICD was induced in vivo by CrO application on the mouse ear and in vitro by incubation of murine macrophages and dendritic cells (DCs) with CrO and ATP. Infiltrating cells were identified by flow cytometry, histology and myeloperoxidase (MPO) determination. Effects of the ATP scavenger apyrase were assessed to investigate further the role of P2X7R in ICD. Animals were also treated with N-1330, a caspase-1 inhibitor, or with clodronate, which induces macrophage apoptosis. CrO application induced severe inflammatory Gr1(+) cell infiltration and increased MPO levels in the mouse ear. Selective P2X7R antagonism with A438079 or genetic P2X7R deletion reduced the neutrophil infiltration. Clodronate administration significantly reduced Gr1(+) cell infiltration and local IL-1ß levels. In vitro experiments confirmed that A438079 or apyrase treatment prevented the increase in IL-1ß that was evoked by macrophage and DC incubation with CrO and ATP. These data support a key role for P2X7 in ICD-mediated inflammation via modulation of inflammatory cells. It is tempting to suggest that P2X7R inhibition might be an alternative ICD treatment.

Altered responsiveness to extracellular ATP enhances acetaminophen hepatotoxicity.

BACKGROUND: Adenosine triphosphate (ATP) is secreted from hepatocytes under physiological conditions and plays an important role in liver biology through the activation of P2 receptors. Conversely, higher extracellular ATP concentrations, as observed during necrosis, trigger inflammatory responses that contribute to the progression of liver injury. Impaired calcium (Ca2+) homeostasis is a hallmark of acetaminophen (APAP)-induced hepatotoxicity, and since ATP induces mobilization of the intracellular Ca2+ stocks, we evaluated if the release of ATP during APAP-induced necrosis could directly contribute to hepatocyte death. RESULTS: APAP overdose resulted in liver necrosis, massive neutrophil infiltration and large non-perfused areas, as well as remote lung inflammation. In the liver, these effects were significantly abrogated after ATP metabolism by apyrase or P2X receptors blockage, but none of the treatments prevented remote lung inflammation, suggesting a confined local contribution of purinergic signaling into liver environment. In vitro, APAP administration to primary mouse hepatocytes and also HepG2 cells caused cell death in a dose-dependent manner. Interestingly, exposure of HepG2 cells to APAP elicited significant release of ATP to the supernatant in levels that were high enough to promote direct cytotoxicity to healthy primary hepatocytes or HepG2 cells. In agreement to our in vivo results, apyrase treatment or blockage of P2 receptors reduced APAP cytotoxicity. Likewise, ATP exposure caused significant higher intracellular Ca2+ signal in APAP-treated primary hepatocytes, which was reproduced in HepG2 cells. Quantitative real time PCR showed that APAP-challenged HepG2 cells expressed higher levels of several purinergic receptors, which may explain the hypersensitivity to extracellular ATP. This phenotype was confirmed in humans analyzing liver biopsies from patients diagnosed with acute hepatic failure. CONCLUSION: We suggest that under pathological conditions, ATP may act not only an immune system activator, but also as a paracrine direct cytotoxic DAMP through the dysregulation of Ca2+ homeostasis.