Microcurrent and adipose-derived stem cells modulate genes expression involved in the structural recovery of transected tendon of rats.

Tendon injuries are common and have a high incidence of re-rupture that can cause loss of functionality. Therapies with adipose-derived stem cells (ASC) and the microcurrent (low-intensity electrical stimulation) application present promising effects on the tissue repair. We analyzed the expression of genes and the participation of some molecules potentially involved in the structural recovery of the Achilles tendon of rats, in response to the application of both therapies, isolated and combined. The tendons were distributed in five groups: normal (N), transected (T), transected and ASC (C) or microcurrent (M) or with ASC, and microcurrent (MC). Microcurrent therapy was beneficial for tendon repair, as it was observed a statistically significant increase in the organization of the collagen fibers, with involvement of the TNC, CTGF, FN, FMDO, and COL3A1 genes as well as PCNA, IL-10, and TNF-α. ASC therapy significantly increased the TNC and FMDO genes expression with no changes in the molecular organization of collagen. With the association of therapies, a significant greater collagen fibers organization was observed with involvement of the FMOD gene. The therapies did not affect the expression of COL1A1, SMAD2, SMAD3, MKX, and EGR1 genes, nor did they influence the amount of collagen I and III, caspase-3, tenomodulin (Tnmd), and hydroxyproline. In conclusion, the application of the microcurrent isolated or associated with ASC increased the organization of the collagen fibers, which can result in a greater biomechanical resistance in relation to the tendons treated only with ASC. Future studies will be needed to demonstrate the biological effects of these therapies on the functional recovery of injured tendons.

PML/RARa leukemia induced murine model for immunotherapy evaluation.

Even though leukemia murine models are valuable tools for new drug therapy studies, most of these models consist of immunocompromised mice, which do not exhibit immune responses. In order to obtain an adequate leukemia model, we established an acute promyelocytic leukemia transplantation-based model (PML/RARa) in immunocompetent BALB/c mice, thus making it possible to study drug-induced cellular immune responses in leukemia. The development of PML/RARa leukemia was confirmed by leukocytosis (76.27 ± 21.8 vs. 3.40 ± 1.06; P < 0.0001), anemia (7.46 ± 1.86 vs. 15.10 ± 0.96; P < 0.0001), and thrombocytopenia (131.85 ± 39.32 vs. 839.50 ± 171.20; P < 0.0001), and the presence of blasts in the peripheral blood of mice (approximately 50% blasts; P < 0.0001), 15 days after the transplants. These findings were corroborated through differential counts, flow cytometry, and in vivo imaging, which indicated increased number of immature cells in the bone marrow (15.75 ± 3.30 vs 6.69 ± 0.55; P < 0.001), peripheral blood (7.88 ± 2.67 vs 1.22 ± 0.89; P < 0.001), and spleen (35.21 ± 4.12 vs 1.35 ± 0.86; P < 0.0001), as well as promyelocytes in the bone marrow (41.23 ± 4.80 vs 5.73 ± 1.50; P < 0.0001), peripheral blood (46.08 ± 7.52 vs 1.10 ± 0.59; P < 0.0001) and spleen (35.31 ± 8.26 vs 2.49 ± 0.29; P < 0.0001) of PML/RARa mice. Compared to basal conditions of untransplanted mice, the PML/RARa mice exhibited frequencies of T lymphocytes CD4 helper = 14.85 ± 2.91 vs 20.77 ± 2.9 in the peripheral blood (P < 0.05); 12.75 ± 1.33 vs 45.90 ± 2.02 in the spleen (P < 0.0001); CD8 cytotoxic = 11.27 ± 3.44 vs 11.05 ± 1.22 in the peripheral blood (P > 0.05); 10.48 ± 1.16 vs 30.02 ± 1.80 in the spleen (P < 0.0001); natural killer (NK) cells = 3.68 ± 1.35 vs 6.84 ± 0.52 in the peripheral blood (P < 0.001); 4.43 ± 0.57 vs 6.40 ± 1.14 in the spleen (P < 0.05); B cells 2.50 ± 0.60 vs 15.20 ± 5.34 in the peripheral blood (P < 0.001); 17.77 ± 4.39 vs 46.90 ± 5.92 in the spleen (P < 0.0001); neutrophils = 5.97% ± 1.88 vs 31.57 ± 9.14 (P < 0.0001); and monocytes = 6.45 ± 2.97 vs 15.85 ± 2.57 (P < 0.001), selected as classical (3.33 ± 3.40 vs 57.80 ± 16.51, P < 0.0001), intermediate (57.42 ± 10.61 vs 21.75 ± 5.90, P < 0.0001), and non-classical monocytes (37.51 ± 10.85 vs 18.08 ± 7.13, P < 0.05) in the peripheral blood; and as classically activated (M1) within in the bone marrow (3.70 ± 0.94 vs 1.88 ± 0.39, P < 0.05) and spleen 15.19 ± 3.32 vs 9.47 ± 1.61, P < 0.05), in addition to alternatively activated (M2) macrophages within the bone marrow (23.06 ± 5.25 vs 1.76 ± 0.74, P < 0.0001) and spleen (46.51 ± 11.18 vs 30.58 ± 2.64, P < 0.05) compartments. All-trans retinoic acid (ATRA) treatment of PML/RARa mice reduced blast (immature cells) in the bone marrow (8.62 ± 1.81 vs 15.76 ± 1.25; P < 0.05) and spleen (8.75 ± 1.31 vs 35.21 ± 1.55; P < 0.0001) with no changes in the peripheral blood (10.13 ± 3.33 vs 7.88 ± 1.01; P > 0.05), as well as reduced promyelocytes in the bone marrow (19.79 ± 4.84 vs 41.23 ± 1.81; P < 0.05), peripheral blood (31.65 ± 3.92 vs 46.09 ± 2.84; P < 0.05) and spleen (24.84 ± 2.03 vs 41.46 ± 2.39; P < 0.001), and increased neutrophils of the peripheral blood (35.48 ± 7.24 vs 7.83 ± 1.40; P < 0.05) which was corroborated by reducing of immature cells and increase of neutrophil in the stained smears from PML/RARa mice, thus confirming that this model can be used in drug development studies. Our results show the effective induction of PML/RARa leukemia in BALB/c mice, thus producing a low-priced and reliable tool for investigating cellular immune responses in leukemia.

The IL-17A-neutrophil axis promotes epithelial cell IL-33 production during nematode lung migration.

The early migratory phase of pulmonary helminth infections is characterized by tissue injury leading to the release of the alarmin interleukin (IL)-33 and subsequent induction of type 2 immune responses. We recently described a role for IL-17A, through suppression of interferon (IFN)-γ, as an important inducer of type 2 responses during infection with the lung-migrating rodent nematode Nippostrongylus brasiliensis. Here, we aimed to investigate the interaction between IL-17A and IL-33 during the early lung migratory stages of N. brasiliensis infection. In this brief report, we demonstrate that deficiency of IL-17A leads to impaired IL-33 expression and secretion early in infection, independent of IL-17A suppression of IFN-γ. Neutrophil-depletion experiments, which dramatically reduce lung injury, revealed that neutrophils are primarily responsible for the IL-17A-dependent release of IL-33 into the airways. Taken together, our results reveal an IL-17A-neutrophil-axis that can drive IL-33 during helminth infection, highlighting an additional pathway by which IL-17A regulates pulmonary type 2 immunity.

Injectable in situ forming nanogel: A hybrid Alginate-NLC formulation extends bupivacaine anesthetic effect.

Finding an ideal anesthetic agent for postoperative pain control, with long action and low side effects, is still a challenge. Local anesthetics have potential for such application if their time of action is improved. This work introduces a new hybrid formulation formed by the association of a nanostructured lipid carrier with a biopolymeric system to encapsulate bupivacaine (BVC). The hybrid formulation was physicochemical and structurally characterized by DLS, TEM, DSC, XRD and FTIR-ATR, and it remained stable for 12 months at room temperature. In vivo analgesia and imaging tests showed that the hybrid system was able to modulate the release, and to increase the concentration of BVC at the site of action, by forming a nanogel in situ. Such nanogel improved over 5 times (>24 h) the anesthesia duration, when compared to free BVC at clinical (0.5%) doses. Therefore, this novel in situ-forming nanogel shows great potential to be used in postsurgical pain control, improving the action of BVC, without losing its versatility of (infiltrative) application.

Low-level laser and adipose-derived stem cells altered remodelling genes expression and improved collagen reorganization during tendon repair.

OBJECTIVES: The cellular therapy using adipose-derived mesenchymal stem cells (ASCs) aims to improve tendon healing, considering that repaired tendons often result in a less resistant tissue. Our objective was to evaluate the effects of the ASCs combination with a low-level laser (LLL), an effective photobiostimulation for the healing processes. MATERIALS AND METHODS: Rats calcaneal tendons were divided into five groups: normal (NT), transected (T), transected and ASCs (SC) or LLL (L), or with ASCs and LLL (SCL). RESULTS: All treated groups presented higher expression of Dcn and greater organization of collagen fibres. In comparison with T, LLL also up-regulated Gdf5 gene expression, ASCs up-regulated the expression of Tnmd, and the association of LLL and ASCs down-regulated the expression of Scx. No differences were observed for the expression of Il1b, Timp2, Tgfb1, Lox, Mmp2, Mmp8 and Mmp9, neither in the quantification of hydroxyproline, TNF-α, PCNA and in the protein level of Tnmd. A higher amount of IL-10 was detected in SC, L and SCL compared to T, and higher amount of collagen I and III was observed in SC compared to SCL. CONCLUSIONS: Transplanted ASCs migrated to the transected region, and all treatments altered the remodelling genes expression. The LLL was the most effective in the collagen reorganization, followed by its combination with ASCs. Further investigations are needed to elucidate the molecular mechanisms involved in the LLL and ASCs combination during initial phases of tendon repair.

Enhanced Immune Response in Immunodeficient Mice Improves Peripheral Nerve Regeneration Following Axotomy.

Injuries to peripheral nerves cause loss of motor and sensory function, greatly affecting life quality. Successful repair of the lesioned nerve requires efficient cell debris removal, followed by axon regeneration and reinnervation of target organs. Such process is orchestrated by several cellular and molecular events in which glial and immune cells actively participate. It is known that tissue clearance is largely improved by macrophages, which activation is potentiated by cells and molecules of the acquired immune system, such as T helper lymphocytes and antibodies, respectively. In the present work, we evaluated the contribution of lymphocytes in the regenerative process of crushed sciatic nerves of immunocompetent (wild-type, WT) and T and B-deficient (RAG-KO) mice. In Knockout animals, we found increased amount of macrophages under basal conditions and during the initial phase of the regenerative process, that was evaluated at 2, 4, and 8 weeks after lesion (wal). That parallels with faster axonal regeneration evidenced by the quantification of neurofilament and a growth associated protein immunolabeling. The motor function, evaluated by the sciatic function index, was fully recovered in both mouse strains within 4 wal, either in a progressive fashion, as observed for RAG-KO mice, or presenting a subtle regression, as seen in WT mice between 2 and 3 wal. Interestingly, boosting the immune response by early adoptive transference of activated WT lymphocytes at 3 days after lesion improved motor recovery in WT and RAG-KO mice, which was not ameliorated when cells were transferred at 2 wal. When monitoring lymphocytes by in vivo imaging, in both mouse strains, cells migrated to the lesion site shortly after transference, remaining in the injured limb up to its complete motor recovery. Moreover, a first peak of hyperalgesia, determined by von-Frey test, was coincident with increased lymphocyte infiltration in the damaged paw. Overall, the present results suggest that a wave of immune cell infiltration takes place during subacute phase of axonal regeneration, resulting in transient set back of motor recovery following peripheral axonal injury. Moreover, modulation of the immune response can be an efficient approach to speed up nerve regeneration.

Infection by the Sylvio X10/4 clone of Trypanosoma cruzi: relevance of a low-virulence model of Chagas' disease.

The physiopathology of Chagas' disease has been largely defined in murine infections with virulent strains which partially represent parasite diversity. This report reviews our studies with Sylvio X10/4 parasites, a Trypanosoma cruzi clone that induces no acute phase but in C3H/He mice leads to chronic myocarditis resembling the human disease.