Chemokine Expression Is Upregulated in Chondrocytes in Diabetic Fracture Healing

Chemokines are thought to play an important role in several aspects of bone metabolism including the recruitment of leukocytes and the formation of osteoclasts. We investigated the impact of diabetes on chemokine expression in normal and diabetic fracture healing. Fracture of the femur was performed in streptozotocin-induced diabetic and matched normoglycemic control mice. Microarray analysis was carried out and chemokine mRNA levels in vivo were assessed. CCL4 were examined in fracture calluses by immunohistochemistry and the role of TNF in diabetes-enhanced expression was investigated by treatment of animals with the TNF-specific inhibitor, pegsunercept. In vitro studies were conducted with ATDC5 chondrocytes. Diabetes significantly upregulated mRNA levels of several chemokines in vivo including CCL4, CCL8, CCL6, CCL11, CCL20, CCL24, CXCL2, CXCL5 and chemokine receptors CCR5 and CXCR4. Chondrocytes were identified as a significant source of CCL4 and its expression in diabetic fractures was dependent on TNF (P \u3c 0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P \u3c 0.05). CCL4 expression at the mRNA and proteins levels was induced by FOXO1 over-expression and reduced by FOXO1 knockdown. The current studies point to the importance of TNF-α as a mechanism for diabetes enhanced chemokine expression by chondrocytes, which may contribute to the accelerated loss of cartilage observed in diabetic fracture healing. Moreover, in vitro results point to FOXO1 as a potentially important transcription factor in mediating this effect

FOXO1 Modulates Osteoblast Differentiation

Forkhead box O1 (FOXO1) is upregulated during bone formation and in response to stimulation by bone morphogenetic proteins. Studies presented here examined the functional role of FOXO1 in a well defined culture system in which pre-osteoblastic cells undergo terminal differentiation in vitro. Mineralizing cultures of MC3T3-E1 cells were examined with or without FOXO1 knockdown by RNAi. Normal cells show the upregulation of FOXO1 and RUNX2 DNA binding activity, alkaline phosphatase activity, and mRNA levels of FOXO1, RUNX2, type 1 collagen, osteocalcin and MMP13 during formation of mineralizing nodules. In FOXO1 depleted cells each of these measurements was significantly reduced compared to values in control cells transfected with scrambled siRNA (P \u3c 0.05). Depletion of FOXO1 also reduced the number of mineralized nodules formed. Moreover, chromatin immunoprecipitation assays revealed a direct interaction of FOXO1 with the RUNX2 promoter. Overexpression of FOXO1 reduced the MC3T3-E1 cell number and the number of PCNA positive cells with little effect on apoptosis. These findings indicate that FOXO1 plays an important role in promoting osteoblast differentiation and suppressing proliferation in differentiating cells

Impaired Wound Healing in Mouse Models of Diabetes Is Mediated by TNF-α Dysregulation and Associated With Enhanced Activation of Forkhead Box O1 (FOXO1)

Aims/hypothesis The role of TNF-α in impaired wound healing in diabetes was examined by focusing on fibroblasts. Methods Small excisional wounds were created in the db/db mice model of type 2 diabetes and normoglycaemic littermates, and in a streptozotocin-induced type 1 diabetes mouse model and control mice. Fibroblast apoptosis was measured by the TUNEL assay, proliferation by detection of proliferating cell nuclear antigen, and forkhead box O1 (FOXO1) activity by DNA binding and nuclear translocation. TNF-α was specifically inhibited by pegsunercept. Results Diabetic wounds had increased TNF-α, fibroblast apoptosis, caspase-3/7 activity and activation of the pro-apoptotic transcription factor FOXO1, and decreased proliferating cell nuclear antigen positive fibroblasts (p \u3c 0.05). TNF-α inhibition improved healing in the diabetic mice and increased fibroblast density. This may be explained by a decrease in fibroblast apoptosis and increased proliferation when TNF-α was blocked (p  \u3c 0.05). Although decreased fibroblast proliferation and enhanced FOXO1 activity were investigated in type 2 diabetes, they may also be implicated in type 1 diabetes. In vitro, TNF-α enhanced mRNA levels of gene sets related to apoptosis and Akt and p53 but not mitochondrial or cell-cycle pathways. FOXO1 small interfering RNA reduced gene sets that regulate apoptosis, Akt, mitochondrial and cell-cycle pathways. TNF-α also increased genes involved in inflammation, cytokine, Toll-like receptor and nuclear factor-kB pathways, which were significantly reduced by FOXO1 knockdown. Conclusions/interpretation These studies indicate that TNF-α dysregulation in diabetic wounds impairs healing, which may involve enhanced fibroblast apoptosis and decreased proliferation. In vitro, TNF-α induced gene sets through FOXO1 that regulate a number of pathways that could influence inflammation and apoptosis

Cactus Cladodes Opuntia or Nopalea and By-Product of Low Nutritional Value as Solutions to Forage Shortages in Semiarid Areas

Simple Summary In the different livestock production systems, forage is the main feed resource. However, the availability and quality of the forage fluctuate throughout the year due to variable environmental conditions, such as temperature, humidity, location, or lack of rainfall. In semiarid regions, this fact is even more critical. The option for forage plants adapted to the semiarid climate, such as cactus cladodes, becomes indispensable for the sustainability of the systems. Nonetheless, it is necessary to combine the cactus with high-fiber-content feeds (silage, hay, and agroindustry residues, among others) to increase fiber contents in the diet to promote ideal rumen conditions. Based on the knowledge that cactus cladodes (Opuntia spp. and Nopalea spp.) are one of the most viable crops in semiarid regions, the association with a by-product rich in NDF proves to be a more feasible alternative in terms of price and availability, with the producer making the final decision. We aimed to evaluate the effect of the cactus cladodes Nopalea cochenillifera (L). Salm-Dyck. (NUB) and cactus cladodes Opuntia stricta (Haw.) Haw. (OUB), both combined with sugarcane bagasse (SB) plus urea, Tifton hay (TH), corn silage (CS), and sorghum silage (SS) plus urea on nutrient intake and digestibility, ruminal dynamics, and parameters. Five male sheep, fistulated in the rumen, were assigned in a 5 x 5 Latin square design. The NUB provided a higher intake of dry matter (DM) and any nutrients than SS. TH provided larger pools of DM and iNDF. The OUB and CS provided a higher DM degradation. CS provided a higher NDF degradation rate. OUB provided a lower ruminal pH. Depending on the collection time, the lowest pH value was estimated at 3.79 h after the morning feeding. There was an interaction between treatments and collection time on VFA concentrations. Due to the high degradation rate, greater energy intake, less change in rumen pH, greater volatile fatty acid production, and feasibility, we recommend using cactus associated with sugarcane bagasse plus urea in sheep diets

A Screen against Leishmania Intracellular Amastigotes: Comparison to a Promastigote Screen and Identification of a Host Cell-Specific Hit

The ability to screen compounds in a high-throughput manner is essential in the process of small molecule drug discovery. Critical to the success of screening strategies is the proper design of the assay, often implying a compromise between ease/speed and a biologically relevant setting. Leishmaniasis is a major neglected disease with limited therapeutic options. In order to streamline efforts for the design of productive drug screens against Leishmania, we compared the efficiency of two screening methods, one targeting the free living and easily cultured promastigote (insect–infective) stage, the other targeting the clinically relevant but more difficult to culture intra-macrophage amastigote (mammal-infective) stage. Screening of a 909-member library of bioactive compounds against Leishmania donovani revealed 59 hits in the promastigote primary screen and 27 in the intracellular amastigote screen, with 26 hits shared by both screens. This suggested that screening against the promastigote stage, although more suitable for automation, fails to identify all active compounds and leads to numerous false positive hits. Of particular interest was the identification of one compound specific to the infective amastigote stage of the parasite. This compound affects intracellular but not axenic parasites, suggesting a host cell-dependent mechanism of action, opening new avenues for anti-leishmanial chemotherapy

First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

Background: in a previous study, we detected the presence of a Mycobacterium avium species-specific insertion sequence, IS1245, in Mycobacterium kansasii. Both species were isolated from a mixed M. avium-M. kansasii bone marrow culture from an HIV-positive patient. the transfer mechanism of this insertion sequence to M. kansasii was investigated here.Methodology/Principal Findings: A linear plasmid (pMA100) was identified in all colonies isolated from the M. avium-M. kansasii mixed culture carrying the IS1245 element. the linearity of pMA100 was confirmed. Other analyses suggested that pMA100 contained a covalently bound protein in the terminal regions, a characteristic of invertron linear replicons. Partial sequencing of pMA100 showed that it bears one intact copy of IS1245 inserted in a region rich in transposase-related sequences. These types of sequences have been described in other linear mycobacterial plasmids. Mating experiments were performed to confirm that pMA100 could be transferred in vitro from M. avium to M. kansasii. pMA100 was transferred by in vitro conjugation not only to the M. kansasii strain from the mixed culture, but also to two other unrelated M. kansasii clinical isolates, as well as to Mycobacterium bovis BCG Moreau.Conclusions/Significance: Horizontal gene transfer (HGT) is one of most important mechanisms leading to the evolution and diversity of bacteria. This work provides evidence for the first time on the natural occurrence of HGT between different species of mycobacteria. Gene transfer, mediated by a novel conjugative plasmid, was detected and experimentally reproduced.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Cooperacion Interuniversitaria UAM-Banco Santander con America Latina (CEAL), UAM, SpainConselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilLab Nacl Comp Cient, Petropolis, BrazilUniv Autonoma Madrid, Fac Med, Dept Prevent Med, Madrid, SpainInst Adolfo Lutz Registro, Nucleo TB & Micobacterioses, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, Escola Paulista Med, São Paulo, BrazilFAPESP: FAPESP - 06/01533-9Web of Scienc