Detection of surfactant proteins A and D in human tear fluid and the human lacrimal system

PURPOSE: To evaluate the expression and presence of surfactant protein (SP) A and SP-D in the lacrimal apparatus, at the ocular surface, and in tears in healthy and pathologic states. METHODS: Expression of mRNA for SP-A and SP-D was analyzed by RT-PCR in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal ducts as well as in a spontaneously immortalized conjunctival epithelial cell line (HCjE; IOBA-NHC) and a SV40-transfected cornea epithelial cell line (HCE). Deposition of SP-A and SP-D was determined by Western blot, dot blot, and immunohistochemistry in healthy tissues, in tears, aqueous humor, and in sections of different corneal abnormalities (keratoconus, herpetic keratitis, and Staphylococcus aureus-based ulceration). Cell lines were stimulated with different cytokines and bacterial components and were analyzed for the production of SP-A and SP-D by immunohistochemistry. RESULTS: The presence of SP-A and SP-D on mRNA and protein levels was evidenced in healthy lacrimal gland, conjunctiva, cornea, and nasolacrimal duct samples. Moreover, both proteins were present in tears but were absent in aqueous humor. Immunohistochemistry revealed the production of both peptides by acinar epithelial cells of the lacrimal gland and epithelial cells of the conjunctiva and nasolacrimal ducts, whereas goblet cells revealed no reactivity. Healthy cornea revealed weak reactivity on epithelial surface cells only. In contrast, SP-A and SP-D revealed strong reactivity in patients with herpetic keratitis and corneal ulceration surrounding lesions and in several immigrated defense cells. Reactivity in corneal epithelium and endothelium was also seen in patients with keratoconus. Cell culture experiments revealed that SP-A and SP-D are produced by both epithelial cell lines without and after stimulation with cytokines and bacterial components. CONCLUSIONS: These results show that SP-A, in addition to SP-D, is a peptide of the tear film. Based on the known direct and indirect antimicrobial effects of collectins, the surfactant-associated proteins A and D seem to be involved in several ocular surface diseases.

Regional comparisons of nano-mechanical properties of the human meniscus; structure and function

Osteoarthritis (OA) is a debilitating disease that is becoming more prevalent in today’s society. OA affects approximately 28 million adults in the United States alone and when present in the knee joint, usually leads to a total knee replacement. Numerous studies have been conducted to determine possible methods to halt the initiation of OA, but the structural integrity of the menisci has been shown have a direct effect on the progression of OA. Menisci are two C-shaped structures that are attached to the tibial plateau and aid in facilitating proper load transmission within the knee. The meniscal cross-section is wedge-like to fit the contour of the femoral condyles and help attenuate stresses on the tibial plateau. While meniscal tears are common, only the outer 1/3 of the meniscus is vascularized and has the capacity to heal, hence tears of the inner 2/3rds are generally treated via meniscectomy, leading to OA. To help combat this OA epidemic, an effective biomimetric meniscal replacement is needed. Numerous mechanical and biochemical studies have been conducted on the human meniscus, but very little is known about the mechanical properties on the nano-scale and how meniscal constituents are distributed in the meniscal cross-section. The regional (anterior, central and posterior) nano-mechanical properties of the meniscal superficial layers (both tibial and femoral contacting) and meniscal deep zone were investigated via nanoindentation to examine the regional inhomogeneity of both the lateral and medial menisci. Additionally, these results were compared to quantitative histological values to better formulate a structure-function relationship on the nano-scale. These data will prove imperative for further advancements of a tissue engineered meniscal replacement.

VISUAL-INERTIAL INTEGRATION FOR HUMAN MOTION TRACKING AND NAVIGATION IN FREE-LIVING ENVIRONMENTS

The aging population has become a burning issue for all modern societies around the world recently. There are two important issues existing now to be solved. One is how to continuously monitor the movements of those people having suffered a stroke in natural living environment for providing more valuable feedback to guide clinical interventions. The other one is how to guide those old people effectively when they are at home or inside other buildings and to make their life easier and convenient. Therefore, human motion tracking and navigation have been active research fields with the increasing number of elderly people. However, motion capture has been extremely challenging to go beyond laboratory environments and obtain accurate measurements of human physical activity especially in free-living environments, and navigation in free-living environments also poses some problems such as the denied GPS signal and the moving objects commonly presented in free-living environments. This thesis seeks to develop new technologies to enable accurate motion tracking and positioning in free-living environments. This thesis comprises three specific goals using our developed IMU board and the camera from the imaging source company: (1) to develop a robust and real-time orientation algorithm using only the measurements from IMU; (2) to develop a robust distance estimation in static free-living environments to estimate people’s position and navigate people in static free-living environments and simultaneously the scale ambiguity problem, usually appearing in the monocular camera tracking, is solved by integrating the data from the visual and inertial sensors; (3) in case of moving objects viewed by the camera existing in free-living environments, to firstly design a robust scene segmentation algorithm and then respectively estimate the motion of the vIMU system and moving objects. To achieve real-time orientation tracking, an Adaptive-Gain Orientation Filter (AGOF) is proposed in this thesis based on the basic theory of deterministic approach and frequency-based approach using only measurements from the newly developed MARG (Magnet, Angular Rate, and Gravity) sensors. To further obtain robust positioning, an adaptive frame-rate vision-aided IMU system is proposed to develop and implement fast vIMU ego-motion estimation algorithms, where the orientation is estimated in real time from MARG sensors in the first step and then used to estimate the position based on the data from visual and inertial sensors. In case of the moving objects viewed by the camera existing in free-living environments, a robust scene segmentation algorithm is firstly proposed to obtain position estimation and simultaneously the 3D motion of moving objects. Finally, corresponding simulations and experiments have been carried out.

MicroRNA-140 is expressed in differentiated human articular chondrocytes and modulates interleukin-1 responses

OBJECTIVE: MicroRNA (miRNA) are a class of noncoding small RNAs that act as negative regulators of gene expression. MiRNA exhibit tissue-specific expression patterns, and changes in their expression may contribute to pathogenesis. The objectives of this study were to identify miRNA expressed in articular chondrocytes, to determine changes in osteoarthritic (OA) cartilage, and to address the function of miRNA-140 (miR-140). METHODS: To identify miRNA specifically expressed in chondrocytes, we performed gene expression profiling using miRNA microarrays and quantitative polymerase chain reaction with human articular chondrocytes compared with human mesenchymal stem cells (MSCs). The expression pattern of miR-140 was monitored during chondrogenic differentiation of human MSCs in pellet cultures and in human articular cartilage from normal and OA knee joints. We tested the effects of interleukin-1beta (IL-1beta) on miR-140 expression. Double-stranded miR-140 (ds-miR-140) was transfected into chondrocytes to analyze changes in the expression of genes associated with OA. RESULTS: Microarray analysis showed that miR-140 had the largest difference in expression between chondrocytes and MSCs. During chondrogenesis, miR-140 expression in MSC cultures increased in parallel with the expression of SOX9 and COL2A1. Normal human articular cartilage expressed miR-140, and this expression was significantly reduced in OA tissue. In vitro treatment of chondrocytes with IL-1beta suppressed miR-140 expression. Transfection of chondrocytes with ds-miR-140 down-regulated IL-1beta-induced ADAMTS5 expression and rescued the IL-1beta-dependent repression of AGGRECAN gene expression. CONCLUSION: This study shows that miR-140 has a chondrocyte differentiation-related expression pattern. The reduction in miR-140 expression in OA cartilage and in response to IL-1beta may contribute to the abnormal gene expression pattern characteristic of OA.

Expression and Function of Siglec-8 in Human Eosinophils, Basophils, and Mast Cells

Siglec-8, the eighth member of the sialic acid-binding, immunoglobulin [Ig]-like lectin family, was initially discovered as a cell surface protein selectively expressed on human eosinophils. It is now know to also be expressed by mast cells and basophils. Siglec-8 engagement with specific antibodies causes apoptosis via caspase and mitochondrial-dependent pathways. For mast cells, inhibition of mediator release, but no apoptosis, is observed. Siglec-F is the closest mouse paralog to Siglec-8, and both selectively bind the sulfated glycan 6’-sulfo-sialyl Lewis X. Antibodies to Siglec-F reduce blood and tissue eosinophil numbers in vivo. This suggests that Siglec-8 may be a useful future therapeutic target for allergic and other eosinophilic disorders.

Infection with human herpesvirus 8 and transplant-associated gammopathy

BACKGROUND: The role of human herpesvirus (HHV)-8 in the pathogenesis of multiple myeloma and its pre-malignant state of monoclonal gammopathy is unclear. HHV-8 is transmitted by organ transplantation, representing a unique model with which to investigate primary HHV-8 infection. METHODS: The authors studied the incidence of clonal gammopathy in renal transplant recipients and correlated it with previous and recent HHV-8 infection. RESULTS: Clonal gammopathy was observed in 31 of 162 (19%) HHV-8-seronegative patients, in 5 of 17 (29%) HHV-8-seropositive patients, and in 9 of 24 (38%) HHV-8 seroconverters within 5 years after transplantation. Gammopathy was often transient, and no progression to myeloma was observed. Two patients with persistent gammopathy developed B-cell lymphoma. In a logistic regression model, HHV-8 serostatus of the graft recipient was significantly associated with subsequent development of gammopathy, with a relative risk (RR) of 1.9 and a 95% confidence interval (CI) of 0.5 to 6.4 for an HHV-8-seropositive recipient and an RR of 2.9 and a 95% CI of 1.01 to 8.0 for seroconverters as compared with baseline (HHV-8 seronegative). Other significant variables were cytomegalovirus (CMV) serostatus and the intensity of immunosuppression (RR of 10.4 and 95% CI of 2.6-41.7 for a CMV-negative recipient with a CMV-positive donor vs. a CMV-negative recipient with a CMV-negative donor and RR of 17.6 and 95% CI of 2.0-150.8 if OKT3 was used vs. no use of antilymphocytic substances). CONCLUSIONS: Transplant recipients with HHV-8 infection are more likely to develop clonal gammopathy. However, this risk is much lower than the risk conferred by CMV infection and antilymphocytic therapy, arguing against a major role of HHV-8 infection in the pathogenesis of clonal plasma cell proliferation.

Suburothelial myofibroblasts in the human overactive bladder and the effect of botulinum neurotoxin type A treatment

BACKGROUND: An increasing body of evidence suggests a possible role of suburothelial myofibroblasts (MFs) in bladder mechanosensation and in the pathophysiology of detrusor overactivity (DO). OBJECTIVE: To determine whether markers of MFs, including gap junction protein connexin43 (Cx43) and c-kit have altered immunohistochemical expression in the suburothelium of patients with neurogenic DO (NDO) or idiopathic DO (IDO) and whether this is affected by successful treatment of DO with botulinum neurotoxin type A (BoNTA). DESIGN, SETTING, AND PARTICIPANTS: Patients with NDO (n=10) or IDO (n=11) were treated in a single-centre, open-label study of intradetrusor BoNTA injections. Control tissue was obtained from 10 patients undergoing pelvic-floor repair procedures who had no overactive bladder (OAB) symptoms. This study is registered with ClinicalTrials.gov, number NCT00662064. INTERVENTIONS: Bladder biopsies performed with flexible cystoscopes were obtained from control subjects and from NDO and IDO patients before BoNTA treatment and at 4 wk and 16 wk after treatment. They were studied with quantitative immunofluorescence using antibodies to connexin 43 (Cx43), vimentin, and c-kit. MEASUREMENTS: Differences in Cx43, vimentin, and c-kit immunoreactivity between control subjects and NDO or IDO patients (primary outcomes). Changes in NDO or IDO, Cx43 immunoreactivity, and c-kit immunoreactivity after BoNTA treatment (secondary outcomes). RESULTS AND LIMITATIONS: Cx43 immunoreactivity was increased in both IDO and NDO patients compared to controls, but remained unchanged after BoNTA treatment. C-kit immunoreactivity was similar in NDO/IDO patients and controls and remained unchanged after BoNTA treatment. CONCLUSIONS: Increased gap junction formation in the suburothelium has been demonstrated in biopsies from humans with DO. It is hypothesised that this change could have a significant role in the pathogenesis of the detrusor abnormality. Successful treatment of NDO or IDO does not appear to be associated with changes in the expression of Cx43 or c-kit on suburothelial MFs.