Mesenchymal Stromal Cell (MSC) Functional Analysis-Macrophage Activation and Polarization Assays.

Stem cell-based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between MSCs and macrophages, we developed a potency assay that directly co-cultures MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies. Key features • The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells-derived macrophages. • The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores. • The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages. • This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.

Bioprinting of exosomes: Prospects and challenges for clinical applications.

453Three-dimensional bioprinting (3DBP) is an additive manufacturing technique that has emerged as a promising strategy for the fabrication of scaffolds, which can successfully recapitulate the architectural, biochemical, and physical cues of target tissues. More importantly, 3DBP offers fine spatiotemporal control and high submicron scale resolution, which can be leveraged for the incorporation and directional gradient release of single or multiple biomimetic cues, including cell-derived exosomes (EXOs). EXOs are extracellular vesicles that originate from the endosomal compartment of various cell types, with sizes ranging from 30 to120 nm. They act as cell mediators and contain discrete cell constituents, including growth factors, cytokines, lipid moieties, nucleic acids, metabolites, and cell surface markers, depending on the cell type. Essentially, owing to their therapeutic potential, EXOs derived from mesenchymal stem cells (MSCs) have been recently investigated in several clinical trials for the treatment of various conditions, including cancer, diabetes, dry eyes, periodontitis, and acute ischemic stroke. The 3DBP strategy of EXOs is especially useful in tissue engineering and regenerative medicine applications, as tissues can be biofabricated to closely mimic the complex microarchitecture and developmental profiles of native heterogeneous tissues for restoring biological functions. Moreover, EXOs can be manipulated to carry exogenous cargo such as genes or proteins of therapeutic interest, confer multifunctional attributes, and further enhance their tissue regenerative potential. However, significant challenges, including the selection of appropriate bioink, pattern resolution, engineering-defined exosomal gradient, spatial presentation and modulation of EXO release kinetics, as well as EXO stability and storage conditions, must be addressed for the successful translation of therapeutic grade EXOs to clinical settings. In this review, we highlight the recent advances and offer future perspectives on the bioprinting of EXOs as regenerative biotherapeutics for the fabrication of complex heterogeneous tissues that are suitable for clinical transplantation.

Streamlined Methods for Processing and Cryopreservation of Cell Therapy Products Using Automated Systems.

Streamlined procedures for processing and cryopreservation of cell therapies using good laboratory practices are integral to biomanufacturing process development and clinical applications. The protocol herein begins with the preparation of human cell types cultured as adherent (i.e., mesenchymal stromal cells, MSCs) or suspension cells (i.e., peripheral blood mononuclear cells, PBMCs) to comprehensively demonstrate procedures that are applicable to commonly used primary cell cultures. Cell processing steps consist of preparing high yields of cells for cryopreservation using instruments routinely used in cell manufacturing, including the Finia® Fill and Finish System and a controlled-rate freezer. The final steps comprise the storage of cells at subzero temperatures in liquid nitrogen vapor phase followed by the analysis of cell phenotypes before and after processing and cryopreservation, along with cell quality metrics for validation. Additionally, the protocol includes important considerations for the implementation of quality control measures for equipment operation and cell handling, as well as Good Laboratory Practices for cell manufacturing, which are essential for the translational use of cell therapies. Key features • The protocol applies to small- or large-scale manufacturing of cell therapy products. • It includes streamlined procedures for processing and cryopreservation of cells cultured as adherent cells (MSCs) and suspension cells (PBMCs). • Provides temperature control and rapid partitioning of sample in cryopreservation solution to maintain high viability of a range of cell types throughout the procedures. • This protocol employs the Finia® Fill and Finish System and a controlled-rate freezer. Graphical overview.

Functionally Graded Bioactive Composites Based on Poly(vinyl alcohol) Made through Thiol-Ene Click Reaction.

Functionally graded materials (FGMs) composed of a polymer matrix embedded with calcium phosphate particles are preferred for bone tissue engineering, as they can mimic the hierarchical and gradient structure of bones. In this study, we report the design and development of a FGM based on thiolated poly(vinyl alcohol) (TPVA) and nano-hydroxyapatite (nano-HA) with graded bioactivity, cell compatibility, and degradability properties that are conducive for bone regeneration. The polymer matrix comprises crosslinked poly(vinyl alcohol) with ester and thioether linkages formed via the thiol-ene click reaction, avoiding undesired additives and byproducts. Freshly precipitated and spray-dried HA was mixed with the TPVA hydrogel, and layers of varying concentrations were cast. Upon lyophilization, the hydrogel structure yielded porous sheets of the graded composite of TPVA and nano-HA. The new FGM showed higher values of tensile strength and degradation in phosphate buffer saline (PBS) in vitro, compared to bare TPVA. The bioactive nature of the FGM was confirmed through bioactivity studies in simulated body fluid (SBF), while cytocompatibility was demonstrated with human periodontal ligament cells in vitro. Cumulatively, our results indicate that based on the composition, mechanical properties, bioactivity, and cytocompatibility, the fabricated TPVA-HA composites can find potential use as guided bone regeneration (GBR) membranes.

Human Cadaveric Donor Cornea Derived Extra Cellular Matrix Microparticles for Minimally Invasive Healing/Regeneration of Corneal Wounds.

Biological materials derived from extracellular matrix (ECM) proteins have garnered interest as their composition is very similar to that of native tissue. Herein, we report the use of human cornea derived decellularized ECM (dECM) microparticles dispersed in human fibrin sealant as an accessible therapeutic alternative for corneal anterior stromal reconstruction. dECM microparticles had good particle size distribution (≤10 µm) and retained the majority of corneal ECM components found in native tissue. Fibrin-dECM hydrogels exhibited compressive modulus of 70.83 ± 9.17 kPa matching that of native tissue, maximum burst pressure of 34.3 ± 3.7 kPa, and demonstrated a short crosslinking time of ~17 min. The fibrin-dECM hydrogels were found to be biodegradable, cytocompatible, non-mutagenic, non-sensitive, non-irritant, and supported the growth and maintained the phenotype of encapsulated human corneal stem cells (hCSCs) in vitro. In a rabbit model of anterior lamellar keratectomy, fibrin-dECM bio-adhesives promoted corneal re-epithelialization within 14 days, induced stromal tissue repair, and displayed integration with corneal tissues in vivo. Overall, our results suggest that the incorporation of cornea tissue-derived ECM microparticles in fibrin hydrogels is non-toxic, safe, and shows tremendous promise as a minimally invasive therapeutic approach for the treatment of superficial corneal epithelial wounds and anterior stromal injuries.

Fluorescence imaging of nitric oxide in living cells using o-phenylenediamine-rhodamine based polymeric nanosensors.

Direct real-time measurement of nitric oxide (NO) in living cells has proven quite challenging, owing in part to the lack of tools that are selective and sensitive to measure intracellular concentrations of NO. Herein we report the synthesis and characterization of polyvinyl alcohol (PVA) based nanosensors for fluorescence imaging of cytosolic NO using an o-phenylenediamine-rhodamine (OPD-RhB) platform. More specifically, thiol-functionalized PVA incorporating RhB conjugated OPD was disulfide crosslinked to yield NO-responsive nanosensors. The polymeric nanosensors were anionic, averaged 170 nm in hydrodynamic size, and exhibited linear increases in fluorescence intensity (FLI) to micro- and nanomolar concentrations of NO in a sodium nitroprusside (SNP) concentration-dependent manner. In the presence of SNP, the engineered nanosensors demonstrated physical stability at extracellular glutathione (GSH) conditions, while favoring NO detection at cytoplasmic GSH conditions. In addition, the PVA-based nanosensors were non-cytotoxic, cell membrane-permeable and demonstrated hydrogen peroxide-dependent FL increases upon incubation with activated synoviocytes in vitro. Most notably, NO-induced cell FLIs correlated strongly with total nitrite/nitrate content of conventional Griess assays with Pearson correlation coefficients of 0.96. Comprehensively, our results show that OPD-RhB-conjugated PVA nanosensors offer real-time imaging of NO with high sensitivity in living cells that can be employed for direct quantification of NO.

Recent Advances in Biomaterials Science and Engineering Research in India: A Minireview.

Biomedical research in health innovation and product development encompasses convergent technologies that primarily integrate biomaterials science and engineering at its core. Particularly, research in this area is instrumental for the implementation of biomedical devices (BMDs) that offer innovative solutions to help maintain and improve quality of life of patients worldwide. Despite achieving extraordinary success, implantable BMDs are still confronted with complex engineering and biological challenges that need to addressed for augmenting device performance and prolonging lifetime in vivo. Biofabrication of tissue constructs, designing novel biomaterials and employing rational biomaterial design approaches, surface engineering of implants, point of care diagnostics and micro/nano-based biosensors, smart drug delivery systems, and noninvasive imaging methodologies are among strategies exploited for improving clinical performance of implantable BMDs. In India, advances in biomedical technologies have dramatically advanced health care over the last few decades and the country is well-positioned to identify opportunities and translate emerging solutions. In this article, we attempt to capture the recent advances in biomedical research and development progressing across the country and highlight the significant research work accomplished in the areas of biomaterials science and engineering.

Bioreducible amino acid-derived polymeric nanoparticles for delivery of functional proteins.

We report the preparation of protein encapsulated amino acid derived redox-responsive nanoparticles (NPs) as effective nanocarriers for intracellular delivery of proteins. More specifically, acryloyl derivatives of lysine, ornithine, cystine and cystamine, were employed as monomers and disulfide crosslinkers for non-covalent encapsulation of model protein bovine serum albumin (BSA) and were interfacially crosslinked via free radical polymerization to form redox-responsive protein NPs. Notably, prepared NPs exhibited high protein loading content between 37 and 45%, averaged ∼400 nm in hydrodynamic size and possessed a mean surface charge of -15 mV. Furthermore, blank polymeric NPs displayed exceptional cytocompatibility with cell viability exceeding 92% at concentrations as high as 4 mg/mL, while redox-responsive protein NPs displayed glutathione (GSH)-dependent BSA release behavior in vitro. Additionally, cellular uptake studies confirmed that protein NPs entered MDA-MA-231 cells predominantly via the endocytic pathway. Upon cellular internalization, redox-responsive NPs delivered protein into cytosol of cells within 60 min demonstrating intrinsic endosomolytic characteristics and efficient protein release under cytoplasmic high GSH conditions. Most importantly, insulin analog-loaded NPs significantly increased glucose consumption in HepG2 cultures confirming protein stability and retention of protein function. Cumulatively, our approach presents a simple yet effective strategy for intracellular delivery of biologically active proteins for various biomedical applications.

Identification of Notch-1 expression in the limbal basal epithelium.

PURPOSE: To determine whether Notch-1, a ligand-activated transmembrane receptor known to maintain cells in an undifferentiated state, primarily progenitor cells in other systems, could be used as a stem cell marker in human limbal epithelium. METHODS: Human corneoscleral tissues obtained from the Doheny Eye & Tissue Transplant Bank were prepared for cross section and whole mount analysis. Tissue for whole mount was incubated in dispase; the epithelial sheet was removed and fixed in 4% paraformaldehyde. Sections and whole mount were stained with antibodies against Notch-1, Notch-2, beta-1 integrin, alpha-6, and the G2 subtype member of the ATP binding cassette transporter (ABCG2). Specificity of the Notch-1 antibody was determined by western blot analysis with Cos-7 cells transfected with Notch-1. Explant culture was performed and only primary cultures were used in this experiment. RESULTS: Notch-1 was found to be expressed in the limbal basal region where stem cells reside. Notch-1 antigenicity was more pronounced in cell clusters, mainly in the palisades of Vogt. The central cornea was almost devoid of Notch-1. The intensity of Notch-1 staining in cultured cells from the limbal explants was high in only a few cells. The Notch-1 signal was diminished in dividing cells. Expression in cultured cells was more cytoplasmic; few cells showed additional nuclear staining. The Notch-1-stained whole mount showed only a few cells in the limbal region. A 300 kDa and a 110 kDa band confirmed the specificity of the antibody in Cos-7 cells transfected with Notch-1. Double staining for ABCG2 and Notch-1 showed some ABCG2-positive cells co-expressing Notch-1 in the limbal basal epithelium, indicating that Notch-1-expressing cells might be a unique subpopulation of cells with stem cell properties. CONCLUSIONS: Immunofluorescence data shows that Notch-1 could be a possible marker for the stem cells in the limbal basal epithelium. Further studies and characterization of the Notch pathway in corneal development will provide valuable clues for the identification of stem cells.

Tissue-engineered tear secretory system: functional lacrimal gland acinar cells cultured on matrix protein-coated substrata.

Dry eye is a general term that refers to a myriad of ophthalmic disorders resulting in the inadequate wetting of the corneal surface by the tear film. Dry eyes are typically treated by the application of artificial tears. However, patients with lacrimal insufficiencies such as Stevens-Johnson syndrome, chemical and thermal injuries, or ocular cicatricial pemphigoid have very limited options because of the short duration and action of lubricating agents. As a therapeutic strategy, we are working to develop a bioengineered tear secretory system for such patients. This article describes the growth and physiological properties of purified rabbit lacrimal gland acinar cells (pLGACs) on several matrix protein-coated polymers such as silicone, collagen I, copolymers of poly-D,L-lactide-co-glycolide (PLGA; 85:15 and 50:50), poly-L-lactic acid (PLLA), and Thermanox plastic cell culture coverslips. Monolayers of acinar cells were established on all of the polymeric substrata. An assay of beta-hexosaminidase activity in the supernatant medium showed significant increases in protein secretion, following stimulation with 100 microM carbachol on matrix protein-coated and uncoated polymers such as silicone, PLGA 85:15, and PLLA. Our study demonstrates that PLLA supported the morphological and physiological properties of purified rabbit lacrimal gland epithelial cells more successfully than the others.

Design, preparation and characterization of pH-responsive prodrug micelles with hydrolyzable anhydride linkages for controlled drug delivery.

We report a new prodrug micelle-based approach in which a model hydrophobic non-steroidal anti-inflammatory drug (NSAID), ibuprofen (Ibu), is tethered to amphiphilic methoxy polyethylene glycol-polypropylene fumarate (mPEG-PPF) diblock copolymer via hydrolytic anhydride linkages for potential controlled release applications of NSAIDs. Synthesized mPEG-PPF-Ibu polymer drug conjugates (PDCs) demonstrated high drug conjugation efficiency (∼90%) and self-assembled to form micellar nanostructures in aqueous medium with critical micelle concentrations ranging between 16 and 30µg/mL. The entrapment efficiency of Ibu in prepared PDC micelles was as high as 18% (w/w). Crosslinking of prodrug micelles with N,N'-dimethylaminoethyl methacrylate conferred pH-responsive characteristics. pH-responsive PDC micelles averaged 100nm in size at pH 7.4 and exhibited concomitant changes in size upon incubation in physiologically relevant mildly acidic conditions. Ibu release was observed to increase with increasing acidic conditions and could be controlled by varying the amount of crosslinker used. Furthermore, the prepared mPEG-PPF-based micelles demonstrated excellent cytocompatibility and cellular internalization in vitro. More importantly, PDC micelles exerted anti-inflammatory effects by significantly decreasing monosodium urate crystal-induced prostaglandin E2 levels in rabbit synoviocyte cultures in vitro. Cumulatively, our results indicate that this new prodrug micelle approach is promising for NSAID-based therapies in the treatment of arthritis and cancer.

Current status of gene delivery and gene therapy in lacrimal gland using viral vectors.

Gene delivery is one of the biggest challenges in the field of gene therapy. It involves the efficient transfer of transgenes into somatic cells for therapeutic purposes. A few major drawbacks in gene delivery include inefficient gene transfer and lack of sustained transgene expression. However, the classical method of using viral vectors for gene transfer has circumvented some of these issues. Several kinds of viruses, including retrovirus, adenovirus, adeno-associated virus, and herpes simplex virus, have been manipulated for use in gene transfer and gene therapy applications. The transfer of genetic material into lacrimal epithelial cells and tissues, both in vitro and in vivo, has been critical for the study of tear secretory mechanisms and autoimmunity of the lacrimal gland. These studies will help in the development of therapeutic interventions for autoimmune disorders such as Sjögren's syndrome and dry eye syndromes which are associated with lacrimal dysfunction. These studies are also critical for future endeavors which utilize the lacrimal gland as a reservoir for the production of therapeutic factors which can be released in tears, providing treatment for diseases of the cornea and posterior segment. This review will discuss the developments related to gene delivery and gene therapy in the lacrimal gland using several viral vector systems.

Tissue engineering: current and future approaches to ocular surface reconstruction.

Although cells have been cultured outside the body for many years, research has only recently begun to develop complex three-dimensional tissue constructs that will, ideally, mature into fully functional tissues and organs. Tissue engineering is an emerging field in the area of biotechnology that combines the principles and methods of life sciences with those of engineering for the purpose of regenerating, repairing, or replacing diseased tissues. In this review, we describe the recent advances and current development of tissue engineering approaches as related to the ocular surface system, which comprises the three main integrated tissue units: conjunctiva, cornea and lacrimal glands.

Injectable in situ forming xylitol-PEG-based hydrogels for cell encapsulation and delivery.

Injectable in situ crosslinking hydrogels offer unique advantages over conventional prefabricated hydrogel methodologies. Herein, we synthesize poly(xylitol-co-maleate-co-PEG) (pXMP) macromers and evaluate their performance as injectable cell carriers for tissue engineering applications. The designed pXMP elastomers were non-toxic and water-soluble with viscosity values permissible for subcutaneous injectable systems. pXMP-based hydrogels prepared via free radical polymerization with acrylic acid as crosslinker possessed high crosslink density and exhibited a broad range of compressive moduli that could match the natural mechanical environment of various native tissues. The hydrogels displayed controlled degradability and exhibited gradual increase in matrix porosity upon degradation. The hydrophobic hydrogel surfaces preferentially adsorbed albumin and promoted cell adhesion and growth in vitro. Actin staining on cells cultured on thin hydrogel films revealed subconfluent cell monolayers composed of strong, adherent cells. Furthermore, fabricated 3D pXMP cell-hydrogel constructs promoted cell survival and proliferation in vitro. Cumulatively, our results demonstrate that injectable xylitol-PEG-based hydrogels possess excellent physical characteristics and exhibit exceptional cytocompatibility in vitro. Consequently, they show great promise as injectable hydrogel systems for in situ tissue repair and regeneration.

In vivo fluorescence imaging of biomaterial-associated inflammation and infection in a minimally invasive manner.

Implant-associated inflammation and bacterial infection severely limit the functional performance of medical devices and are a major cause of implant failure. Therefore, it is crucial to develop methodologies to monitor/image implant-associated aseptic inflammation and bacterial infection in a minimally invasive manner. Here, we exploited near-infrared fluorescence (NIRF) molecular probes injected locally at the implant site to perform minimally invasive, simultaneous imaging of inflammation, and infection associated with implanted polymer disks. The hydro-sulfo-Cy5 (H-s-Cy5) probe detected reactive oxygen species associated with inflammatory responses to both aseptic and biofilm-containing implants, whereas diaminocyanine sulfonate selectively detected nitric oxide associated with a biofilm on the biomaterial at acute time points (<4 days). This imaging modality also allows longitudinal monitoring because of high specificity and fast clearance rate of the fluorescent probes. Taken together, these NIRF molecular probes represent a useful tool to directly image inflammatory responses and infections associated with implanted devices for the diagnosis of device-associated inflammation and infection as well as the development of effective therapies.

PHACOS, a functionalized bacterial polyester with bactericidal activity against methicillin-resistant Staphylococcus aureus.

Biomaterial-associated infections represent a significant clinical problem, and treatment of these microbial infections is becoming troublesome due to the increasing number of antibiotic-resistant strains. Here, we report a naturally functionalized bacterial polyhydroxyalkanoate (PHACOS) with antibacterial properties. We demonstrate that PHACOS selectively and efficiently inhibits the growth of methicillin-resistant Staphylococcus aureus (MRSA) both in vitro and in vivo. This ability has been ascribed to the functionalized side chains containing thioester groups. Significantly less (3.2-fold) biofilm formation of S. aureus was detected on PHACOS compared to biofilms formed on control poly(3-hydroxyoctanoate-co-hydroxyhexanoate) and poly(ethylene terephthalate), but no differences were observed in bacterial adhesion among these polymers. PHACOS elicited minimal cytotoxic and inflammatory effects on murine macrophages and supported normal fibroblast adhesion. In vivo fluorescence imaging demonstrated minimal inflammation and excellent antibacterial activity for PHACOS compared to controls in an in vivo model of implant-associated infection. Additionally, reductions in neutrophils and macrophages in the vicinity of sterile PHACOS compared to sterile PHO implant were observed by immunohistochemistry. Moreover, a similar percentage of inflammatory cells was found in the tissue surrounding sterile PHACOS and S. aureus pre-colonized PHACOS implants, and these levels were significantly lower than S. aureus pre-colonized control polymers. These findings support a contact active surface mode of antibacterial action for PHACOS and establish this functionalized polyhydroxyalkanoate as an infection-resistant biomaterial.

Diverse mediators modulate the chloride ion fluxes that drive lacrimal fluid production.

PURPOSE: To learn whether locally expressed and systemic mediators might modulate the cholinergically induced transepithelial Cl(-) fluxes that underlie lacrimal fluid production. METHODS: Reconstituted epithelial monolayers were exposed to a submaximal dose of the muscarinic agonist, carbachol (CCh), or to one of several paracrine mediators for 18 hours, then acutely stimulated with an optimal dose of CCh. Secretory Cl(-) fluxes were assessed as negative short-circuit currents (ISC). RESULTS: Exposure to IL-6 at concentrations of 1 and 10 ng/mL and IL-1ß at 10 ng/mL significantly decreased CCh-induced Cl(-) secretion. Prolactin decreased CCh-induced Cl(-) secretion, but the extent of the decrease diminished as the prolactin concentration increased from 20 to 200 ng/mL. CCh, 10 µM, prevented CCh, 100 µM, from eliciting Cl(-) secretion. Exposure to histamine, 10 mM, prevented formation of confluent monolayers. Exposure to histamine, 1 mM, decreased CCh-induced Cl(-) secretion, whereas exposure to 5-HT, 1 mM, potentiated CCh-induced Cl(-) secretion. CONCLUSIONS: Chronic exposure to inflammatory cytokines may significantly impair cholinergically induced lacrimal fluid production. Concentrations of prolactin within the high range of normal values also may impair fluid production, but this effect is reversed at levels associated with pregnancy. Autonomic neurotransmitters and paracrine mediators that signal through different G protein-coupled receptors appear to exert varying influences, which range from complete suppression to potentiation of cholinergically induced fluid production. Thus, some hormones and paracrine mediators may impair secretion in apparently homeostatic glands as well as diseased glands, whereas mediators produced by certain immune cell infiltrates may actually enhance fluid formation.

Minimally invasive, longitudinal monitoring of biomaterial-associated inflammation by fluorescence imaging.

Implant-associated inflammation is a major cause for the reduced performance/lifetime and failure of numerous medical devices. Therefore, the ability to non-invasively and quantitatively monitor implant-associated inflammation is critically important. Here we show that implant-associated inflammation can be imaged via fluorescence imaging using near-infrared hydrocyanine dyes delivered either locally or intravenously in living mice. This imaging strategy allowed quantitative longitudinal monitoring of inflammation by detecting reactive oxygen species (ROS) released by inflammatory cells in response to implanted poly(ethylene terephthalate) (PET) disks or injected poly (lactic-co-glycolic acid) (PLGA) microparticles, and exhibited a strong correlation to conventional analysis of inflammation. Furthermore, modulation of inflammatory responses via controlled release of the anti-inflammatory agent dexamethasone was detected using this sensitive imaging approach. Thus, hydrocyanine-based fluorescence imaging of ROS could serve as a surrogate measure for monitoring implant-associated inflammation as well as evaluating the efficacy of therapeutic approaches to modulate host responses to implanted medical devices.

Distinct dacryoadenitides autoadoptively transferred to rabbits by different subpopulations of lymphocytes activated ex vivo.

PURPOSE: To test whether CD4+ T cells proliferate in mixed cell reactions with autologous lacrimal gland (LG) acinar cells and whether these cells can autoadoptively transfer disease. METHODS: Purified acinar cells were gamma irradiated and cocultured with peripheral blood lymphocytes. Activated CD4+ T cells were sorted by fluorescence-activated cell sorting (FACS). Unfractionated activated peripheral blood lymphocytes (UF), CD4+-enriched and CD4+-depleted T cells from an autologous mixed cell reaction were injected into the donor rabbit's remaining LG. After 4 weeks, ocular examinations were performed, and the rabbits were euthanized; LGs were removed for histopathology, immunohistochemistry, and real-time reverse transcription-polymerase chain reaction studies. RESULTS: CD4 T cells increased in the autologous mixed cell reaction from 20% to 80%. Tear production decreased in the induced disease/UF (ID/UF) group and declined even more in the ID/CD4+-enriched group. Tear breakup times decreased and rose bengal staining increased in all groups. All LGs exhibited significant histopathology and increased messenger RNAs for tumor necrosis factor α. The ID/UF group exhibited the largest increases of CD4+ and rabbit T-lymphocyte antigen-positive cells. The ID/CD4+-enriched group contained fewer infiltrating CD4 cells but more eosinophils, severely altered acinar morphology, and increased fibrosis. LG of the ID/CD4+-depleted group exhibited large increases of CD18, major histocompatibility complex II, and CD4+ cells. Messenger RNAs for interleukin 2, interleukin 4, and CD4+ increased in the ID/CD4+-enriched group compared with the CD4+-depleted group. CONCLUSIONS: Autoreactive CD4+ effector cells activated ex vivo and autoadoptively transferred, caused what seems to be a distinct dacryoadenitis. The CD4+-depleted cell fraction also contained pathogenic effector cells capable of inducing disease.

Adeno-associated virus-mediated IL-10 gene transfer suppresses lacrimal gland immunopathology in a rabbit model of autoimmune dacryoadenitis.

PURPOSE: To evaluate the effect of adeno-associated virus (AAV) vector-mediated viral (v)IL-10 gene expression on lacrimal gland (LG) immunopathology and ocular surface disease in a rabbit model of induced autoimmune dacryoadenitis (ID). METHODS: Autologous peripheral blood lymphocytes, activated in a mixed-cell reaction when cocultured with purified rabbit lacrimal epithelial cells, induce a Sjögren's-like autoimmune dacryoadenitis when injected directly back into the donor animal's inferior LG. Four weeks after disease induction, AAV vector expressing the vIL-10 gene under control of a tetracycline-inducible promoter was injected into the inferior LG of the treatment group (ID/Rx), and doxycycline was fed orally to induce transgene expression. The ID group serving as control also received doxycycline. All LGs were removed 16 weeks after disease induction. RESULTS: Clinical symptoms showed overall improvement in the ID/Rx group compared with the ID group. Histopathologic examination of the ID group's LG revealed scattered large lymphocytic foci and areas of altered or distorted acini, whereas the ID/Rx group had scattered small lymphocytic foci. The number of CD18(+) cells was almost fivefold lower in the ID/Rx group than in the ID group. Although the total number of RTLA(+) cells did not differ between the groups, the CD4/CD8 ratio was 16-fold smaller in the ID/Rx group. CONCLUSIONS: Animals with experimentally induced autoimmune dacryoadenitis appeared to benefit from AAV-mediated vIL-10 gene transfer therapy. Quantitative immunohistochemical analysis suggested that the therapy might not have been simply immunosuppressive but rather supported the induction of CD8(+) regulatory cells.