Danilo Lopes Ferrucci

Sequential IL-23 and IL-17 and increased Mmp8 and Mmp14 expression characterize the progression of an experimental model of periodontal disease in type 1 diabetes

Molecular mechanisms responsible for periodontal disease (PD) and its worsening in type 1 Diabetes Mellitus (DM1) remain unknown. Cytokine profile and expression levels of collagenases, Mmp14, and tissue inhibitors were determined, as were the numbers of neutrophils and macrophages in combined streptozotocin‐induced DM1 and ligature‐induced PD models. Increased IL‐23 (80‐fold) and Mmp8 expression (25‐fold) was found in DM1. Ligature resulted in an IL‐1β/IL‐6 profile, increased expression of Mmp8, Mmp13, and Mmp14 (but not Mmp1), and transient expression of Timp1 and Reck in non‐diabetics. PD in DM1 involved IL‐1β (but not IL‐6) and IL‐23/IL‐17, reduced IL‐6 and IL‐10, sustained Mmp8 and Mmp14, increased Mmp13 and reduced Reck expression in association with 20‐fold higher counts of neutrophils and macrophages. IL‐23 and Mmp8 expression are hallmarks of DM1. In association with the IL‐1/IL‐6 (Th1) response in PD, one found a secondary IL‐17 (Th17) pathway in non‐diabetic rats. Low IL‐6/TNF‐α suggest that the Th1 response was compromised in DM1, while IL‐17 indicates a prevalence of the Th17 pathway, resulting in high neutrophil recruitment. Mmp8, Mmp13, and Mmp14 expression seems important in the tissue destruction during PD in DM1. PD‐associated IL‐1/IL‐6 (Th1), IL‐10, and Reck expression are associated with the acute‐to‐chronic inflammation transition, which is lost in DM1. In conclusion, IL‐23/IL‐17 are associated with the PD progression in DM1. J. Cell. Physiol. 227: 2441–2450, 2012.

Periodontal Disease-Associated Compensatory Expression of Osteoprotegerin Is Lost in Type 1 Diabetes Mellitus and Correlates with Alveolar Bone Destruction by Regulating Osteoclastogenesis

Alveolar bone resorption results from the inflammatory response to periodontal pathogens. Systemic diseases that affect the host response, such as type 1 diabetes mellitus (DM1), can potentiate the severity of periodontal disease (PD) and accelerate bone resorption. However, the biological mechanisms by which DM1 modulates PD are not fully understood. The aim of this study was to determine the influence of DM1 on alveolar bone resorption and to evaluate the role of receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin (OPG) in osteoclastogenesis in rats. PD was induced by means of ligature in nondiabetic and in streptozotocyn-induced DM1 rats. Morphological and morphometric analyses, stereology and osteoclast counting were performed. RANKL and OPG mRNA levels, protein content, and location were determined. PD caused alveolar bone resorption, increased the number of osteoclasts in the alveolar bone crest and also promoted changes in RANKL/OPG mRNA expression. DM1 alone showed alveolar bone destruction and an increased number of osteoclasts at the periapical and furcal regions. DM1 exacerbated these characteristics, with a greater impact on bone structure, resulting in a low OPG content and a higher RANKL/OPG ratio, which correlated with prominent osteoclastogenesis. This work demonstrates that the effects of PD and DM1 enhance bone destruction, confirms the importance of the RANKL signaling pathway in bone destruction in DM1 in animal models and suggests the existence of alternative mechanisms potentiating bone degradation in PD.

Expression of matrix metalloproteinases, type IV collagen, and interleukin-10 in rabbits treated with morphine after lamellar keratectomy

PURPOSE To study the effects of topical administration of 1% morphine on corneal analgesia in rabbits submitted to lamellar keratectomy and to assess the expression of matrix metalloproteinase-1, metalloproteinase-2, metalloproteinase-9 (MMPs), type IV collagen, and interleukin-10 (IL-10) during the treatment. METHODS Morphine group (MG) received 50 μL of topical 1% morphine four times daily, while the control group received saline instead. Corneal touch threshold (CTT) and the wound area were assessed until corneal healing. Corneal samples were processed for routine histology, immunohistochemistry, zymography, and ELISA. RESULTS Following keratectomy, CTT increased significantly from 6 to 96 h time points. Mean corneal re-epithelization rate and scores of leukocyte infiltration did not differ significantly between treatment groups. Immunolabeling pattern for MMP-1, MMP-9, and type IV collagen was similar in both treatment groups. In the MG, zymography indicated significantly higher levels of active MMP-2 on days 6 and 12; and in the latent MMP-9, on days 3 and 6, and in the active MMP-9, on day 6. Latent MMP-2 and MMP-9, and active MMP-9 decreased to values close to those of healthy corneas on day 12, but levels of active MMP-2 remained significantly elevated in the MG. IL-10 levels measured on days 1-6 were reduced as compared to those of healthy corneal tissue and returned to levels close to those of healthy corneas on day 12. CONCLUSION Topical morphine promoted corneal analgesia for up to 4 days and did not delay corneal re-epithelization. The re-establishment of MMPs and IL-10 to levels close to baseline values at the end of the study and the expression of type IV collagen in both groups reinforce that, with caution, 1% morphine can be used after lamellar keratectomy in rabbits.

Thymic alterations induced by Plasmodium berghei: expression of matrix metalloproteinases and their tissue inhibitors.

The thymus plays a crucial role in the generation of T-cells, and so our laboratory has been interested in the study of the intrathymic events that occur during infection diseases and may cause disruption in its functions. Previously, we showed that thymus from experimentally Plasmodium berghei-infected mice present histological alterations with high levels of apoptosis, changes in cell migration-related molecules, and premature egress of immature thymocytes to periphery. In addition, parasites were found inside the thymus. In this work we investigated alterations in the expression pattern and activity of matrix metalloproteinases MMP-2 and -9, and their tissue inhibitors, TIMP-1 and TIMP-2. Our results show enhanced expression and widespread distribution of these molecules in thymus from infected animals. Also, the presence of active MMP-2 was detected. These data are suggestive of MMPs and TIMPs importance in the earlier observed changes in the extracellular matrix during thymic alterations after plasmodium infection.

Implication Of Rho-kinase And Soluble Guanylyl Cyclase Enzymes In Prostate Smooth Muscle Dysfunction In Middle-aged Rats.

Aging is highly associated with benign prostate hyperplasia (BPH). We investigated here the alterations of the contractile and relaxant machinery in prostates of middle‐aged rats, focusing on the Rho‐kinase, nitric oxide (NO)‐soluble guanylyl cyclase (sGC), α1‐ and β‐adrenoceptor pathways.

Exhaustive Exercise With Different Rest Periods Changes the Collagen Content and MMP-2 Activation on the Calcaneal Tendon

Tendons adapt to different mechanical stimuli through a remodeling process involving metalloproteinases (MMPs) and collagen synthesis. The purpose of this study was to investigate the activities of MMP‐2 and MMP‐9 and the collagen content in tendons after exhaustive acute exercise sessions over the course of 1, 3, or 6 days, with 1‐hr or 3‐hr rest periods between each session. Wistar rats were grouped into control (C), trained with 1‐hr (groups 1d1h, 3d1h, and 6d1h) and trained with 3‐hr (groups 1d3h, 3d3h and 6d3h) groups with rest periods between the treadmill running sessions, for 1, 3, and 6 days. The analysis of MMP‐2 showed a larger presence of the latent isoform in the 1d3h group and a larger presence of the active isoform in the 6d3h group compared to the control. No differences were detected for MMP‐9. A lower concentration of hydroxyproline was found in the 6d3h group compared to the 6d1h group. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed more prominent collagen bands in the 6d3h group, which was confirmed by Western blotting for collagen type I. A higher concentration of glycosaminoglycans was observed in the 3d3h group compared to the 3d1h group, and the 6d3h group presented the highest value for non‐collagenous proteins compared to other groups. In conclusion, different rest periods between exercise sessions had different effects on the composition of the calcaneal tendon because a greater activation of MMP‐2 and a reduction of total collagen were observed on day 6 of exercise with 3‐hr rest periods compared to 1‐hr rest periods. Anat Rec, 297:281–288, 2014.

Expression of matrix metalloproteases-2 and -9 in horse hoof laminae after intestinal obstruction, with or without Hydrocortisone treatment

Twenty horses were used in the experiment, for composed control group, (Cg) instrumented group, (Ig;without intestinal obstruction), treated group (Tg;submitted to intestinal obstruction and hydrocortisone treatment) and non-treated group (Ntg;submitted to intestinal obstruction without treatment). Immunohistochemistry and zymography techniques were used for researches on MMPs 2 and 9 in horse hoof laminae. There was an increase in the expression of MMP-2 in animals of Tg and Ntg. MMP-9 increased on animals from groups Ntg and Ig, however there was no rise of this MMP on the Tg when compared to the other groups in the immunohistochemistry analysis. Based on the results, it was observed that the intestinal injury caused by enterotomy and intestinal obstruction raise the quantities of MMPs in the hoof laminae.

Lipocalina associada à gelatinase de neutrófilos (NGAL) e calprotectina no tecido laminar de equinos após obstrução jejunal, tratados ou não com hidrocortisona

Laminitis is a severe hoof condition in horses that may cause intense suffering. In this study, leukocyte infiltration in hoof laminar tissue was investigated in horses subject to intestinal obstruction using immunohistochemistry to detect calprotectin, and zymography to detect neutrophil gelatinase associated lipocalin (NGAL). There were four groups: the Control Group (Gc), with seven horses, without surgical procedures; the Sham-operated Group (Gi), with five horses that were subjected to surgical procedure without intestinal obstruction; the No Treat group (Gnt), with four horses subjected to intestinal obstruction (jejunal distention using an intraluminal balloon) without treatment; and Treated group (Gt), with four horses subjected to intestinal obstruction and treated with hydrocortisone. Positive calprotectin imunostaining was detected in all experimental groups, with increase cell counts in horses of the distended group compared with the control group. NGAL expression was increased in Gd compared with Gc e Gi. The Gt did not differ from the others. In conclusion, small intestine distension can promote leukocyte infiltration in equine hoof laminar tissue, and NGAL zymography was considered a useful method for leukocyte tissue detection in horses. New studies will be conducted to verify the possible beneficial anti-inflammatory effects of hydrocortisone in hoof of horses with intestinal obstruction.

Injured Achilles Tendons Treated with Adipose-Derived Stem Cells Transplantation and GDF-5

Tendon injuries represent a clinical challenge in regenerative medicine because their natural repair process is complex and inefficient. The high incidence of tendon injuries is frequently associated with sports practice, aging, tendinopathies, hypertension, diabetes mellitus, and the use of corticosteroids. The growing interest of scientists in using adipose-derived mesenchymal stem cells (ADMSC) in repair processes seems to be mostly due to their paracrine and immunomodulatory effects in stimulating specific cellular events. ADMSC activity can be influenced by GDF-5, which has been successfully used to drive tenogenic differentiation of ADMSC in vitro. Thus, we hypothesized that the application of ADMSC in isolation or in association with GDF-5 could improve Achilles tendon repair through the regulation of important remodeling genes expression. Lewis rats had tendons distributed in four groups: Transected (T), transected and treated with ADMSC (ASC) or GDF-5 (GDF5), or with both (ASC+GDF5). In the characterization of cells before application, ADMSC expressed the positive surface markers, CD90 (90%) and CD105 (95%), and the negative marker, CD45 (7%). ADMSC were also differentiated in chondrocytes, osteoblast, and adipocytes. On the 14th day after the tendon injury, GFP-ADMSC were observed in the transected region of tendons in the ASC and ASC+GDF5 groups, and exhibited and/or stimulated a similar genes expression profile when compared to the in vitro assay. ADMSC up-regulated Lox, Dcn, and Tgfb1 genes expression in comparison to T and ASC+GDF5 groups, which contributed to a lower proteoglycans arrangement, and to a higher collagen fiber organization and tendon biomechanics in the ASC group. The application of ADMSC in association with GDF-5 down-regulated Dcn, Gdf5, Lox, Tgfb1, Mmp2, and Timp2 genes expression, which contributed to a lower hydroxyproline concentration, lower collagen fiber organization, and to an improvement of the rats’ gait 24 h after the injury. In conclusion, although the literature describes the benefic effect of GDF-5 for the tendon healing process, our results show that its application, isolated or associated with ADMSC, cannot improve the repair process of partial transected tendons, indicating the higher effectiveness of the application of ADMSC in injured Achilles tendons. Our results show that the application of ADMSC in injured Achilles tendons was more effective in relation to its association with GDF-5.

Desquamation takes center stage at the origin of proliferative inflammatory atrophy, epithelial–mesenchymal transition, and stromal growth in benign prostate hyperplasia

In this commentary, we propose a relationship between desquamation, initially described as the collective detachment and deletion of epithelial cell in the prostate gland after castration, and proliferative inflammatory atrophy (PIA) and stromal growth in benign prostate hyperplasia (BPH). First, in response to diverse stimuli, including inflammatory mediators, epithelial cells desquamate and leave a large surface of the luminal side of the basement membrane (BM) exposed. Basal cells are activated into intermediate‐type cells, which change morphology to cover and remodel the exposed BM (simple atrophy) to a new physiological demand (such as in the hypoandrogen environment, simulated by surgical and/or chemical castration) and/or to support re‐epithelialization (under normal androgen levels). In the presence of inflammation (that might be the cause of desquamation), the intermediate‐type cells proliferate and characterize PIA. Second, in other circumstances, desquamation is an early step of epithelial‐to‐mesenchymal transition (EMT), which contributes to stromal growth, as suggested by some experimental models of BPH. The proposed associations correlate unexplored cell behaviors and reveal the remarkable plasticity of the prostate epithelium that might be at the origin of prostate diseases.