Carla Martins Kaneto

Anti-inflammatory effects of carvacrol: Evidence for a key role of interleukin-10

Carvacrol, a phenolic monoterpene, has been reported to possess anti-inflammatory properties. However, the mechanisms involved in its pharmacological properties are currently not well understood. In the present study, the contribution of cytokine modulation to the anti-inflammatory effects of carvacrol was investigated in a classical inflammation model: the complete Freunds adjuvant (CFA)-induced paw inflammation in mice. The paw edema was measured using a plesthismometer. Paw tissue was removed 2h after the inflammatory stimulus to determine the levels of prostaglandin E(2) (PGE(2)) by enzyme immunoassay, the levels of interleukin-1 β (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin-10 (IL-10) by ELISA or the mRNA expression of cyclooxygenase-2 (COX-2), IL-1β, TNF-α, and IL-10 by real-time PCR. Administration of carvacrol produced anti-inflammatory effects against CFA-induced inflammation in mice. Treatment of mice with carvacrol at 50 and 100mg/kg attenuated the paw edema and reduced the IL-1β and PGE(2), but not TNF-α, local levels. Similarly, carvacrol (100mg/kg) reduced the COX-2 and IL-1β mRNA expression. The levels of IL-10, an anti-inflammatory cytokine, and the IL-10 mRNA expression in the inflamed paw were enhanced by carvacrol. In addition, the treatment with carvacrol did not reduce the CFA-induced paw edema in IL-10 knockout mice. The present results suggest that carvacrol causes anti-inflammatory effects by reducing the production of inflammatory mediators, such as IL-1β and prostanoids, possibly through the induction of IL-10 release.

Safety and neurological assessments after autologous transplantation of bone marrow mesenchymal stem cells in subjects with chronic spinal cord injury.

IntroductionThe administration of stem cells holds promise as a potential therapy for spinal cord injury (SCI). Mesenchymal stem cells have advantages for clinical applications, since they can be easily obtained, are suitable for autologous transplantation and have been previously shown to induce regeneration of the spinal cord in experimental settings. Here we evaluated the feasibility, safety and potential efficacy of autologous transplantation of mesenchymal stem cells in subjects with chronic complete SCI.MethodWe conducted a phase I, non-controlled study in 14 subjects of both genders aging between 18 to 65 years, with chronic traumatic SCI (>6 months), at thoracic or lumbar levels, classified as American Spinal Injury Association (ASIA) A - complete injury. Baseline somatosensory evoked potentials (SSEP), spinal magnetic resonance imaging (MRI) and urodynamics were assessed before and after treatment. Pain rating was performed using the McGill Pain Questionnaire and a visual analogue score scale. Bone marrow-derived mesenchymal stem cells were cultured and characterized by flow cytometry, cell differentiation assays and G-band karyotyping. Mesenchymal stem cells were injected directly into the lesion following laminectomy and durotomy.ResultsCell transplantation was an overall safe and well-tolerated procedure. All subjects displayed variable improvements in tactile sensitivity and eight subjects developed lower limbs motor functional gains, principally in the hip flexors. Seven subjects presented sacral sparing and improved American Spinal Injury Association impairment scale (AIS) grades to B or C – incomplete injury. Nine subjects had improvements in urologic function. One subject presented changes in SSEP 3 and 6 months after mesenchymal stem cells transplantation. Statistically significant correlations between the improvements in neurological function and both injury size and level were found.ConclusionIntralesional transplantation of autologous mesenchymal stem cells in subjects with chronic, complete spinal cord injury is safe, feasible, and may promote neurological improvements.Trial registrationClinicalTrials.gov NCT01325103 – Registered 28 March 2011

Transplantation of bone marrow mononuclear cells decreases seizure incidence, mitigates neuronal loss and modulates pro-inflammatory cytokine production in epileptic rats

Approximately 30% of patients with mesial temporal lobe epilepsy do not respond to treatment with antiepileptic drugs. We have previously shown that transplantation of mononuclear bone marrow cells (BMC) has an anticonvulsant effect in acute epilepsy. Here, we used pilocarpine to induce epilepsy in rats and studied the effects of BMC injected intravenously either at the onset of seizures or after 10 months of recurrent seizures. BMC effectively decreased seizure frequency and duration. In addition, decreased levels of proinflammatory cytokines (TNF-α, IL-1β and IL-6) and increased levels of anti-inflammatory cytokine (IL-10) were observed in the brain and serum of BMC-treated rats. Transplants performed at seizure-onset protected against pilocarpine-induced neuronal loss and gliosis and stimulated the proliferation of new neurons in epileptic rats. Our data demonstrate that BMC transplantation has potent therapeutic effects and could be a potential therapy for clinically intractable epilepsies.

Transplantation of stem cells obtained from murine dental pulp improves pancreatic damage, renal function, and painful diabetic neuropathy in diabetic type 1 mouse model

Diabetes mellitus (DM) is one of the most common and serious chronic diseases in the world. Here, we investigated the effects of mouse dental pulp stem cell (mDPSC) transplantation in a streptozotocin (STZ)-induced diabetes type 1 model. C57BL/6 mice were treated intraperitoneally with 80 mg/kg of STZ and transplanted with 1 × 106 mDPSCs or injected with saline, by an endovenous route, after diabetes onset. Blood and urine glucose levels were reduced in hyperglycemic mice treated with mDPSCs when compared to saline-treated controls. This correlated with an increase in pancreatic islets and insulin production 30 days after mDPSC therapy. Moreover, urea and proteinuria levels normalized after mDPSC transplantation in diabetic mice, indicating an improvement of renal function. This was confirmed by a histopathological analysis of kidney sections. We observed the loss of the epithelial brush border and proximal tubule dilatation only in saline-treated diabetic mice, which is indicative of acute renal lesion. STZ-induced thermal hyperalgesia was also reduced after cell therapy. Three days after transplantation, mDPSC-treated diabetic mice exhibited nociceptive thresholds similar to that of nondiabetic mice, an effect maintained throughout the 90-day evaluation period. Immunofluorescence analyses of the pancreas revealed the presence of GFP+ cells in, or surrounding, pancreatic islets. Our results demonstrate that mDPSCs may contribute to pancreatic β-cell renewal, prevent renal damage in diabetic animals, and produce a powerful and long-lasting antinociceptive effect on behavioral neuropathic pain. Our results suggest stem cell therapy as an option for the control of diabetes complications such as intractable diabetic neuropathic pain.

Administration of granulocyte colony-stimulating factor induces immunomodulation, recruitment of T regulatory cells, reduction of myocarditis and decrease of parasite load in a mouse model of chronic Chagas disease cardiomyopathy

Chagas disease, caused by Trypanosoma cruzi infection, is a leading cause of heart failure in Latin American countries. In a previous study, we showed beneficial effects of granulocyte colony‐stimulating factor (G‐CSF) administration in the heart function of mice with chronic T. cruzi infection. Presently, we investigated the mechanisms by which this cytokine exerts its beneficial effects. Mice chronically infected with T. cruzi were treated with human recombinant G‐CSF (3 courses of 200 μg/kg/d for 5 d). Inflammation and fibrosis were reduced in the hearts of G‐CSF‐treated mice, compared with the hearts of vehicle‐treated mice, which correlated with decreased syndecan‐4, intercellular adhesion molecule‐1, and galectin‐3 expressions. Marked reductions in interferon‐γ and tumor necrosis factor‐α and increased interleukin‐10 and transforming growth factor‐β were found after G‐CSF administration. Because the therapy did not induce a Th1 to Th2 immune response deviation, we investigated the role of regulatory T (Treg) cells. A significant increase in CD3+Foxp3+ cells was observed in the hearts of G‐CSF‐treated mice. In addition, a reduction of parasitism was observed after G‐CSF treatment. Our results indicate a role of Treg cells in the immunosuppression induced by G‐CSF treatment and reinforces its potential therapeutic use for patients with Chagas disease.—Vasconcelos, J. F., Souza, B. S. F., Lins, T. F. S., Garcia, L. M. S., Kaneto, C. M., Smapaio, G. P., de Alcântara, A. C., Meira, C. S., Macambira, S. G., Ribeiro‐dos‐Santos, R., Soares, M. B. P., Administration of granulocyte colony‐stimulating factor induces immunomodulation, recruitment of T regulatory cells, reduction of myocarditis and decrease of parasite load in a mouse model of chronic Chagas disease cardiomyopathy. FASEB J. 27, 4691–4702 (2013). www.fasebj.org

Reduction of galectin-3 expression and liver fibrosis after cell therapy in a mouse model of cirrhosis

BACKGROUND AIMS Cirrhosis, end-stage liver disease, is caused by different mechanisms of injury, associated with persistent inflammation. Galectin-3 is an important regulator of fibrosis that links chronic inflammation to fibrogenesis. We investigated the role of bone marrow cell (BMC) transplantation in chronic inflammation and hepatic fibrosis. METHODS Liver cirrhosis was induced by administration of carbon tetrachloride and ethanol to wild-type C57BL/6 or bone marrow chimeric mice. Bone marrow chimeras were generated by lethal irradiation and transplantation with BMC obtained from green fluorescent protein (GFP(+) )donors. Wild-type cirrhotic mice were transplanted with BMC without irradiation. Livers from chimeras and cirrhotic transplanted mice were obtained for evaluation of inflammation, fibrosis and regulatory factors [galectin-3, matrix metallopeptidase (MMP)-9, tissue inhibitor of metalloproteinase (TIMP)-1 and transforming growth factor (TGF)-β]. RESULTS The development of cirrhosis was associated with increased expression of galectin-3 by F4/80(+) cells and intense migration of BMC to the liver. Furthermore, when transplanted after the establishment of cirrhosis, BMC also migrated to the liver and localized within the fibrous septa. Two months after BMC therapy, cirrhotic mice had a significant reduction in liver fibrosis and expression of type I collagen. We did not find any difference in levels of TGF-β, TIMP-1 and MMP-9 between saline and BMC groups. However, the numbers of inflammatory cells, phagocytes and galectin-3(+) cells were markedly lower in the livers of cirrhotic mice treated with BMC. CONCLUSIONS Our results demonstrate an important role for BMC in the regulation of liver fibrosis and that transplantation of BMC can accelerate fibrosis regression through modulatory mechanisms.

Evidence for the involvement of spinal cord-inhibitory and cytokines-modulatory mechanisms in the anti-hyperalgesic effect of hecogenin acetate, a steroidal sapogenin-acetylated, in mice.

Hecogenin is a steroidal sapogenin largely drawn from the plants of the genus Agave, commonly known as ‘sisal’, and is one of the important precursors used by the pharmaceutical industry for the synthesis of steroid hormones. Hecogenin acetate (HA) is a steroidal sapogenin-acetylated that produces antinociceptive activity. Thus, we evaluate the antihyperalgesic profile of HA in mice in inflammatory models, as well as its possible involvement with c-fos expression on spinal cord area and cytokines to produces analgesic profile. Acute pretreatment with HA (5, 10, or 20 mg/kg; i.p.) inhibited the development of mechanical hyperalgesia induced by carrageenan, TNF-α, dopamine and PGE2. Additionally, the immunofluorescence data demonstrated that acute pretreatment with HA, at all doses tested, significantly inhibited Fos-like expression in the spinal cord dorsal horn normally observed after carrageenan-inflammation. Moreover, HA did not affect the motor performance of the mice as tested in the Rota rod test. This antinociceptive profile seems to be related, at least in part, to a reduction of pro-inflammatory cytokines, as IL-1β. The present results suggest that HA attenuates mechanical hyperalgesia by blocking the neural transmission of pain at the spinal cord levels and by cytokines-inhibitory mechanisms.

Transplantation of bone marrow cells decreases tumor necrosis factor-α production and blood–brain barrier permeability and improves survival in a mouse model of acetaminophen-induced acute liver disease

BACKGROUND AIMS Acute liver failure (ALF), although rare, remains a rapidly progressive and frequently fatal condition. Acetaminophen (APAP) poisoning induces a massive hepatic necrosis and often leads to death as a result of cerebral edema. Cell-based therapies are currently being investigated for liver injuries. We evaluated the therapeutic potential of transplantation of bone marrow mononuclear cells (BMC) in a mouse model of acute liver injury. METHODS ALF was induced in C57Bl/6 mice submitted to an alcoholic diet followed by fasting and injection of APAP. Mice were transplanted with 10(7) BMC obtained from enhanced green fluorescent protein (GFP) transgenic mice. RESULTS BMC transplantation caused a significant reduction in APAP-induced mortality. However, no significant differences in serum aminotransferase concentrations, extension of liver necrosis, number of inflammatory cells and levels of cytokines in the liver were found when BMC- and saline-injected groups were compared. Moreover, recruitment of transplanted cells to the liver was very low and no donor-derived hepatocytes were observed. Mice submitted to BMC therapy had some protection against disruption of the blood-brain barrier, despite their hyperammonemia, and serum metalloproteinase (MMP)-9 activity similar to the saline-injected group. Tumor necrosis factor (TNF)-α concentrations were decreased in the serum of BMC-treated mice. This reduction was associated with an early increase in interleukin (IL)-10 mRNA expression in the spleen and bone marrow after BMC treatment. CONCLUSIONS BMC transplantation protects mice submitted to high doses of APAP and is a potential candidate for ALF treatment, probably via an immunomodulatory effect on TNF-α production.

Transplantation of adipose tissue mesenchymal stem cells in experimental chronic chagasic cardiopathy.

BACKGROUND Chagas disease, caused by the protozoan Trypanosoma cruzi, is a major cause of heart failure in Latin America. Tissue therapy has been investigated as a possible therapeutic option for patients with cardiovascular disease. OBJECTIVE This study evaluated the effects of therapy with mesenchymal stem cells in an experimental model of chronic Chagasic cardiomyopathy. 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 mesenchymal human stem cells from adipose tissue (STAT) or with Dulbecco/Vogt modified Eagles minimal essential medium - DMEM (control). The treated group received two intraperitoneal injections of STAT (1x10(6) cells/dose), with a month interval between the two doses. Before and after the first and second months of treatment, the chagasic and normal control animals underwent cardiopulmonary exercise testing and electrocardiography. All animals were sacrificed under anesthesia after two months of treatment for histopathological analysis of the heart. RESULTS No improvement was observed in arrhythmias and cardiovascular function in the group of animals treated with STAT; however, sections of mice hearts in this group revealed a significant reduction in the number of inflammatory cells (p<0.0001) and areas of fibrosis (p<0.01) in comparison with chagasic animals treated with DMEM. CONCLUSION Thus, it is concluded that administration of intraperitoneal STAT can reduce inflammation and fibrosis in the heart of mice chronically infected with T. cruzi; however, there were no effects on the cardiac function two months after transplantation.

Bone marrow cells migrate to the heart and skeletal muscle and participate in tissue repair after Trypanosoma cruzi infection in mice

Infection by Trypanosoma cruzi, the aetiological agent of Chagas disease, causes an intense inflammatory reaction in several tissues, including the myocardium. We have previously shown that transplantation of bone marrow cells (BMC) ameliorates the myocarditis in a mouse model of chronic Chagas disease. We investigated the participation of BMC in lesion repair in the heart and skeletal muscle, caused by T. cruzi infection in mice. Infection with a myotropic T. cruzi strain induced an increase in the percentage of stem cells and monocytes in the peripheral blood, as well as in gene expression of chemokines SDF‐1, MCP1, 2, and 3 in the heart and skeletal muscle. To investigate the fate of BMC within the damaged tissue, chimeric mice were generated by syngeneic transplantation of green fluorescent protein (GFP+) BMC into lethally irradiated mice and infected with Trypanosoma cruzi. Migration of GFP+ BMC to the heart and skeletal muscle was observed during and after the acute phase of infection. GFP+ cardiomyocytes and endothelial cells were present in heart sections of chimeric chagasic mice. GFP+ myofibres were observed in the skeletal muscle of chimeric mice at different time points following infection. In conclusion, BMC migrate and contribute to the formation of new resident cells in the heart and skeletal muscle, which can be detected both during the acute and the chronic phase of infection. These findings reinforce the role of BMC in tissue regeneration.