Th17-Derived Cytokines Synergistically Enhance IL-17C Production by the Colonic Epithelium.

Tightly regulated communication between the gastrointestinal epithelium and immune cells in the underlying lamina propria is critical for immune homeostasis and inflammation. IL-17C, produced by epithelial cells after exposure to inflammatory stimuli, facilitates cell-to-cell communication by promoting inflammatory responses in Th17 cells. In this study, we demonstrate that Th17-derived cytokines TNF-α, IL-17A, and IL-22 synergistically enhance IL-17C expression in both human-transformed colonic epithelial cell lines and primary non-inflammatory bowel disease colonic epithelial spheroids. This synergistic expression requires activation of the transcription factor NF-κB downstream of the TNF-α stimulus, evidenced by the reduction of IL-17C expression in the presence of an IκBα inhibitor. IL-17A and IL-22 enhance IL-17C expression through the activation of the transcription factor AP-1 in a p38 MAPK-dependent manner. Colonic spheroids derived from uninvolved epithelial of ulcerative colitis patients stimulated with TNF-α, IL-17A, and IL-22 show muted responses compared with non-inflammatory bowel disease spheroids, and inflamed spheroids yielded more IL-17C expression in the presence of TNF-α, and no response to IL-22 stimulation. Altogether, a role for IL-17C in activating Th17 cells combined with our findings of Th17-derived cytokine-driven synergy in the expression of IL-17C identifies a novel inflammatory amplification loop in the gastrointestinal tract between epithelial cells and Th17 cells.

Nanomedicine platform for targeting activated neutrophils and neutrophil-platelet complexes using an α1-antitrypsin-derived peptide motif.

Targeted drug delivery to disease-associated activated neutrophils can provide novel therapeutic opportunities while avoiding systemic effects on immune functions. We created a nanomedicine platform that uniquely utilizes an α1-antitrypsin-derived peptide to confer binding specificity to neutrophil elastase on activated neutrophils. Surface decoration with this peptide enabled specific anchorage of nanoparticles to activated neutrophils and platelet-neutrophil aggregates, in vitro and in vivo. Nanoparticle delivery of a model drug, hydroxychloroquine, demonstrated significant reduction of neutrophil activities in vitro and a therapeutic effect on murine venous thrombosis in vivo. This innovative approach of cell-specific and activation-state-specific targeting can be applied to several neutrophil-driven pathologies.

Injectable cellulose-based hydrogels as nucleus pulposus replacements: Assessment of in vitro structural stability, ex vivo herniation risk, and in vivo biocompatibility.

Current treatments for intervertebral disc degeneration and herniation are palliative only and cannot restore disc structure and function. Nucleus pulposus (NP) replacements are a promising strategy for restoring disc biomechanics and height loss. Cellulose-based hydrogel systems offer potential for NP replacement since they are stable, non-toxic, may be tuned to match NP material properties, and are conducive to cell or drug delivery. A crosslinked, carboxymethylcellulose-methylcellulose dual-polymer hydrogel was recently formulated as an injectable NP replacement that gelled in situ and restored disc height and compressive biomechanical properties. The objective of this study was to investigate the translational potential of this hydrogel system by examining the long-term structural stability in vitro, the herniation risk and fatigue bending endurance in a bovine motion segment model, and the in vivo biocompatibility in a rat subcutaneous pouch model. Results showed that the hydrogels maintained their structural integrity over a 12-week period. AF injury significantly increased herniation risk and reduced fatigue bending endurance in bovine motion segments. Samples repaired with cellulosic hydrogels demonstrated restored height and exhibited herniation risk and fatigue endurance comparable to samples that underwent the current standard treatment of nucleotomy. Lastly, injected hydrogels elicited a minimal foreign body response as determined by analysis of fibrous capsule development and macrophage presence over 12 weeks. Overall, this injectable cellulosic hydrogel system is a promising candidate as an NP substitute. Further assessment and optimization of this cellulosic hydrogel system in an in vivo intradiscal injury model may lead to an improved clinical solution for disc degeneration and herniation.

Fibrin-Genipin Hydrogel for Cartilage Tissue Engineering in Nasal Reconstruction.

OBJECTIVES: Nasal reconstruction is limited by the availability of autologous cartilage. The aim is to investigate an adhesive biomaterial for tissue engineering of nasal cartilage by evaluating mechanical properties of hydrogels made of fibrin crosslinked with genipin as compared to native tissue. METHODS: Hydrogels of fibrin, fibrin-genipin, and fibrin-genipin with extracellular matrix (ECM) particles were created and evaluated with mechanical testing to determine compression, tensile, and shear properties. Rabbit nasal septal cartilage was harvested and tested in these modalities for comparison. Transmission electron microscopy characterized hydrogel structure. RESULTS: Fibrin-genipin gels had higher compressive, tensile, and shear moduli compared to fibrin alone or fibrin-genipin with ECM. However, all hydrogel formulations had lower moduli than the rabbit nasal septal cartilage. Electron microscopy showed genipin crosslinking increased structural density of the hydrogel and that cartilage ECM created larger structural features with lower crosslinking density. CONCLUSION: The addition of genipin significantly improved mechanical properties of fibrin hydrogels by increasing the compressive, tensile, and shear moduli. The addition of cartilage ECM, which can add native structure and composition, resulted in decreased moduli values. Fibrin-genipin is a bioactive and biomechanically stable hydrogel that may offer promise as a scaffold for cartilage tissue engineering in nasal reconstruction, yet further augmentation is required to match material properties of native nasal cartilage.

Cell-Seeded Adhesive Biomaterial for Repair of Annulus Fibrosus Defects in Intervertebral Discs.

Defects in the annulus fibrosus (AF) of intervertebral discs allow nucleus pulposus tissue to herniate causing painful disability. Microdiscectomy procedures remove herniated tissue fragments, but unrepaired defects remain allowing reherniation or progressive degeneration. Cell therapies show promise to enhance repair, but methods are undeveloped and carriers are required to prevent cell leakage. To address this challenge, this study developed and evaluated genipin-crosslinked fibrin (FibGen) as an adhesive cell carrier optimized for AF repair that can deliver cells, match AF material properties, and have low risk of extrusion during loading. Part 1 determined that feasibility of bovine AF cells encapsulated in high concentration FibGen (F140G6: 140 mg/mL fibrinogen; 6 mg/mL genipin) for 7 weeks could maintain high viability, but had little proliferation or matrix deposition. Part 2 screened tissue mechanics and in situ failure testing of nine FibGen formulations (fibrin: 35-140 mg/mL; genipin: 1-6 mg/mL). F140G6 formulation matched AF shear and compressive properties and significantly improved failure strength in situ. Formulations with reduced genipin also exhibited satisfactory material properties and failure behaviors warranting further biological screening. Part 3 screened AF cells encapsulated in four FibGen formulations for 1 week and found that reduced genipin concentrations increased cell viability and glycosaminoglycan production. F70G1 (70 mg/mL fibrinogen; 1 mg/mL genipin) demonstrated balanced biological and biomechanical performance warranting further testing. We conclude that FibGen has potential to serve as an adhesive cell carrier to repair AF defects with formulations that can be tuned to enhance biomechanical and biological performance; future studies are required to develop strategies to enhance matrix production.

Structural and Chemical Modification to Improve Adhesive and Material Properties of Fibrin-Genipin for Repair of Annulus Fibrosus Defects in Intervertebral Disks.

Annulus fibrosus (AF) defects from intervertebral disk (IVD) herniation and degeneration are commonly associated with back pain. Genipin-crosslinked fibrin hydrogel (FibGen) is an injectable, space-filling AF sealant that was optimized to match AF shear properties and partially restored IVD biomechanics. This study aimed to enhance mechanical behaviors of FibGen to more closely match AF compressive, tensile, and shear properties by adjusting genipin crosslink density and by creating a composite formulation by adding Poly(D,L-lactide-co-glycolide) (PDLGA). This study also evaluated effects of thrombin concentration and injection technique on gelation kinetics and adhesive strength. Increasing FibGen genipin concentration from 1 to 36 mg/mL significantly increased adhesive strength (∼5 to 35 kPa), shear moduli (∼10 to 110 kPa), and compressive moduli (∼25 to 150 kPa) with concentration-dependent effects, and spanning native AF properties. Adding PDLGA to FibGen altered the material microstructure on electron microscopy and nearly tripled adhesive strength, but did not increase tensile moduli, which remained nearly 5× below native AF, and had a small increase in shear moduli and significantly decreased compressive moduli. Increased thrombin concentration decreased gelation rate to < 5 min and injection methods providing a structural FibGen cap increased pushout strength by ∼40%. We conclude that FibGen is highly modifiable with tunable mechanical properties that can be formulated to be compatible with human AF compressive and shear properties and gelation kinetics and injection techniques compatible with clinical discectomy procedures. However, further innovations, perhaps with more efficient fiber reinforcement, will be required to enable FibGen to match AF tensile properties.

Rat mesenchymal stem cell secretome promotes elastogenesis and facilitates recovery from simulated childbirth injury.

Vaginal delivery is a risk factor for stress urinary incontinence (SUI). Mesenchymal stem cells (MSCs) home to injured organs and can facilitate repair. The goal of this study was to determine if MSCs home to pelvic organs after simulated childbirth injury and facilitate recovery from SUI via paracrine factors. Three experiments were performed. Eighteen female rats received vaginal distension (VD) or sham VD and labeled intravenous (IV) MSCs to investigate if MSCs home to the pelvic organs. Whole-organ imaging and immunofluorescence were performed 1 week later. Thirty-four female rats received VD and IV MSCs, VD and IV saline, or sham VD and IV saline to investigate if MSCs accelerate recovery of continence. Twenty-nine female rats received VD and periurethral concentrated conditioned media (CCM), VD and periurethral control media, or sham VD and periurethral control media to investigate if factors secreted by MSCs accelerate recovery from VD. Urethral histology and function were assessed 1 week later. Significantly more MSCs were observed in the urethra, vagina, and spleen after VD compared to sham VD. Continence as measured by leak point pressure (LPP) was significantly reduced after VD in rats treated with saline or control media compared to sham VD but not in those given MSCs or CCM. External urethral sphincter (EUS) function as measured by electromyography (EMG) was not improved with MSCs or CCM. Rats treated with MSCs or CCM demonstrated an increase in elastin fibers near the EUS and urethral smooth muscle more similar to that of sham-injured animals than rats treated with saline or control media. MSCs homed to the urethra and vagina and facilitated recovery of continence most likely via secretion of paracrine factors. Both MSCs and CCM have promise as novel noninvasive therapies for SUI.

Electrical stimulation of anal sphincter or pudendal nerve improves anal sphincter pressure.

OBJECTIVE: Stimulation of the pudendal nerve or the anal sphincter could provide therapeutic options for fecal incontinence with little involvement of other organs. The goal of this project was to assess the effects of pudendal nerve and anal sphincter stimulation on bladder and anal pressures. DESIGN: Ten virgin female Sprague Dawley rats were randomly allocated to control (n = 2), perianal stimulation (n = 4), and pudendal nerve stimulation (n = 4) groups. A monopolar electrode was hooked to the pudendal nerve or placed on the anal sphincter. Aballoon catheter was inserted into the anus to measure anal pressure, and a catheter was inserted into the bladder via the urethra to measure bladder pressure. Bladder and anal pressures were measured with different electrical stimulation parameters and different timing of electrical stimulation relative to spontaneous anal sphincter contractions. RESULTS: Increasing stimulation current had the most dramatic effect on both anal and bladder pressures. An immediate increase in anal pressure was observed when stimulating either the anal sphincter or the pudendal nerve at stimulation values of 1 mA or 2 mA. No increase in anal pressure was observed for lower current values. Bladder pressure increased at high current during anal sphincter stimulation, but not as much as during pudendal nerve stimulation. Increased bladder pressure during anal sphincter stimulation was due to contraction of the abdominal muscles. CONCLUSION: Electrical stimulation caused an increase in anal pressures with bladder involvement only at high current. These initial results suggest that electrical stimulation can increase anal sphincter pressure, enhancing continence control.

Stem cell therapy for incontinence: where are we now? What is the realistic potential?

A significant number of women experience stress urinary incontinence (SUI), which greatly affects their quality of life. Recent research investigating utilization of stem cells and their derivatives for the prevention and treatment of SUI has been performed to test the effect of cell source and method of administration in several animal models of SUI. The type of stem cell, timing of optimal dose or doses after injury, mechanism of action of stem cells, and route of administration must be investigated both preclinically and clinically before stem cell therapy becomes a possible treatment for SUI, although the future of this therapy looks promising. This article reviews the progress in stem cell research for incontinence and describes areas of future work as suggested by research in other fields.

High-risk sexual and drug using behaviors among male injection drug users who have sex with men in 2 Mexico-US border cities.

OBJECTIVES: The population of Latino men who have sex with men (MSM) and who are also injection drug users (IDUs) is understudied. We explored risk behaviors of MSM/IDUs compared with other male IDUs in 2 Mexican border cities. STUDY DESIGN: In 2005, IDUs who had injected within the previous 30 days were recruited using respondent-driven sampling (RDS) in Tijuana and Ciudad Juárez. They underwent antibody testing for HIV, HCV, and syphilis and interviewer-administered surveys. Men were categorized as MSM if they reported > or =1 lifetime male partners. Logistic regression was used to compare MSM/IDUs with non-MSM/IDUs. RESULTS: A third (31%) of 377 male IDUs were categorized as MSM (47% in Tijuana and 13% in Ciudad Juárez, P <0.01). Combined RDS-adjusted prevalence of HIV and Hepatitis C was 3% (95% CI: 1, 5) and 96%, (95% CI: 94, 99) respectively, while 17% (95% CI: 2, 36) of MSM and 8% (95% CI: 3, 12) of non-MSM tested positive for syphilis antibody. In multivariate logistic regression adjusted for site, MSM/IDUs were more likely than non-MSM/IDUs to have ever used inhalants (OR: 3.4; 95% CI: 1.8, 6.2) or oral tranquilizers (OR: 2.4; 95% CI: 1.3, 4.6), received treatment for a drug problem (OR:1.9; 95% CI: 1.1, 3.2) shared needles in the last six months (OR: 2.1; 95% CI: 1.0, 4.2) and also had higher numbers of lifetime female partners (log-transformed continuous variable, OR: 1.6; 95% CI: 1.2, 2.1). CONCLUSIONS: In these Mexican cities, the proportion of MSM among male IDUs was high. Compared with other male IDUs, MSM/IDUs were more likely to engage in behaviors placing them at risk of acquiring HIV/STIs. Culturally appropriate interventions targeting Latino MSM/IDUs are warranted.

Respondent-driven sampling of injection drug users in two U.S.-Mexico border cities: recruitment dynamics and impact on estimates of HIV and syphilis prevalence.

Respondent-driven sampling (RDS), a chain referral sampling approach, is increasingly used to recruit participants from hard-to-reach populations, such as injection drug users (IDUs). Using RDS, we recruited IDUs in Tijuana and Ciudad (Cd.) Juárez, two Mexican cities bordering San Diego, CA and El Paso, TX, respectively, and compared recruitment dynamics, reported network size, and estimates of HIV and syphilis prevalence. Between February and April 2005, we used RDS to recruit IDUs in Tijuana (15 seeds, 207 recruits) and Cd. Juárez (9 seeds, 197 recruits), Mexico for a cross-sectional study of behavioral and contextual factors associated with HIV, HCV and syphilis infections. All subjects provided informed consent, an anonymous interview, and a venous blood sample for serologic testing of HIV, HCV, HBV (Cd. Juárez only) and syphilis antibody. Log-linear models were used to analyze the association between the state of the recruiter and that of the recruitee in the referral chains, and population estimates of the presence of syphilis antibody were obtained, correcting for biased sampling using RDS-based estimators. Sampling of the targeted 200 recruits per city was achieved rapidly (2 months in Tijuana, 2 weeks in Cd. Juárez). After excluding seeds and missing data, the sample prevalence of HCV, HIV and syphilis were 96.6, 1.9 and 13.5% respectively in Tijuana, and 95.3, 4.1, and 2.7% respectively in Cd. Juárez (where HBV prevalence was 84.7%). Syphilis cases were clustered in recruitment trees. RDS-corrected estimates of syphilis antibody prevalence ranged from 12.8 to 26.8% in Tijuana and from 2.9 to 15.6% in Ciudad Juárez, depending on how recruitment patterns were modeled, and assumptions about how network size affected an individual's probability of being included in the sample. RDS was an effective method to rapidly recruit IDUs in these cities. Although the frequency of HIV was low, syphilis prevalence was high, particularly in Tijuana. RDS-corrected estimates of syphilis prevalence were sensitive to model assumptions, suggesting that further validation of RDS is necessary.