Comparative Effects of Intra-Articular versus Intravenous Mesenchymal Stromal Cells Therapy in a Rat Model of Osteoarthritis by Destabilization of Medial Meniscus.

Transplanted mesenchymal stromal cells (MSCs) exhibit a robust anti-inflammatory and homing capacity in response to high inflammatory signals, as observed in studies focused on rheumatic diseases that target articular cartilage (AC) health. However, AC degradation in osteoarthritis (OA) does not necessarily coincide with a highly inflammatory joint profile. Often, by the time patients seek medical attention, they already have damaged AC. In this study, we examined the therapeutic potential of a single bone marrow MSC transplant (2 × 106 cells/kgbw) through two different routes: intra-articular (MSCs-IAt) and intravenous (MSCs-IVt) in a preclinical model of low-grade inflammatory OA with an established AC degeneration. OA was induced through the destabilization of the medial meniscus (DMM) in female Wistar Kyoto rats. The animals received MSCs 9 weeks after surgery and were euthanized 4 and 12 weeks post-transplant. In vivo and ex vivo tracking of MSCs were analyzed via bioluminescence and imaging flow cytometry, respectively. Cytokine/chemokine modulation in serum and synovial fluid was measured using a multiplex panel. AC degeneration was quantified through histology, and hindlimb muscle balance was assessed with precision weighing. To our knowledge, we are the first group to show the in vivo (8 h) and ex vivo (12 h) homing of cells to the DMM-OA joint following MSCs-IVt. In the case of MSCs-IAt, the detection of cellular bioluminescence at the knee joint persisted for up to 1 week. Intriguingly, intra-articular saline injection (placebo-IAt) resulted in a worse prognosis of OA when compared to a non-invasive control (placebo-IVt) without joint injection. The systemic cytokines/chemokines profile exhibited a time-dependent variation between transplant routes, displaying a transient anti-inflammatory systemic response for both MSCs-IVt and MSCs-IAt. A single injection of MSCs, whether administered via the intra-articular or intravenous route, performed 9 weeks after DMM surgery, did not effectively inhibit AC degeneration when compared to a non-invasive control.

The proinflammatory protein HMGB1 is a substrate of transglutaminase-2 and forms high-molecular weight complexes with autoantigens.

High-mobility group box 1 (HMGB1) is a chromatin-associated protein that, in response to stress or injury, translocates from the nucleus to the extracellular milieu, where it functions as an alarmin. HMGB1's function is in part determined by the complexes (HMGB1c) it forms with other molecules. However, structural modifications in the HMGB1 polypeptide that may regulate HMGB1c formation have not been previously described. In this report, we observed high-molecular weight, denaturing-resistant HMGB1c in the plasma and peripheral blood mononuclear cells of individuals with systemic lupus erythematosus (SLE) and, to a much lesser extent, in healthy subjects. Differential HMGB1c levels were also detected in mouse tissues and cultured cells, in which these complexes were induced by endotoxin or the immunological adjuvant alum. Of note, we found that HMGB1c formation is catalyzed by the protein-cross-linking enzyme transglutaminase-2 (TG2). Cross-link site mapping and MS analysis revealed that HMGB1 can be cross-linked to TG2 as well as a number of additional proteins, including human autoantigens. These findings have significant functional implications for studies of cellular stress responses and innate immunity in SLE and other autoimmune disease.

Determinants of alveolar ridge preservation differ by anatomic location.

AIM: To investigate and compare outcomes following alveolar ridge preservation (ARP) in posterior maxilla and mandible. METHODS: Twenty-four patients (54 ± 3 years) with single posterior tooth extraction were included. ARP was performed with freeze-dried bone allograft and collagen membrane. Clinical parameters were recorded at extraction and re-entry. Harvested bone cores were analysed by microcomputed tomography (micro-CT), histomorphometry and immunohistochemistry. RESULTS: In both jaws, ARP prevented ridge height loss, but ridge width was significantly reduced by approximately 2.5 mm. Healing time, initial clinical attachment loss and amount of keratinized tissue at extraction site were identified as determinants of ridge height outcome. Buccal plate thickness and tooth root length were identified as determinants of ridge width outcome. In addition, initial ridge width was positively correlated with ridge width loss. Micro-CT revealed greater mineralization per unit volume in new bone compared with existing bone in mandible (p < 0.001). Distributions of residual graft, new cellular bone and immature tissue were similar in both jaws. CONCLUSION: Within the limitations of this study, the results indicate that in different anatomic locations different factors may determine ARP outcomes. Further studies are needed to better understand determinants of ARP outcomes.

The basic science of continuous passive motion in promoting knee health: a systematic review of studies in a rabbit model.

PURPOSE: To determine whether the basic science evidence supports the use of continuous passive motion (CPM) after articular cartilage injury in the knee. METHODS: A systematic review was performed identifying and evaluating studies in animal models that focused on the basic science of CPM of the knee. Databases included in this review were PubMed, Biosis Previews, SPORTDiscus, PEDro, and EMBASE. All functional, gross anatomic, histologic, and histochemical outcomes were extracted and analyzed. RESULTS: Primary outcomes of CPM analyzed in rabbit animal models (19 studies) included histologic changes in articular cartilage (13 studies), biomechanical changes and nutrition of intra-articular tissue (3 studies), and anti-inflammatory biochemical changes (3 studies). Nine studies specifically examined osteochondral defects, 6 of which used autogenous periosteal grafts. Other pathologies included were antigen-induced arthritis, septic arthritis, medial collateral ligament reconstruction, hemarthrosis, and chymopapain-induced proteoglycan destruction. In comparison to immobilized knees, CPM therapy led to decreased joint stiffness and complications related to adhesions while promoting improved neochondrogenesis with formation and preservation of normal articular cartilage. CPM was also shown to create a strong anti-inflammatory environment by effectively clearing harmful, inflammatory particles from within the knee. CONCLUSIONS: Current basic science evidence from rabbit studies has shown that CPM for the knee significantly improves motion and biological properties of articular cartilage. This may be translated to potentially improved outcomes in the management of articular cartilage pathology of the knee. CLINICAL RELEVANCE: If the rabbit model is relevant to humans, CPM may contribute to improved knee health by preventing joint stiffness, preserving normal articular tissue with better histologic and biologic properties, and improving range of motion as compared with joint immobilization and intermittent active motion.

Transcriptome-wide gene regulation by gentle treadmill walking during the progression of monoiodoacetate-induced arthritis.

OBJECTIVE: Physiotherapies are the most widely recommended conservative treatment for arthritic diseases. The present study was undertaken to examine the molecular mechanisms underlying the effects of gentle treadmill walking (GTW) on various stages of monoiodoacetate-induced arthritis (MIA) to elucidate the basis for the success or failure of such therapies in joint damage. METHODS: Knees were obtained from untreated control rats, rats with MIA that did not undergo GTW, rats with MIA in which GTW regimens were started 1 day post-MIA induction, and rats with MIA in which GTW regimens were started after cartilage damage had progressed to grade 1 or grade 2. The cartilage was examined macroscopically, microscopically, and by microfocal computed tomography imaging. Transcriptome-wide gene expression analysis was performed, and microarray data were assessed by Ingenuity Pathways Analysis to identify molecular functional networks regulated by GTW. RESULTS: GTW intervention started on day 1 post-MIA induction significantly prevented the progression of MIA, but its efficacy was reduced when implemented on knees exhibiting close to grade 1 cartilage damage. GTW accelerated cartilage damage in knees with close to grade 2 damage. Transcriptome-wide gene expression analysis revealed that GTW intervention started 1 day post-MIA inception significantly suppressed inflammation-associated genes and up-regulated matrix-associated gene networks. However, delayed GTW intervention after grade 1 damage had occurred was less effective in suppressing proinflammatory genes or up-regulating matrix synthesis. CONCLUSION: The present findings suggest that GTW suppresses proinflammatory gene networks and up-regulates matrix synthesis to prevent progression of cartilage damage in MIA-affected knees. However, the extent of cartilage damage at the initiation of GTW may be an important determinant of the success or failure of such therapies.

Mechanical signals activate vascular endothelial growth factor receptor-2 to upregulate endothelial cell proliferation during inflammation.

Signals generated by the dynamic mechanical strain critically regulate endothelial cell proliferation and angiogenesis; however, the molecular basis remains unclear. We investigated the mechanisms by which human dermal microvascular endothelial cells (HDMECs) perceive mechanical signals and relay them intracellularly to regulate gene expression and endothelial cell proliferation. HDMECs were exposed to low/physiologic levels of dynamic strain and probed for the differential activation/inhibition of kinases in the mechanosignaling cascade associated with endothelial cell gene activation. Because angiogenesis is important at inflammatory sites, we also assessed the mechanisms of mechanosignaling in the presence of an proinflammatory cytokine IL-1beta. In this article, we demonstrate that the mechanosignaling cascade is initiated by vascular endothelial growth receptor-2 (VEGFR2) activation. Mechanoactivation of VEGFR2 results in its nuclear translocation and elevation of PI3K-dependent Ser473-Akt phosphorylation. Subsequently, activated Akt inactivates the kinase activity of the serine/threonine kinase, glycogen synthase kinase-3beta (GSK3beta), via its Ser9 phosphorylation. Thus, inactive GSK3beta fails to phosphorylate cyclin D1 and prevents its proteosomal degradation and, consequently, promotes endothelial cell survival and proliferation. In the presence of IL-1beta, cyclin D1 is phosphorylated and degraded, leading to inhibition of cell proliferation. However, mechanical signals repress cyclin D1 phosphorylation and upregulate cell proliferation, despite the presence of IL-1beta. The data indicate that the VEGFR2/Akt/GSK3beta signaling cascade plays a critical role in sensing and phospho-relaying mechanical stimuli in endothelial cells. Furthermore, mechanical forces control highly interconnected networks of proinflammatory and Akt signaling cascades to upregulate endothelial cell proliferation.

Fabrication of skeletal muscle constructs by topographic activation of cell alignment.

Skeletal muscle fiber construction for tissue-engineered grafts requires assembly of unidirectionally aligned juxtaposed myotubes. To construct such a tissue, a polymer microchip with linearly aligned microgrooves was fabricated that could direct myoblast adaptation under stringent conditions. The closely spaced microgrooves fabricated by a modified replica molding process guided linear cellular alignment. Examination of the myoblasts by immunofluorescence microscopy demonstrated that the microgrooves with subcellular widths and appropriate height-to-width ratios were required for practically complete linear alignment of myoblasts. The topology-dependent cell alignment encouraged differentiation of myoblasts into multinucleate, myosin heavy chain positive myotubes. The monolayer of myotubes formed on the microstructured chips allowed attachment, growth and differentiation of subsequent layers of linearly arranged myoblasts, parallel to the primary monolayer of myotubes. The consequent deposition of additional myoblasts on the previous layer of myotubes resulted in three-dimensional multi-layered structures of myotubes, typical of differentiated skeletal muscle tissue. The findings demonstrate that the on-chip device holds promise for providing an efficient means for guided muscle tissue construction.

Changes in surface topologies of chondrocytes subjected to mechanical forces: an AFM analysis.

The cartilage is composed of chondrocytes embedded in a matrix of collagen fibrils interspersed within a network of proteoglycans and is constantly exposed to biomechanical forces during normal joint movement. Characterization of the surface morphology, cytoskeletal structure, adherance and elastic properties of these mechanosensitive cells are crucial in understanding the effects of mechanical forces around a cell and how a cell responds to changes in its physical environment. In this work, we employed the atomic force microscope (AFM) to image cultured chondrocytes before and after subjecting them to mechanical forces in the presence or absence of interleukin-1beta to mimic inflammatory conditions. Nanoscale imaging and quantitative measurements from AFM data revealed that there are distinct changes in cell-surface topology and cytoskeleton arrangement in the cells following treatment with mechanical forces, IL-1beta or both. Our findings for the first time demonstrate that cultured chondrocytes are amenable to high-resolution AFM imaging and dynamic tensile forces may help overcome the effect of inflammatory factors on chondrocyte response.

Regulation of chondrocytic gene expression by biomechanical signals.

Cartilage is a mechanosensitive tissue, which means that it can perceive and respond to biomechanical signals. Despite the known importance of biomechanical signals in the etiopathogenesis of arthritic diseases and their effectiveness in joint restoration, little is understood about their actions at the cellular level. Recent molecular approaches have revealed that specific biomechanical stimuli and cell interactions generate intracellular signals that are powerful inducers or suppressors of proinflammatory and reparative genes in chondrocytes. Biomechanical signals are perceived by cartilage in magnitude-, frequency-, and time-dependent manners. Static and dynamic biomechanical forces of high magnitudes induce proinflammatory genes and inhibit matrix synthesis. Contrarily, dynamic biomechanical signals of low/physiologic magnitudes are potent antiinflammatory signals that inhibit interleukin-1beta (IL-1beta)-induced proinflammatory gene transcription and abrogate IL-1beta/tumor necrosis factor-alpha-induced inhibition of matrix synthesis. Recent studies have identified nuclear factor-kB (NF-kB) transcription factors as key regulators of biomechanical signal-mediated proinflammatory and antiinflammatory actions. These signals intercept multiple steps in the NF-kappaB signaling cascade to regulate cytokine gene expression. Taken together, these findings provide insight into how biomechanical signals regulate inflammatory and reparative gene transcription, underscoring their potential in enhancing the ability of chondrocytes to curb inflammation in diseased joints.

Cyclic compressive loading facilitates recovery after eccentric exercise.

PURPOSE: To assess the biologic basis of massage therapies, we developed an experimental approach to mimic Swedish massage and evaluate this approach on recovery from eccentric exercise-induced muscle damage using a well-controlled animal model. METHODS: Tibialis anterior muscles of six New Zealand White rabbits were subjected to one bout of damaging, eccentric contractions. One muscle was immediately subjected to cyclic compressive loads, and the contralateral served as the exercised control. RESULTS: We found that commencing 30 min of cyclic compressive loading to the muscle, immediately after a bout of eccentric exercise, facilitated recovery of function and attenuated leukocyte infiltration. In addition, fiber necrosis and wet weight of the tissue were also reduced by compressive loading. CONCLUSION: We conclude that subjecting muscle to compressive loads immediately after exercise leads to an enhanced recovery of muscle function and attenuation of the damaging effects of inflammation in the rabbit model. Although these observations suggest that skeletal muscle responds to cyclic compressive forces similar to those generated by clinical approaches, such as therapeutic massage, further research is needed to assess the translational efficacy of these findings.

Compressive forces induce osteogenic gene expression in calvarial osteoblasts.

Bone cells and their precursors are sensitive to changes in their biomechanical environment. The importance of mechanical stimuli has been observed in bone homeostasis and osteogenesis, but the mechanisms responsible for osteogenic induction in response to mechanical signals are poorly understood. We hypothesized that compressive forces could exert an osteogenic effect on osteoblasts and act in a dose-dependent manner. To test our hypothesis, electrospun poly(epsilon-caprolactone) (PCL) scaffolds were used as a 3-D microenvironment for osteoblast culture. The scaffolds provided a substrate allowing cell exposure to levels of externally applied compressive force. Pre-osteoblasts adhered, proliferated and differentiated in the scaffolds and showed extensive matrix synthesis by scanning electron microscopy (SEM) and increased Young's modulus (136.45+/-9.15 kPa) compared with acellular scaffolds (24.55+/-8.5 kPa). Exposure of cells to 10% compressive strain (11.81+/-0.42 kPa) resulted in a rapid induction of bone morphogenic protein-2 (BMP-2), runt-related transcription factor 2 (Runx2), and MAD homolog 5 (Smad5). These effects further enhanced the expression of genes and proteins required for extracellular matrix (ECM) production, such as alkaline phosphatase (Akp2), collagen type I (Col1a1), osteocalcin/bone gamma carboxyglutamate protein (OC/Bglap), osteonectin/secreted acidic cysteine-rich glycoprotein (ON/Sparc) and osteopontin/secreted phosphoprotein 1 (OPN/Spp1). Exposure of cell-scaffold constructs to 20% compressive strain (30.96+/-2.82 kPa) demonstrated that these signals are not osteogenic. These findings provide the molecular basis for the experimental and clinical observations that appropriate physical activities or microscale compressive loading can enhance fracture healing due in part to the anabolic osteogenic effects.

Regulation of biomechanical signals by NF-kappaB transcription factors in chondrocytes.

Physical therapies and exercise are beneficial not only for physiological recovery in inflamed or injured joints, but also for promoting a homeostatic equilibrium in healthy joints. Human joints provide the pivot points and physiological hinges essential for ambulation and movement to the body, and it is this mobility that in return promotes the health of the joints. But how mobilization regulates the joint microenvironment at the molecular level has remained enigmatic for many years. Recent advances in joint biomechanics and molecular approaches have facilitated an enriched understanding of how joints operate. Consequently, the mechanisms active during joint inflammation that lead to arthritic conditions, both in vivo in animal models, and in vitro at cell and tissue levels, have become increasingly detailed and defined. These efforts have produced mounting evidences supporting the premise that biomechanical signals play a fundamental role in both the etiopathogenesis of arthritic diseases and in the physiological restoration of joints. This report aims to summarize current peer-reviewed literature and available experimental data to explain how the signals generated by mechanical forces/joint mobilization generate beneficial effects on inflamed articular cartilage, and to propose the basis for using appropriate physical therapies for the optimal benefit to the patient suffering from joint associated injuries.

Improved cellular infiltration in electrospun fiber via engineered porosity.

Small pore sizes inherent to electrospun matrices can hinder efficient cellular ingrowth. To facilitate infiltration while retaining its extracellular matrix-like character, electrospinning was combined with salt leaching to produce a scaffold having deliberate, engineered delaminations. We made elegant use of a specific randomizing component of the electrospinning process, the Taylor Cone and the falling fiber beneath it, to produce a uniform, well-spread distribution of salt particles. After 3 weeks of culture, up to 4 mm of cellular infiltration was observed, along with cellular coverage of up to 70% within the delaminations. To our knowledge, this represents the first observation of extensive cellular infiltration of electrospun matrices. Infiltration appears to be driven primarily by localized proliferation rather than coordinated cellular locomotion. Cells also moved from the salt-generated porosity into the surrounding electrospun fiber matrix. Given that the details of salt deposition (amount, size, and number density) are far from optimized, the result provides a convincing illustration of the ability of mammalian cells to interact with appropriately tailored electrospun matrices. These layered structures can be precisely fabricated by varying the deposition interval and particle size conceivably to produce in vivo-like gradients in porosity such that the resulting scaffolds better resemble the desired final structure.

Biomechanical strain regulates TNFR2 but not TNFR1 in TMJ cells.

We sought to examine whether cyclic tensile strain (CTS) regulates the gene expression of tumor necrosis factor (TNF)-alpha, its receptors TNFR1 and TNFR2, and inducible nitric oxide synthase (iNOS) under inflammatory conditions, and whether these effects of CTS are sustained. Rat temporomandibular joint disc cells (TDC) were exposed to CTS in the presence or absence of interleukin (IL)-1beta for 4 and 24h. Cells were also stimulated with IL-1beta for 24h while being subjected to CTS only for the initial 1, 2, 4, 8, and 12h or the entire 24h incubation time. Furthermore, cells were incubated with IL-1beta for 24, 36, or 48 h while being exposed to CTS only for the initial 8h. Gene expression of TNF-alpha, its receptors, and iNOS was analyzed by RT-PCR, whereas protein synthesis was determined by ELISA for TNF-alpha, immunofluorescence for TNFRs, and Griess reaction for nitric oxide. CTS inhibited the IL-1beta-stimulated synthesis of TNF-alpha, TNFR2, and iNOS. TNFR1 was constitutively expressed but not regulated by IL-1beta or CTS. Application of CTS for only 1 or 2h during a 24h incubation with IL-1beta was sufficient to inhibit IL-1beta-induced upregulation of TNF-alpha, TNFR2, and iNOS. However, for maximal inhibition of these genes a longer exposure of CTS was required. These findings are the first to show that biomechanical signals regulate the expression of TNFR2 but not TNFR1 under inflammatory conditions. Furthermore, the antiinflammatory effects of biomechanical signals on TDC are maintained for prolonged periods of time but are transient.

Dynamic biomechanical strain inhibits IL-1beta-induced inflammation in vocal fold fibroblasts.

Despite the fact that vocal folds are subjected to extensive mechanical forces, the role of mechanical strain in vocal fold wound healing has been overlooked. Recent studies on other tissues have demonstrated that low physiological levels of mechanical forces are beneficial to injured tissues, reduce inflammation, and induce synthesis of matrix-associated proteins essential for enhanced wound healing. In this study, we speculated that mechanical strain of low magnitudes also attenuates the production of inflammatory mediators and alters the extracellular matrix synthesis to augment wound healing in cultured vocal fold fibroblasts. To test this hypothesis, fibroblasts from rabbit vocal folds were isolated and exposed to various magnitudes of cyclic tensile strain (CTS) in the presence or absence of interleukin-1beta (IL-1beta). Results suggest that IL-1beta activates proinflammatory gene transcription in vocal fold fibroblasts. Furthermore, CTS abrogates the IL-1beta-induced proinflammatory gene induction in a magnitude-dependent manner. In addition, CTS blocks IL-1beta-mediated inhibition of collagen type I synthesis, and thereby upregulates collagen synthesis in the presence of IL-1beta. These findings are the first to reveal the potential utility of low levels of mechanical signals in vocal fold wound healing, and support the emerging on vivo data suggesting beneficial effects of vocal exercise on acute phonotrauma.

Is fine needle aspiration cytology (FNAC) useful in skin adnexal masses? A study on 5 cases of pilomatixoma.

Superficial cutaneous/subcutaneous nodules, caused by a variety of inflammatory, benign and malignant pathology of diverse origin, are tempting lesion for fine needle aspiration cytology (FNAC). Amongst these, adnexal tumor show considerable overlap, both in clinical manifestation as well as in histopathology. Archieval records of clinical findings, FNAC smears and reports pertaining to 5 histologically proved cases of pilomatricoma (PMX) were analyzed. Different cytological findings were graded + to +++. Pre FNAC clinical diagnoses were sebaceous cyst, tuberculous lymphadenopathy, dermatofibroma, reactive lymphadenopathy and lipoma. PMX was diagnosed on FNAC in 3 cases on finding groups of basaloid cells, ghost epithelial cells, pink fibrillary material and calcium deposits. Other cases were diagnosed as epidermal inclusion cyst with the differential diagnosis of well differentiated squamous cell carcinoma and skin appendageal tumor of undetermined origin in one case each. In all the cases, FNAC established epithelial nature of the lesion, excluding clinically mimicking inflammatory/neoplastic lesions of other origin. FNAC should be followed by excision biopsy to accurately type the epithelial neoplasm.

The RhoGAP Myo9b Promotes Bone Growth by Mediating Osteoblastic Responsiveness to IGF-1.

The Ras homolog A (RhoA) subfamily of Rho guanosine triphosphatases (GTPases) regulates actin-based cellular functions in bone such as differentiation, migration, and mechanotransduction. Polymorphisms or genetic ablation of RHOA and some of its regulatory guanine exchange factors (GEFs) have been linked to poor bone health in humans and mice, but the effects of RhoA-specific GTPase-activating proteins (GAPs) on bone quality have not yet been identified. Therefore, we examined the consequences of RhoGAP Myo9b gene knockout on bone growth, phenotype, and cellular activity. Male and female mice lacking both alleles demonstrated growth retardation and decreased bone formation rates during early puberty. These mice had smaller, weaker bones by 4 weeks of age, but only female KOs had altered cellular numbers, with fewer osteoblasts and more osteoclasts. By 12 weeks of age, bone quality in KOs worsened. In contrast, 4-week-old heterozygotes demonstrated bone defects that resolved by 12 weeks of age. Throughout, Myo9b ablation affected females more than males. Osteoclast activity appeared unaffected. In primary osteogenic cells, Myo9b was distributed in stress fibers and focal adhesions, and its absence resulted in poor spreading and eventual detachment from culture dishes. Similarly, MC3T3-E1 preosteoblasts with transiently suppressed Myo9b levels spread poorly and contained decreased numbers of focal adhesions. These cells also demonstrated reduced ability to undergo IGF-1-induced spreading or chemotaxis toward IGF-1, though responses to PDGF and BMP-2 were unaffected. IGF-1 receptor (IGF1R) activation was normal in cells with diminished Myo9b levels, but the activated receptor was redistributed from stress fibers and focal adhesions into nuclei, potentially affecting receptor accessibility and gene expression. These results demonstrate that Myo9b regulates a subset of RhoA-activated processes necessary for IGF-1 responsiveness in osteogenic cells, and is critical for normal bone formation in growing mice. © 2017 American Society for Bone and Mineral Research.

Daily Moderate Exercise Is Beneficial and Social Stress Is Detrimental to Disease Pathology in Murine Lupus Nephritis.

Daily moderate exercise (DME) and stress management are underemphasized in the care of patients with lupus nephritis (LN) due to a poor comprehensive understanding of their potential roles in controlling the inflammatory response. To investigate these effects on murine LN, disease progression was monitored with either DME or social disruption stress (SDR) induction in NZM2410/J mice, which spontaneously develop severe, early-onset LN. SDR of previously established social hierarchies was performed daily for 6 days and DME consisted of treadmill walking (8.5 m/min for 45 min/day). SDR significantly enhanced kidney disease when compared to age-matched, randomly selected control counterparts, as measured by histopathological analysis of H&E staining and immunohistochemistry for complement component 3 (C3) and IgG complex deposition. Conversely, while 88% of non-exercised mice displayed significant renal damage by 43 weeks of age, this was reduced to 45% with exercise. DME also reduced histopathology in kidney tissue and significantly decreased deposits of C3 and IgG complexes. Further examination of renal infiltrates revealed a macrophage-mediated inflammatory response that was significantly induced with SDR and suppressed with DME, which also correlated with expression of inflammatory mediators. Specifically, SDR induced IL-6, TNF-α, IL-1ß, and MCP-1, while DME suppressed IL-6, TNF-α, IL-10, CXCL1, and anti-dsDNA autoantibodies. These data demonstrate that psychological stressors and DME have significant, but opposing effects on the chronic inflammation associated with LN; thus identifying and characterizing stress reduction and a daily regimen of physical activity as potential adjunct therapies to complement pharmacological intervention in the management of autoimmune disorders, including LN.

Regulation of RANKL by biomechanical loading in fibrochondrocytes of meniscus.

OBJECTIVE: We sought to determine whether fibrochondrocytes from menisci express receptor activator of NF-kappaB (RANK), its ligand (RANKL), or osteoprotegerin (OPG) and, if so, whether their expression is modulated by dynamic mechanical loading under inflammatory and normal conditions. METHODS: Fibrochondrocytes from rat menisci were subjected to cyclic tensile strain (CTS) at various magnitudes and frequencies in the presence or absence of interleukin (IL)-1beta for up to 24 h. In order to determine whether a possible regulatory effect of mechanical loading on RANKL and its receptors under inflamed conditions is sustained, cells were stimulated with IL-1beta for 24 h while being subjected to CTS only for the initial 4 and 8h, respectively. Regulation of RANKL, RANK, and OPG expression and synthesis were determined by semiquantitative and real-time PCR, Western blotting, and immunofluorescence. RESULT: Fibrochondrocytes constitutively expressed low levels of RANKL and RANK but marked levels of OPG. IL-1beta upregulated expression and synthesis of RANKL and RANK significantly (p<0.05), whereas expression of OPG was unaffected following 4 and 24 h. When fibrochondrocytes were simultaneously subjected to CTS and IL-1beta, expression of RANKL and RANK was significantly (p<0.05) downregulated as compared to that of IL-1beta-stimulated unstretched cells. The inhibitory effect of CTS on the IL-1beta-induced upregulation of RANKL and RANK was sustained as well as magnitude and frequency dependent. CONCLUSIONS: Our study provides evidence that RANKL and its receptors are expressed in fibrochondrocytes from meniscus. These data also demonstrate that dynamic mechanical loading can modify the expression of RANKL and RANK in inflammatory conditions.

Evaluation of Polymerase Chain Reaction (PCR) with Slit Skin Smear Examination (SSS) to Confirm Clinical Diagnosis of Leprosy in Eastern Nepal.

BACKGROUND: Detection of Mycobacterium leprae in slit skin smear (SSS) is a gold standard technique for the leprosy diagnosis. Over recent years, molecular diagnosis by using PCR has been increasingly used as an alternative for its diagnosis due to its higher sensitivity. This study was carried out for comparative evaluation of PCR and SSS microscopy in a cohort of new leprosy cases diagnosed in B. P. Koirala Institute of health Sciences, Dharan, Nepal. METHODOLOGY/PRINCIPAL FINDINGS: In this prospective crossectional study, 50 new clinically diagnosed cases of leprosy were included. DNA was extracted from SSS and PCR was carried out to amplify 129 bp sequence of M. leprae repetitive element. Sensitivity of SSS and PCR was 18% and 72% respectively. Improvement of 54% case detection by PCR clearly showed its advantage over SSS. Furthermore, PCR could confirm the leprosy diagnosis in 66% of AFB negative cases indicating its superiority over SSS. In the paucibacillary (PB) patients, whose BI was zero; sensitivity of PCR was 44%, whereas it was 78% in the multibacillary patients. CONCLUSIONS/SIGNIFICANCE: Our study showed PCR to be more sensitive than SSS microscopy in diagnosing leprosy. Moreover, it explored the characteristic feature of PCR which detected higher level of early stage(PB) cases tested negative by SSS. Being an expensive technique, PCR may not be feasible in all the cases, however, it would be useful in diagnosis of early cases of leprosy as opposed to SSS.