Mesenchymal stem cell based therapies for uveitis: a systematic review of preclinical studies.

Cell therapy has shown promising results for treating uveitis in preclinical studies. As the field continues to grow towards clinical translation, it is important to review and critically appraise existing studies. Herein, we analysed and critically appraised all preclinical studies using cell therapy or cell derived extracellular vesicles (EVs) for uveitis, and provided insight into mechanisms regulating ocular inflammation. We used PubMed, Medline, and Embase to search for preclinical studies examining stem cell therapy (e.g., mesenchymal stem cells [MSC]) and secreted EVs. All included studies were assessed for quality using the SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) checklist. Sixteen preclinical studies from 2011 to 2022 were analysed and included in this review of which 75% (n = 12) focused only on cell therapy, 18.7% (n = 3) studies focused on EVs, and 6.3% (n = 1) study focused on both cells and EVs. MSCs were the most common type of cells used in preclinical studies (n = 15) and EVs were commonly isolated from MSCs (n = 3). Overall, both MSCs and EVs showed improvements in ocular inflammation (seen on fundoscopy/slit lamp and histology) and electroretinogram outcomes. Overall, MSC and MSC-derived EVs shown great potential as therapeutic agents for treating uveitis. Unfortunately, small sample size, risk of selection/performance bias, and lack of standardized cell harvesting or delivery protocols are some factors which limits clinical translation. Large scaled, randomized preclinical studies are required to understand the full potential of MSCs for treating uveitis.

Prevention of atrial fibrillation after open-chest surgery with extracellular vesicle therapy.

Almost half of patients recovering from open-chest surgery experience atrial fibrillation (AF) that results principally from inflammation in the pericardial space surrounding the heart. Given that postoperative AF is associated with increased mortality, effective measures to prevent AF after open-chest surgery are highly desirable. In this study, we tested the concept that extracellular vesicles (EVs) isolated from human atrial explant-derived cells can prevent postoperative AF. Middle-aged female and male rats were randomized to undergo sham operation or induction of sterile pericarditis followed by trans-epicardial injection of human EVs or vehicle into the atrial tissue. Pericarditis increased the probability of inducing AF while EV treatment abrogated this effect in a sex-independent manner. EV treatment reduced infiltration of inflammatory cells and production of pro-inflammatory cytokines. Atrial fibrosis and hypertrophy seen after pericarditis were markedly attenuated by EV pretreatment, an effect attributable to suppression of fibroblast proliferation by EVs. Our study demonstrates that injection of EVs at the time of open-chest surgery shows prominent antiinflammatory effects and prevents AF due to sterile pericarditis. Translation of this finding to patients might provide an effective new strategy to prevent postoperative AF by reducing atrial inflammation and fibrosis.

Primary meningococcal conjunctivitis in an adult patient.

Primary meningococcal conjunctivitis from Neisseria meningitidis is a rare cause for acute, purulent conjunctivitis most commonly presenting in children. Here we present a case of primary meningococcal conjunctivitis in an adult patient with mild signs/symptoms mimicking non-gonococcal bacterial conjunctivitis. The patient was immediately treated with topical and systemic antibiotics. Here we highlight that an early diagnosis of a mild case can be missed thus, clinicians need to keep a high index of suspension as prompt recognition is important to initiate appropriate systemic antimicrobial therapy to prevent systemic disease.

Sequential central retinal artery occlusions associated with cryoglobulinemia.

BACKGROUND: Cryoglobulinemia, the presence of serum cryoglobulins which are immunoglobulins or complement components that precipitate at temperatures below 37 °C, commonly present with cutaneous manifestations initially, but are more rarely associated with ocular manifestations. To our knowledge, we report the first case of a patient presenting with sequential central retinal artery occlusion (CRAO) associated with cryoglobulinemia. CASE PRESENTATION: A 69-year-old female with a history of indolent B-cell lymphoma associated cryoglobulinemia, treated hepatitis B infection and CRAO in the left eye presented with acute vision loss and diffuse retinal whitening with a cherry red spot in her right eye, suggestive of sequential CRAO. Laboratory studies revealed a cryocrit of 55% (normal < 1%), elevated titres of cryoglobulin IgG at 1.98 g/L and cryoglobulin IgM at 3.78 g/L (normal < 0.3 g/L)9, and elevated kappa free light chain at 283.5 mg/L (normal < 0.06 g/L). Such elevated tires of cryoglobulins in the context of the patient's CRAO raised suspicion of cryoglobulinemia associated CRAO. The patient was promptly referred to rheumatology and oncology and was admitted for treatment including intravenous methylprednisone, rituximab and bendamustine chemotherapy. CONCLUSIONS: We report a patient with a complex medical history presenting with significant vision loss due to a sequential CRAO likely associated with cryoglobulinemia. Although a direct relationship between cryoglobulinemia and CRAO cannot be confirmed in this case, it highlights the importance of considering cryoglobulinemia in high-risk patients with prior history of hematological malignancy or chronic hepatitis infection.

Pathophysiology of central serous chorioretinopathy: a literature review with quality assessment.

The pathogenesis of central serous chorioretinopathy (CSCR), a pachychoroid disease, is poorly understood. While choroid hyperpermeability and retinal pigment epithelium dysfunction are cornerstones for developing CSCR, the mechanisms at the retinal, vascular, retinal pigment epithelium, and cellular level continue to be an enigma. A few preclinical studies and the development of small-sized, poorly controlled clinical trials have resulted in limited insight into the disease mechanism. Effective treatments for CSCR are still lacking as current trials have produced inconsistent results for functional and structural gains. Thus, critically evaluating the literature to explore disease mechanisms and provide an up-to-date understanding of pathophysiology can provide valuable information and avenues to new treatments. In this study, a comprehensive summary of the mechanistic insight into CSCR is presented while highlighting the shortcomings of current literature. The mechanism was divided into seven sub-categories including mechanical obstruction, inflammation, oxidative stress, paracrine factors, autonomic dysfunction, mineralocorticoid receptors activation, and medications. We implemented validated tools like the JBI and CAMARADES to objectively analyze the quality of both clinical and preclinical studies, respectively. Overall, our analysis of the literature showed that no single mechanism was populated with a large number of sufficiently sized and good-quality studies. However, compiling these studies gave hints not only to CSCR pathogenesis but also pachychoroid disease in general while providing suggestions for future exploration.

A novel 5-ring multifocal electroretinography stimulus for detecting hydroxychloroquine retinal toxicity.

PURPOSE: Multifocal electroretinogram (mfERG) shows great utility as a screening tool to detect early hydroxychloroquine (HCQ) retinopathy, but its widespread use is limited by the lack of accessibility and long test duration. In this study, we evaluated a novel concentric 5-ring mfERG stimulus to provide a simplified and rapid protocol for screening HCQ toxicity. METHODS: Patients referred for HCQ retinopathy screening were consented to this observational cross-sectional study. Patients with amblyopia, high refractive error (more than 8 diopters), other retinal diseases precluding appropriate evaluation or history of retinal surgery were excluded. The data were collected from patients undergoing HCQ screening at a single center from July 2019 to March 2020. Patients were tested with the new concentric 5-ring mfERG stimulus, standard 61-hexagon mfERG stimulus, spectral domain optical coherence tomography and automated 10-2 visual fields. For the main outcome, the 5-ring mfERG was compared to 61-hexagon stimulus to determine the time-to-test completion and assess the association between ring (R1-R5) amplitude and ring ratio compared against cumulative dose, dose by real body weight and duration of therapy using Pearson correlation. RESULTS: In total, 52 patients (104 eyes; 5 males and 47 females) were recruited with a mean age of 59 years (range 23-85 years). The 5-ring protocol was markedly quicker to perform (1.3 ± 0.2 min; mean (SD)) compared to the 61-hexagon protocol (5.2 ± 0.6 min), p < 0.0001; n = 10 patients. The new R2/R5 ring ratio showed a moderate correlation with daily dose (r = - 0.640), cumulative dose (r = - 0.581) and duration of therapy (r = - 0.417). Similar correlations were observed with the new R2/R4 ring ratio which were not significantly different from the new R2/R5 correlation coefficients. The new R2/R5 ring ratio demonstrated a stronger correlation with daily (p = 0.002) and cumulative dose (p = 0.0001) compared to the 61-hexagon stimulus. CONCLUSIONS: In this exploratory study, our novel 5-ring mfERG protocol significantly shortened data acquisition time while providing comparable results to the standard 61-hexagon stimulus for detecting HCQ-induced electrophysiological changes that are correlated with HCQ dosages and treatment duration. Our protocol has the potential to be more clinically practical by simplifying routine screening.

Electrophysiological engineering of heart-derived cells with calcium-dependent potassium channels improves cell therapy efficacy for cardioprotection.

We have shown that calcium-activated potassium (KCa)-channels regulate fundamental progenitor-cell functions, including proliferation, but their contribution to cell-therapy effectiveness is unknown. Here, we test the participation of KCa-channels in human heart explant-derived cell (EDC) physiology and therapeutic potential. TRAM34-sensitive KCa3.1-channels, encoded by the KCNN4 gene, are exclusively expressed in therapeutically bioactive EDC subfractions and maintain a strongly polarized resting potential; whereas therapeutically inert EDCs lack KCa3.1 channels and exhibit depolarized resting potentials. Somatic gene transfer of KCNN4 results in membrane hyperpolarization and increases intracellular [Ca2+], which boosts cell-proliferation and the production of pro-healing cytokines/nanoparticles. Intramyocardial injection of EDCs after KCNN4-gene overexpression markedly increases the salutary effects of EDCs on cardiac function, viable myocardium and peri-infarct neovascularization in a well-established murine model of ischemic cardiomyopathy. Thus, electrophysiological engineering provides a potentially valuable strategy to improve the therapeutic value of progenitor cells for cardioprotection and possibly other indications.

Deterministic paracrine repair of injured myocardium using microfluidic-based cocooning of heart explant-derived cells.

While encapsulation of cells within protective nanoporous gel cocoons increases cell retention and pro-survival integrin signaling, the influence of cocoon size and intra-capsular cell-cell interactions on therapeutic repair are unknown. Here, we employ a microfluidic platform to dissect the impact of cocoon size and intracapsular cell number on the regenerative potential of transplanted heart explant-derived cells. Deterministic increases in cocoon size boosted the proportion of multicellular aggregates within cocoons, reduced vascular clearance of transplanted cells and enhanced stimulation of endogenous repair. The latter being attributable to cell-cell stimulation of cytokine and extracellular vesicle production while also broadening of the miRNA cargo within extracellular vesicles. Thus, by tuning cocoon size and cell occupancy, the paracrine signature and retention of transplanted cells can be enhanced to promote paracrine stimulation of endogenous tissue repair.

Physiologic expansion of human heart-derived cells enhances therapeutic repair of injured myocardium.

BACKGROUND: Serum-free xenogen-free defined media and continuous controlled physiological cell culture conditions have been developed for stem cell therapeutics, but the effect of these conditions on the relative potency of the cell product is unknown. As such, we conducted a head-to-head comparison of cell culture conditions on human heart explant-derived cells using established in vitro measures of cell potency and in vivo functional repair. METHODS: Heart explant-derived cells cultured from human atrial or ventricular biopsies within a serum-free xenogen-free media and a continuous physiological culture environment were compared to cells cultured under traditional (high serum) cell culture conditions in a standard clean room facility. RESULTS: Transitioning from traditional high serum cell culture conditions to serum-free xenogen-free conditions had no effect on cell culture yields but provided a smaller, more homogenous, cell product with only minor antigenic changes. Culture within continuous physiologic conditions markedly boosted cell proliferation while increasing the expression of stem cell-related antigens and ability of cells to stimulate angiogenesis. Intramyocardial injection of physiologic cultured cells into immunodeficient mice 1 week after coronary ligation translated into improved cardiac function and reduced scar burden which was attributable to increased production of pro-healing cytokines, extracellular vesicles, and microRNAs. CONCLUSIONS: Continuous physiological cell culture increased cell growth, paracrine output, and treatment outcomes to provide the greatest functional benefit after experimental myocardial infarction.

Glyoxalase 1 Prevents Chronic Hyperglycemia Induced Heart-Explant Derived Cell Dysfunction.

Decades of work have shown that diabetes increases the risk of heart disease and worsens clinical outcomes after myocardial infarction. Because diabetes is an absolute contraindication to heart transplant, cell therapy is increasingly being explored as a means of improving heart function for these patients with very few other options. Given that hyperglycemia promotes the generation of toxic metabolites, the influence of the key detoxification enzyme glyoxalase 1 (Glo1) on chronic hyperglycemia induced heart explant-derived cell (EDC) dysfunction was investigated. Methods: EDCs were cultured from wild type C57Bl/6 or Glo1 over-expressing transgenic mice 2 months after treatment with the pancreatic beta cell toxin streptozotocin or vehicle. The effects of Glo1 overexpression was evaluated using in vitro and in vivo models of myocardial ischemia. Results: Chronic hyperglycemia reduced overall culture yields and increased the reactive dicarbonyl cell burden within EDCs. These intrinsic cell changes reduced the angiogenic potential and production of pro-healing exosomes while promoting senescence and slowing proliferation. Compared to intra-myocardial injection of normoglycemic cells, chronic hyperglycemia attenuated cell-mediated improvements in myocardial function and reduced the ability of transplanted cells to promote new blood vessel and cardiomyocyte growth. In contrast, Glo1 overexpression decreased oxidative damage while restoring both cell culture yields and EDC-mediated repair of ischemic myocardium. The latter was associated with enhanced production of pro-healing extracellular vesicles by Glo1 cells without altering the pro-healing microRNA cargo within. Conclusions: Chronic hyperglycemia decreases the regenerative performance of EDCs. Overexpression of Glo1 reduces dicarbonyl stress and prevents chronic hyperglycemia-induced dysfunction by rejuvenating the production of pro-healing extracellular vesicles.

Systematic review of biological therapies for atrial fibrillation.

Biological therapies that increase or suppress the expression of transcripts underlying atrial fibrillation (AF) progression are increasingly being explored to create novel treatment paradigms beyond simply suppressing or destroying tissue. To date, there has been no systematic overview of the preclinical evidence exploring manipulation of fundamental biological principles in the treatment of AF. As such, the objective of this study was to establish the effect of biological approaches used in the treatment of AF within large and small animals. We performed a systematic search using predefined terms, which yielded 25 studies. We determined the effect of biological approaches on primary efficacy outcomes and assessed the quality of included studies or possible bias in the treatment of AF. Compared with non-transduced or transduced controls, biological therapies reduced AF inducibility (85% less AF; odds ratio 0.15; 95% confidence interval [CI] 0.07-0.35; P < .01) and atrial scar burden (6.7% smaller scars; 95% CI 4.2-9.2; P < .01) or increased number of days in sinus rhythm (6.4 more days in sinus rhythm; 95% CI 5.83-6.97; P < .01). Similar effects were seen in both large and small animals, while a minor tendency to higher risk of bias was observed in small animal studies. In conclusion, treatment with any biological therapy significantly improved AF in preclinical animal models compared with controls. Although biological therapies target markedly different fundamental mechanisms, we observed a consistent difference in their effect on AF outcomes.

Deterministic Encapsulation of Human Cardiac Stem Cells in Variable Composition Nanoporous Gel Cocoons To Enhance Therapeutic Repair of Injured Myocardium.

Although cocooning explant-derived cardiac stem cells (EDCs) in protective nanoporous gels (NPGs) prior to intramyocardial injection boosts long-term cell retention, the number of EDCs that finally engraft is trivial and unlikely to account for salutary effects on myocardial function and scar size. As such, we investigated the effect of varying the NPG content within capsules to alter the physical properties of cocoons without influencing cocoon dimensions. Increasing NPG concentration enhanced cell migration and viability while improving cell-mediated repair of injured myocardium. Given that the latter occurred with NPG content having no detectable effect on the long-term engraftment of transplanted cells, we found that changing the physical properties of cocoons prompted explant-derived cardiac stem cells to produce greater amounts of cytokines, nanovesicles, and microRNAs that boosted the generation of new blood vessels and new cardiomyocytes. Thus, by altering the physical properties of cocoons by varying NPG content, the paracrine signature of encapsulated cells can be enhanced to promote greater endogenous repair of injured myocardium.

Interleukin-6 Mediates Post-Infarct Repair by Cardiac Explant-Derived Stem Cells.

Although patient-sourced cardiac explant-derived stem cells (EDCs) provide an exogenous source of new cardiomyocytes post-myocardial infarction, poor long-term engraftment indicates that the benefits seen in clinical trials are likely paracrine-mediated. Of the numerous cytokines produced by EDCs, interleukin-6 (IL-6) is the most abundant; however, its role in cardiac repair is uncertain. In this study, a custom short-hairpin oligonucleotide lentivirus was used to knockdown IL-6 in human EDCs, revealing an unexpected pro-healing role for the cytokine. METHODS: EDCs were cultured from atrial appendages donated by patients undergoing clinically indicated cardiac surgery. The effects of lentiviral mediated knockdown of IL-6 was evaluated using in vitro and in vivo models of myocardial ischemia. RESULTS: Silencing IL-6 in EDCs abrogated much of the benefits conferred by cell transplantation and revealed that IL-6 prompts cardiac fibroblasts and macrophages to reduce myocardial scarring while increasing the generation of new cardiomyocytes and recruitment of blood stem cells. CONCLUSIONS: This study suggests that IL-6 plays a pivotal role in EDC-mediated cardiac repair and may provide a means of increasing cell-mediated repair of ischemic myocardium.

Cellular mechanisms underlying cardiac engraftment of stem cells.

INTRODUCTION: Over the past decade, it has become clear that long-term engraftment of any ex vivo expanded cell product transplanted into injured myocardium is modest and all therapeutic regeneration is mediated by stimulation of endogenous repair rather than differentiation of transplanted cells into working myocardium. Given that increasing the retention of transplanted cells boosts myocardial function, focus on the fundamental mechanisms limiting retention and survival of transplanted cells may enable strategies to help to restore normal cardiac function. Areas covered: This review outlines the challenges confronting cardiac engraftment of ex vivo expanded cells and explores means of enhancing cell-mediated repair of injured myocardium. Expert opinion: Stem cell therapy has already come a long way in terms of regenerating damaged hearts though the poor retention of transplanted cells limits the full potential of truly cardiotrophic cell products. Multifaceted strategies directed towards fundamental mechanisms limiting the long-term survival of transplanted cells will be needed to enhance transplanted cell retention and cell-mediated repair of damaged myocardium for cardiac cell therapy to reach its full potential.

Translating bacterial detection by DNAzymes into a litmus test.

Microbial pathogens pose serious threats to public health and safety, and results in millions of illnesses and deaths as well as huge economic losses annually. Laborious and expensive pathogen tests often represent a significant hindrance to implementing effective front-line preventative care, particularly in resource-limited regions. Thus, there is a significant need to develop low-cost and easy-to-use methods for pathogen detection. Herein, we present a simple and inexpensive litmus test for bacterial detection. The method takes advantage of a bacteria-specific RNA-cleaving DNAzyme probe as the molecular recognition element and the ability of urease to hydrolyze urea and elevate the pH value of the test solution. By coupling urease to the DNAzyme on magnetic beads, the detection of bacteria is translated into a pH increase, which can be readily detected using a litmus dye or pH paper. The simplicity, low cost, and broad adaptability make this litmus test attractive for field applications, particularly in the developing world.

Silver deposited polystyrene (PS) microspheres for surface-enhanced Raman spectroscopic-encoding and rapid label-free detection of melamine in milk powder.

Silver nanoparticles coated amino modified polystyrene microspheres (PS-NH2/Ag NPs) with extremely high surface enhanced Raman scattering (SERS) activity and uniform surface morphology were created by precise controlling of deposition time. Nanojets that were formed underneath the individual microspheres could be used for nondestructive analysis of species adsorbed on the smooth gold or glass surface. A 10 fold enhancement of SERS was observed between PS-NH2/Ag NPs and gold surface compared to glass surface due to more effective coupling of surface plasmonic resonance. These microsphere SERS substrates could detect 2-Mercaptopyridine down to 10(-9)M. Four different thiol compounds have been successfully utilized as tags to prepare SERS encoded PS-NH2/Ag NPs microspheres. Furthermore, the potential application of these SERS substrate for rapid detection of melamine in milk powder was explored. A linear relationship was observed between SERS intensity and logarithm of melamine concentrations with the limit of detection (LOD) of 1.9 × 10(-8)mol/L. The promising advantages of easy sample pretreatment, low protein background interference, short detection time and low cost makes the PS-NH2/Ag NPs substrate a potential detection tool in the field of food safety.

Lighting Up RNA-Cleaving DNAzymes for Biosensing.

The development of the in vitro selection technique has allowed the isolation of functional nucleic acids, including catalytic DNA molecules (DNAzymes), from random-sequence pools. The first-ever catalytic DNA obtained by this technique in 1994 is a DNAzyme that cleaves RNA. Since then, many other RNase-like DNAzymes have been reported from multiple in vitro selection studies. The discovery of various RNase DNAzymes has in turn stimulated the exploration of these enzymatic species for innovative applications in many different areas of research, including therapeutics, biosensing, and DNA nanotechnology. One particular research topic that has received considerable attention for the past decade is the development of RNase DNAzymes into fluorescent reporters for biosensing applications. This paper provides a concise survey of the most significant achievements within this research topic.

Detection of bacteria using fluorogenic DNAzymes.

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.(1) PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.(2-4) Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.(5) DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.(6-8) These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 10(16) individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).(9-16) These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.(6-8) Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.(17-29) RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity. More recently, we developed a method of isolating RFDs for bacterial detection.(5) These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets. Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).(5) This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3). Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.