In vivo induced allo-reactive natural killer cells.

We have previously shown that rat allo-selective cells of the CD2+CD5- phenotype were generated in Brown Norway (BN) rats after immunization with allogeneic Wistar/Furth (WF) cells, whereas immunization with semi-allogeneic F1 (WF/BN) cells generated CD2+CD5+ effector T cells. We now report that the allo-selective CD2+CD5- lymphocytes lacked expression of intact CD3 complexes and expressed NKR-P1 molecules although lower as compared to classical NK cells, implicating that these lymphocytes constitute a subset of NK cells. The CD5+ T cells were not cytolytically active in BN rats immunized with WF cells indicating an intersubset regulation with mutually exclusive activation of either allo-selective T cells or allo-selective NK cells. Cold target inhibition showed that lysis of both allogeneic target cells and NK-sensitive target cells was mediated by the same NKR-P1 intermediate effector cells. These NK cells lysed WF but not allogeneic Fischer 344 or autologous BN target cells, indicating selective recognition of an allogeneic determinant. Semiallogeneic F1 (WF/BN) target cells were not lysed. Furthermore, target cells from F1 (WF/BN) x WF back-cross hybrids lacking expression of RT1n (self-MHC class I) were susceptible to lysis, whereas back-cross hybrids expressing RT1n were protected from lysis, indicating that self-MHC molecules conferred protection from lysis. These findings implicate the existence of NKR-P1intermediate and NKR-P1high NK cell subsets with different regulation and function in vivo.     

Effects of inflammatory cytokines on bone/cartilage repair

Many inflammatory factors can affect cell behaviors and work as a form of inter-regulatory networks through the inflammatory pathway. Inflammatory cytokines are critical for triggering bone regeneration after fracture or bone injury. Also, inflammatory cytokines play an important role in cartilage repair. The synergistic or antagonistic effects of both proinflammatory and anti-inflammatory cytokines have a great influence on fracture healing. This review discusses key inflammatory cytokines and signaling pathways involved in bone or cartilage repair.     

In vivo measurement of translational stiffness of rabbit knees

This paper describes the design, evaluation, and preliminary results of a specialized testing device and surgical protocol to determine translational stiffness of a rabbit knee, replicating the clinical anterior drawer test. Coronal-plane transverse pins are inserted through the rabbit leg, two in the tibia and one in the distal femur, to hold and reproducibly position the leg in the device for tests at multiple time points. A linear stepper motor draws the tibia upward then returns to the home position, and a load cell measures the resisting force; force-displacement knee stiffness is then calculated. Initial evaluation of this testing device determined the effects of preconditioning, intra-operator repeatability, rabbit-to-rabbit variability, knee flexion angle (90 degrees vs. 135 degrees ), and anterior cruciate ligament (ACL) sectioning (0%, 25%, 50%, 75%, 100%). Knee stiffness generally decreased as ACL sectioning increased. This testing device and surgical protocol provide an objective and efficient method of determining translational rabbit knee stiffness in vivo, and are being used in an ongoing study to evaluate the effect of knee instability (via partial to complete ACL sectioning) on the development of post-traumatic osteoarthritis.     

In vivo tibiofemoral cartilage deformation during the stance phase of gait

The knowledge of articular cartilage contact biomechanics in the knee joint is important for understanding the joint function and cartilage pathology. However, the in vivo tibiofemoral articular cartilage contact biomechanics during gait remains unknown. The objective of this study was to determine the in vivo tibiofemoral cartilage contact biomechanics during the stance phase of treadmill gait. Eight healthy knees were magnetic resonance (MR) scanned and imaged with a dual fluoroscopic system during gait on a treadmill. The tibia, femur and associated cartilage were constructed from the MR images and combined with the dual fluoroscopic images to determine in vivo cartilage contact deformation during the stance phase of gait. Throughout the stance phase of gait, the magnitude of peak compartmental contact deformation ranged between 7% and 23% of the resting cartilage thickness and occurred at regions with thicker cartilage. Its excursions in the anteroposterior direction were greater in the medial tibiofemoral compartment as compared to those in the lateral compartment. The contact areas throughout the stance phase were greater in the medial compartment than in the lateral compartment. The information on in vivo tibiofemoral cartilage contact biomechanics during gait could be used to provide physiological boundaries for in vitro testing of cartilage. Also, the data on location and magnitude of deformation among non-diseased knees during gait could identify where loading and later injury might occur in diseased knees.     

In vivo patellar tracking and patellofemoral cartilage contacts during dynamic stair ascending

The knowledge of normal patellar tracking is essential for understanding the knee joint function and for diagnosis of patellar instabilities. This paper investigated the patellar tracking and patellofemoral joint contact locations during a stair ascending activity using a validated dual-fluoroscopic imaging system. The results showed that the patellar flexion angle decreased from 41.9° to 7.5° with knee extension during stair ascending. During first 80% of the activity, the patella shifted medially about 3.9mm and then slightly shifted laterally during the last 20% of the ascending activity. Anterior translation of 13mm of the patella was measured at the early 80% of the activity and the patella slightly moved posteriorly by about 2mm at the last 20% of the activity. The path of cartilage contact points was slightly lateral on the cartilage surfaces of patella and femur. On the patellar cartilage surface, the cartilage contact locations were about 2mm laterally from heel strike to 60% of the stair ascending activity and moved laterally and reached 5.3mm at full extension. However, the cartilage contact locations were relatively constant on the femoral cartilage surface (～5mm lateral). The patellar tracking pattern was consistent with the patellofemoral cartilage contact location pattern. These data could provide baseline knowledge for understanding of normal physiology of the patellofemoral joint and can be used as a reference for clinical evaluation of patellofemoral disorders.     

In vivo selective cytoskeleton dynamics quantification in interphase cells induced by pulsed ultraviolet laser nanosurgery

We report on the manipulation of intracellular filaments using a nanosurgery system based on a subnanosecond pulsed UV laser optimized for the localized severing of biological polymers. By inducing artificial catastrophe of selected microtubules (MTs), we perform shrinkage-rate measurements in interphase Ptk-2 cells throughout the entire cell. We quantify the impact of two labeling methods and three fluorescent markers, showing a 25% faster depolymerization with Alexa-488 tubulin compared with Rhodamine and yellow fluorescent protein (YFP) tubulins and a 20% higher variability induced by microinjection compared with stable transfection. Using EB3-GFP as a tip marker, we establish a new protocol to measure shrinkage rate, growth rate and rescue frequency simultaneously with high temporal and spatial specificity in live cells. As our analysis shows, laser-induced MT dynamics are physiologically relevant. The high statistical efficiency that the method offers in terms of numbers of measured events and therefore reduced standard deviations represents an important quantitative improvement in the measurement of dynamic instability parameters in vivo. We extend the application of the method by demonstrating induced dynamic behavior of actin-stress fibers after severing. This new method enables the quantitative investigation of cytoskeleton dynamics in a local confinement.     

Keratinocyte-derived chemotactic cytokines: expressional modulation by nitric oxide in vitro and during cutaneous wound repair in vivo

Inhibition of inducible nitric oxide-synthase (iNOS) enzymatic activity during cutaneous wound repair leads to severely impaired tissue regeneration. To assess whether disturbed leukocyte infiltration might participate in impaired repair, we determined expressional kinetics of neutrophil-attracting macrophage inflammatory protein-2 (MIP-2), and monocyte-attracting macrophage chemoattractant protein-1 (MCP-1) using an excisional wound healing model in mice. MCP-1 was induced in epithelial keratinocytes upon wounding, and our data indicate that NO serves a negative regulatory role for MCP-1 expression in vivo, as clearly reduced numbers of wound margin keratinocytes associated with NO-deficient repair compensate for high MCP-1 expression levels observed during normal healing. MIP-2 expression was restricted to hair follicles which were not reduced in number during NO-deficient repair. In vitro studies confirmed a regulatory role of NO for keratinocyte-derived chemokine expression, as NO attenuated IL-1beta- and TNF-alpha-induced MCP-1 mRNA expression, whereas NO augmented IL-1beta-induced IL-8 (functional human homolog to murine MIP-2) mRNA expression in the human keratinocyte cell line HaCaT.     

Biomechanical assessment of tissue retrieved after in vivo cartilage defect repair: tensile modulus of repair tissue and integration with host cartilage

Failure to restore the mechanical properties of tissue at the repair site and its interface with host cartilage is a common problem in tissue engineering procedures to repair cartilage defects. Quantitative in vitro studies have helped elucidate mechanisms underlying processes leading to functional biomechanical changes. However, biomechanical assessment of tissue retrieved from in vivo studies of cartilage defect repair has been limited to compressive tests. Analysis of integration following in vivo repair has relied on qualitative histological methods. The objectives of this study were to develop a quantitative biomechanical method to assess (1) the tensile modulus of repair tissue and (2) its integration in vivo, as well as determine whether supplementation of transplanted chondrocytes with IGF-I affected these mechanical properties. Osteochondral blocks were obtained from a previous 8 month study on the effects of IGF-I on chondrocyte transplantation in the equine model. Tapered test specimens were prepared from osteochondral blocks containing the repair/native tissue interface and adjacently located blocks of intact native tissue. Specimens were then tested in uniaxial tension. The tensile modulus of repair tissue averaged 0.65 MPa, compared to the average of 5.2 MPa measured in intact control samples. Integration strength averaged 1.2 MPa, nearly half the failure strength of intact cartilage samples, 2.7 MPa. IGF-I treatment had no detectable effects on these mechanical properties. This represents the first quantitative biomechanical investigation of the tensile properties of repair tissue and its integration strength in an in vivo joint defect environment.     

Clinically translatable cell tracking and quantification by MRI in cartilage repair using superparamagnetic iron oxides

BACKGROUND: Articular cartilage has very limited intrinsic regenerative capacity, making cell-based therapy a tempting approach for cartilage repair. Cell tracking can be a major step towards unraveling and improving the repair process of these therapies. We studied superparamagnetic iron oxides (SPIO) for labeling human bone marrow-derived mesenchymal stem cells (hBMSCs) regarding effectivity, cell viability, long term metabolic cell activity, chondrogenic differentiation and hBMSC secretion profile. We additionally examined the capacity of synovial cells to endocytose SPIO from dead, labeled cells, together with the use of magnetic resonance imaging (MRI) for intra-articular visualization and quantification of SPIO labeled cells. METHODOLOGY/PRINICIPAL FINDINGS: Efficacy and various safety aspects of SPIO cell labeling were determined using appropriate assays. Synovial SPIO re-uptake was investigated in vitro by co-labeling cells with SPIO and green fluorescent protein (GFP). MRI experiments were performed on a clinical 3.0T MRI scanner. Two cell-based cartilage repair techniques were mimicked for evaluating MRI traceability of labeled cells: intra-articular cell injection and cell implantation in cartilage defects. Cells were applied ex vivo or in vitro in an intra-articular environment and immediately scanned. SPIO labeling was effective and did not impair any of the studied safety aspects, including hBMSC secretion profile. SPIO from dead, labeled cells could be taken up by synovial cells. Both injected and implanted SPIO-labeled cells could accurately be visualized by MRI in a clinically relevant sized joint model using clinically applied cell doses. Finally, we quantified the amount of labeled cells seeded in cartilage defects using MR-based relaxometry. CONCLUSIONS: SPIO labeling appears to be safe without influencing cell behavior. SPIO labeled cells can be visualized in an intra-articular environment and quantified when seeded in cartilage defects.     

In vivo assembly and disassembly of Rad51 and Rad52 complexes during double-strand break repair

Assembly and disassembly of Rad51 and Rad52 complexes were monitored by immunofluorescence during homologous recombination initiated by an HO endonuclease-induced double-strand break (DSB) at the MAT locus. DSB-induced Rad51 and Rad52 foci colocalize with a TetR-GFP focus at tetO sequences adjacent to MAT. In strains in which HO cleaves three sites on chromosome III, we observe three distinct foci that colocalize with adjacent GFP chromosome marks. We compared the kinetics of focus formation with recombination intermediates and products when HO-cleaved MATalpha recombines with the donor, MATa. Rad51 assembly occurs 1 h after HO cleavage. Rad51 disassembly occurs at the same time that new DNA synthesis is initiated after single-stranded (ss) MAT DNA invades MATa. We present evidence for three distinct roles for Rad52 in recombination: a presynaptic role necessary for Rad51 assembly, a synaptic role with Rad51 filaments, and a postsynaptic role after Rad51 dissociates. Additional biochemical studies suggest the presence of an ssDNA complex containing both Rad51 and Rad52.     

Basic fibroblast growth factor (FGF) promotes cartilage repair in vivo

Although it has been clearly established that basic fibroblast growth factor (FGF) is a potent mitogen for chondrocytes in vitro, there is little evidence that it can stimulate this cell type in vivo. In an effort to address this problem, we examined the effect of an intraarticular administration of basic FGF. Alzet osmotic pumps delivering the mitogen to the site of injury promotes the healing of intra-chondrial lesions by stimulating chondrocyte proliferation and the formation of extracellular matrix. The observation that chronic infusions of basic FGF can elicit a repair response at the site of injury suggests that this growth factor may have therapeutic applications that extend beyond its capacity to induce neovascularization. The results also suggest that one of the ways that the perichondrium mediates cartilage repair may be by the local production of FGF-like mitogens.     

Isolation and ex vivo expansion of synovial mesenchymal stromal cells for cartilage repair

Background aimsHyaline articular cartilage is a highly specialized tissue that offers a low-friction and wear-resistant interface for weight-bearing surface articulation in diarthrodial joints, but it lacks vascularity. It displays an inherent inability to heal when injured in a skeletally mature individual. Joint-preserving treatment procedures such as mosaicplasty, débridement, perichondrium transplantation and autologous chondrocyte implantation have shown variable results, and the average long-term result is sub-standard. Because of these limitations of the treatment methods and lack of intrinsic repair capacity of mature cartilage tissue, an alternative treatment approach is needed, and synovial mesenchymal stromal cells (SMSCs) represent an attractive therapeutic alternative because of their ex vivo proliferation capacity, multipotency and ability to undergo chondrogenesis.MethodsSMSCs were isolated from tissues obtained by arthroscopy using two types of biopsies. Ex vivo cell expansion was accomplished under static and dynamic culture followed by characterization of cells according to the International Society for Cellular Therapy guidelines. Kinetic growth models and metabolite analysis were used for understanding the growth profile of these cells.ResultsFor the first time, SMSCs were expanded in stirred bioreactors and achieved higher cell density in a shorter period of time compared with static culture or with other mesenchymal stromal cell sources.ConclusionsIn this study we were able to achieve (8.8 ± 0.2) × 10⁵ cells within <2 weeks in dynamic culture under complete xeno-free conditions. Our results also provided evidence that after dynamic culture these cells had an up-regulation of chondrogenic genes, which can be a potential factor for articular cartilage regeneration in clinical settings.     

In vivo assessment of natural killer cell responses during chronic feline immunodeficiency virus infection

Accumulating evidence suggests that natural killer (NK) cells may have an important role in HIV-1 disease pathogenesis; however, in vivo studies are lacking. Feline immunodeficiency virus (FIV) infection of cats provides a valuable model to study NK cell function in vivo. The immune response against Listeria monocytogenes (Lm) is well characterized, allowing its use as an innate immune probe. We have previously shown that locally delivered IL-15 can improve Lm clearance in FIV-infected animals, and this correlated with an increase in NK cell number. In the present study, chronically FIV-infected and SPF-control cats were challenged with Lm by unilateral subcutaneous injection next to the footpad and then treated with 5-bromo-2'-deoxyuridine (BrdU). The Lm draining and contralateral control lymph nodes were evaluated for NK, NKT, CD4+ and CD8+ T cell number, proliferation, apoptosis, and NK cell function. Listeria monocytogenes burden was also assessed in both control and Lm draining lymph nodes. NK, NKT, CD4+ T and CD8+ T cells in the Lm-challenged lymph node of FIV-infected cats did not increase in number. In addition, after Lm challenge, NK cells from FIV-infected cats did not increase their proliferation rate, apoptosis was elevated, and perforin expression was not upregulated when compared to SPF-control cats. The failure of the NK cell response against Lm challenge in the draining lymph node of FIV-infected cats correlates with the delayed control and clearance of this opportunistic bacterial pathogen.     

In vivo cartilage contact strains in patients with lateral ankle instability

Damage to the anterior talofibular ligament (ATFL) and cacaneofibular ligament (CFL) during an ankle sprain may be linked to the development of osteoarthritis. Although altered tibiotalar kinematics have been demonstrated, the effects of lateral ankle instability (LAI) on in vivo cartilage strains have not been described. We hypothesized that peak cartilage strains increase, and the location is shifted in patients with ATFL injuries. We used 3-D MRI models and biplanar fluoroscopy to evaluate in vivo cartilage contact strains in seven patients with unilateral LAI. Subjects had chronic unilateral ATFL injury or combined ATFL and CFL injury, and were evaluated with increasing load while stepping onto a force plate. Peak cartilage strain and the location of the peak strain were measured using the contralateral normal ankle as a control. Ankles with LAI demonstrated significantly increased peak strain when compared with ATFL-intact controls. For example, at 100% body weight, peak strain was 29±8% on the injured side compared to 21±5% on the intact side. The position of peak strain on the injured ankle also showed significant anterior translation and medial translation. At 100% body weight, the location of peak strain in the injured ankle translated anteriorly by 15.5±7.1 mm and medially by 12.9±4.3 mm relative to the intact ankle. These changes correspond to the region of clinically observed osteoarthritis. Chronic LAI, therefore, may contribute to the development of tibiotalar cartilage degeneration due to altered cartilage strains.     

In vivo natural killer cell depletion during primary simian immunodeficiency virus infection in rhesus monkeys

The contribution of natural killer (NK) cells to the immune containment of human immunodeficiency virus infection remains undefined. To directly assess the role of NK cells in an AIDS animal model, we depleted rhesus monkeys of >88% of CD3(-) CD16(+) CD159a(+) NK cells at the time of primary simian immunodeficiency virus (SIV) infection by using anti-CD16 antibody. During the first 11 days following SIV inoculation, when NK cell depletion was most profound, a trend toward higher levels of SIV replication was noted in NK cell-depleted monkeys compared to those in control monkeys. However, this treatment did not result in significant changes in the overall levels or kinetics of plasma viral RNA or affect the SIV-induced central memory CD4(+) T-lymphocyte loss. These findings are consistent with a limited role for cytotoxic CD16(+) NK cells in the control of primary SIV viremia.     

In vivo quantification of cell coupling in plants with different phloem-loading strategies

Uptake of photoassimilates into the leaf phloem is the key step in carbon partitioning and phloem transport. Symplasmic and apoplasmic loading strategies have been defined in different plant taxa based on the abundance of plasmodesmata between mesophyll and phloem. For apoplasmic loading to occur, an absence of plasmodesmata is a sufficient but not a necessary criterion, as passage of molecules through plasmodesmata might well be blocked or restricted. Here, we present a noninvasive, whole-plant approach to test symplasmic coupling and quantify the intercellular flux of small molecules using photoactivation microscopy. Quantification of coupling between all cells along the prephloem pathways of the apoplasmic loader Vicia faba and Nicotiana tabacum showed, to our knowledge for the first time in vivo, that small solutes like sucrose can diffuse through plasmodesmata up to the phloem sieve element companion cell complex (SECCC). As expected, the SECCC was found to be symplasmically isolated for small solutes. In contrast, the prephloem pathway of the symplasmic loader Cucurbita maxima was found to be well coupled with the SECCC. Phloem loading in gymnosperms is not well understood, due to a profoundly different leaf anatomy and a scarcity of molecular data compared with angiosperms. A cell-coupling analysis for Pinus sylvestris showed high symplasmic coupling along the entire prephloem pathway, comprising at least seven cell border interfaces between mesophyll and sieve elements. Cell coupling together with measurements of leaf sap osmolality indicate a passive symplasmic loading type. Similarities and differences of this loading type with that of angiosperm trees are discussed.     

In vivo microdialysis sampling of cytokines produced in mice given bacterial lipopolysaccharide

Cytokines are proteins that mediate communication between cells of the immune system as well as certain other non-immune host cells. These proteins are produced by many cell types and they mediate immune and inflammatory responses. However, the direct site analysis of these critical proteins is hampered by the lack of site-specific tools available for such direct measurements. In this study, both in vitro and in vivo microdialysis sampling of different cytokines (tumor necrosis factor-alpha [TNF-alpha], interferon-gamma [IFN-gamma], interleukin-6 [IL-6], IL-12p70, and macrophage chemoattractant protein-1 [MCP-1]) was performed. A mouse model of bacterial lipopolysaccharide (LPS) administration and response pattern was used for in vivo studies. Three cytokines, TNF-alpha, IL-6, and MCP-1 were quantified in the serum from mice given LPS. In vivo studies demonstrated the ability to monitor increasing levels of these cytokines (TNF-alpha, IL-6, and MCP-1) via microdialysis probes placed in the peritoneal cavity of mice given LPS. All three cytokines were quantified simultaneously in 15 muL of dialysate using a multiplexed bead-based immunoassay for flow cytometry. The detected dialysate cytokine concentrations varied between 200 pg/mL and 1500 pg/mL for TNF-alpha, between 600 pg/mL and 3000 pg/mL for MCP-1, and between 2700 pg/mL and more than 5000 pg/mL for IL-6. The detected serum cytokine concentrations ranged from 5700 pg/mL to 35,000 pg/mL for TNF-alpha, from 40,000 pg/mL to 65,000 pg/mL for MCP-1, and greater than than 100,000 pg/mL for IL-6. This work demonstrates that microdialysis sampling can be used in vivo to collect temporal profiles of cytokine production.     

DNA repair synthesis and chromosomal aberrations induced in vivo by triethylenemelamine

The alkylating agent, triethylenemelamine (TEM), was studied for its ability to induce unscheduled DNA (repair) synthesis (UDS) in vivo in rat lymphocytes. Somatic cytogenetic alterations were analyzed (in bone marrow) and compared with UDS as a function of TEM dosage. UDS was evaluated through the use of autoradiography; cytogenetic alterations were studied in metaphase bone marrow chromosome preparations.Data indicated that the degree of UDS is a direct function of TEM dosage up to a rate-limiting concentration, at which point it ceases to be dose dependent. Except for a deviation at the highest dose level tested, the extent of cytogenetic damage was directly and linearly related to TEM dose. Between the control and intermediate (0.2 mg/kg) dose levels, UDS response increased II-fold while cytogenetic damage showed only a 4-fold increase; this disparity diminished with increasing TEM dose. In the lower dose levels, therefore, the greater relative sensitivity of UDS evaluation in the detection of genetic activity may be indicated. Patterns of UDS response observed through the in vivo assay developed in this study were found to be analogous to those established in in vitro studies.